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Effects of corn and molasses supplements with and without feed additives on performance, voluntary intake, and digestive function in cattle fed bermudagrass hay

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Effects of corn and molasses supplements with and without feed additives on performance, voluntary intake, and digestive function in cattle fed bermudagrass hay
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Balbuena, Osvaldo, 1955-
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vii, 315 leaves : ill. ; 29 cm.

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Ammonia ( jstor )
Cattle ( jstor )
Corn ( jstor )
Digestion ( jstor )
Feed conversion ratio ( jstor )
Feed intake ( jstor )
Forage ( jstor )
Molasses ( jstor )
Propionates ( jstor )
Rumen ( jstor )
Animal Science thesis, Ph. D ( lcsh )
Dissertations, Academic -- Animal Science -- UF ( lcsh )
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theses ( marcgt )
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Thesis (Ph. D.)--University of Florida, 1996.
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Includes bibliographical references (leaves 286-314).
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Also available online.
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Typescript.
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Vita.
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by Osvaldo Balbuena.

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EFFECTS OF CORN AND MOLASSES SUPPLEMENTS WITH AND WITHOUT FEED
ADDITIVES ON PERFORMANCE, VOLUNTARY INTAKE, AND DIGESTIVE
FUNCTION IN CATTLE FED BERMUDAGRASS HAY















By

OSVALDO BALBUENA



















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996

























This dissertation is dedicated to my wife Elena for the years of love, help, and encouragement and to my children Gonzalo, Nicolas and Maria Daniela.














ACKNOWLEDGMENTS


I wish to express sincere gratitude to the supervisory committee consisting of Drs. W.E. Kunkle, D.B. Bates, J.E. Moore, A.C. Hammond, and L.E. Sollenberger. Special gratitude is extended to Drs. Kunkle and G. Hembry for financial support during the last six months of my project. I am very thankful to the Instituto Nacional de Tecnologia Agropecuaria (INTA) for financial support during my first thirty months at UF. Special thanks to the efforts of Jerry Wasdin, Dan Price, the Pine Acres and Santa Fe crew, and Dane Bernis (Feed Mill). Special thanks is extended to Jeanette Filer and Angelita Mariano for their help in the lab and during the experiments. I am in debt to John Funk, Pamela Miles, and Nancy Wilkinson from Nutrition Lab and to Sandra Armantrout and Christa Jenssen from Extension. I am also in debt to E. Bowers from STARS-Brooksville, Drs. R.C. Hill and K. Scott for allowing me to use their GC, and Dr. C.H. Courtney and Qiyun Jeng for helping me with parasitology analysis. I am very thankful to Kevin Downs, Diane Campbell, and Francisco Olbrich for their help during the experiments. I would also like to extend special thanks to Diego Rochinotti for his friendship and help, and thanks to Pedro Garces-Yepez for sharing his experience with me.

iii












TABLE OF CONTENTS


ACKNOWLEDGMENTS ... . .. . .

ABSTRACT . . ....................... vi

CHAPTERS

I INTRODUCTION . . . . 1

II LITERATURE REVIEW .... ........... 3

Introduction . . . 3
Antibiotic Feed Additives in Ruminants .... 5
Mechanisms of Action ... ........ 6
Importance of Gut Metabolism . . 12
Nonionophore Antibiotics . . 14
Bambermycins . . . . 15
Ruminant Performance . . . 17
Digestive Function . . . 26
Post-ruminal Effects . . . 31
Summary of Effects of Bambermycins . 35
Ionophore Antibiotics . . ... 36
Effects on Performance . ... .38 Ionophore Modes of Action . . 44
Ruminal effects . . . 45
Total tract digestibility . . 52 Metabolism of the host animal . 53 Animal health .. . . . 56
Interaction with Minerals ......... 57 Frequency of Feeding . . . 60
Summary of Effects of Ionophores . 62
Considerations in Feeding Molasses . 63
Digestive Function . . . 64
Intake and Performance .......... 77
Summary of Feeding Molasses ........ 79

III EFFECT OF BAMBERMYCINS AND MONENSIN IN CORN
OR MOLASSES SUPPLEMENTS ON PERFORMANCE OF
GROWING CATTLE . . . . 82

Introduction . . . . 82
Materials and Methods . . . 83
Results and Discussion . . . 91
Animal Performance ............ 93
Volatile Fatty Acids . . 113



iv









Plasma Urea Nitrogen ......... 121
Parasites ... . . 123


IV EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES
SUPPLEMENTS ON INTAKE, DIGESTIBILITY, AND DIGESTION
KINETICS IN HEIFERS ........... 125

Introduction . . . . 125
Materials and Methods . . . 126
Results and Discussion . . 135
Pre-trial . . . . 136
Latin Square Intake and Digestibility 138 Latin Square Digestion Kinetics. .... 149


V EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES
SUPPLEMENTS ON RUMEN FUNCTION . . 162

Introduction . . . . 162
Materials and Methods . . . 163
Digesta Kinetics .. .. ............ 164
Characteristics of Ruminal Fluid . 167 Ruminal and Total Tract Digestion . 168 Nitrogen Flow and Microbial Efficiency 170
Results and Discussion. . . 173
Digesta Kinetics .... ............ 175
Characteristics of Ruminal Fluid . 181 Ruminal and Total Tract Digestion . 204 Nitrogen Flow and Microbial Efficiency .. 220

VI SUMMARY AND CONCLUSIONS ...... ..... 233

Experiment 1 Animal Performance (Chapter III) 233
Experiment 2 Intake and
Digestibility (Chapter IV) ........ 237
Experiment 3 Rumen Function and
Digestibility (Chapter V) ......... 238
Conclusions . . . . 244

APPENDICES

A TABLES . . . . .. .. 247

B RAW DATA . . . . . 256

LITERATURE CITED. ......... ..... 286

BIOGRAPHICAL SKETCH ................. 315



v














Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

EFFECTS OF CORN AND MOLASSES SUPPLEMENTS WITH AND WITHOUT FEED ADDITIVES ON PERFORMANCE, VOLUNTARY INTAKE, AND
DIGESTIVE FUNCTION IN CATTLE FED BERMUDAGRASS HAY

By

Osvaldo Balbuena

December, 1996


Chairperson: W.E. Kunkle
Major Department: Animal Science

Ionophores and bambermycins have improved gain of

growing cattle fed forage based diets when mixed in grain or mineral supplements. In limited research monensin has not improved gain of growing cattle when fed in molasses based liquid feeds offered at 2 kg/d or more and the efficacy of bambermycins in this supplement has not been evaluated.

Growing cattle fed bermudagrass hay were supplemented

with 1.57 kg TDN from corn-urea (CU) or molasses-corn gluten meal (MCG), without and with monensin or bambermycins. Cattle fed supplements without antibiotics gained .62 kg/d. Monensin increased gain .035 kg/d in CU and decreased gain .029 kg/d in MCG. Bambermycins increased gain .106 kg/d in CU and .042 kg/d in MCG. Monensin decreased hay intake .14% of BW while bambermycins increased hay intake .14% of BW in


vi








vii

Year 1 and had no effect in Year 2. Monensin increased the difference between observed and predicted gains (an estimator of feed efficiency) .102 and .026 kg/d in CU and MCG, while bambermycins increased that difference .063 and .041 kg/d in CU and MCG, respectively.

Effects of CU or MCG with and without bambermycins on

feed intake and digestive function were evaluated in two 4 x

4 Latin squares with ad libitum and restricted intake. Bambermycins increased total DM intake .08% of BW but did not affect digestibility. Bambermycins increased ruminal pH (6.63 vs 6.52), decreased butyrate molar proportions (9.8 vs 10.6), and did not affect acetate:propionate ratio (C2:C3) and microbial N efficiency. Ruminal pH, total VFA, VFA molar proportions, and C2:C3 exhibited a supplement by time postfeeding interaction (P < .07). Steers fed CU had higher (P < .033) apparent (3.59 vs 2.99 g N/100 g OM) and true (2.47 vs 2.19 g N/100 g OM) microbial N efficiency, and higher ruminal feed CP degradability (69.2 vs 58.9%) than those fed MCG.

Monensin improved gain in cattle fed corn but did not

improve gain in cattle fed molasses supplement. Bambermycins improved gain in cattle fed corn and molasses supplements but this effect was greater in corn than in molasses. Increased gain due to bambermycins was not explained by changes in digestive function.














CHAPTER 1
INTRODUCTION


Warm-season grasses are the main feed resource for cowcalf and backgrounding production systems. Animal production based on warm season forages is usually lower than on temperate forages because intake and nutritive value of C4 grasses is lower. Excess biomass production during the warm and humid season is conserved as hay, haylage or stockpiled forage to be used as the basal diet during the winter months. High animal performance is not possible with this feed resource alone because intake of digestible energy often is only enough to meet maintenance requirements or support low gains. Supplemental feeds to meet animal requirements for energy, protein, and minerals are recommended in Florida. Feed additives that enhance animal production and therefore improve the biological and economical response to supplemental feeding are desirable. Molasses is a locally available energy source competitively priced compared to traditional feeds. Researchers at University of Florida have evaluated high levels of molasses supplementation with the addition of dry ingredients containing natural protein sources (molasses slurries). An effective feed additive to improve gain in cattle fed


1








2

molasses slurries at levels of 30 to 40% of the total diet is not available. Limited research has suggested that ionophores are not effective when fed in molasses slurries but more evidence is necessary.

Objectives of this research are as follows:

1. Evaluate the efficacy of ionophore (monensin) and nonionophore (bambermycins) antibiotics to improve gain in growing cattle fed high forage diets (bermudagrass hay) supplemented with a corn- or molasses-based feeds.

2. Evaluate the effects of feeding bambermycins on

voluntary hay intake and digestive function of cattle fed high forage diets supplemented with corn- or molasses-based feeds.

3. Compare the digestive function in cattle fed high

forage-based diets supplemented with corn- or molasses-based feeds.














CHAPTER 2
LITERATURE REVIEW



Introduction



Animal performance (growth, reproduction) is often suboptimal when only warm-season forages are fed. Consequently, some type of protein and(or) energy supplementation is often needed. Molasses-based supplements are often fed to cattle consuming warm-season forage diets. Feed additives that enhance growth may improve biological and economical response to supplementation.

Competitive feedstuffs are defined as those that can also be used as human food, while complementary feedstuffs are not used directly for human consumption. Rumsey (1984) and Hammond (1991) discussed the noncompetitive nature of ruminant production using the protein conversion ratio (Table 2-1). Relative input/output for ruminant protein is less efficient than protein produced by nonruminant animals. However, the use of competitive feedstuffs inputs is lower in ruminant production systems. The advantage of ruminants is the use of complementary feeds. The challenge is, therefore, to increase the animal output using noncompetitive feeds.

3








4

Table 2-1. Conversions of total and competitive feed
protein into animal protein

Protein conversion ratioa
Production unit Totalb Competitivec Ruminant
Beef 7.11 2.30 Sheep 14.50 1.90 Dairy 4.10 .95 Average 8.57 2.05 Nonruminant
Swine 5.92 5.50 Broilers 3.91 2.50 Layers 3.91 2.20 Average 4.58 3.40
a Rumsey (1984).
b Total input per unit produced.
c Competitive input per unit produced.


When considering intensification of ruminant production to improve its competitiveness, research focusing on the following items should be beneficial:

a) Improve the utilization of high fiber feeds to take advantage of the comparative advantage of ruminants. In developing countries the feed input in ruminant production is almost exclusively rangelands, improved pastures and crop residues.

b) Use supplementation strategies, when pasture quality and(or) quantity is decreased, as an effective way to increase digestible energy intake and(or) deliver protein and other essential nutrients needed to balance the diet. As part of these strategies, the use of feed additives could improve and potentiate the response to supplements, both biologically and economically. Supplementation programs vary








5

widely from the more traditional corn-soybean meal to a variety of by-product feeds.

This review will focus on the use of antibiotics as feed additives in ruminants. Molasses supplementation in cattle fed high roughage diets will be briefly discussed.


Antibiotic Feed Additives in Ruminants



Feed additives that enhance animal performance through increased growth rate and(or) feed conversion in clinically healthy and nutritionally normal animals are termed growth promoters. According to Armstrong (1986), growth promoters can be defined as substances, other than a dietary nutrient, that increase growth rate and(or) feed efficiency in healthy animals fed a balanced diet. In contrast, feed additives that act prophylactically as disease suppressants are not growth promoters. Sometimes the same compound may serve both roles. For example, ionophores are coccidiostats in poultry and growth promoters in cattle. In practice it is often difficult to differentiate which role is more prevalent in a given situation. Other names for these effects found in the literature include nutrition improvers, growth permittants, growth effectors, and rumen additives (Muir, 1985).

Hays (1991) presented an overview of the beneficial

response to antibiotics used at subtherapeutic levels (Table 2-2). These data illustrate that the response in young calves and pigs is greater than that in older animals, which








6

is consistent with one of the mechanisms proposed (disease control). Improvement in gain and feed efficiency account for the major portion of economic benefits and have reduced food cost (beef, pork, chicken and poultry) to consumers by more than 3.5 billion dollars annually in the USA (CAST, 1981). It is estimated that 45 to 55% of the antibacterial agents produced in the USA are administered to animals. Such estimates include the ionophores (Hays, 1991). Table 2-2. Beneficial responses to subtherapeutic levels
of antibiotics by several species

Percentage improvement
Number of
Speciesa experiments Gain Feed:gain Broiler chick 286 2.94 2.48 Turkey 126 7.03 3.83 Beef cattle 65 4.92 5.27 Layer hens 244 4.01 4.72 Pigs:
Starter stage 378 16.09 6.90 Growth stage 276 10.68 4.47 Grower-finisher 279 3.97 2.08 Young calves 85 14.29 a From Hays (1991).


Mechanisms of Action


The mechanisms by which antibiotics improve animal

production in healthy animals has not been fully clarified. Three modes of action has been postulated for both, monogastric and ruminant species (Hays, 1991):

Metabolic effect. A direct effect has been shown for antibiotics that are absorbed, for example, inclusion of








7

chlortetracycline in the diet altered water and nitrogen excretion in pigs. However, antibiotics that are not absorbed can have indirect effects through nutrients available for absorption. Data that support a metabolic effect also tend to support a disease control or nutritional effect as the metabolic processes may be influenced by systemic or gastrointestinal infections, or by absorption of microbial metabolites.

Nutrient sparing effect. An increased response to

antibiotics in the presence of nutritional deficiencies can be of major economic significance. Antibiotics may partially bridge the gap between nutritionally optimal and economically practical diets. Nutrient sparing effects can occur by one or more of the following:

a. Bacteria with similar requirements for critical
nutrients (vitamins and amino acids) are
inhibited.
b. Improved absorption of those nutrients that are
available in limited quantities (changes in gut
wall thickness).
c. Interaction of protein levels (quantity) and
source (quality) and antibiotics may occur.
Beneficial effects of adding antibiotics have been
enhanced in animals fed protein deficient diets
(quantity) and when feeding vegetable protein
diets, compared to milk protein diets (quality). Interpretation is difficult because these effects can be confounded with intake change. Enhancement of growth and feed efficiency is frequently associated with an increase in feed intake.

Disease control effect. Mainly related with control of subclinical diseases (analogous to subclinical parasitosis








8

of the gastrointestinal tract). The major benefit from subtherapeutic use of antibiotics results from the suppression of harmful microorganisms.

In a summary of the effect of the gastrointestinal

micro flora, Visek (1978) stated that they a) affect growth and development of the host, b) influence nutritional requirements, c) affect morphogenesis of the gastrointestinal tract, d) modify metabolic activity of endogenous and exogenous substances introduced into the gastrointestinal lumen and e) play an active role in preventing foreign microorganisms from becoming established. The most convincing evidence is the lack of improved growth under germ free-conditions. Additional evidence includes the following: a) inactivated antibiotics do not have any effect on growth or feed utilization, b) injected antibiotics promote growth to the extent that they are secreted into the gut after injection, c) antibacterial agents are generally more effective growth promoters in quarters where hygiene is poor than in new or clean environments (Visek, 1978).

It has been suggested that the overall effect of

antibiotics in ruminants is likely to be a composite of the effect of the antibiotics on the micro flora and fauna within the reticulorumen, and that resulting from any subsequent effect of the antibiotic within the small intestine and probably in the cecum and colon (Armstrong, 1984). Ruminal effect will be covered when discussing








9

specific drugs. Direct and indirect effects at the intermediate metabolism level are difficult to separate from the above, but cannot be ruled out. For post-ruminal effects to occur, the antibiotic included in the feed must survive the ruminal environment and not be absorbed or excreted post-ruminally.

Events in the small intestine. It may be useful to review effects at the small intestine level, even though most information was generated with monogastric species. Most of the comments are extracted from Visek (1978), Coates (1980), and Parker and Armstrong (1987). In germ-free animals there are specific changes in the histology of small intestinal villi with a reduction in the rate of enterocyte migration up the villus. In addition, there are changes in enzyme activity and rates of nutrient absorption.

A finding that was observed early and reported by

several laboratories is a reduction in weight of the small intestine in antibiotic-fed chickens, swine and rats. There is evidence, from studies in pigs, that inclusion of antibiotics in the diet resulted in changes in morphology of the small intestine. Elongated villi and higher villus:crypt ratio were reported which is indicative of a lower rate of enterocyte migration. It has been suggested that a reduction in the production of toxic by-products normally arising from microbial activity in the digestive tract could reduce enterocyte damage and therefore lower cell renewal rates.








10

Degradation of endogenous urea to ammonia has been proposed as one of the negative effects of the microbial flora. Ammonia concentration in the intestinal lumen is above the concentration required to kill cells, alter nucleic acid synthesis, and depress immune response. Depression of bacterial ammonia production in the small intestine may therefore be one mechanism by which antibiotics stimulate growth (Visek, 1978). Antimicrobial compounds reduce ammonia concentrations and the production of amines, particularly cadaverine (Parker and Armstrong, 1987).

In germ-free animals the intestine and associated

lymphoid structures contain less tissue, and cells of the intestinal mucosa are replaced at a slower rate (Coates, 1980). Their intestines are thinner and nutrients pass through them more rapidly in vitro. Germ-free animals also have a lower basal metabolic rate (Visek, 1978).

Reduction of microbial activity has also been

suggested as playing a role in bile acid metabolism, enzyme activity and efficiency of absorption. Data discussed by Armstrong (1986) showed that uptake of methionine and glucose was increased in germ-free compared with conventional chickens. However, this increase was significant when expressed per gram of intestinal tissue but was not significant when expressed per unit length of the intestine. Intestinal tissues of conventional animals fed diets supplemented with antibacterial agents develop










characteristics similar to those of germ-free animals (Visek, 1978; Coates, 1980).

Inclusion of avoparcin in rat diets resulted in

increased aminopeptidase activity in the illeal mucosa. Microbes within the small intestine of poultry are able to deconjugate bile acids with impairment of lipid absorption but it is uncertain to what extent the process is of significance for the bird. Antimicrobial feed additives in pig diets resulted in increased sucrase activity throughout the length of the small intestine. It has also been suggested that bacterial protease activity may play a role in the turnover of brush-border proteins, in which case reduction of bacterial numbers might increase mucosal enzyme activity. In pigs, virginiamycin enhanced uptake (9%) of free amino acids from a temporarily isolated intestinal loop (Parker and Armstrong, 1987). In sheep, avoparcin increased the number or activity of glucose receptors in brush border membrane vesicles in vitro (Parker, 1990).

A population of approximately 106 bacteria/g of small intestine content has been reported. Most of the isolates were gram-positive and were able to utilize starch. Possible competition with the host for starch digestion and other ruminal bypass nutrients has been suggested (Nicoletti et al., 1984).








12

Also, improved animal performance may be due to reduced energy cost of gut metabolism (Visek, 1978; Parker and Armstrong, 1987; Parker, 1990). Importance of Gut Metabolism


Gastrointestinal tract (GIT) is one of the most active of the organ masses. In a week-old pre-ruminant lamb the fractional synthesis rate of protein (FSR) was 69%/d which is three times the FSR in skeletal muscle of the same animal. In older animals, the GIT may contribute up to onethird of total protein synthesis in the animal and equal or exceed muscle synthesis by up to 250%. The passage from the pre-ruminant to ruminant increases protein synthesis not only in the reticulo-rumen but also in the intestines (Lobley, 1993). Contribution of different tissues to total protein synthesis is summarized in Table 2-3.

Protein FSR of more than 100%/d has been observed for jejunum and duodenum in 8-wk-old weaned lamb, which was equivalent to a mean half-life of 14 to 18 h. Epithelial cell renewal times did not explain the rapid turnover. Renewal times (estimated from cell migration rate) were 60 to 90 h in comparable lambs. Intracellular synthesis of secretory proteins may account for the difference. In his review, Lobley (1993) concluded that the larger GIT mass and the substantial contribution this tissue makes to both the whole-body synthesis and to the overall protein economy of








13

ruminants makes this a particular target for potential manipulation.


Table 2-3. Contribution of major tissues to whole body
protein synthesis (% of total) Tissue or organ
Rest of
Animal (age)a Muscle Skin GIT tract Liver body Lamb (1 wk) 29 13 12 (4) 12 34 Lamb (8 month) 18 20 26 (6-8) 8 28 Cattle (1-8 yr) 20 14 35 (6-8) 4 27 a From Lobley (1993). Values in brackets are percentages of total body weight (wt/wt).

A manipulation of N partitioning towards muscle and away from the gastrointestinal tract should give two advantages to the animal. First, a significant drain to the N economy of the animal relates to secretions and desquamations occurring in the gastrointestinal tract. The desquamated epithelial cell protein, excreted mucosal proteins, and other digestive secretions are digested and the amino acids are absorbed, but this resorption is unlikely to approach 100%. Second, the amino acid composition of gastrointestinal protein and other tissues (muscle, wool) are different. The match in need versus supply is superior in muscle relative to other tissues. Gastrointestinal tract secretions have high demand for valine, threonine, serine and proline (MacRae and Lobley, 1991).

Data derived from fluxes across the portal-drained viscera (PDV, includes gut, pancreas, spleen and omentum)








14

and liver of multi-catheterized cattle provided measurements of energy metabolism of gut tissues. Oxygen uptake by the PDV gave an estimate of heat energy (HE) attributable to those tissues. The PDV accounted for 18 to 25% and the liver 17 to 25% of the whole body oxygen uptake, or energy lost as HE (Huntington, 1990). He concluded that PDV and liver are metabolically active at rates disproportionately greater than their contribution to body mass, together they account for half the HE. McBride and Kelly (1990) reviewed the contributions of various biochemical processes to overall energy in the GIT (Table 2-4). These data suggested that due to the large contribution of GIT and liver to whole-animal energy expenditure, their manipulation could alter the energetic efficiency of ruminant production. Table 2-4. Metabolic energy expenditures pertaining to
the ruminant gastrointestinal tract (GIT)

GIT energy Whole-body energy Itema expenditure, % expenditure, % Na, K-ATPase 29 to 62 5.7 to 12.4 Protein synthesis 20 to 23 4.0 to 4.6 Protein degradation 4.3 .9 Total 53 to 90 10.6 to 17.9 From McBride and Kelly (1990).


NonionoDhore Antibiotics



Numerous antibiotics have been or are in use for growth promotion (Table 2-5). They represent a diverse group differing in chemistry, primary antibacterial spectrum, mode








15

of action of bacterial inhibition, molecular weight, and absorption from the GIT. Antibiotics that are not absorbed from the gut or poorly absorbed at the low dosage used are desirable as feed additives, because of the absence of residues in milk and meat and because there is no need for a withdrawal period before slaughter (Nagaraja, 1995).

Bacitracin, chlortetracycline, oxytetracycline, and

tylosin have been approved for control of liver abscesses in the USA. Tylosin has proven to be more efficacious than other antibiotics for this purpose and it is used routinely with monensin as an additive in feedlot diets (Nagaraja, 1995).

Avoparcin, a glycopeptide antibiotic produced by 1.j

candidus, interferes with bacterial cell wall biosynthesis. It is effective against gram-positive organisms and gramnegative species with a gram-positive structure (Armstrong, 1984). It is used as a feed additive in Europe and Australia, but it is not approved for use in the USA.


Bambermycins



Bambermycins (GainproT) is a fermentation product of a variety of Streptomyces spp., including S.bambergiensis, S. ahanaensis, S. ederensis and S. geysiriensis (HoechstRoussel, 1993). The first antibiotic complex of this group was moenomycin (flavomycin, bambermycins), reported in 1965 (Huber, 1979). They are classified as sugar lipid










Table 2-5. Characteristics of nonionophore antibiotics used in ruminants

Antibacterial Bacterial Molecular Absorption Antibioticsa Chemistry spectrum inhibition weight from gut Avoparcin Glycopeptide Narrow, Gram + Cell wall 1500 No synthesis
Bacitracin Polypeptide Narrow, Gram + Cell wall 1488 No synthesis
Chlortetracycline Tetracyclines Broad, Gram + Protein 479 Yes and Gram synthesis

Flavomycin or Phosphorus Narrow, Gram + Cell wall 1582 No Bambermycins containing synthesis
glycolipid

Neomycin Aminoglucoside Narrow, Gram + Protein 909 No synthesis
Oxytetracycline Tetracyclines Broad, Gram + Protein 499 Yes and Gram synthesis

Spiramycin Macrolide Narrow, Gram + Protein 842-898 Yes synthesis

Tylosin Macrolide Narrow, Gram + Protein 915 Yes synthesis
Viginiamycin Peptolide and Narrow, Gram + Protein 525 and 823 Yes
macrocyclic synthesis
lactone
aAdpated from Nagaraja (1995).








17

antibiotics (Berdy, 1980). These antibiotics are characterized by a high molecular weight from 1,600 to 2,100 daltons and a phosphorus concentration of about 2%. They consist of an oligosaccharide part, phosphoglyceric acid, a C25-lipid alcohol, and an UV chromophore (Huber, 1979). These glycolipid antibiotics exhibit bacteriostatic activity at very low concentrations. At higher concentrations, they are bactericidal only against actively multiplying grampositive bacteria (Huber, 1979). The mode of action of these antibiotics involves the inhibition of bacterial cell-wall biosynthesis by preventing formation of the cell wall component peptidoglycan (Huber, 1979; Berdy, 1980). Ruminant Performance


Relatively few reports are available on the effect of bambermycins in ruminants. It has been in use as feed additive (FlavomycinT) for cattle and sheep for several years in Europe and Australia.

Bambermycins tested at 0 to 80 mg/d showed no

improvement in ADG at dosages above 20 mg/d. Pooled analysis of the five studies presented in Table 2-6 for treatments 0, 5, 10 and 20 mg bambermycins demonstrated that bambermycins fed at 10 and 20 mg/d increased (P < .05) ADG of steers and heifers consuming pasture by .09 kg when compared with control cattle (Hoechst-Roussel, 1993). Grant et al. (1974) reported 5% average response to flavomycin in both gain and feed efficiency, when dosed at 0, 2.5, 5, 10, and 20 mg/d in








18

nine trials with cattle fed medium- to high-energy diets. Based on nine feedlot dose titration studies, a range of 10 to 20 mg bambermycins/d was approved to increase ADG and feed efficiency in feedlot cattle (Table 2-6, HoechstRoussel, 1993). Huber (1979) recommended 25 to 50 mg flavomycin for beef cattle. European studies used 40 to 50 mg flavomycin for beef cattle.

Two trials with growing and finishing feedlot cattle compared bambermycins with ionophores (Table 2-7). Bambermycins did not appear to depress intake and intake was higher than when monensin and lasalocid were added. Feed:gain was similar to lasalocid and ADG was similar to that achieved with monensin in one trial (Hoechst-Roussel, 1994). In the other trial, however, response to monensin was better than bambermycins for ADG and feed efficiency (Burris and Randolph, 1996). A pooled summary of four pasture trials (Table 2-7) showed that bambermycins and lasalocid had greater ADG than control, and bambermycins had greater ADG than monensin or lasalocid (Keith et al., 1995).

Flavomycin improved gain and feed efficiency when

cattle were fed beet pulp but not when fed corn silage and had no effect on DM intake in either diet (Table 2-8, De Schrijver et al., 1990). Bambermycins increased gain and feed efficiency in heifers fed high forage growing diets (Dhuyvetter et al., 1996). Bambermycins combined with A. oryzae decreased fed intake (Table 2-8). Bambermycins was








19

also effective in improving gain in cattle on pasture

offered a self fed supplement (Kunkle et al., unpublished

results, Table 2-8).


Table 2-6. Gain (kg/d) in titration studies for cattle
consuming pasture and feedlot diets, and gain
and feed efficiency for feedlot cattle

Bambermycins, mg/d

Pasture 0 5 10 20 40 60 80

1. Buffalo
grass .762 1.033 .927 .927 .973 .893
2. Smooth
brome grass .690 .742 .793 .771 .812 .788
3. Fescue/
ladino
clover .777 .773 .842 .843 .863 .856

4. Bermuda/
bahiagrass .465 .469 .505 .602
5. Wheat/
ryegrass .876 .884 .903 .945

Pooled 1-3a .78 .86e .89e .87e .91e .87e

Pooled 4&5b .67d .67d .70d .77e
Feedlot:c
ADG 1.14 1.17 1.18 1.18 1.18 Feed:gain 8.90 8.76 8.70 8.97 8.67
a Deetz et al. (1990). Yearling cattle received .45 or .9 kg control supplement or containing bambermycins. Quadratic effect (P < .0005)6 plateau was 18.5 mg of bambermycins.
Deetz et al. (1992). Yearling cattle received .45 kg corn control or with bambermycins.
C Hoechst-Roussel (1993). Cattle dosed 5 mg bambermycins/d gained
1.15 kg (different from control, P < .05) and had 8.9 feed:gain (similar to control). Cattle dosed with 10 mg or more had higher (P < .05) gain and feed efficiency than control.
Means within a row with different superscripts differ (P < .05).









20

Table 2-7. Performance data for 112-day feeding
experiments comparing bambermycins, monensin
and lasalocid


Item Control Bamberm Monen Lasal Ref

Feedlot:
ADG, kg 1.16a 1.10ab 1.05b 1
Intake, kg 8.14a 7.70b 7.75b Feed:gain 7.00ab 6.96a 7.28b

ADG, kg 1.34 1.39 1.45 1.37 2
Intake, kg 9.04 9.05 8.85 8.96 Feed:gain 6.77 6.52 6.14 6.57

Pasture:
Fescue .85b .94a .83b .84b 3
Crested wheat .61b .75a .69a .72a Bermudagrass .73a .73a .63b .76a Orchardgrass .51c .63ab .66a .56

Pooled .67Y .76x .70z .72yz
ab Means in a row with different superscript differ (P < .05).
wyz Means in a row with different superscript differ (P < .08).
1 = Hoechst-Roussel (1994). First 56 days, ration composition was 52.6 % TDN and 57.1 % NDF (brome hay 44%, corn silage 52% and a proteinenergy supplement); rest of the trial ration composition was 60.6 % TDN and 44.6 % NDF (alfalfa hay 17%, corn silage 63%, and a protein-energy supplement).
2 = Burris and Randolph (1996). Diet was corn silage (6.7% CP; 69% TDN) and 7% cracked corn ad libitum, along with .9 kg of a pelleted supplement (45% CP) which provided no additive (control), 20 mg bambermycins, 200 mg monensin or 300 mg lasalocid. Feed additives higher ADG than control (P < .11), monensin higher (P < .01) than bambermycins or lasalocid. Bambermycins higher (P < .09) feed intake than monensin or lasalocid. Monensin higher (P < .02) feed efficiency than bambermycins or lasalocid.
3 = Keith et al. (1995). All steers fed .9 kg supplement, nonmedicated (control), with 20 mg bambermycins, and 150 mg monensin or lasalocid.








21

Table 2-8. Feed intake, gain, and feed efficiency in
cattle fed bambermycins (flavomycin)

Intake, Gain, Feed to
Reference and diets kg DM kg gain
De Schrijver et al. (1990)a
Basal-1 : corn silage + 1%
BW concentrate 9.22 1.44 6.41 Basal-1 + 43 mg flavomycin 9.13 1.46 6.25

Basal-2: beet pulp 50%,
concentrate 50% 10.15 1.31 7.69 Basal-2 + 53 mg flavomycin 10.55 1.51 6.99

Pooled analysis:b
Basal-1 and 2 9.67 1.34 7.21 Flavomycin 9.80 1.43 6.84
Dhuyvetter et al. (1996)c
Basal: corn silage 38%, oat
hay 25%, barley grain
30.5%, protein supplement
5.6% 7.19 .98 7.37 Basal + 20 mg bambermycins 7.25 1.04 6.91 Basal + A. oryzae 7.17 1.03 6.92
Basal + bambermycins and
A. oryzae 7.07 NR NR
Kunkle et al. (unpublished)
Bahiagrass pastured .45
Block supplement control .58
Block supp. + flavomycin
Bahiagrass pasturee
Loose mineral control .39 Loose mineral + flavomycin .47
White-blue bulls 350 kg initial BW. Trial duration was 12 wk for each bgsal diet.
Animals were shifted diets over a 4-wk period. Probability
values for intake, gain and feed:gain were: .67, .81, and .71 (basal-l); .23, .002, and .03 (basal-2); and .36, .09, and .13 (pooled analysis).
Charolais crossbred heifers 260 initial BW. Bambermycins by A..
orvzae interaction (P = .03) for DM intake. Animals fed bambermycins and A. orvzae combined had lower (P < .05) intake than all other treatments. Gain and feed:gain reported are main effects of bambermycins and A& orvzae. Both feed additives increased ADG (P < .02) and feed efficiency (P < .93). NR = not reported.
Holstein steers and heifers of various breeds 250 kg initial BW. Supplement consumption was .29 kg/d. Gain higher (P < .05) with flavomycin.
Holstein steers and heifers of various breeds 230 kg initial BW. Supplement consumption was .27 and .38 kg/d for control and flavomycin mineral supplement. Gain higher (P < .05) with flavomycin.








22

Other reports include those of Flachowsky and Richter (1991) in which flavomycin did not affect feed intake but increased ADG (10.5%) and reduced feed and energy required per unit gain (10.6%) in heifers. Alert et al. (1993) reported 3.5% higher gain and 3.2% better energy utilization in fattening Friesian bulls supplemented with 50 mg/d flavomycin. Carcass composition or organ weight were not affected by the additive. Feed intake was increased in the first 56 d of the trial. Poppe et al. (1993) reported no influence of flavomycin on feed intake of heifers. Scott and Kay (1984) reported no effect of 40 mg of flavomycins in gain or feed efficiency of cattle fed grass silage and rolled barley supplement while monensin and salinomycin improved feed efficiency. Kay et al. (1983) reported gain increases of .15, .07, and .05 kg in three trials in cattle fed grass silage and rolled barley supplement with avoparcin, monensin, salinomycin, and 40 mg of flavomycin. Galbraith et al. (1983) reported increased gain in cattle fed 40 mg of flavomycin on barley diets (1.66 vs 1.50 kg). In young calves flavomycin tended to (P > .05) increase gain by 5 to 8% and had no effect on feed intake (El-Jack et al., 1986; Fallon et al., 1986).

The limited information available show that

bambermycins increased ADG by 1 to 29% and improved feed efficiency by 2 to 10%. Bambermycins did not affect or tended to increase feed intake when compared with control








23

diets, and tended to increase intake when compared with ionophores, especially monensin.

The effect of bambermycins on feed intake in sheep is not clear (Table 2-9). Aitchison et al. (1989b) and Murray et al. (1992) reported no effect of flavomycin on feed intake. Murray et al. (1992) reported slower rate of eating in sheep supplemented with flavomycin. The eating rate was dependent on the type of diet; it was decreased in animals fed hay-fishmeal and supplemented with flavomycins (Murray et al., 1990). In addition, sheep fed lupin seed twice a week with flavomycin and methionine ate less (P < .05) chaff on the day after feeding on the lupins and then ate more (P < .05) on the following day than sheep not fed any sulfur supplement (Murray et al., 1991).

Gain and wool production response to bambermycins appear related to diet (Table 2-9). Murray et al. (1992) suggested that the lack of wool production response to flavomycin in growing sheep may be related to partitioning of dietary protein towards tissue rather than wool growth. Maturity (age) and nutritional history of sheep may have an important effect on wool production. They also suggested that the best response to flavomycin will be obtained in adult sheep in areas where the feed supply is reasonably constant.

In summary, bambermycins increased gain of cattle fed a variety of diets, and it appears especially indicated in








24

Table 2-9. Effect of flavomycin and other feed additives
in sheep

Daily
Feed Daily wool
Intake, gain, growth,
Reference and diets g/d g/d g/m2

Aitchison et al., (1989a)
Oaten chaffa
Control 726
Lasalocid 27 5.11 Avoparcin 27 4.40 Flavomycin 21 4.89 43 5.52
Pelleta 591 Control -3 6.40 Lasalocid -9 6.37 Avoparcin -7 5.75 Flavomycin 9 6.82

Aitchison et al., (1989b)
Run I (weeks 1 to 4)
Wheaten chaffb
Control 865 149 5.5 Flavomycin, 10 ppm 895 165 5.9 Flavomycin, 20 ppm 853 174 6.8 Tetronasin, 5 ppm 893 179 5.7 Tetronasin, 10 ppm 843 104 5.7

Pelletb
Control 1503 352 11.5 Flavomycin, 10 ppm 1573 359 12.7 Flavomycin, 20 ppm 1500 344 14.1 Tetronasin, 5 ppm 1548 390 13.0 Tetronasin, 10 ppm 1519 367 11.8

Run II (weeks 5 to 9)
Wheaten chaffb
Control 926 38 6.2 Flavomycin, 10 ppm 920 25 5.8 Flavomycin, 20 ppm 962 30 5.8 Tetronasin, 5 ppm 965 7 7.0 Tetronasin, 10 ppm 912 25 5.5

Pelletb
Control 1727 231 11.6 Flavomycin, 10 ppm 1875 289 11.8 Flavomycin, 20 ppm 1835 303 13.3 Tetronasin, 5 ppm 1800 278 14.0 Tetronasin, 10 ppm 1720 276 13.4








25

Table 2-9 --continued

Daily
Feed Daily wool
Intake, gain, growth,
Reference and diets g/d g/d g/m2
Murray et al. (1990)
Lucerne-lupinsc NR
Control 138 13.4 Flavomycin, 10 ppm 144 13.0
Flavomycin, 20 ppm 162d 13.8 Flavomycin, 30 ppm 152 13.5
Hay-fishmealc NR
Control 161 13.2 Flavomycin, 10 ppm 151 14.6 Flavomycin, 20 ppm 142d 15.5d Flavomycin, 30 ppm 130d 15.4d a Diets fed at maintenance to Merino wethers, 37 kg BW. Oaten chaff 6% CP; pellet diet (59% lucerne, 25% lupins, and 15% barley) had 20% CP. Lasalocid: 30, 50, and 70 ppm; avoparcin: 25, 50, and 75 ppm, flavomycin: 5, 15, and 30 ppm. Reported values are means of the three levels because no effect of level. Flavomycin higher (P < .05) gain than all others treatments. Flavomycin higher (P < .01) wool production than other additives and higher (P < .1) wool production than control. Pellet greater (P < .001) wool production than chaff diets.
Weaner Merino wethers, 29 kg BW, given ad libitum access to diets. Chaff diet had 7.3% CP; pellet diet (59% lucerne, 25% lupins, and 15% barley) had 19% CP. Difference due to additive: both additives increased (P < .001) gain of sheep eating pellet in Run II, and both additives increased (P < .01) wool growth in sheep fed pellet in both Runs. C Twenty-month old merino ewes fed at 3.5% of BW. Lucerne-lupin diet (58% lucerne, 25% lupin, 15% barley) had 18% CP; hay-fishmeal (24.5% lucerne, 51.5% wheat chaff, 10% lupin, 12% fishmeal) had 17.3% CP. Flavomycins linearly (P < .001) depressed gain in animals fed hayfishmeal.
d Indicates value different from control (P < .005).


high roughage diets. Bambermycins appears to have little

effect on feed intake when compared with control. Animals

supplemented with bambermycins appear to have higher feed

intake than the ones supplemented with ionophores,

especially monensin. How bambermycins affects intake of

warm-season grasses (pasture or hay) is unknown. Other feed

additives, such as monensin, have been shown to have a

variable effect on forage intake (Ellis et al., 1984).








26

Because bambermycins is targeted for use with high fiber diets it is a high research priority to evaluate its effect on intake in this type of diet. Bambermycins also improved efficiency of feed utilization. This information, however, has the same limitations indicated for intake.

Limited information suggests that bambermycins did not affect amount of feed intake in sheep. Reports only mentioned effect on rate of eating and it is not clear if this effect translated into effects on performance. Information from sheep research did little to clarify the effect of bambermycins on intake, gain and feed efficiency.

There are no available data on the effect of

bambermycins with high roughage diets supplemented with molasses. Limited information suggests that ionophores may not be efficacious in those diets. Research is needed to test the efficacy of bambermycins for improving gain, and to evaluate its effects on feed intake and efficiency of feed utilization with cattle fed this type of diet. Digestive Function


In vitro. Bambermycins included at 8 or 20 ppm in media had very little or no effect on several variables. Substrates included casein, amino acids, cellobiose-maltose, neutral detergent fiber, and starch. Variables measured included proportion of volatile fatty acids produced from different substrates, methane and ammonia production, efficiency of bacterial growth, and fiber and starch








27

digestion. The only effect of flavomycin was a higher proportion of acetic acid and lower proportion of butyric acid when neutral detergent fiber was the substrate. Flavomycin, one of 15 additives tested, had less effect on variables studied than most of the other additives (Van Nevel and Demeyer, 1990; Van Nevel and Demeyer, 1992).

Ruminal pH. Bambermycins inclusion in the diet

increased ruminal pH at the end of week 4; however, by the end of week 9 there was no effect on pH (Murray et al., 1992). Using high (pellet) and low (chaff) quality diets at maintenance level, Aitchison et al. (1989a) found that inclusion of bambermycins increased ruminal pH (6.94 vs

6.57, average of both diets). When the same diets were given ad libitum, there was a trend toward a similar effect (Aitchison et al., 1989b). No effect of bambermycins on pH was found in diets supplemented with different sources of sulfur (Murray et al., 1991), or 10, 20 and 40 ppm bambermycins (Murray et al., 1990). Because samples were obtained with stomach tube in these trial, the sampling technique may have contributed to the variability observed.

Ruminal ammonia. Bambermycins increased ruminal ammonia concentrations in weaner lambs (147 vs 117 mg N/L) and adult sheep (180 vs 173 mg N/L) on a diet of alfalfa chaff (30%), chopped wheaten hay (52%), fishmeal (6%) and lupin grain (10%), containing 16.4% CP (Murray et al., 1992). Other work reported by the same researchers showed no effect of








28

bambermycins on ruminal ammonia with diets of different qualities and supplemented with several sources of sulfur (Murray et al., 1991). When alfalfa-lupin and hay-fishmeal diets were supplemented with 10, 30 and 40 ppm bambermycins, ruminal ammonia concentrations were depressed only in the hay-fishmeal diet (Murray et al. 1990). Bambermycins produced opposite effects on ruminal ammonia concentration depending upon the level of feed intake. Bambermycins added to high or low quality diets fed at maintenance level increased ruminal ammonia (349 vs 291 mg N/L) in the high quality diet only (Aitchison et al., 1989a). However, when those diets were fed ad libitum bambermycins reduced ruminal ammonia concentration (168 vs 216 mg N/L) in the high quality diet (Aitchison et al., 1989b). Bambermycins inclusion in concentrate fed to young calves had no effect on ruminal ammonia concentrations (El-Jack et al., 1986; Fallon et al., 1986). Rowe et al. (1982) also did not find an effect of flavomycin on ruminal ammonia in cattle.

Ruminal volatile fatty acids. Inclusion of bambermycins in the diet decreased the total VFA concentration (65.3 vs 78.5 mM/L) by the end of wk 4, but there was no effect of bambermycins by wk 9. Molar proportions of propionate were increased at both sampling times from 25 to 28 mol/100 mol (Murray et al., 1992). In another trial, bambermycins increased total VFA when the diet was supplemented with methionine, but it had no effect on total VFA or propionate








29

molar proportion when the diet was supplemented with other sulfur sources (Murray et al., 1991). In lucerne-lupin and hay-fishmeal based diets, addition of 10, 20 and 40 ppm bambermycins did not affect total VFA or acetate proportions. However, propionate proportion was increased, while butyrate was decreased in both diets. The level of 20 ppm of bambermycins was the most consistent in producing these effects (Murray et al., 1990).

Bambermycins did not affect total VFA or molar

proportions of individual VFA when added to low or high quality diets fed at maintenance levels (Aitchison et al., 1989a). When these diets were fed ad libitum, 10 ppm bambermycins decreased total VFA in both diets. Addition of 20 ppm bambermycins increased propionate proportion in the high quality diet only (Aitchison et al., 1989b).

In fattening cattle, bambermycins did not affect total VFA or VFA molar proportions (Flachowsky and Richter, 1991; Alert et al., 1993). Also, in young calves, flavomycin inclusion in the dry feed had no effect on VFA proportions in ruminal fluid (El-Jack et al., 1986; Fallon et al., 1986). In contrast, Earley et al. (1996) reported that bambermycins lowered the acetate:propionate ratios in steers fed alfalfa-grass hay. Steers fed monensin, however, had lower acetate:propionate ratios than steers fed bambermycins. In a similar experiment, DelCurto et al. (1996) reported higher total VFA in steers fed a 90%








30

concentrate diet with bambermycins. In this trial, steers fed bambermycins had lower acetate:propionate ratios than the ones fed lasalocid.

Ruminal Drotozoa. Bambermycins in the diet did not affect the number of protozoa in a nine-week trial with sheep (Murray et al., 1992). Alert et al. (1993) reported similar findings in cattle.

In summary, bambermycins has not had consistent effects on ruminal pH, ammonia, total VFA concentrations or molar proportions of VFA. This may be related to experimental conditions and(or) bambermycins may not have a specific action on ruminal fermentation, in contrast to ionophores.

Ruminal digestion. Bambermycins did not affect

cellulose degradation, total VFA or VFA proportions in the rumen in cattle (Rowe et al., 1982), even though they used a high dose (20 mg/100 kg body weight). This was interpreted as no effect of bambermycins on ruminal fermentation. In situ rate of digestion was not affected by addition of bambermycins or ionophores in steers on alfalfa-hay diets (Early et al., 1996). On the other hand, bambermycins decreased OM, CP and CF digestibility in the rumen (Poppe et al., 1993). Bacterial microbial protein production was also reduced. However, 30 g/d more amino acid, apparently of dietary origin, reached the gut in bulls supplemented with bambermycins.








31

Total tract digestion. Bambermycins did not affect

total tract digestibility of DM, CP, fat, CF, ash and N-free extract in wethers offered a diet of 50% beet pulp and 50% concentrate at maintenance level (De Schrijver et al., 1991). Flachowsky and Richter (1991) also reported no effect of 5 or 10 mg of bambermycins/d on apparent OM digestibility in wethers. In an experiment with fattening Friesian bulls, 50 mg of bambermycins/d increased apparent DM, CF and N-free extract digestibility (Alert et al., 1993). Increased total tract OM digestibility was also reported by Poppe et al. (1993) in cattle. In young cattle, addition of flavomycin increased total tract CP digestibility (Fallon et al., 1986). Total tract digestibility tended to increase in steers supplemented with bambermycins or ionophores on alfalfa-hay diets (Earley et al., 1996). However, no effect of feed additive was observed in 90% concentrate diets (DelCurto et al., 1996).

There is no consistent effect of bambermycins on ruminal or total tract digestibility. Post-ruminal Effects


Rowe et al. (1982) measured the post-ruminal

antibacterial activity of bambermycins by inhibition bioassay using Bacillus subtilis. There appeared to be no loss of antibacterial activity of bambermycins in the sheep digestive tract. The presence of active antibiotic in the intestine prompted suggestions that bambermycins may act at








32

the post-ruminal level, similar to the mechanism of action proposed in monogastrics (MacRae, 1989; Rowe et al., 1991).

Bambermycins increased whole body protein accretion (Table 2-10) when given alone or in combination with clenbuterol. These findings support the notion that antibiotics active in the gastrointestinal tract reduce the mucosal cell turnover by reducing microbial invasion, thereby allowing a greater net partitioning of amino acids towards other body tissues (MacRae, 1989; MacRae and Lobley, 1991).

Post-ruminal effects have also been proposed for

avoparcin. MacGregor and Armstrong (1984) used mature sheep fitted with ruminal cannulas and re-entrant cannulas at the proximal duodenum and the terminal ileum. Avoparcin and(or) saline solution were continuously infused into the proximal duodenum and subsequently into the rumen of four sheep. Results of their study and those of a previous one where avoparcin was incorporated in the diet (MacGregor and Armstrong, 1982) are presented in Table 2-11. Table 2-10. Protein accretion (g/d) in sheep given
clenbuterol (1.5 mg/d) and(or) bambermycins
(20 mg/d)

Treatmenta Control Clenbuterolb Control 20.6 35.0 Bambermycinsc 25.0 39.4
From MacRae and Lobley (1991). Sheep (n = 12) were fed pelleted dried crass at twice maintenance level of energy intake.
SEffect of clenbuterol, P <.001.
C Effect of bambermycins, P <.05.








33

Table 2-11. Effect of avoparcin on the net uptake of
amino acids from the small intestine (g amino
acid N/g amino acid N entering the small
intestine)

Duodenal Ruminal Included in infusion infusion feed Amino acida Con Avop Con Avop Con Avop Total .648 .697 .634 .692 .598b .687c Essential .613 .679 .664 .734 .553 .682 Non-Essential .628 .689 .673 .747 .580 .718 MacGregor and Armstrong (1982).
b,c P <.05, trends for individual amino acids were in the same direction. Con = control; Avop = avoparcin.


Avoparcin increased net uptake of amino acids when

included in the diet but not when ruminally or duodenally infused. Nevertheless, the authors felt that the trends were consistent with avoparcin enhancing net uptake of amino acids from the small intestine and that this effect was independent of any effects the antibiotic may have on digestion occurring in the rumen.

Further work in rats showed that increasing levels of avoparcin in the diet increased intestinal dipeptidase activity (units/g fresh weight of mucosa) and specific activity (units/mg protein). If the hydrolysis of dipeptides to amino acids by the action of dipeptidases was the rate limiting step in the transfer of amino-N from the intestinal lumen to the portal blood then an increase in dipeptidase activity stimulated by avoparcin could account for at least part of its growth-promoting effect (Parker et al., 1984).








34

Predominant bacteria in the small intestine are grampositive which are sensitive to many antibiotic additives, whereas those in the large intestine are gram-negative. Therefore it is expected that the small intestine is the more likely site for antimicrobial effects (Parker, 1990).

Avoparcin was associated with increased N retention,

probably due to lower turnover of gut mucosa (MacRae, 1989; (Parker, 1990). To test the hypothesis that avoparcin will affect gut tissue metabolism of ruminants, Parker (1990) conducted a trial in which weaned lambs were fed a pelleted diet containing either 0, 19 or 28 ppm avoparcin for 6 wk. At the end of the trial, five sheep from each group were anesthetized and injected with vincristine, which causes any cell entering into mitosis to be arrested at metaphase. Rate of cell division in the crypts of duodenal tissue after 90 min was significantly lower with avoparcin treatment, providing evidence for a nutrient-sparing effect in the small intestine. Thus, it appears that the effect of avoparcin in the small intestine of ruminants could be similar to those observed in monogastric species consuming diets supplemented with antibiotics.

Other effects. Bambermycins reduced the duration and

prevalence of Salmonella shedding in calves and reduced the number of Salmonella resistant to other antibiotics (Dealy and Moeller, 1977a). Reduction of Escherichia coli resistant to other antibiotics has also been reported (Dealy and








35

Moeller, 1977b). Flavomycin exhibited a preferential inhibition of E. coli and S. typhimurium bearing plasmids (Huber, 1979).

Summary of Effects of Bambermycins


The limited information available suggests that

bambermycins improves animal performance, but in some cases, this effect depends upon the nature of the basal diets. Effects on ruminal fermentation are not consistent. Some researchers have reported increased propionate, while most others reported no difference in VFA concentrations or molar proportions. Total tract digestibility has been increased in several reports, but not in others. The presence of active antibiotic in the small intestine suggests possible postruminal effects, and modes of action may be similar to those reported for avoparcin. Bambermycins improved gain in several experiments, but it was seldom associated with changes in ruminal fermentation that would suffice to explain this effect. Few ruminal effects support the hypothesis of post-ruminal effects. Research is needed to test this hypothesis in cattle. Techniques described by Jin et al. (1994) to evaluate intestinal growth, cell proliferation, and morphology may be useful for such purposes. However, this type of research is beyond the scope of this project. The effect of bambermycins on ruminal function and nutrient digestibility is not completely elucidated, and there is no information with high roughage








36

diets supplemented with molasses. Therefore this research will address the effect of bambermycins on digestive function in cattle fed these diets.


IonoDhore Antibiotics



Ionophores have been defined as substances capable of

interacting with metal ions, thereby serving as a carrier by which these ions can be transported across a bimolecular lipid membrane. Monensin can be described as a cation-proton antiporter while lasalocid does not display an obligatory cation-proton antiporter mechanism. Ionophores do not display the same affinity for all cations. Monensin mediates primarily Na'-H' exchange, because the affinity of monensin for Na* is ten times higher than that for K'. Lasalocid displays a higher affinity for K' (Bergen and Bates, 1984).

Accepted mechanisms by which ionophores negatively

affect bacteria include nonphysiological ion leak caused by ionophores and consequently ATP depletion. This effect is greater in gram-positive bacteria. Gram-negative bacteria have a cellular envelope (outer membrane) that serves as a protective barrier, excluding ionophore complexes (Bergen and Bates, 1984; Russell and Strobel, 1989).

Bergen and Bates (1984) summarized the effects of

monensin as follows: the ionophore acts on the flux of ions through membranes dissipating cation and proton gradients and interfering with the uptake of solutes and the primary








37

transport system in the cells. The organisms try to maintain primary transport by expending metabolic energy. Because gram-negative bacteria are able to produce ATP through electron transport, they can survive better, so there is a shift to these organisms in the rumen. It is this shift that is responsible for the final effect of an ionophore on ruminal metabolism.

Ionophores are generally bacteriostatic and not

bactericidal (Nagaraja and Taylor, 1987). Bergen and Bates (1984) suggested that monensin would cause entry of protons into ruminal bacteria in exchange for Na'. However, Russell (1987) using S. bovis as a model, showed that direction of Na' was opposite to this. Monensin produced a decrease in intracellular K' concentration and influx of protons, resulting in lower intracellular pH. Once intracellular pH was acidic, monensin produced an efflux of protons in exchange for Na'. The inhibition of S. bovis was attributed to futile cycling of ions across the cell membrane resulting in loss of intracellular K', accumulation of intracellular Na' and depletion of ATP (Russell, 1987; Strobel et al., 1989). The postulated mechanism of inhibition may be affected by high mineral concentration, as will be discussed later.

A list of ionophores used or under investigation is given in Table 2-12, taken from Nagaraja (1995).








38

Table 2-12. Ionophores used or under investigation for
use in ruminant diets

Molecular
Ionophorea weight Cation Selectivity Sequence Monensin 671 Na'>K+, Li>Rb >Cs Lasalocid 591 Ba", K >Rb*>Na >Cs >Li Laidlomycin 721 NDb Lysocellin 660 Na'>K Ca", Mg+" Narasin 765 Na'>K', Rb Cs*, Li Salinomycin 751 Rb*, Na+>K>>Cs*, Sr Ca", Mg+ Tetronasin 628 Ca">Mg +>Na', K>Rb
a From Nagaraja (1995).
ND = Not determined.


Effects on Performance


A review by Goodrich et al. (1984) summarized the

results of 228 feedlot trials and 28 pasture studies (Table 2-13). In feedlot diets, the most significant effect of monensin is an improvement of feed efficiency, as a result of little effect on gain and a reduced feed intake. A more recent review (Owens et al. 1991) showed the same trend with feedlot cattle fed monensin.

Effects of other ionophores in concentrate diets are summarized in Table 2-14, adapted from Owens et al. (1991). The effect on feed intake is dependent on the ionophore used in feedlot diets. Monensin and laidlomycin appear to represent the extreme effects of ionophores on intake. Intake decreased with increasing levels of monensin. Lasalocid showed similar trends with less depression of intake, while laidlomycin increased feed intake (Owens et al., 1991).








39

Table 2-13. Summary of effect of monensin on intake and
performance

Change,
Diet type Variable Control Monensin % Ref.a
Concentrate ADG, kg 1.09 1.10 1.6 1
Intake, kg 8.27 7.73 -6.4 Feed/gain 8.09 7.43 -7.5

Concentrate ADG, kg 1.26 1.27 .6 2
Intake, kg 8.97 8.47 -5.6 Feed/gain 7.29 6.83 -7.5

Pasture ADG, kg .609 .691 13.5 3

Pasture ADG, kg .786 .893 13.7 4

Pasture ADG, kg .540 .630 17 5

Pasture ADG, kg .560 .650 16.3 6 Pasture ADG, kg .590 .680 15.5 6

Harvested ADG, kg .612 .698 14.1 6 Forage Intake, kg 7.39 7.18 -3.1
Feed/gain 12.4 10.5 -15.3

Small Grain ADG, kg .540 .620 15 7 Pasture ADG, kg .540 .605 12
ADG, kg 1.04 1.12 7.8 ADG, kg 1.15 1.24 7.8

Pasture ADG, kg .600 .640 6.7 8
ADG, kg .860 .880 2.3 ADG, kg 1.16 1.25 7.8 ADG, kg .970 1.02 5.2 ADG, kg .610 .690 13.1
References:
1 = Goodrich et al. (1984), summary of 228 trials.
2 = Owens et al. (1991), summary of 137 trials.
3 = Goodrich et al. (1984), summary of 24 trials.
4 = Wilkinson et al. (1980), 12 trials.
5 = Potter et al. (1976), 4 trials.
6 = Potter et al. (1986), 24, 11, and 12 trials respectively.
7 = Ellis et al. (1984), each mean is one trial.
8 = Parrot et al. (1990), 8, 8, 4, 4, and 4 trials respectively.
Monensin delivered by ruminal bolus.












Table 2-14. Influence of ionophores on performance

Itema Control Monensin Lasalocid Laidlomycin Salinomycin Tetronasin Lysocellin Trials 156 137 33 44 37 13 6 ADG, kg 1.26 1.27e 1.31 b 1.35be 1.28d 1.29 1.31d Feed, kg 8.97 8.47b 8.70b 9.07c 8.74b 8.47c 8.17b Feed:gain 7.29 6.83ce 6.74bf 6.88be 6.90bf 7.30 7.29

a Owens et al. (1991). Adjusted performance measurement for each ionophores was calculated for
ionophore dosage at the mean dosage level used based on linear or, when quadratic effect was detected (P < .1), linear and quadratic regression coefficients relative to the mean of cattle fed the control diet.
b Linear change (P < .01) with level of ionophores.
Linear change (P < .05) with level of ionophores.
d Quadratic effect (P < .01) in addition to the above linear effect of ionophore level.
Quadratic effect (P < .05) in addition to the above linear effect of ionophore level.
Quadratic effect (P < .05) in addition to the above linear effect of ionophore level.















Ic








41

Effect of monensin on high roughage diets is also presented in Table 2-13. In general, the effect is translated into increased ADG and feed efficiency. The effect on feed intake appears variable. Under grazing conditions, ionophores usually improve ADG. Feed efficiency data are rarely available because inherent difficulty in measurement of feed intake in grazing animals. A more subtle effect such as no change in ADG with decreased pasture intake may occur. In this case, the benefit of feeding an ionophore will be realized only if stocking rate is increased. However, this effect is difficult to measure and may not have a real economic value (Rowe et al., 1991). Horton et al. (1992) reported an increased ADG in yearlings supplemented with ionophores (lasalocid or monensin) while grazing subtropical grass forages, but responses were inconsistent and appeared to be associated with forage quality and environmental conditions. This variable effect of ionophores (monensin) in grazing animals was related to the interaction of monensin with pasture digestibility and digestive function (Ellis et al. 1984). In grazing animals monensin generally reduces the turnover rate of undigested forage residues and thereby increases the digestibility of fiber. Intake of forages (45 to 65% OMD) was increased with monensin apparently as a result of increased undigested fill. Intake of poor quality forages (< 45% OMD) is decreased by monensin caused by a reduced turnover of








42

undigested dry matter combined with the animals' inability to accommodate further increases in fill of undigested dry matter. Intake of higher quality forages (> 65% OMD) appears to be decreased by monensin perhaps through a metabolic intake regulation, analogous to the lower intake observed with high concentrate diets. Thus the expected gain response to monensin decreases as the quality of forage consumed increases (Ellis et al., 1984). Rowe et al. (1991) suggested that in animals grazing low quality pastures, maintaining or losing weight, there is less chance of a positive response to ionophores. This opinion would agree with the depressed intake observed when ionophores are fed with low quality forage (Ellis et al., 1984).

Pond and Ellis (1979) reported three trials where the response to monensin was evaluated in cattle grazing bermudagrass pastures. Monensin increased intake (3.4%) in one trial, but it decreased intake in the other two trials (4.6 and 19.4%). Although intake was decreased, monensin increased ADG. This effect was explained by a reduction in rate of passage of digesta. The resulting increased residence time in the rumen increased digestibility of forage.

Gains were .460, .565, and .780 kg for cattle on

bermudagrass pasture alone, pasture plus .9 kg corn, and pasture plus corn and 100 mg of monensin, respectively (Oliver, 1975). Monensin increased ADG by 38%. In a similar








43

experiment, cattle on bermudagrass pasture gained .42 and .52 kg for pasture plus .9 kg corn and pasture plus corn and 200 mg of monensin, respectively. Forage to gain ratios estimated for control and monensin were 19:1 and 15:1, respectively (Rouquette et al., 1980). In a different trial, ADG were .45, .47, and .68 for steers on pasture alone, pasture plus .9 kg corn, and pasture plus corn and 200 mg of monensin, respectively. Estimates of forage to gain ratios were 20.5:1, 19:1, and 13:1, respectively (Rouquette et al., 1980). Monensin increased gain by 24 to 45% in this trial.

Byers and Schelling (1984) used an isotope dilution technique to measure body composition. They reported that digestive tract fill in cattle grazing high-quality pastures was decreased by monensin or lasalocid. When cattle grazed more mature, low-quality forage, lasalocid reduced fill, while monensin had no effect on fill. Thus, fill may be different not only under the conditions as discussed by Ellis et al. (1984), but also with different ionophores.

Ionophores also can increase the response to

supplements. This effect is particularly important because in high roughage diets ionophores are generally fed daily in .5 to 1 kg of a carrier supplement. Pasture plus supplement (no monensin) supported gains of cattle ranging from .24 to .96 kg/d (Potter et al., 1986). The addition of 200 mg monensin/d to the supplement increased gain by .09 kg/d (16.3%) across the 24 trials. In a different series of 11








44

trials, cattle on pasture alone gained .50 kg. Supplementation with .9 kg/d of energy supplement increased ADG by .09 kg, and the addition of monensin to supplement further increased ADG by .09 kg/d (Potter et al., 1986). In a series of 12 trials, monensin supplementation of harvested forage fed in confinement reduced feed intake by 3.1%, improved ADG by .09 kg (14.4%) and improved feed efficiency by 15.3%. Efficiencies of supplemental feed to extra gain (kg supplement:kg gain) were 10.1:1 and 5.0:1 for the supplemented only and the supplemented plus monensin groups, respectively (Potter et al., 1986).

Monensin can be administered through intraruminal

devices, avoiding the need for a carrier supplement. Parrot et al. (1990) reported increased ADG in steers and heifers under different environmental conditions (Table 2-13). Response to monensin may be related to stocking rate and pasture quality (Cochran et al., 1990).

Ionophore feeding has also been shown to benefit cowcalf production systems. Monensin feeding has been shown to increase ADG and reduce age at puberty in beef heifers (Sprott et al., 1988). This effect of monensin appears to be independent of growth rate (Lalman et al., 1993). Ionophore Modes of Action


The mechanisms by which ionophores improve performance or feed efficiency have been attributed principally to alterations in ruminal fermentation. However, because








45

ionophores have activity in both prokaryotic and eukaryotic cells, part of the performance may be due to effects outside the rumen (Nagaraja, 1995). According to Bergen and Bates (1984) ionophore feeding affects ruminal fermentation in three major areas: a) Increased production of propionate and decreased production of methane, b) decreased protein degradation and deamination of amino acids, c) decreased lactic acid production and froth formation in the rumen.

A summary of known effects of bambermycins, avoparcin, monensin, and lasalocid is presented in Table 2-15. Ruminal effects

Ruminal fermentation of carbohydrates, protein and

glycerol result in anaerobic oxidation to acetate, carbon dioxide and ammonia. Methane, propionate, and butyrate are produced mainly as a result of electron and proton transfer reactions (hydrogen sinks). Methanogenesis keeps the partial pressure of hydrogen very low avoiding the formation of lactate or ethanol as major end products and allowing more acetate to be produced (Van Nevel and Demeyer, 1995).

Volatile fatty acids. The most consistent fermentation alteration when ionophores are fed is the increased molar proportion of propionic acid produced in the rumen (Table 216). Increased propionate production results in improved fermentation efficiency because of a greater recovery of metabolic hydrogen (Chalupa, 1984). Furthermore, Armstrong








46

Table 2-15. Effect of antibiotic feed additives on
digestive function and disorders

Itema Monenb Lasal Avopd Bambe
Ruminal
NH3 ncg +h/i/nc Total VFA nc/- nc nc/Acetic -/nc +/-/nc Propionic + + + +/nc Butyric nc/-/+ A:P -/nc Methane nc/Rumen fill + nc nc/+ Liquid turnover nc/- nc/Solid turnover

Rumen bacteria
Yield -- nc -/nc Gram positive Lactate utilizers nc nc Rumen protozoa nc Rumen fungi

Rumen digestibility
DM nc/- + -/nc Fiber nc nc/+ nc/Protein +/Starch nc/Total tract
digestibility
DM nc/+ + + nc/+ Fiber nc/+ + nc/+ Starch nc nc Protein + + nc

Other
Bloat
Coccidia nc Lactate 3-Methylindol
Chalupa, (1984); Van Nevel and Demeyer, (1988, 1992); Bergen and Bates, (1984); Schelling, (1984); Faulkner et al., (1985); Spears, (1990); Owens et al., (1991), De Schrijver et al., (1990); Murray et al., (1990); Murray et al., (1992); Aitchison et al. (1989a, 1989b); Galbraith et al., (1983); Alert et al., (1983); Poppe et al., (1993); MacGregor and Armstrong, (1982); Chalupa et al., (1981); Froetschel et al., (1983); Nagaraja, (1995); Nagaraja et al., (1987); Early et al. (1996)i DelCurto et al. (1996). d
Monen = monensin, C Lasal = lasalocid, Avop = avoparcin, and e Bamb = bambermycin.
References: = decrease; nc = no change; h+ = increase; / = or.








47

Table 2-16. Effect of monensin of ruminal volatile fatty
acids in cattle

Dieta Variable Control Monensin % change
70:30b Concentration, mM Acetate 70 61e -13 Propionate 19 23e +21 Butyrate 10 8 -20
Proportion, %
Acetate 71.0 66.8 -6 Propionate 19.1 24.7e +29 Butyrate 9.9 8.5e -14
Production, M/d
Propionate 7.74 11.2e +46

50:50c Concentration, mM Acetate 65.8 55.9 -15 Propionate 41.1 41.9 +2 Butyrate 13.5 9.1 -33
Proportion, %
Acetate 53.5 51.3e -4 Propionate 33.4 38.4e +15 Butyrate 11.0 8.3 -25
Production, M/d
Acetate 7.32 8.68 +19 Propionate 4.82 7.30 +52 Butyrate 2.12 1.76 -17

Production, g/d
70:30d Propionate 441 659e +49 20:80d Propionate 510 899e +76 Source: Nagaraja (1995).
a Roughage:concentrate.
Prange et al. (1978).
d Rogers and Davis (1982).
d Van Maanen et al. (1978).
e Different from control, P <.05.


and Blaxter (1957) showed that the efficiency of utilization

of acetate was low when infused into the rumen as the only

energy source, but was increased by the addition of

propionate. The heat increment of acetate is reduced by

addition of glucose precursors to the diet (Tyrrell et al.,

1979). Acetate clearance rate is increased by an incresed








48

ruminal propionate:acetate ratio (Cronje et al., 1991). Propionate can be used in gluconeogenesis or oxidation, while acetate can not be used for gluconeogenesis. The increased molar proportion of propionate in ruminal fluid may represent a conservative estimate of the amount available for subsequent metabolism. It has been shown in steers fed a high roughage diet with added monensin that propionate production rate was increased by 49% while the molar proportion of propionate in the ruminal liquor only increased by 15%; comparable figures for steers fed low roughage diets were 76% and 25% (Van Maanen et al., 1978).

Methane. Methane production can be as great as 12 L/h

in beef cattle (Thorton and Owens, 1981). Methane production can represent as much as 12% loss of feed energy. Ionophores can decrease methane loses by 30% (Schelling, 1984). The effect of monensin on methane production in vivo is variable. Reduction of 16 to 31% in methane production has been reported according to research reviewed by Van Nevel and Demeyer (1995). About half of the decrease in methane production when monensin was fed was associated with the reduced feed intake. When cattle were fed hourly no effect of monensin on methane production was observed (O'Kelly and Spiers, 1992). However, depression of methane production may be transitory, with methane production returning to normal within two weeks (Rumpler et al., 1986). Monensin is not directly toxic to ruminal methanogens, but inhibits








49

organisms converting formate to carbon dioxide and hydrogen. Thus, monensin reduces the supply of substrate for methanogenesis (Van Nevel and Demeyer, 1995).

Ruminal nitrogen metabolism. Ruminal ammonia production often exceeds the needs of ammonia-utilizing species. Excess ammonia in the rumen is absorbed and converted to urea in the liver. Although some urea is recycled back to the rumen, much of it is lost in the urine (Russell and Strobel, 1989). Monensin decreases ammonia production in vitro (Van Nevel and Demeyer, 1977) and in vivo (Dinius et al., 1976; Hanson and Klopfenstein, 1979; Poos et al., 1979). Ionophores appear to affect ruminal degradation of peptides and deamination of amino acids to a greater extent than proteolysis. An apparent contradiction in the nitrogen sparing effect of ionophores was that the most active ammonia-producing bacteria (gram-negative) were resistant to ionophores. Three new gram-positive species with high activity of ammonia production have been isolated. These new isolates were sensitive to ionophores and had a 20-fold greater ammonia production than previously identified ruminal bacteria species (Russell, 1991; Russell et al., 1991). Decreased peptide degradation and amino acid deamination caused by ionophores is attributed to inhibition of these ammonia-producing bacteria (Yang and Russell, 1993b). Monensin decreased by 10-fold the number of highly active amino acid-fermenting ruminal bacteria in vivo. These








50

bacteria utilize peptides and amino acids, but not carbohydrates for growth. As a result, there was less amino acid deamination and less ammonia production. In this study monensin did not increase soluble protein, peptides or amino acids in ruminal fluid. Monensin, however, increased the concentration of bacterial protein in ruminal fluid, which would provide additional protein for the animal. They suggested that monensin-resistant bacteria utilized the peptides and amino acids from dietary protein (soybean meal) that were spared from deamination and converted these to microbial protein. Some ruminal bacteria, such as Prevotella ruminicola showed increased growth efficiency when peptides and amino acids were their source of N (Russell, 1984). In another experiment Yang and Russell (1993a) added protein hydrolysates directly to the rumen. Peptide and amino acid concentrations in ruminal fluid decreased at a logarithmic rate after addition. When cows were fed 350 mg monensin/d the rate of peptide and amino acid disappearance and ammonia concentration were decreased. Monensin increased the ruminal outflow of peptides and amino acids from infused hydrolysates. The effect was dependent on the source: greater ruminal outflow for gelatin than soybean hydrolysates. These reports suggest that monensin increased passage of amino-N out of the rumen, although by a different mechanism (increased bacterial-N and dietary amino-N flow, respectively). It is noteworthy that the experimental model,








51

cows fed at frequent intervals to achieve steady state conditions, was the same in both experiments.

Monensin decreased ruminal urease activity by 66%

(Starnes et al., 1984). This effect would have a beneficial effect on urea utilization in ruminants, because the rate of urea hydrolysis is faster than the rate of ammonia assimilation by ruminal bacteria (Starnes et al., 1984).

The net effect of ionophores on the nitrogen economy of the animal will depend upon specific dietary situations. Increased flow of amino acids to the small intestine could improve production when diets are: a) marginal in crude protein, b) the crude protein is high quality true protein, c) the rate of proteolysis is rapid and d) the rate of carbohydrate fermentation is slow (Russell, 1991). Thus, it is conceivable that the effect of ionophores on metabolizable protein for the host animal will depend upon the net result of all these processes.

Other effects. Other effects of ionophores on rumen function include reduction of turnover rate of solids and liquids, modification in ruminal fill and retention time, and depression of ruminal motility (Lemenager et al., 1978a; Ellis et al., 1984; Deswysen et al., 1987). These changes may explain changes in feed intake, especially in high roughage diets.








52

Total tract digestibility

Spears (1990) suggested that the higher energy

digestion could be explained by increased fiber digestion (Table 2-17). This higher fiber digestibility may result from longer solid retention time in the rumen, thus allowing greater time for microbial digestion of fiber.

Total tract digestibility of starch was not affected by ionophores. However, lasalocid and monensin reduced ruminal digestibility of starch and increased the quantity of starch digested in the intestine. This shift in site of digestion should have resulted in more energy absorbed from starch as glucose rather than as VFA and improved energy utilization (Muntifering et al., 1981; Spears, 1990).

Higher apparent nitrogen digestibility could be

explained by: a) a higher ratio of dietary to microbial protein entering the small intestine because feed protein is usually more digestible than microbial protein, b) fecal endogenous nitrogen losses may be reduced, by decreased microbial protein synthesis in the large intestine and cecum or by decreased sloughing of intestinal cells (Spears, 1990).

Ionophores improve absorption of several minerals. In his review, Spears (1990) concluded that ionophore supplementation increased apparent absorption of Mg, P, Zn and Se, whereas absorption of Ca, K and Na were affected inconsistently by ionophore feeding.








53

Table 2-17. Apparent digestibility of energy and nitrogen
in cattle fed ionophores

Digestibility, %
Nutrient/ Percent Number of Ionophorea Control Ionophore change trials Energy
Monensin 70.3 72.4 -.9 to 9.2 17 Lasalocid 75.7 77.7b 1.9 to 2.2 8 Nitrogen
Monensin 62.2 65.7b .3 to 8.0 15 Lasalocid 70.8 76.4b .2 to 7.2 3

Adapted from Spears (1990).
b Means for control and treated animals differ (P<.05) when analyzed by analysis of variance using experiment as replicate.


Chalupa (1984) summarized the increased retention of energy and protein produced by addition of monensin in the diet (Table 2-18). In those experiments the increase in energy retention was related to an increase in the amount of metabolizable energy by decreasing fecal and methane energy losses.

Metabolism of the host animal

Inhibition of methane production in monensin-treated animals is responsible for about one-third of the improvement in energy utilization (Van Nevel and Demeyer, 1988). There are several studies where significant increases in propionate molar proportions were measured without any improvement in feed conversion efficiency, and there are also studies in which the increase in propionate was too small to explain the magnitude of the improvement observed (Rowe et al., 1991).








54

Table 2-18. Energy and nitrogen partitioning in animals
supplemented with monensin

Trial
Itema Sheepb Sheepc Steerd Energy, % of control
Feces 98 93 90 Digested 101 103 104 Urine 92 84 99 Methane 74 69 74 Metabolized 105 108 107 Heat 102 105 104 Retained 111 115 119 Nitrogen, % of control
Feces 97 98 88 Digested 102 101 107 Urine 92 87 99 Retained 127 138 120
a Data summarized by Chalupa, 1984.
b Monensin at 10 ppm in 50% grain diet.
c Monensin at 20 ppm in 50% grain diet.
d Monensin at 3 mg/kg BW75, 80% grain diet.


In cattle and sheep, about half the dose of monensin is absorbed, metabolized, excreted in the bile, and eliminated in feces (Donoho, 1984). This suggests that monensin may have systemic effects. Plasma concentration of minerals (Mg, Na, K) has been altered with ionophores (Spears, 1990; Owens et al., 1991). Depression of heat increment and amino acid sparing effects have also been cited (Bergen and Bates, 1984).

A second "protein sparing effect" can occur in the

host. Increased propionate production in the rumen and its subsequent absorption may reduce catabolism of amino acids for gluconeogenesis (Van Nevel and Demeyer, 1988). These








55

investigators also reported effects of monensin beyond the rumen, including changes in blood concentrations of several metabolites, hormones, and minerals.

Evidence of an effect of monensin on metabolism in

ruminants independent of alterations in ruminal microbial metabolism have been provided (Armstrong and Spears, 1988). Intravenous administration of monensin depressed plasma concentrations of K, P, and Mg, and increased glucose and free fatty acids concentrations. Changes in plasma mineral concentrations were suggested as indices of the cellular effect of monensin.

Other indications that effects of monensin on animal performance may not be totally explained by changes in ruminal fermentation are provided by measurement of net nutrient flux (Harmon and Avery, 1987; Harmon et al., 1993). These investigators suggested that changes in the products of ruminal fermentation may not be translated into the products appearing in the portal circulation. Urea recycling was reduced in both concentrate- and forage-fed cattle. Changes in VFA net absorption from feeding monensin in forage fed animals were not consistent with its role in increasing ruminal propionate production, because total net energy flux did not change. They questioned the role of monensin in improving feed efficiency solely through increased ruminal propionate production.








56

Animal health

Altered ruminal fermentation associated with ionophore feeding reduces the incidence of acidosis, bloat, and acute bovine pulmonary edema and emphysema (Nagaraja, 1995). Probably the more important effect is the reduction in lactate production, resulting in reduced incidence of lactic acidosis in high concentrate diets. Reduction of lactic acid production resulted from a direct antibiotic effect on the gram-positive bacteria which are the more important lactate producers (S. boyis, Lactobacillus sop). Lactate utilizers are not sensitive to ionophores, providing an additional way of lowering lactic acid concentration (Bergen and Bates, 1984; Nagaraja, 1995).

Ionophores in the diet had positive effects on blood glucose levels, reduced blood 3-hydroxybutyrate concentrations in late pregnancy and eliminated signs of pregnancy toxemia in ewes (Parker and Armstrong, 1987).

Subclinical coccidiosis in lambs has been reduced when an ionophore was added to the diet. The problem appears to be particularly severe in lambs between 3 and 10 wk of age after which natural immunity builds up. In intensive lamb production, the use of ionophores could give a distinctive advantage over other feed additives that have no effect on coccidia (Armstrong, 1986).

Growth promotion has also been observed in young calves. Monensin fed to young calves (7 to 10 d of age)








57

resulted in 10 to 47% higher gains during the suckling period (30 d) and of 6 to 17% higher gains during the next 90 d. Feed intake was increased during suckling period and decreased thereafter. These effects were independent of coccidia control because no coccidia were found (Ilan et al., 1981). In a separate trial they found increased dry matter digestibility when monensin was administered either in the milk replacer or directly into the rumen. Interaction with Minerals


It has been shown that elevated dietary concentrations of Na and K may decrease the response of cattle to lasalocid and monensin (Rumpler et al., 1986; Russell, 1987; Russell and Strobel, 1989; Schwingel et al., 1989). High dietary K appears to inhibit the antibacterial effect of lasalocid more than that of monensin. Research at the University of Florida evaluating monensin and lasalocid in molasses slurries consumed at 2 to 3 kg/d has not shown improvement in gains of grazing cattle (Kunkle and Bates, personal communication). It was suspected that the high concentration of minerals in molasses, especially K (3 to 4%) and the high consumption of the molasses contributed to the lack of efficacy of ionophores in these studies.

The interaction with minerals has not been completely clarified. Greene et al. (1986) has suggested that monensin appears more effective in decreasing the acetate:propionate ratio in lambs when fed with high dietary K. High dietary Na








58

may also decrease the effectiveness of ionophore supplementation of cattle (Bergen and Bates, 1984; Rogers and Davis, 1982). High dietary Na reversed the depression of microbial synthesis (urinary allantoin) induced by monensin in sheep (Dewhurst et al., 1992). Increasing K concentration in the growth medium of pure cultures of ruminal bacteria increased the resistance of these organisms to ionophore. High extracellular K increased the minimum inhibitory concentration of lasalocid in several species. The effect of K on minimum inhibitory concentrations of monensin was similar to, but not as great as, the effect on lasalocid (Dawson and Boling, 1987). Funk et al. (1986) noted an interaction of lasalocid and K (1 and 2.5% K in the diet) for plasma urea N, acetate:propionate ratio, and NDF digestion in lambs. These interactions, however, were not reflected in lamb gains or feed intake.

Research conducted at the University of Florida has

shown that changes in the concentration of K and Na resulted in altered in vitro VFA production (Schwingel et al., 1989). Important findings were: a) high K concentration increased acetate:propionate ratio when lasalocid was fed, b) high Na concentration reduced VFA production when either lasalocid or monensin were fed. This research suggested potential problems associated with high dietary K and lasalocid, and high dietary Na and either lasalocid or monensin. The nature of the interaction appears complex, as Bates and Schwingel








59

(unpublished results) have shown different effects of lasalocid and monensin on S. bovis and R. albus. Monensin was less toxic to S. bovis than lasalocid, but more toxic to R. albus. Increasing Na in the growth medium allowed 5. bovis to proliferate in the presence of ionophores, especially monensin. High Na concentration, however, increased toxicity of ionophores to R. albus. Increasing K permitted R. albus to survive in the presence of lasalocid, whereas no appreciable effect was observed with monensin.

Because high dietary K potentially decreases the

efficacy of lasalocid more than that of monensin, it has been hypothesized that monensin will be more effective in supplementation programs utilizing molasses slurries. However, there are reports of improved ADG in cattle on wheat pasture supplemented with monensin (Horn et al., 1981; Davenport et al., 1989) and lasalocid (Andersen and Horn, 1987). Potassium concentration in wheat pasture is usually high, between 2 to 4% of DM (Grunes et al., 1984). Response to monensin feeding in cattle grazing wheat pasture may also be related with reduction of bloat (Horn et al., 1981). It was suggested that monensin may be useful in neutralizing Krelated depression of Mg absorption in ruminants consuming diets high in K (Greene et al, 1986). Hypomagnesemic tetany is a metabolic disorder common in cattle grazing small-grain pastures (Grunes et al., 1984).








60

Garret et al. (1989) reported improved gain in feedlot cattle consuming a diet containing 30% sugar beet molasses and with monensin added. Feed efficiency was improved by monensin in cattle consuming 30 and 60% molasses in the diet. Increasing levels of molasses in the control diets depressed animal performance. Apparently monensin was effective in overcoming bloat in supplemented cattle. Frequency of Feeding


Molasses supplements are usually delivered 2 to 3 d/wk in most production situations. It is therefore relevant to address the issue of frequency of feeding on the efficacy of ionophores. Efficacy of monensin administration on alternate days compared to daily feeding was evaluated in five trials involving 342 cattle in 32 pastures (Muller et al., 1986). Pooled ADG were .544, .621, and .626 kg for control supplement fed daily, monensin supplement fed daily, and monensin supplement fed on alternate days, respectively. This response was similar to other trials where control and monensin supplements were fed daily (Potter et al., 1976). These results demonstrate that monensin can be effectively administered to pasture cattle in dry supplements that are fed on alternate days. Research conducted at Ona (Horton et al., 1992) with lasalocid fed daily or three times a week in dry supplement showed higher ADG in cattle fed three times a week (.64 vs .56 kg). Monensin was administrated in a dry supplement (average .7 kg/d) fed every other day to stocker








61

cattle on wheat pasture (Andrae et al., 1994). Improvement in gain response to supplements containing monensin were similar to previous trials conducted under similar pasture conditions, but with daily feeding of supplement monensin.

Soybean meal or corn gluten meal, with or without

monensin, was fed on a daily or alternate day schedule to cattle consuming ground corn stalk basal diets (Collin and Pritchard, 1992). Interactions of feeding interval by monensin, and protein source by monensin were observed for ADG and feed intake. Monensin fed at 48-h intervals reduced feed intake in steers. Monensin added to diets supplemented with corn gluten meal reduced ADG by .21 kg but monensin increased ADG in diets supplemented with soybean meal. Monensin fed on alternate days in a high ruminal escape protein supplement was not recommended based on this study. It is noteworthy that protein sources with low ruminal degradability are recommended for molasses slurries (Stateler et al., 1995; Kunkle et al., 1994; Pate et al., 1995). Therefore, it is relevant to test the effect of monensin included in molasses supplements formulated with protein sources of low ruminal degradability.

Based on the literature reviewed, it appears that

frequency of feeding is unlikely to be involved in observed lack of response of ionophore fed in molasses supplements. Inherent differences in the energy substrate in the supplement carrier do exist (starch vs sugar) in addition to








62

the mineral composition. Interactions of energy substrate and ionophore can not be ruled out. Summary of Effects of IonoDhores


Monensin is perhaps the most researched antibiotic feed additive for ruminants. Yet, the mechanism by which it improves animal performance has not been completely elucidated. Effects of monensin on feed intake and gain in cattle fed high roughage diets appears variable. Research has shown that interactions of ionophores with different dietary mineral concentrations are complex.

Limited information suggests that monensin is not efficacious to improve gain when fed in molasses supplements. High molasses mineral concentration, especially K, has been suggested as the probable cause of this lack of efficacy. However, monensin has increased gain in diets high in K, such as small grain pastures. More research is needed to clarify this issue. Energy substrate (sugars), protein level and degradability may also play a role and future research may need to consider these factors.

The present research was undertaken to evaluate the efficacy of monensin to improve gain in cattle fed a high roughage diet and supplemented with different sources of energy (molasses or corn).








63

Considerations in Feeding Molasses.



Animal responses to feeding molasses have been

extensively reviewed. Effects of molasses on rumen function, feed intake, digestibility, animal performance, and metabolic disorders were summarized from trials that covered many different dietary situations (Pate, 1983). More recently, the value of liquid supplements for animals on low quality forage has been addressed (Bowman et al., 1994). Kunkle et al. (1994) and Kunkle et al. (1996) summarized research conducted at the University of Florida comparing sources of N (urea and natural protein of different degradability in molasses slurries). Moore et al. (1995) gathered a data base from different dietary situations and analyzed feed intake and animal performance response to liquid supplements. Factors that affect liquid supplement consumption by grazing ruminants were also reviewed (Bowman and Sowell, 1995b). Effects of low levels of molasses on rumen function of high producing animals, especially dairy cows, have also been summarized (Emanuele, 1996).

Because recent reviews have summarized many aspects of molasses-based supplements these topics will not be discussed. Rather, the effect of molasses on rumen function will be emphasized and highlights on intake and animal performance will be summarized.








64

Digestive Function


Ruminal microorganisms. Most of the ruminal bacteria that degrade complex carbohydrates are also capable of fermenting some of the simple sugars. In addition, Treponema bryantii, T. saccharophilum, Lactobacillus vitulinus and L. ruminus have been identified as sugar fermenters in the rumen (Stewart and Bryant, 1988). The utilization of soluble sugars is thought to be the major role of the large bacteria Quins's Oval, which has been found to proliferate in the rumen when sugar-rich diets are fed (Stewart and Bryant, 1988). When molasses is fed in high amounts, methanol can be produced from the breakdown of pectin by pectinesterase (Russell, 1984). Eubacterium limosum, which is capable of using ethanol, was found in rumen of sheep fed a molassesbased diet. Secondary fermentations in the rumen of cattle and sheep on high-molasses diets have been reported (Rowe et al., 1979b). The bacteria Methanosarcina bakerii is capable of transforming acetate to methane and carbon dioxide. They suggested that this finding may explain the low acetate concentration found in ruminal fluid when high molasses diets are fed. This bacteria is found in mud and sludge, and because it has a slow growth rate, its survival would be possible only under low dilution rate, a condition in the rumen which is known to occur in high-molasses diets (Rowe et al., 1979a). Pate (1983) concluded that a somewhat different microbial population would be expected in the








65

rumen of cattle fed molasses diets in view of fermentation patterns (see below) and the substrate requirements of different microorganisms. He also suggested that more research is needed to identify the microbial population in molasses-fed cattle.

Protozoal densities in molasses- and sugar cane-based diets are similar (1 to 5 x 10S/mL ruminal fluid) but their species population differs. Protozoal biomass is larger with sugar cane diets because large holotrichs predominate. With molasses based diets the smaller entodinia predominate. It appears that at feeding, protozoa are distributed through the rumen more uniformly. After feeding, the large isotrichs quickly store carbohydrate and through increased density they settle in the ruminal fluid and congregate. This results in selective retention of protozoa in the rumen. In slaughtered cattle on sugar cane diets, large isotrichs were not found in omasal fluid. On molasses diets, approximately 20% of the small entodinia left the rumen (Preston and Leng, 1980). Because considerable engulfment and breakdown of bacteria by protozoa took place in the rumen, a reduction in bacterial protein available to the animal occurred. Estimations suggest that the hourly turnover rate due to protozoal predation is higher than that of ruminal fluid turnover in most cases (Ushida et al., 1991). In a summary of 11 experiments (Ushida et al., 1991), defaunation under different dietary conditions resulted in increased flow of








66

microbial CP and efficiency of bacterial CP synthesis (3.17 vs 4.78 g N/100 g OM digested). Defaunation consistently resulted in higher animal performance in animals on high energy (molasses or sugars) and low protein (urea) diets (Bird and Leng, 1978; Bird et al., 1979; Bird et al., 1984). Defaunation appeared to have greater effects on wool production than on growth, reflecting perhaps a specific sulfur amino acid requirement for wool production.

Increased protozoal numbers with inclusion of sugar in the diet was not always shown. Chamberlain et al. (1985) reported an increased protozoal population in starchsupplemented rather than in sucrose-supplemented diets. Khalili and Huhtanen (1991a) could not find differences in protozoal populations between a grass silage basal diet and basal diet plus 1 kg (16% of DM) of sucrose, infused either continuously or two times a day. Bird (1989), cited by Leng (1990), suggested that response to defaunation may not be related to protozoal population densities, a low density being as detrimental as a relatively high density. Defaunation improved animal performance on molasses-urea diets but the mechanism was not clear. Improved microbial protein supply to the host, changed protein:energy ratios in absorbed products of digestion, and improved efficiency of feed utilization have been offered as explanations (Leng, 1990). On the other hand, defaunation did not result in improved animal performance on higher quality diets high in








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true protein. In a review, Veira (1986) stated that the major nutritional effect of protozoa is to change the ratio of protein to energy in the nutrients absorbed, with faunated animals having lower protein and higher energy availabilities compared with defaunated ruminants.

Ryle and Orskov (1987) suggested that the positive response to defaunation in molasses-fed animals may be related with the particular population of protozoa. Because holotrics are more sensitive to pH fluctuations, entodinia predominate and they are more active predators. They suggested that increasing dietary fiber (long hay) may create favorable conditions for holotrics. They also noted that holotrics were associated with high propionate concentrations, while entodinia where associated with higher butyrate concentrations.

Defaunation under Florida conditions, where the basal diet (medium to low quality hay or stockpiled pasture) is supplemented with molasses slurries (often containing true protein) may not be beneficial. If urea is used as the major source of N, then the protozoal population may become relevant.

Ruminal volatile fatty acids. Feeding molasses to

cattle increases the molar proportion of butyric acid in the rumen. This increase appeared to be at the expense of propionic acid when molasses is substituted for grain, or at the expense of acetic acid when molasses is fed as a








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supplement in forage-based diets (Pate 1983). Feeding of molasses did not appear to have a consistent effect on the total VFA concentration or ruminal pH.

Beever (1993) summarized three dietary scenarios (Table 2-19). These suggest that an acetate-inducing fermentation is more efficient with respect to both VFA production and ATP yield than high propionate or butyrate fermentation. However, the yield of methane is higher in acetate fermentation. With the high-cereal diet, more energy is recovered in the end products of fermentation (VFA and VFA plus ATP). Net ATP production is important because it will be used for microbial growth and maintenance. Russell and Wallace (1988) suggested, from the pathways of VFA production, that the net ATP production is 4, 4, and 3 mol/mol of hexose fermented for acetate, propionate and butyrate, respectively. Only 2 mol of ATP will be produced if propionate is synthesized by the acrylate pathway.

Because VFA absorption rates may change with pH or VFA concentrations, VFA molar proportions in ruminal fluid may not reflect the actual VFA proportions in which they are produced (Dijkstra, 1994).

Protozoal contribution to VFA production in the rumen varied between 16 and 37%. End products of protozoal fermentation are mainly acetic and butyric acids, while only trace amounts of propionic acids are produced. Thus, starch and sugars fermented by bacteria would yield more propionic








69

acid and less acetic and butyric acids than would fermentation of the same substrate by protozoa (Dijkstra, 1994). This investigator stressed that VFA produced is not only related to type of substrate, but also the characteristics of the diets. Stoichiometric yield parameters for VFA production from soluble carbohydrate, derived from a large data set, were 1.38, .41, and .10 for acetate, propionate and butyrate in high roughage diets. For a high concentrate diet, the estimated yields were .90, .42, and .30, for acetate, propionate and butyrate, respectively.


Table 2-19. Fermentation of 1 mol of contrasting
carbohydrate sources

High High High Itema forage cereal molasses VFA produced, mol 1.90 1.80 1.67 Acetate 1.34 .90 .94 Propionate .45 .70 .40 Butyrate .11 .20 .33 Methane produced, mol .61 .38 .54 ATP produced, mol 4.62 4.38 4.54 Energy from original
substrate:
VFA energy, % 73 80 75 VFA + ATP energy, % 85 92 87 VFA, mol/100 mol
Acetate 70.5 50.0 56.2 Propionate 23.7 38.9 24.0 Butyrate 5.8 11.1 19.8 Based on estimations of Beever (1993).








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Table 2-20. Effects of molasses level on volatile fatty
acid concentrations, ammonia and pH

No Low Medium High Item molasses molasses molasses molasses
Molasses levela,
kg DM 0 1.5 3.0 4.5 % of total DM 0 13.9 26.0 35.2
Total VFA, mM 103.2 102.4 89.4 100.6
Molar proportion
Acetate 72.6 67.7 64.8 57.3 Propionate 16.0 18.4 17.4 20.5 Butyrate 10.6 12.8 16.4 20.5
Ammonia, mM 9.45 7.71 6.29 5.75 pH 6.62 6.57 6.57 6.24

Molasses levelb,
kg DM 0 1.0 2.0 3.0 % of total DM 0 12.9 24.5 36.5
Total VFA, mM 101.3 117.8 117.7 109.6
Molar proportion
Acetate 70.0 69.2 67.7 63.8 Propionate 16.2 16.9 18.3 21.7 Butyrate 11.4 12.0 12.5 12.8
Ammonia, mM 7.65 7.50 7.14 9.71 pH 6.43 6.31 6.32 6.34
a Khalili (1993). Basal diet: grass hay ad libitum and 2 kg of
cottonseed cake. Linear contrast (P < .05) for individual VFA, ammonia and pH.
b Osuji and Khalili (1994). Basal diet: grass hay ad libitum and 4 kg DM of wheat bran. In the other diets wheat bran was replaced with molasses. Linear contrast (P < .05) for individual VFA.


The increase in butyrate concentration in ruminal fluid

appeared to be related to the level of molasses in the diet.

In fattening systems using 77% molasses in the diet, molar

percents were 31, 19 and 41 for acetic, propionic and

butyric acids respectively (Marty and Preston, 1970). Two

experiments where molasses was added to a basal diet (or

substituted for other ingredient) are summarized in Table 220. Increasing molasses from none to 4.5 kg doubled the

proportion of butyrate in the first experiment. In the








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second, however, increasing molasses from none to 3 kg had small effects on butyrate molar percent. It is noteworthy that 2 to 3 kg molasses (representing 30 to 40% of diet DM) have beeb supplemented in Florida.

Data summarized by Pate (1983) showed that sugars, not the ash, in molasses are responsible for the increase in butyrate. Inclusion of molasses or sugars in the diet almost always increased the proportion of butyrate in ruminal fluid. The magnitude of the increase, however, was variable and not always related to level of inclusion of molasses or sugars in the diet.

Nitrogen utilization. Much of the N in molasses is nonprotein. Stateler (1993) estimated from an in vitro semicontinuous fermentation trial that between 75 and 85% of total N was available for bacterial growth. Urea is usually added to molasses to increase the CP content. Because molasses is low in P, phosphoric acid is usually added. When urea and phosphoric acid are combined, a urea-phosphate salt is formed. Urea-phosphate given to sheep resulted in lower ruminal pH and blood ammonia than when urea alone was given (Perez et al., 1967). Addition of 3% phosphoric acid to a 10% urea liquid supplement prevented ammonia toxicity, apparently due to decreased ruminal pH caused by the phosphoric acid addition, reducing absorption of free ammonia (Davidovich et al., 1977). Increasing urea levels in molasses depressed molasses intake. Urea and monensin,








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independently or combined, have been used to regulate intake of molasses. Intake was regulated effectively with 30 g urea per kg of molasses or with 120 mg of monensin per kg of molasses (Gulbransen and Elliot, 1990).

The addition of sugar-based or starch-based supplements to a basal diet almost always resulted in a decrease in ammonia concentration in ruminal fluid. The lowered ruminal ammonia levels in energy-supplemented animals is associated with an increased rate of fermentation. Intake of energy supplements are often associated with an increased influx of urea into the rumen, but ruminal ammonia levels are decreased because of increased uptake of ammonia by microbes (Obara et al., 1991). In sheep fed a lucerne hay basal diet, infusion of 200 g sucrose (17% of the DM intake) improved N balance, reduced ruminal ammonia and plasma urea N concentrations, increased transfer of urea to the gut and rumen and increased ammonia capture into microbial N. In a similar experiment (sucrose infusion, 20% of DM intake) using sheep fed fresh lucerne, the results were similar (Obara et al., 1991).

The use of molasses has been proposed in diets with a high concentration of non-protein N such as silage. Increased ruminal microbial protein synthesis has been reported when silage was supplemented with sugar (Khalili and Huhtanen, 1991a) or molasses (Huhtanen, 1988). Supplementing silage with a source of readily available








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energy has been found to reduce ruminal ammonia concentration and increase the flow of microbial protein to the small intestine (Rooke et al., 1987; Huhtanen, 1988). These studies were conducted with restricted feeding. When Petit and Veira (1994) fed ad libitum timothy silage mixed with 7.5 or 15% molasses, they found that molasses decreased ruminal ammonia concentrations. Nitrogen retention or plasma urea concentration, however, were not affected by molasses addition to silage diet (Petit et al., 1994). They suggested that sugar supplementation in animals fed ad libitum would decrease ruminal ammonia N concentration as a result of decreased degradability of silage CP, whereas sugar supplementation in feed-restricted animals would reduce ruminal ammonia N concentration as a result of increased microbial CP synthesis in the rumen. Supplementation of silage diets reviewed by Emanuele (1996) suggested that molasses and sugar can be used to replace corn or barley without detrimental effects at low levels of inclusion in the diet. He concluded that molasses fed with protein sources that supply amino acids and peptides to ruminal bacteria supports a higher level of performance than molasses alone or molasses and urea combinations.

According to data summarized by Pate (1983), ureanitrogen was less efficiently utilized in forage diets supplemented with molasses than those supplemented with starch or corn. Bates et al. (1988) found that N retention








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was lower with molasses-urea than with aeschynomene hay or alfalfa meal supplementation of a basal diet low in CP and digestibility. The inefficient use of supplemental nonprotein N occurred because much of the N absorbed from the gastrointestinal tract was excreted in the urine. They suggested that efficient N recycling in ruminants may limit the effectiveness of supplements which contribute primarily to the ruminally available N pool.

Pate (1983) suggested that the feeding of moderate to high levels of molasses reduced the apparent digestibility of CP by 5 to 15%. Practical implications would be an increase in CP requirement above the levels recommended at that time, with the old CP system (NRC, 1976). The fact that young bulls gaining 1 to 1.1 kg/d needed 30 to 60% more CP (as fish meal supplement) than recommended by NRC (1976) may indeed reflect high ruminal N losses as ammonia and(or) low microbial yield.

The finding that 15 to 25% of N from molasses may be unavailable (Stateler, 1993) may provide a partial explanation to lower CP digestibility. Molasses may also depress protein digestibility of the basal diet. It was shown that sucrose or molasses supplementation depressed ruminal degradability of silage CP (Huhtanen, 1988; Petit and Veira, 1994).

Efficiency of microbial synthesis was low on molassesbased diets, and efficiency was increased with the addition








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of a starch source (Rowe et al., 1980). They suggested that addition of starch provided a more uniform supply of fermentable energy for the ruminal bacteria. Barley-urea increased duodenal N flow more than molasses-urea when sheep were given cereal straw, suggesting better efficiency of ammonia capture in microbial protein when starch was the energy source (Oldham et el., 1977). Obara et al. (1991) infused sucrose (20% of the DM intake) in the rumen of sheep fed fresh alfalfa. Nitrogen balance was improved and ruminal ammonia concentration was reduced by sucrose infusion. An unexpected result was that there was no increase in ammonia incorporation into microbial N. Calculation from data presented shows that microbial efficiency was 4.03 and 2.75 g N/100 g OM digested, with basal and basal plus sucrose infusion, respectively. No difference was observed in the protozoal population.

In his review, Pate (1983) found evidence that sugars, and particularly sucrose, were less effective than starch in promoting microbial synthesis from urea. Nitrogen retention was also lower for molasses-urea than for corn-urea diets. He inferred that if biological value of all microbial protein is similar, then the higher urinary-N losses observed in animals fed molasses-urea indicate that urea-N was less efficiently synthesized into microbial protein.

Pulse of glucose added to glucose-limited cultures of S. ruminantium and B. ruminicola caused an immediate








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doubling of heat (energy spilling) production and little increase in cell protein (Russell, 1986). Van Kessel and Russell (1996) reported that when ammonia was the growth limiting nutrient of predominant ruminal bacteria, the impact of energy spilling was very great, and additional ammonia caused a large increase in yield. However, when energy-excess batch cultures were provided with amino N, the growth rate increased and less energy was spilled (Van Kessel and Russell, 1996). Ruminal conditions created by feeding molasses and non-protein N with low quality forage may be similar to those described for energy-excess cultures. This may explain, at least partially, the low efficiency of N utilization.

Digestive associative effects. Pate (1983) concluded

that molasses increased the digestibility of the total diet, but depressed forage DM and fiber digestibility, particularly low quality forages. The degree of depression was dependent upon the level of molasses in the diet and the crude protein balance. With properly balanced forage-based diets, molasses increased DM digestibility and did not appear to severely depress the digestibility of fiber.

Brown et al. (1987) found no effect on OM digestibility and depression of NDF digestibility when limpograss hay was supplemented with 25% DM molasses. The same effect was seen when the basal diet was rice straw. Kalmbacher et al. (1995) found that molasses-based supplements increased the apparent








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OM digestibility of total diet (creeping bluestem basal diet), but decreased NDF digestibility. Similar results were obtained by Brown (1993) using ammoniated stargrass as the basal diet.

Mould et al. (1983) reported that reduction of fiber digestion by molasses supplementation appeared related to the presence of highly fermentable carbohydrate (carbohydrate effect) rather than to low ruminal pH. Ruminal infusion of sugars depressed fiber digestion although pH was not affected (Huhtanen, 1988; Rooke et al., 1987). Increasing levels of molasses supplements (1.5 to 4.5 kg molasses) caused a linear increase in DM and OM apparent digestibility and a decrease in NDF digestibility with increasing level of molasses (Khalili, 1993). Addition of bicarbonate with the higher level of molasses (37% of the diet) did not affect DM, OM or NDF ruminal digestibility. He suggested that the depressed fiber digestibility may have been associated with a preference by ruminal microbes for soluble carbohydrates, as previously observed in vitro (Russell, 1984).

Intake and Performance


Feed intake. Moore et al. (1995) analyzed the effect of liquid supplements on forage intake based on 151 comparisons of voluntary forage intake when fed alone and with supplement. When the forage was balanced (DOM:CP < 7) supplements almost always decreased forage intake. When the








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forage was very unbalanced (DOM:CP > 12) all types and levels of supplements increased forage intake. When forage DOM:CP was between 7 and 12, forage intake was both increased and decreased by supplements.

When intake of forage fed alone was >1.75% of BW,

supplements decreased forage intake; when forage intake was <1.75% BW, supplements increased forage intake. The level of supplement was also important: forage intakes were depressed by liquid supplements when supplement intake was >.8% BW. Supplement CP concentration also affected forage intake. Forage intake was increased when liquid supplement CP was >25% of OM.

Animal performance. Moore et al. (1995) analyzed the

effect of liquid supplements on animal performance based on 148 comparisons of non-supplemented control (grazed or fed forage) and supplemented with molasses. They concluded that daily gains were generally, but not always, increased by feeding liquid supplements. When a source of N was added, gains were greater than when molasses alone was fed. When supplement CP concentrations were above 15% of OM, gains were almost always increased. When supplemental CP intake was greater than .1% of BW, gains were always increased.

Forage quality was also important. When forage intake was low and DOM:CP was unbalanced, liquid supplements increased both intake and gain, but gain was still low or even negative. When forage intake was high and the DOM:CP








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was balanced, liquid supplements decreased forage intake generally, but increased gains if the supplement contained meal or combination of meal and non-protein N. Pate (1983) concluded that a source of N should be provided in molasses when supplemented to low quality forage diets. He also recognized that natural protein was superior to non-protein N sources.

Kunkle et al. (1994) reviewed experiments where

molasses slurries were fed as supplements on basal diets of subtropical pastures or hays. They found that supplemental ruminal undegraded protein (feather, blood and/or corn gluten meal) increased gains in growing cattle from .08 to .30 kg/day and averaged .15 kg/day. They recommended that a source of protein of low ruminal degradability be included after the requirements for ruminal degraded protein are met.

Pate et al. (1995), and Stateler et al. (1995) obtained a good response of ADG in growing cattle when the molasses slurries contained part of the total CP as ruminally undegradable protein.

Summary of Feeding Molasses


When high levels of molasses are fed, ruminal

fermentation is characterized by high butyrate molar proportion, increased population of entodinia protozoa, and lower ammonia concentration. Secondary fermentation (sludgetype fermentation) and low ruminal motility has been reported with high molasses diets. Molasses supplementation








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at about 30% of the diet, as recommended in Florida, is not expected to produce dramatic changes in ruminal fermentation.

In silage diets, a low level of molasses

supplementation appeared to improve N utilization. However, provision of ruminal degradable protein appears necessary.

Addition of non-protein N was better than molasses alone when the basal diet was low in CP. Research with molasses slurries showed that natural protein sources improved performance over non-protein N. Provision of additional protein sources with low ruminal degradability in molasses slurries increased gain in growing cattle fed forage diets.

Several reports suggested that cattle used N

(especially non-protein N) less efficiently with molasses than with grain. Research conducted with silage diets suggested that ruminal feed N degradability may be depressed by molasses supplementation. Furthermore, between 15 to 25% of N in molasses may be unavailable.

There is no direct measurement of provision of non

ammonia N (an estimator of metabolizable protein supply) in cattle fed high roughage diets supplemented with molasses. More information is needed to understand the supply and utilization of nutrients, especially protein, when molasses is fed.








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The present research will evaluate the effects of moderate levels of molasses and corn supplementation on characteristics of ruminal fermentation, efficiency of microbial growth, and nutrient supply in cattle fed bermudagrass hay.














CHAPTER III
EFFECT OF BAMBERMYCINS AND MONENSIN IN CORN
OR MOLASSES SUPPLEMENTS ON PERFORMANCE OF GROWING CATTLE


Introduction



Florida has approximately 1 million beef cows and some of the weaned calves are stockered after weaning. Molasses is usually the lowest cost energy source available for supplementing grazing beef cattle in Central and South Florida. Liquid feeds require less labor to feed than grainbased supplements which reduces supplementation costs.

Researchers at University of Florida developed molasses slurries by adding 10 to 25% dry ingredients such as cottonseed meal, feather meal, blood meal and(or) wheat midds. Molasses slurries are consumed at higher levels than traditional liquid supplements. These higher levels of intake are usually needed to reach the desired performance in growing calves grazing perennial forages in Florida during the fall and winter. Molasses slurries are often limit-fed 3 d/wk in tubs or troughs. Molasses slurries formulated with natural protein that is undegraded in the rumen have been shown to improve the performance of growing cattle (Pate et al., 1995; Stateler et al., 1995).



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Ionophores such as monensin (RumensinT) and lasalocid (BovatecTm) have been effective in improving gains of grazing cattle. Limited research at the University of Florida evaluating monensin and lasalocid in molasses slurries has not shown improvements in gains of grazing cattle (Kunkle et al., 1990; Pate, 1995). However, more evidence is needed to corroborate this finding.

Bambermycins (GainproT) is a feed additive that has

improved gains of grazing cattle (Deetz et al., 1990). Its efficacy in molasses-based supplements is not known, at least under Florida conditions.

A feed additive that improves gains when delivered in

molasses supplements fed at high levels is needed to improve the cost effectiveness of supplementation. The objective of this experiment is to evaluate the efficacy of monensin and bambermycins in corn and molasses slurry supplements.



Materials and Methods



Performance trials were conducted at the University of Florida Pine Acres Research Unit located in northern Marion County from December 1, 1994 to March 23, 1995 (Year 1, 112 d), and at the Santa Fe Research Unit located in northern Alachua County from December 20, 1995 to April 3, 1996 (Year 2, 106 d).








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Year 1. Seventy-six Angus and Brahman x Angus steers and 92 heifers of varying percentages of each breed, weighing from 190 to 320 kg (average 244 kg for all cattle) and 7- to 12-months-old at the beginning of the trial were used. Cattle were balanced by sex and breed type in each pen. Each pen (experimental unit) had three heifers and three steers, except for eight pens which had four heifers and two steers. Seven treatments were completely randomized across the 28, .9-ha paddocks, dormant bahiagrass (Pasualum notatum) frosted before the trial. All animals were dewormed and deloused at the beginning of the trial (IvomecTM pour on).

Full weights were taken on d 0, 28, 56, 84 and 112. Shrunk weights were measured on d 1 and 113 after an overnight feed and water withdrawal. Body condition score (BCS) was evaluated by a single evaluator on d 1 and 113 using a 1 to 9 scoring system (Herd and Sprott, 1986). Initial hip height was calculated as the average of measurements made on d 0 and 1, and final hip height was the average of measurements made on d 112 and 113. Blood samples from all animals were collected via jugular venipuncture on d 28, 56, 84 and 112 for plasma urea N (PUN) analysis. Blood was collected with polypropylene syringes containing 1.6 mg potassium EDTA/mL of blood as an anticoagulant (Monovette, Sarstedt Inc., Newton, NC). Ruminal fluid was obtained from two animals per pen on d 28, 56, 84 and 112 using a stomach








85

tube. The tube was fitted with a ruminal strainer at the end and introduced via the mouth through a Frick speculum. Ruminal fluid was aspirated with an electric vacuum pump. Ruminal fluid samples were filtered trough four layers of cheesecloth and acidified with 5 mL of 20% sulfuric acid/100 mL of ruminal fluid. Samples were obtained between 3 to 5 h after feeding corn supplements. Sampling was conducted the day after new molasses supplements were offered (it was not possible to control sampling time after feeding in this case). Rectal grab samples of feces were collected from all animals on d 28, 56, 84 and 112 for coccidia oocyte and nematoda egg counts. All samples were stored on ice until being translated to Gainesville for processing and storage. Blood samples were centrifuged at 1,500 g the morning after sampling. Ruminal fluid was frozen upon arrival. Fecal samples were refrigerated until analysis were conducted within one week.

Year 2. The breed types of steers and heifers were

similar to those used in Year 1. Initial weight ranged from 245 to 275 kg (average 260 kg for all cattle). Animal allocation to pen and treatment was similar, except that sex was confounded with pen, so that in any given pen only one sex was present. Different animal allocation was decided based on animal availability and the need to gather data useful for modeling work (e.g., intake and performance data by sex).








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Animal management and sampling were conducted as

described for Year 1, with the following differences: Full weights were taken on d 0, 28, 56, 84 and 106; shrunk weights were measured on d 1 and 107; BCS was evaluated on d 1 and 107; initial hip height was measured on d 0 and 1, and final hip height was taken on d 106 and 107. Blood samples for PUN were obtained from 3 animals per pen on d 28, 56, and 84. Rumen fluid was obtained from 2 animals per pen on d 28 and 84. Fecal samples were collected from 2 animals per pen on d 28, 56, 84 and 106.

Diets. Diets consisted of bermudagrass (Cynodon dactylon) hay (large round bales harvested during the previous summer) fed alone or with corn or molasses supplements. Treatments were:

1. Hay + corn meal (CC)
2. Hay + corn meal + monensin (200 mg/day)(CM)
3. Hay + corn meal + bambermycins(20 mg/day) (CB)
4. Hay + molasses slurry (MC)
5. Hay + molasses slurry + monensin (200 mg/day) (MM)
6. Hay + molasses slurry + bambermycins(20 mg/day) (MB)
7. Hay alone (HAY)

Details of supplement formulation (projected intake and ingredients) are described in Table 3-1.

The source of bypass protein in both molasses slurry and corn-urea was corn protein. Ruminal degradable protein was balanced using urea. Eighty-five percent of CP in molasses was assumed to be available in the rumen (Stateler, 1993); the rest was considered unavailable. Amount of








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supplement delivered as fed was calculated to provide the

same quantity of TDN from the supplements.


Table 3-1. Supplement formulation and estimated
composition

Supplement

Item Corn Molasses

Ingredient, % as fed:
Corn meal 93.8 0
Cane molassesb 0 89.6 Corn gluten meal 0 10.0 Urea 2.8 .4
Limestone 1.0 0 Dicalcium phosphate 1.8 0 Dynamate .6 0

Estimated composition
TDN, % DM 84.4 73.4 CP, % DM 18.4 16.7
DIP, % CP 74 74 UIP, % CP 26 26
Ca, % DM .86 .90 P, % DM .66 .67
RumensinTM and GainproTm added in the corresponding treatments to deliver 200 and 20 mg/an/d of monensin and bambermycins, respectively.
Blackstrap molasses not less than 40% inverted sugars, fortified with phosphoric acid and 25,000 U.S.P. units vit A, 33,000 U.S.P. units vit D, and 22 Int. units vit E per kg, and .0005% Cu, .00001% Co, .02% Fe, .001% Mn, .0025% Zn, and .00007% I. Sulfur concentration no less than 1%, as fed basis (U.S. Sugar Corporation, FL).
C Calculated from tables (NRC, 1984) and Stateler (1993). Assumes 85% of molasses N available for microbial growth.


Feed additives were diluted in a carrier and mixed with

the total supplement (corn-urea) or with the dry ingredients

(corn gluten meal-urea) for the molasses supplement.

A mineral supplement containing 17.2 to 20.6% salt,

17.2 to 20.6% Ca, 9% P, 1% Fe, .2% Mn, .01% I, .01% Co, .2%

Mg, .12% F, 1,500 ppm Cu, 20 ppm Se, and 4,000 ppm Zn was

offered free choice in mineral feeders in all pens.








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Feeding procedures. Hay bales were weighed, coresampled with a forage sampler 2.5 cm in diameter, and offered free choice in round-bale feeders. Corn supplements were offered daily while molasses supplements were offered on Monday, Wednesday and Friday of each week. Molasses and dry ingredients were weighed and mixed mechanically in open trough feeders in each pen on each feeding day. Uneaten molasses supplements were weighed and recorded the next delivery day, but uneaten supplement was found only during the first 3 wk of the trial. Following the consumption of every third bale (Year 1), hay orts were collected, weighed, and sampled for dry matter determination. A visual estimation of hay waste not collected was recorded each time orts were collected. Weights of hay offered, orts, and waste estimates were used to estimate hay intake. During the last

3 to 4 d of the experiment a fresh bale was delivered to each pen which had less than a third bale of hay left in the feeder, attempting to avoid bias due to fill in the final weights. In Year 2, hay orts were collected after feeding every 5 to 6 bales because hay refusal was small, resulting in lower weigh back expressed as percent of offered hay.

Laboratory analysis. Hay and dry supplement ingredients DM was determined at 1050 C for 18 h in a forced air oven and OM at 5500 C for 6 h in a muffle furnace. Nitrogen concentration in hay, corn mix, corn gluten mix, and base molasses samples were determined by the method of Gallaher








89

et al. (1975) (aluminum-block digestion), and colorimetric analysis (Technicon AutoAnalyzer, Technicon Instruments Corp., Tarrytown, NY; Technicon, 1978). Neutral detergent fiber was determined in hay, corn, and corn gluten meal following the procedures of Goering and Van Soest (1970), modified by Moore and Foster (1986), with the addition of alpha amylase to concentrate samples only. Hay in vitro OM (IVOMD) digestibility was determined by the procedure of Moore and Mott (1974). Molasses DM was determined by freeze drying. Molasses samples were diluted (weight/volume) in 20 parts of distillate water and pH measured with a portable pH meter (Corning M90, Corning, Inc. NY). Whole ort samples (about 50 to 100 g) were analyzed only for DM at 650 C for 48 h in paper bags.

Mineral concentration of supplement was determined

following the procedure of Fick et al. (1979). Calcium, Mg, Na, and K were determined by flame atomic absorption spectrophotometry using a Perkin-Elmer AAS 5000 (PerkinElmer, Norvalk, Connecticut). Phosphorus was analyzed by a colorimetric procedure (Harris and Popat, 1954). Plasma urea N was analyzed as described by Hammond et al. (1994), using an automated colorimetric procedure (Technicon AutoAnalyzer II Industrial Method no. 339-01, Technicon Instruments Corp., Tarrytown, NY) based on the diacetyl monoxime method of Marsh et al. (1965). Samples of ruminal fluid were thawed, centrifuged, and supernatant filtered through .45 um








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microcel filters (Gelman Sciences, Ann Arbor, MI). Volatile fatty acids were analyzed by gas chromatography (Perkin Elmer AutoSystem XL, Norwalk, CT) using a packed column (Supelco, 1990b). Fecal nematoda eggs and coccidia oocyte counts were determined using the Wisconsin flotation technique (Benbrook and Sloss, 1948). For Year 2, coccidia oocyte were not counted but given a score from 0 (no coccidia present) to 4 (more than 40 cysts per field).

Statistical analysis. Statistical analysis was

conducted using the GLM procedure of SAS PC (SAS, 1987), as a completely randomized design using the pen as the experimental unit. The model included the following effects: year, treatment, and year x treatment. Repeated measurements (PUN, VFA, parasites) were also analyzed by year using the repeated statement in the GLM procedure. Probability level for time and time by treatment interaction were obtained from F using adjusted degrees of freedom (G-G test, Littel, 1989). When no time by treatment interaction was detected, data were pooled and analyzed with treatment as the only effect in the model. In addition, six single degree of freedom preplanned contrasts were evaluated. Coefficients for all contrasts are presented in Table 3-2. These partition the five degrees of freedom in the 3 x 2 factorial arrangement of the six supplement treatments (supplements, additives and the interaction of each additive by supplement), the remaining degree of freedom was used to








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compare hay alone vs all supplements. All probabilities levels for treatment contrasts are presented in tables grouped for related variables. Table 3-2. Coefficients for preplanned comparisons for
treatment effects

Treatments
Contrasts CC CM CB MC MM MB HAY C1-Corn vs molasses 1 1 1 -1 -1 -1 0 C2-Monensin vs ctrl 1 -1 0 1 -1 0 0 C3-Bamberm vs ctrl 1 0 -1 1 0 -1 0 C4-Monensin x supp 1 -1 0 -1 1 0 0 C5-Bamberm x supp 1 0 -1 -1 0 1 0 C6-All supp vs hay 1 1 1 1 1 1 -6



Results and Discussion



Composition of supplements used each year is presented in Table 3-3. Average supplement CP concentrations analyzed were 16.6 and 16.3% of DM for corn- and molasses-based supplements, respectively. Calculated TDN concentrations were 84.4 and 73.4% of DM for corn- and molasses-based supplements, respectively (Table 3-2). The CP concentration of the molasses in this trial was higher than tabular values (NRC 1996), but consistent with values reported by Chapman et al. (1965) for molasses produced from cane grown on organic soils in Florida (7 to 10% CP). Phosphorus concentration in blackstrap molasses is typically .1% of DM.








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Phosphoric acid was added to increase the P concentration of the molasses base. Analyzed concentrations of monensin and bambermycins in supplements are presented in Appendix Tables A-I to A-3. Apparently, there was difficulty with the bambermycins analysis. All mix formulations were recalculated and found to be accurate. The original premix was still 100% efficacious 4 months after the experiment was finished.


Table 3-3. Composition of supplements by analysis Corn gluten
Corn mix meal mixa Molasses basea Item Year 1 Year 2 Year 1 Year 2 Year 1 Year 2 DM 86.1 86.1 89.9 91.5 77.5 77.5 As % DM:
OM 95.2 94.6 98.3 98.3 83.4 83.7 CP 16.3 16.9 67.0 75.2 9.3 8.5
NDF 9.3 9.3 5.2 5.2
Ca .65 .76 .03 .04 .71 .74 P .67 .71 .44 .46 .88 .74 Mg .25 .29 .07 .09 .49 .49 K .45 .51 .13 .16 4.28 4.26 Na .01 .01 .03 .02 .11 .08 pH - 4.51 4.71 a Calculated CP in offered molasses slurries (10.4% corn gluten
mix, 89.6% base molasses) were 16.1 and 16.5% of DM for Year 1 and Year 2, respectively.


Composition of bermudagrass hay is given in Table 3-4. As expected, there were no differences (P > .3) for any component due to treatment. All bales were kept in a barn and delivered as needed, therefore a random assignment was presumed and composition data were pooled across treatments.








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Table 3-4. Composition of bermudagrass hay Variable Mean Std Dev Minimum Maximum Year 1 (n = 265)
Dry matter, % 90.5 .32 89.6 91.6
Percent in DM
Organic matter 93.8 .62 91.8 95.1 Crude protein 9.62 1.74 5.96 15.96
NDF 81.9 1.88 76.2 87.3
IVOMD, % 42.7 2.98 34.1 49.7 Year 2 (n = 218)
Dry matter, % 88.8 1.10 82.6 90.4
Percent in DM
Organic matter 94.8 .58 92.1 96.3
Crude protein 9.90 1.56 6.37 14.50
NDF 79.4 2.16 73.9 87.7
IVOMD, % 46.6 2.59 39.6 53.4



The model level 1 (NRC, 1996) was used to estimate the TDN concentration of the hay from observed gains of animals fed hay alone. Predicted and observed gains were similar when 54% TDN was used in both Year 1 and Year 2. Therefore, a value of 54% TDN (DM basis) for the hay offered was used for estimation of efficiency of feed utilization. Animal Performance


There was no interaction of year by treatments for measures of animal performance, therefore means are discussed by year (Table 3-5) and by treatments (Table 3-6). Probability values (F test) for differences due to year, year by treatment interactions, and contrasts are presented in Table 3-7. Results for each year are summarized in Appendix Tables A-6 and A-9.




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EFFECTS OF CORN AND MOLASSES SUPPLEMENTS WITH AND WITHOUT FEED ADDITIVES ON PERFORMANCE, VOLUNTARY INTAKE, AND DIGESTIVE FUNCTION IN CATTLE FED BERMUDAGRASS HAY By OSVALDO BALBUENA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

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,1 1 This dissertation is dedicated to my wife Elena for the years of love, help, and encouragement and to my children Gonzalo, Nicolas and Maria Daniela.

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ACKNOWLEDGMENTS I wish to express sincere gratitude to the supervisory committee consisting of Drs W.E. Kunkle, D.B. Bates, J.E. Moore, A.C. Hammond, and L.E. Sollenberger Special gratitude is extended to Drs. Kunkle and G. Hembry for financial support during the last six months of my project. I am very thankful to the Institute Nacional de Tecnologia Agropecuaria (INTA) for financial support during my first thirty months at UP. Special thanks to the efforts of Jerry Wasdin, Dan Price, the Pine Acres and Santa Fe crew, and Dane Bernis (Feed Mill) Special thanks is extended to Jeanette Filer and Angelita Mariano for their help in the lab and during the experiments. I am in debt to John Funk, Pamela Miles, and Nancy Wilkinson from Nutrition Lab and to Sandra Armantrout and Christa Jenssen from Extension. I am also in debt to E. Bowers from STARS-Brooksville, Drs. R.C. Hill and K. Scott for allowing me to use their GC, and Dr. C.H. Courtney and Qiyun Jeng for helping me with parasitology analysis. I am very thankful to Kevin Downs, Diane Campbell, and Francisco Olbrich for their help during the experiments. I would also like to extend special thanks to Diego Rochinotti for his friendship and help, and thanks to Pedro Garces-Yepez for sharing his experience with me. iii

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TABLE OF CONTENTS ACKNOWLEDGMENTS ABSTRACT CHAPTERS 1 I INTRODUCTION 1 II LITEI^TURE REVIEW 3 Introduction 3 Antibiotic Feed Additives in Ruminants .... 5 Mechanisms of Action 6 Importance of Gut Metabolism 12 Nonionophore Antibiotics 14 BambentiYcins 15 Ruminant Performance 17 Digestive Function 26 Post-ruminal Effects 31 Summary of Effects of Bamberm^cins 35 lonophore Antibiotics 36 Effects on Performance 38 lonophore Modes of Action 44 Ruminal effects 45 Total tract digestibility 52 Metabolism of the host animal 53 Animal health 56 Interaction with Minerals 57 Frequency of Feeding 60 Summary of Effects of lonophores 62 Considerations in Feeding Molasses 63 Digestive Function 64 Intake and Performance 77 Summary of Feeding Molasses 79 III EFFECT OF BAMBERMYCINS AND MONENSIN IN CORN OR MOLASSES SUPPLEMENTS ON PERFORMANCE OF GROWING CATTLE 82 Introduction 82 Materials and Methods 83 Results and Discussion 91 Animal Performance 93 Volatile Fatty Acids 113 iv

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Plasma Urea Nitrogen Parasites 121 123 IV EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES SUPPLEMENTS ON INTAKE, DIGESTIBILITY, AND DIGESTION KINETICS IN HEIFERS 125 Introduction 125 Materials and Methods 126 Results and Discussion 135 Pre-trial 136 Latin Square Intake and Digestibility 138 Latin Square Digestion Kinetics 149 V EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES SUPPLEMENTS ON RUMEN FUNCTION 162 Introduction 162 Materials and Methods 163 Digesta Kinetics 164 Characteristics of Ruminal Fluid 167 Ruminal and Total Tract Digestion 168 Nitrogen Flow and Microbial Efficiency 170 Results and Discussion 173 Digesta Kinetics 175 Characteristics of Ruminal Fluid 181 Ruminal and Total Tract Digestion 204 Nitrogen Flow and Microbial Efficiency 22 0 VI SUMMARY AND CONCLUSIONS 233 Experiment 1 Animal Performance (Chapter III) 233 Experiment 2 Intake and Digestibility (Chapter IV) 237 Experiment 3 Riomen Function and Digestibility (Chapter V) 238 Conclusions 244 APPENDICES A TABLES 247 B RAW DATA 256 LITERATURE CITED 286 BIOGRAPHICAL SKETCH 315 V

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF CORN AND MOLASSES SUPPLEMENTS WITH AND WITHOUT FEED ADDITIVES ON PERFORMANCE, VOLUNTARY INTAKE, AND DIGESTIVE FUNCTION IN CATTLE FED BERMUDAGRASS HAY By Osvaldo Balbuena December, 1996 Chairperson: W.E. Kunkle Major Department: Animal Science lonophores and bambermycins have improved gain of growing cattle fed forage based diets when mixed in grain or mineral supplements. In limited research monensin has not improved gain of growing cattle when fed in molasses based liquid feeds offered at 2 kg/d or more and the efficacy of bambermycins in this supplement has not been evaluated. Growing cattle fed bermudagrass hay were supplemented with 1.57 kg TDN from corn-urea (CU) or molasses-corn gluten meal (MCG) without and with monensin or bamberirrycins Cattle fed supplements without antibiotics gained .62 kg/d. Monensin increased gain .035 kg/d in CU and decreased gain .029 kg/d in MCG. Bambermycins increased gain .106 kg/d in CU and .042 kg/d in MCG. Monensin decreased hay intake .14% of BW while bambermycins increased hay intake .14% of BW in vi

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vii Year 1 and had no effect in Year 2, Monensin increased the difference between observed and predicted gains (an estimator of feed efficiency) .102 and .026 kg/d in CU and MCG, while bambemrycins increased that difference .063 and .041 kg/d in CU and MCG, respectively. Effects of CU or MCG with and without bambermycins on feed intake and digestive function were evaluated in two 4 x 4 Latin squares with ad libitum and restricted intake. Bambennycins increased total DM intake .08% of BW but did not affect digestibility. Bambennycins increased ruminal pH (6.63 vs 6,52), decreased butyrate molar proportions (9.8 vs 10.6), and did not affect acetate :propionate ratio (C2:C3) and microbial N efficiency. Ruminal pH, total VFA, VFA molar proportions, and CjtCj exhibited a supplement by time post feeding interaction (P < .07). Steers fed CU had higher (P < .033) apparent (3.59 vs 2.99 g N/100 g OM) and true (2.47 vs 2.19 g N/100 g OM) microbial N efficiency, and higher ruminal feed CP degradability (69.2 vs 58.9%) than those fed MCG. Monensin improved gain in cattle fed corn but did not improve gain in cattle fed molasses supplement. Bambermycins improved gain in cattle fed corn and molasses supplements but this effect was greater in corn than in molasses. Increased gain due to bambermycins was not explained by changes in digestive function. I

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CHAPTER 1 INTRODUCTION Warm-season grasses are the main feed resource for cowcalf and backgrounding production systems. Animal production based on warm season forages is usually lower than on temperate forages because intake and nutritive value of C4 grasses is lower. Excess biomass production during the warm and humid season is conserved as hay, haylage or stockpiled forage to be used as the basal diet during the winter months. High animal performance is not possible with this feed resource alone because intake of digestible energy often is only enough to meet maintenance requirements or support low gains Supplemental feeds to meet animal requirements for energy, protein, and minerals are recommended in Florida. Feed additives that enhance animal production and therefore improve the biological and economical response to supplemental feeding are desirable. Molasses is a locally available energy source competitively priced compared to traditional feeds. Researchers at University of Florida have evaluated high levels of molasses supplementation with the addition of dry ingredients containing natural protein sources (molasses slurries) An effective feed additive to improve gain in cattle fed 1

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2 molasses slurries at levels of 30 to 40% of the total diet is not available. Limited research has suggested that ionophores are not effective when fed in molasses slurries but more evidence is necessary. Objectives of this research are as follows: 1. Evaluate the efficacy of ionophore (monensin) and nonionophore (bambermycins ) antibiotics to improve gain in growing cattle fed high forage diets (bermudagrass hay) supplemented with a cornor molasses-based feeds. 2. Evaluate the effects of feeding bambermycins on voluntary hay intake and digestive function of cattle fed high forage diets supplemented with cornor molasses-based feeds. 3. Compare the digestive function in cattle fed high forage-based diets supplemented with cornor molasses-based feeds

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CHAPTER 2 LITERATURE REVIEW Tntrodnrtion Animal performance (growth, reproduction) is often suboptimal when only warm-season forages are fed. Consequently, some type of protein and (or) energy supplementation is often needed. Molasses-based supplements are often fed to cattle consuming warm-season forage diets. Feed additives that enhance growth may improve biological and economical response to supplementation. Competitive feedstuffs are defined as those that can also be used as human food, while complementary feedstuffs are not used directly for human consumption. Rumsey (1984) and Hammond (1991) discussed the noncompetitive nature of ruminant production using the protein conversion ratio (Table 2-1) Relative input/output for ruminant protein is less efficient than protein produced by nonruminant animals. However, the use of competitive feedstuffs inputs is lower in ruminant production systems. The advantage of ruminants is the use of complementary feeds. The challenge is, therefore, to increase the animal output using noncompetitive feeds. 3

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4 Table 2-1. Conversions of total and competitive feed protein into animal protein Production unit Protein conversion ratio^ i o cax \_01lL^ti U JL. JV C Ruminant i_J ^ZZ ^ZX. 7.11 2.30 Sheep 14.50 1.90 Dairy4.10 .95 Average 8.57 2 05 Nonruminant Swine 5.92 5.50 Broilers 3.91 2.50 Layers 3.91 2.20 Average 4.58 3 .40 ^ Rumsey (1984) Total input per unit produced. Competitive input per unit produced. When considering intensification of ruminant production to improve its competitiveness, research focusing on the following items should be beneficial: a) Improve the utilization of high fiber feeds to take advantage of the comparative advantage of riiminants. In developing countries the feed input in ruminant production is almost exclusively rangelands, improved pastures and crop residues. b) Use supplementation strategies, when pasture quality and (or) quantity is decreased, as an effective way to increase digestible energy intake and (or) deliver protein and other essential nutrients needed to balance the diet. As part of these strategies, the use of feed additives could improve and potentiate the response to supplements, both biologically and economically. Supplementation programs vary

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5 widely from the more traditional corn-soybean meal to a variety of by-product feeds. This review will focus on the use of antibiotics as feed additives in ruminants. Molasses supplementation in cattle fed high roughage diets will be briefly discussed. Antibiotic Feed Addj tives in Puminants Feed additives that enhance animal performance through increased growth rate and (or) feed conversion in clinically healthy and nutritionally normal animals are termed growth promoters. According to Armstrong (1986), growth promoters can be defined as substances, other than a dietary nutrient, that increase growth rate and (or) feed efficiency in healthy animals fed a balanced diet. In contrast, feed additives that act prophylactically as disease suppressants are not growth promoters. Sometimes the same compound may serve both roles. For example, ionophores are coccidiostats in poultry and growth promoters in cattle. In practice it is often difficult to differentiate which role is more prevalent in a given situation. Other names for these effects found in the literature include nutrition improvers, growth permittants, growth effectors, and rumen additives (Muir, 1985) Hays (1991) presented an overview of the beneficial response to antibiotics used at subtherapeutic levels (Table 2-2) These data illustrate that the response in young calves and pigs is greater than that in older animals, which

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6 is consistent with one of the mechanisms proposed (disease control) Improvement in gain and feed efficiency account for the major portion of economic benefits and have reduced food cost (beef, pork, chicken and poultry) to consumers by more than 3.5 billion dollars annually in the USA (CAST, 1981) It is estimated that 45 to 55% of the antibacterial agents produced in the USA are administered to animals. Such estimates include the ionophores (Hays, 1991) Table 2-2. Beneficial responses to subtherapeutic levels of antibiotics by several species Percentage improvement Number of Species^ experiments Gain Feed: gain Broiler chick 286 2.94 2 .48 Turkey 126 7.03 3.83 Beef cattle 65 4.92 5.27 Layer hens 244 4.01 4.72 Pigs : Starter stage 378 16.09 6.90 Growth stage 276 10.68 4.47 Growerfinisher 279 3.97 2.08 Young calves 85 14.29 ^ From Hays (1991) Mechanis ms of Action The mechanisms by which antibiotics improve animal production in healthy animals has not been fully clarified. Three modes of action has been postulated for both, monogastric and ruminant species (Hays, 1991) : Metabolic effect A direct effect has been shown for antibiotics that are absorbed, for example, inclusion of

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7 chlortetracycline in the diet altered water and nitrogen excretion in pigs. However, antibiotics that are not absorbed can have indirect effects through nutrients available for absorption. Data that support a metabolic effect also tend to support a disease control or nutritional effect as the metabolic processes may be influenced by systemic or gastrointestinal infections, or by absorption of microbial metabolites. Nutrient sparing effect. An increased response to antibiotics in the presence of nutritional deficiencies can be of major economic significance. Antibiotics may partially bridge the gap between nutritionally optimal and economically practical diets. Nutrient sparing effects can occur by one or more of the following: a. Bacteria with similar requirements for critical nutrients (vitamins and amino acids) are inhibited. b. Improved absorption of those nutrients that are available in limited quantities (changes in gut wall thickness) c. Interaction of protein levels (quantity) and source (quality) and antibiotics may occur. Beneficial effects of adding antibiotics have been enhanced in animals fed protein deficient diets (quantity) and when feeding vegetable protein diets, compared to milk protein diets (quality) Interpretation is difficult because these effects can be confounded with intake change. Enhancement of growth and feed efficiency is frequently associated with an increase in feed intake. Disease control effect. Mainly related with control of subclinical diseases (analogous to subclinical parasitosis

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of the gastrointestinal tract) The major benefit from subtherapeutic use of antibiotics results from the suppression of harmful microorganisms. In a summary of the effect of the gastrointestinal micro flora, Visek (1978) stated that they a) affect growth and development of the host, b) influence nutritional requirements, c) affect morphogenesis of the gastrointestinal tract, d) modify metabolic activity of endogenous and exogenous substances introduced into the gastrointestinal lumen and e) play an active role in preventing foreign microorganisms from becoming established. The most convincing evidence is the lack of improved growth under germ free-conditions. Additional evidence includes the following: a) inactivated antibiotics do not have any effect on growth or feed utilization, b) injected antibiotics promote growth to the extent that they are secreted into the gut after injection, c) antibacterial agents are generally more effective growth promoters in quarters where hygiene is poor than in new or clean environments (Visek, 1978) It has been suggested that the overall effect of antibiotics in ruminants is likely to be a composite of the effect of the antibiotics on the micro flora and fauna within the reticulorumen, and that resulting from any subsequent effect of the antibiotic within the small intestine and probably in the cecum and colon (Armstrong, 1984) Ruminal effect will be covered when discussing

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9 specific drugs. Direct and indirect effects at the intermediate metabolism level are difficult to separate from the above, but cannot be ruled out. For post-ruminal effects to occur, the antibiotic included in the feed must survive the ruminal environment and not be absorbed or excreted post-ruminally Events in the small intestine. It may be useful to review effects at the small intestine level, even though most information was generated with monogastric species. Most of the comments are extracted from Visek (1978) Coates (1980), and Parker and Armstrong (1987). In germ-free animals there are specific changes in the histology of small intestinal villi with a reduction in the rate of enterocyte migration up the villus. In addition, there are changes in enzyme activity and rates of nutrient absorption. A finding that was observed early and reported by several laboratories is a reduction in weight of the small intestine in antibiotic-fed chickens, swine and rats. There is evidence, from studies in pigs, that inclusion of antibiotics in the diet resulted in changes in morphology of the small intestine. Elongated villi and higher villus: crypt ratio were reported which is indicative of a lower rate of enterocyte migration. It has been suggested that a reduction in the production of toxic by-products normally arising from microbial activity in the digestive tract could reduce enterocyte damage and therefore lower cell renewal rates.

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10 Degradation of endogenous urea to ammonia has been proposed as one of the negative effects of the microbial flora. Ammonia concentration in the intestinal lumen is above the concentration required to kill cells, alter nucleic acid synthesis, and depress immune response. Depression of bacterial ammonia production in the small intestine may therefore be one mechanism by which antibiotics stimulate growth (Visek, 1978) Antimicrobial compounds reduce ammonia concentrations and the production of amines, particularly cadaverine (Parker and Armstrong, 1987) In germ-free animals the intestine and associated lymphoid structures contain less tissue, and cells of the intestinal mucosa are replaced at a slower rate (Coates, 1980) Their intestines are thinner and nutrients pass through them more rapidly in vitro. Germfree animals also have a lower basal metabolic rate (Visek, 1978) Reduction of microbial activity has also been suggested as playing a role in bile acid metabolism, enzyme activity and efficiency of absorption. Data discussed by Armstrong (1986) showed that uptake of methionine and glucose was increased in germ-free compared with conventional chickens. However, this increase was significant when expressed per gram of intestinal tissue but was not significant when expressed per unit length of the intestine. Intestinal tissues of conventional animals fed diets supplemented with antibacterial agents develop

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11 characteristics similar to those of germ-free animals (Visek, 1978; Coates, 1980). Inclusion of avoparcin in rat diets resulted in increased aminopeptidase activity in the illeal mucosa. Microbes within the small intestine of poultry are able to deconjugate bile acids with impairment of lipid absorption but it is uncertain to what extent the process is of significance for the bird. Antimicrobial feed additives in pig diets resulted in increased sucrase activity throughout the length of the small intestine. It has also been suggested that bacterial protease activity may play a role in the turnover of brush-border proteins, in which case reduction of bacterial numbers might increase mucosal enzyme activity. In pigs, virginiamycin enhanced uptake (9%) of free amino acids from a temporarily isolated intestinal loop (Parker and Armstrong, 1987) In sheep, avoparcin increased the number or activity of glucose receptors in brush border membrane vesicles in vitro (Parker, 1990) A population of approximately 10^ bacteria/g of small intestine content has been reported. Most of the isolates were gram-positive and were able to utilize starch. Possible competition with the host for starch digestion and other nominal bypass nutrients has been suggested (Nicoletti et al., 1984).

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12 Also, improved animal performance may be due to reduced energy cost of gut metabolism (Visek, 1978; Parker and Armstrong, 1987; Parker, 1990). Im portance of Gut Met abolism Gastrointestinal tract (GIT) is one of the most active of the organ masses. In a week-old pre-riaminant lamb the fractional synthesis rate of protein (FSR) was 69%/d which is three times the FSR in skeletal muscle of the same animal. In older animals, the GIT may contribute up to onethird of total protein synthesis in the animal and equal or exceed muscle synthesis by up to 250%. The passage from the pre-ruminant to ruminant increases protein synthesis not only in the reticulo-rumen but also in the intestines (Lobley, 1993) Contribution of different tissues to total protein synthesis is summarized in Table 2-3. Protein FSR of more than 100%/d has been observed for jejunum and duodenum in 8-wk-old weaned lamb, which was equivalent to a mean half-life of 14 to 18 h. Epithelial cell renewal times did not explain the rapid turnover. Renewal times (estimated from cell migration rate) were 60 to 90 h in comparable lambs. Intracellular synthesis of secretory proteins may account for the difference. In his review, Lobley (1993) concluded that the larger GIT mass and the substantial contribution this tissue makes to both the whole-body synthesis and to the overall protein econoiny of

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ruminants makes this a particular target for potential manipulation. Table 2-3. Contribution of major tissues to whole body protein synthesis (% of total) Animal (age)" Tissue or organ Rest of body Muscle Skin GIT tract Liver Lamb (1 wk) 29 13 12 (4) 12 34 Lamb (8 month) 18 20 26 (6-8) 8 28 Cattle (1-8 yr) 20 14 35 (6-8) 4 27 From Lobley (1993). Values in brackets are percentages of total body weight (wt/wt) A manipulation of N partitioning towards muscle and away from the gastrointestinal tract should give two advantages to the animal. First, a significant drain to the N economy of the animal relates to secretions and desquamations occurring in the gastrointestinal tract. The desquamated epithelial cell protein, excreted mucosal proteins, and other digestive secretions are digested and the amino acids are absorbed, but this resorption is unlikely to approach 100%. Second, the amino acid composition of gastrointestinal protein and other tissues (muscle, wool) are different. The match in need versus supply is superior in muscle relative to other tissues. Gastrointestinal tract secretions have high demand for valine, threonine, serine and proline (MacRae and Lobley, 1991) Data derived from fluxes across the portal-drained viscera (PDV, includes gut, pancreas, spleen and omentum)

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14 and liver of multi-catheterized cattle provided measurements of energy metabolism of gut tissues. Oxygen uptake by the PDV gave an estimate of heat energy (HE) attributable to those tissues. The PDV accounted for 18 to 25% and the liver 17 to 25% of the whole body oxygen uptake, or energy lost as HE (Huntington, 1990) He concluded that PDV and liver are metabolically active at rates disproportionately greater than their contribution to body mass, together they account for half the HE. McBride and Kelly (1990) reviewed the contributions of various biochemical processes to overall energy in the GIT (Table 2-4) These data suggested that due to the large contribution of GIT and liver to whole-animal energy expenditure, their manipulation could alter the energetic efficiency of ruminant production. Table 2-4. Metabolic energy expenditures pertaining to the ruminant gastrointestinal tract (GIT) Item^ GIT energy expenditure, % Whole-body energy expenditure, % Na, K-ATPase 29 to 62 5.7 to 12.4 Protein synthesis 20 to 23 4.0 to 4.6 Protein degradation 4.3 .9 Total — r-: : — rr— 53 to 90 10.6 to 17.9 From McBride and Kelly (1990) Noniono phore Antibiotics Numerous antibiotics have been or are in use for growth promotion (Table 2-5) They represent a diverse group differing in chemistry, primary antibacterial spectrum, mode

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15 of action of bacterial inhibition, molecular weight, and absorption from the GIT. Antibiotics that are not absorbed from the gut or poorly absorbed at the low dosage used are desirable as feed additives, because of the absence of residues in milk and meat and because there is no need for a withdrawal period before slaughter (Nagaraja, 1995) Bacitracin, chlortetracycline, oxytetracycline, and tylosin have been approved for control of liver abscesses in the USA. Tylosin has proven to be more efficacious than other antibiotics for this purpose and it is used routinely with monensin as an additive in feedlot diets (Nagaraja, 1995) Avoparcin, a glycopeptide antibiotic produced by 2^ candidus interferes with bacterial cell wall biosynthesis. It is effective against gram-positive organisms and gramnegative species with a gram-positive structure (Armstrong, 1984) It is used as a feed additive in Europe and Australia, but it is not approved for use in the USA. Bambermycins Bambermycins (Gainpro™) is a fermentation product of a variety of Streptomyces spp. including S .banibergiensis 2^ ghanaensis S. ederensis and S. geysiriensis (HoechstRoussel, 1993). The first antibiotic complex of this group was moenomycin ( f lavonrycin, bambern^cins) reported in 1965 (Huber, 1979) They are classified as sugar lipid

PAGE 23

16 m (d •H •H -d 0) m ::i w u •H 4-) O -H ^ -H 4-) C (d 0) u o a o c o •H c o c 4-1 o m u -H 4.) CQ -H ^ Q) 4J U Id ^ (d U in I CN 0) iH X) 03 Eh O 4J •H :i 4J Di g o o m s-i Xi < u 03 rH u 0) rH o 5.2 4J 03 H 0) 4J u 03 -H JJ d o is o o in e 03 U O o i-l u 03 o :2: o IS o 10 (0 to Q) 0) 00 00 en 00 in 00 cy\ 00 in o 1 cn CN cn 00 rH CO rH CO CO rH CO CO rH •H rH -H •H rH •H -H 03 CO 03 CO d CO 03 CO d CO ^ •H 0) OJ •H (U ^ 0) ^ Xi (1) ^ rH 4J rH 4J JJ JJ rH JJ JJ JJ rH c rH C o a rH o d Q) 0) SH >i 0) >i iH >, U CO U CO Oi CO U CO 0^ CO + + + + CO H d CO H 0) 0) ^ JJ JJ 0 d ^ >^ 01 CO CO -rl d CO H 0) (U ^ JJ JJ 0 d u >i 01 CO CO -H CO QJ 0) ^ JJ O u JJ d D^ CO g 03 U O 5 O >H u 03 g 03 U O g 03 U 03 o n u d pa 03 g 03 U O o u u 03 •z g 03 U O 5 o u u 03 IS m 00 'd d 03 in CN in CO •rl CO CO 0) X jj o JJ d Oj CO + + + + g g g g 03 03 OS 03 1 U U U O O o O g 03 U O O o 0 03 u iH u 0 TJ u u u U d 03 03 03 DQ 03 2 to CO (U -rl 0) T3 1 o d -H rH >1 -H •H •H >i QJ CO a 0) U ,d -H rH Di u rH rH rH U d H 0 a 03 a 03 o 0 03 0 o 0 O 0 g u >i ^1 CO JJ u d U iH JJ in JJ Q) rH JJ 0 d >i H JJ U u a u u rH O Q) ^ 0 rH Q) 03 03 0) 03 03 U O u di e S CU g rH 0) -9 JJ U 0 -H JJ 0 U rH 03 g o -H rH d > 03 rH 03 ^ a -H < < CQ u O w > D) (0 2; 6 o •0 4J (0

PAGE 24

17 antibiotics (Berdy, 1980) These antibiotics are characterized by a high molecular weight from 1,600 to 2,100 daltons and a phosphorus concentration of about 2%. They consist of an oligosaccharide part, phosphoglyceric acid, a CjB-lipid alcohol, and an UV chromophore (Ruber, 1979) These glycolipid antibiotics exhibit bacteriostatic activity at very low concentrations. At higher concentrations, they are bactericidal only against actively multiplying grampositive bacteria (Huber, 1979) The mode of action of these antibiotics involves the inhibition of bacterial cell-wall biosynthesis by preventing formation of the cell wall component peptidoglycan (Huber, 1979; Berdy, 1980) Ruminant PerfQimance Relatively few reports are available on the effect of bambermycins in ruminants. It has been in use as feed additive (Flavomycin™) for cattle and sheep for several years in Europe and Australia. Bambermycins tested at 0 to 80 mg/d showed no improvement in ADG at dosages above 20 mg/d. Pooled analysis of the five studies presented in Table 2-6 for treatments 0, 5, 10 and 2 0 mg bambermycins demonstrated that bambermycins fed at 10 and 2 0 mg/d increased (P < .05) ADG of steers and heifers consuming pasture by .09 kg when compared with control cattle (Hoechst-Roussel 1993). Grant et al, (1974) reported 5% average response to flavomycin in both gain and feed efficiency, when dosed at 0, 2.5, 5, 10, and 20 mg/d in

PAGE 25

18 nine trials with cattle fed mediumto high-energy diets. Based on nine feedlot dose titration studies, a range of 10 to 2 0 mg bambermycins/d was approved to increase ADG and feed efficiency in feedlot cattle (Table 2-6, HoechstRoussel, 1993) Huber (1979) recommended 2 5 to 5 0 mg flavomycin for beef cattle. European studies used 40 to 50 mg flavomycin for beef cattle. Two trials with growing and finishing feedlot cattle compared bambermycins with ionophores (Table 2-7) Bambermycins did not appear to depress intake and intake was higher than when monensin and lasalocid were added. Feed: gain was similar to lasalocid and ADG was similar to that achieved with monensin in one trial (Hoechst-Roussel 1994) In the other trial, however, response to monensin was better than bambermycins for ADG and feed efficiency (Burris and Randolph, 1996) A pooled summary of four pasture trials (Table 2-7) showed that bambermycins and lasalocid had greater ADG than control, and bambermycins had greater ADG than monensin or lasalocid (Keith et al., 1995). Flavomycin improved gain and feed efficiency when cattle were fed beet pulp but not when fed corn silage and had no effect on DM intake in either diet (Table 2-8, De Schrijver et al., 1990). Bambermycins increased gain and feed efficiency in heifers fed high forage growing diets (Dhuyvetter et al., 1996). Bambermycins combined with A. oryzae decreased fed intake (Table 2-8) Bambermycins was

PAGE 26

19 also effective in improving gain in cattle on pasture offered a self fed supplement (Kunkle et al., unpublished results, Table 2-8) Table 2-6 Gain (kg/d) in titration studies for cattle consuming pasture and feedlot diets, and gain and feed efficiency for feedlot cattle Bamberm/cins mg/d Pasture 0 5 10 20 40 60 80 1. Buffalo grass .762 1. 033 927 927 973 893 2 Smooth brome grass .690 .742 .793 .771 .812 .788 3. Fescue/ ladino clover .777 .773 .842 .843 .863 .856 4. Bermuda/ bahiagrass .465 .469 .505 602 5. Wheat/ ryegrass .876 .884 .903 .945 Pooled 1-3^ .78^ .86^ .89^ .87^ .91" .87^ Pooled 4&5^ .67'' .67^" .70'' .IT Feedlot ADG Feed: gain 1.14 8.90 1.17 8.76 1.18 8.70 1.18 8.97 1.18 8. 67 Deetz et al. (1990). Yearling cattle received .45 or .9 kg control supplement or containing bambermycins Quadratic effect (P < .0005)jj plateau was 18.5 mg of bambermycins. Deetz et al. (1992). Yearling cattle received .45 kg corn control or with bambermycins. Hoechst-Roussel (1993). Cattle dosed 5 mg bambermycins/d gained 1.15 kg (different from control, P < .05) and had 8.9 feed:gain (similar to control). Cattle dosed with 10 mg or more had higher (P < .05) gain and fe^d efficiency than control. Means within a row with different superscripts differ (P < .05)

PAGE 27

20 Table 2-7 Performance data for 112 -day feeding experiments comparing bambenrr/cins, monensin and lasalocid Item Control Bamberm Monen Lasal Ref Feedlot : ADG, kg Intake, kg Feed: gain 1.16" 8.14" 7.00"" 1.10"" 7.70" 6.96" 1. 05" 7.75" 7.28" 1 /vjjvj Kg Intake, kg Feed: gain 9.04 6.77 1 ?Q 9.05 6.52 1 45 8.85 6.14 1.37 8.96 6.57 2 Pasture: Fescue Crested wheat Bermudagrass Orchardgrass .85" .61" .73" .51 .94" .75" .73" .63"" .83" .69" .63" 66" .84" .72* .76" .56" 3 Pooled .67^ .76^^ .70' .72^== Means in a row with different superscript differ (P < .05). Means in a row with different superscript differ (P < .08). 1 = Hoechst-Roussel (1994) First 56 days, ration composition was 52.6 % TDN and 57.1 % NDF (brome hay 44%, corn silage 52% and a proteinenergy supplement); rest of the trial ration composition was 60.6 % TDN and 44.6 % NDF (alfalfa hay 17%, corn silage 63%, and a protein-energy supplement) 2 = Burris and Randolph (1996). Diet was corn silage (6.7% CP; 69% TDN) and 7% cracked corn ad libitum, along with .9 kg of a pelleted supplement (45% CP) which provided no additive (control), 20 mg bambermycins, 200 mg monensin or 300 mg lasalocid. Feed additives higher ADG than control (P < .11), monensin higher (P < .01) than bambermycins or lasalocid. Bambermycins higher (P < .09) feed intake than monensin or lasalocid. Monensin higher (P < .02) feed efficiency than bambermycins or lasalocid. 3 = Keith et al (1995). All steers fed .9 kg supplement, nonmedicated (control), with 20 mg bambermycins, and 150 mg monensin or lasalocid.

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21 Table 2-8. Feed intake, gain, and feed efficiency in cattle fed bambenrtycins (flavomycin) Intake, Gain, Feed to Reference and diets kg DM kg gain De Schrijver et al. (1990)^ Basal-1 : com silage + 1% BW concentrate y 2.1 1 A A 44 c o /1 1 41 Basal-1 + 43 mg flavomycin Q y 1 c D Z D Basal-2: beet pulp 50%, concentrate 50% 10 lb i 3 1 1 b y Basal-2 + 53 mg flavoitiycin 1 A 10 55 1 51 r D y y Pooled analysis:'' Basal-1 and 2 9 67 1 .34 7 21 Flavomycin 9 80 1 .43 6 84 Dnuyvetter et al. (1996) Basal: corn silage 38%, oat nay zd*, Dariey gram 3 0.5%, protein supplement 5 6% 7 19 98 7 37 Basal + 20 mg bambermycins 7. 25 1 04 6. 91 Basal + A. oryzae 7, 17 1 03 6. 92 Basal + bambermycins and A. oryzae 7. 07 NR NR Kunkle et al (unpublished) Bahiagrass pasture"^ 45 Block supplement control 58 Block supp. + flavomycin Bahiagrass pasture^ Loose mineral control 39 Loose mineral + flavort^cin 47 White-blue bulls 350 kg initial BW. Trial duration was 12 wk for each bgsal diet. Animals were shifted diets over a 4-wk period. Probability values for intake, gain and feed:gain were: .67, .81, and .71 (basal-1); .23, .002, and .03 (basal-2); and .36, .09, and .13 (pooled analysis). Charolais crossbred heifers 260 initial BW. Bambermycins by A. orvzae interaction (P = .03) for DM intake. Animals fed bambermycins and A. orvzae combined had lower (P < .05) intake than all other treatments. Gain and feed:gain reported are main effects of bambermycins and A. orvzae Both feed additives increased ADG (P < .02) and feed efficiency (P < .§3). NR = not reported. Holstein steers and heifers of various breeds 250 kg initial BW. Supplement consumption was .29 kg/d. Gain higher (P < .05) with flavomycin Holstein steers and heifers of various breeds 230 kg initial BW. Supplement consumption was .27 and .38 kg/d for control and flavomycin mineral supplement. Gain higher (P < .05) with flavomycin.

PAGE 29

other reports include those of Flachowsky and Richter (1991) in which flavorrr/cin did not affect feed intake but increased ADG (10.5%) and reduced feed and energy required per unit gain (10.6%) in heifers. Alert et al (1993) reported 3.5% higher gain and 3.2% better energy utilization in fattening Friesian bulls supplemented with 50 mg/d flavomycin. Carcass composition or organ weight were not affected by the additive. Feed intake was increased in the first 56 d of the trial. Poppe et al. (1993) reported no influence of flavomycin on feed intake of heifers. Scott and Kay (1984) reported no effect of 40 mg of flavomycins in gain or feed efficiency of cattle fed grass silage and rolled barley supplement while monensin and salinomycin improved feed efficiency. Kay et al. (1983) reported gain increases of .15, .07, and .05 kg in three trials in cattle fed grass silage and rolled barley supplement with avoparcin, monensin, salinomycin, and 40 mg of flavomycin. Galbraith et al. (1983) reported increased gain in cattle fed 40 mg of flavomycin on barley diets (1.66 vs 1.50 kg). In young calves flavomycin tended to (P > .05) increase gain by 5 to 8% and had no effect on feed intake (ElJack et al., 1986; Fallon et al 1986). The limited information available show that bambermycins increased ADG by 1 to 29% and improved feed efficiency by 2 to 10%. Bambermycins did not affect or tended to increase feed intake when compared with control

PAGE 30

23 diets, and tended to increase intake when compared with ionophores, especially monensin. The effect of bambernr/cins on feed intake in sheep is not clear (Table 2-9). Aitchison et al. (1989b) and Murray et al. (1992) reported no effect of flavomycin on feed intake. Murray et al. (1992) reported slower rate of eating in sheep supplemented with flavomycin. The eating rate was dependent on the type of diet; it was decreased in animals fed hay-fishmeal and supplemented with flavomycins (Murray et al., 1990). In addition, sheep fed lupin seed twice a week with flavomycin and methionine ate less (P < .05) chaff on the day after feeding on the lupins and then ate more (P < .05) on the following day than sheep not fed any sulfur supplement (Murray et al., 1991). Gain and wool production response to bamberniycins appear related to diet (Table 2-9). Murray et al. (1992) suggested that the lack of wool production response to flavonr^cin in growing sheep may be related to partitioning of dietary protein towards tissue rather than wool growth. Maturity (age) and nutritional history of sheep may have an important effect on wool production. They also suggested that the best response to flavomycin will be obtained in adult sheep in areas where the feed supply is reasonably constant In summary, bamberirycins increased gain of cattle fed a variety of diets, and it appears especially indicated in

PAGE 31

24 Table 2-9. Effect of flavoniycin and other feed additives in sheep Daily Feed Daily wool Intake, gain. growth. Reference and diets g/d g/d g/m' Aitchison et al., {1989a) Oaten chaff^ Control 726 Lasalocid 27 5.11 Avoparcin 27 4.40 Flavomycin 21 4.89 43 5.52 Pellet^ 591 Control -3 6.40 Lasalocid -9 6 .37 Avoparcin -7 5.75 Flavomycin q Aitchison et al., (1989b) Run I (weeks 1 to 4) Wheaten chaff Control 865 149 5 5 Flavomycin, 10 ppm 895 165 C A 5 9 Flavomycin, 2 0 ppm 853 174 6.8 Tetronasin, 5 ppm 893 179 5.7 Tetronasin, 10 ppm 843 104 5 7 Pellet^ • Control T rA O o c o 1 D Flavomycin, 10 ppm lb / J o c o 1 O 1 Flavomycin, 2 0 ppm 1 C A A 1500 344 1/1 1 14 1 Tetronasin, 5 ppm 1548 390 13.0 Tetronasin, 10 ppm 1519 367 11.8 Run II (weeks 5 to 9) Wheaten chaff Control 926 38 6 2 Flavomycin, 10 ppm 920 25 5 8 Flavomycin, 2 0 ppm _7 U ^ S ft Tetronasin, 5 ppm 965 7 7.0 Tetronasin, 10 ppm 912 25 5.5 Pellet'' Control 1727 231 11. 6 Flavomycin, 10 ppm 1875 289 11.8 Flavomycin, 2 0 ppm 1835 303 13 .3 Tetronasin, 5 ppm 1800 278 14.0 Tetronasin, 10 ppm 1720 276 13.4

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25 Table 2-9 --continued Daily \t7r\f~\ 1 Intake ^a m yiuwuii, Kererence ana aieus y / U a /m^ y / Murray et al. (1990) Lucernelupins^ NR Control 138 13 .4 Flavoittycin, 10 ppm 144 13 .0 Flavomycin, 2 0 ppm 162"^ 13.8 Flavomycin, 3 0 ppm 152 13.5 Hay-f ishmeal"^ NR Control 161 13 .2 Flavomycin, 10 ppm 151 14.6 Flavomycin, 2 0 ppm 142^" 15. 5"" Flavomycin, 3 0 ppm 130"^ 15.4^ ^ Diets fed at maintenance to Merino wethers, 37 kg BW. Oaten chaff 6% CP; pellet diet (59% lucerne, 25% lupins, and 15% barley) had 20% CP. Lasalocid: 30, 50, and 70 ppm; avoparcin: 25, 50, and 75 ppm, flavomycin: 5, 15, and 30 ppm. Reported values are means of the three levels because no effect of level. Flavomycin higher (P < .05) gain than all others treatments. Flavomycin higher (P < .01) wool production than other additives and higher (P < .1) wool production than control. Pellet greater (P < .001) wool production than chaff diets. ^ Weaner Merino wethers, 29 kg BW, given ad libitum access to diets. Chaff diet had 7.3% CP; pellet diet (59% lucerne, 25% lupins, and 15% barley) had 19% CP. Difference due to additive: both additives increased (P < .001) gain of sheep eating pellet in Run II, and both additives increased (P < .01) wool growth in sheep fed pellet in both Runs. Twenty-month old merino ewes fed at 3.5% of BW. Lucerne-lupin diet (58% lucerne, 25% lupin, 15% barley) had 18% CP; hay-fishmeal (24.5% lucerne, 51.5% wheat chaff, 10% lupin, 12% fishmeal) had 17.3% CP. Flavomycins linearly (P < .001) depressed gain in animals fed hayfishmeal Indicates value different from control (P < .005). high roughage diets. Bambermycins appears to have little effect on feed intake when compared with control. Animals supplemented with bambermycins appear to have higher feed intake than the ones supplemented with ionophores, especially monensin. How bambermycins affects intake of warm-season grasses (pasture or hay) is unknown. Other feed additives, such as monensin, have been shown to have a variable effect on forage intake (Ellis et al., 1984).

PAGE 33

26 Because bamberinycins is targeted for use with high fiber diets it is a high research priority to evaluate its effect on intake in this type of diet. Bamberirtycins also improved efficiency of feed utilization. This information, however, has the same limitations indicated for intake. Limited information suggests that bambermycins did not affect amount of feed intake in sheep. Reports only mentioned effect on rate of eating and it is not clear if this effect translated into effects on performance. Information from sheep research did little to clarify the effect of bambermycins on intake, gain and feed efficiency. There are no available data on the effect of bambermycins with high roughage diets supplemented with molasses. Limited information suggests that ionophores may not be efficacious in those diets. Research is needed to test the efficacy of bambermycins for improving gain, and to evaluate its effects on feed intake and efficiency of feed utilization with cattle fed this type of diet. Digestive Function In vitro Bambermycins included at 8 or 2 0 ppm in media had very little or no effect on several variables. Substrates included casein, amino acids, cellobiose-maltose, neutral detergent fiber, and starch. Variables measured included proportion of volatile fatty acids produced from different substrates, methane and ammonia production, efficiency of bacterial growth, and fiber and starch

PAGE 34

27 digestion. The only effect of flavomycin was a higher proportion of acetic acid and lower proportion of butyric acid when neutral detergent fiber was the substrate. Flavorrtycin, one of 15 additives tested, had less effect on variables studied than most of the other additives (Van Nevel and Demeyer, 1990; Van Nevel and Demeyer, 1992) Rnminal pH. Bamberimycins inclusion in the diet increased ruminal pH at the end of week 4; however, by the end of week 9 there was no effect on pH (Murray et al., 1992) Using high (pellet) and low (chaff) quality diets at maintenance level, Aitchison et al. (1989a) found that inclusion of bambermycins increased ruminal pH (6.94 vs 6.57, average of both diets). When the same diets were given ad libitum, there was a trend toward a similar effect (Aitchison et al., 1989b), No effect of bambermycins on pH was found in diets supplemented with different sources of sulfur (Murray et al., 1991), or 10, 20 and 40 ppm bambermycins (Murray et al., 1990). Because samples were obtained with stomach tube in these trial, the sampling technique may have contributed to the variability observed. Ruminal ammonia Bambermycins increased nominal ammonia concentrations in weaner lambs (147 vs 117 mg N/L) and adult sheep (180 vs 173 mg N/L) on a diet of alfalfa chaff (30%), chopped wheat en hay (52%) fishmeal (6%) and lupin grain (10%), containing 16.4% CP (Murray et al., 1992). Other work reported by the same researchers showed no effect of

PAGE 35

28 bamberiiT/cins on ruminal ammonia with diets of different qualities and supplemented with several sources of sulfur (Murray et al., 1991). When alfalfa-lupin and hay-fishmeal diets were supplemented with 10, 3 0 and 40 ppm bambermycins ruminal ammonia concentrations were depressed only in the hay-fishmeal diet (Murray et al. 1990). Bambermycins produced opposite effects on ruminal ammonia concentration depending upon the level of feed intake. Bambenrr/cins added to high or low quality diets fed at maintenance level increased ruminal ammonia (349 vs 291 mg N/L) in the high quality diet only (Aitchison et al., 1989a). However, when those diets were fed ad libitum bambermycins reduced ruminal ammonia concentration (168 vs 216 mg N/L) in the high quality diet (Aitchison et al., 1989b). Bambermycins inclusion in concentrate fed to young calves had no effect on ruminal ammonia concentrations (El-Jack et al., 1986; Fallon et al., 1986). Rowe et al. (1982) also did not find an effect of flavomycin on ruminal ammonia in cattle. Ruminal volatile fatty acids Inclusion of bambermycins in the diet decreased the total VFA concentration (65.3 vs 78.5 mM/L) by the end of wk 4, but there was no effect of bambermycins by wk 9. Molar proportions of propionate were increased at both sampling times from 25 to 28 mol/100 mol (Murray et al 1992). In another trial, bambermycins increased total VFA when the diet was supplemented with methionine, but it had no effect on total VFA or propionate I

PAGE 36

29 molar proportion when the diet was supplemented with other sulfur sources (Murray et al., 1991). In lucerne-lupin and hay-fishmeal based diets, addition of 10, 20 and 40 ppm bambermycins did not affect total VFA or acetate proportions. However, propionate proportion was increased, while butyrate was decreased in both diets. The level of 2 0 ppm of bambermycins was the most consistent in producing these effects (Murray et al., 1990). Bambermycins did not affect total VFA or molar proportions of individual VFA when added to low or high quality diets fed at maintenance levels (Aitchison et al., 1989a) When these diets were fed ad libitum, 10 ppm bambermycins decreased total VFA in both diets. Addition of 2 0 ppm bambermycins increased propionate proportion in the high quality diet only (Aitchison et al., 1989b). In fattening cattle, bambermycins did not affect total VFA or VFA molar proportions (Flachowsky and Richter, 1991; Alert et al., 1993). Also, in young calves, flavomycin inclusion in the dry feed had no effect on VFA proportions in ruminal fluid (El-Jack et al., 1986; Fallon et al., 1986). In contrast, Barley et al. (1996) reported that bambermycins lowered the acetate:propionate ratios in steers fed alfalfa-grass hay. Steers fed monensin, however, had lower acetate : propionate ratios than steers fed bambermycins. In a similar experiment, DelCurto et al. (1996) reported higher total VFA in steers fed a 90%

PAGE 37

30 concentrate diet with bambermycins In this trial, steers fed bambermycins had lower acetate: propionate ratios than the ones fed lasalocid. Ruminal protozoa Bamberitry-cins in the diet did not affect the number of protozoa in a nine-week trial with sheep (Murray et al., 1992). Alert et al. (1993) reported similar findings in cattle. In summary, bambermycins has not had consistent effects on ruminal pH, ammonia, total VFA concentrations or molar proportions of VFA. This may be related to experimental conditions and (or) bambermycins may not have a specific action on ruminal fermentation, in contrast to ionophores. Ruminal digestion Bambermycins did not affect cellulose degradation, total VFA or VFA proportions in the rumen in cattle (Rowe et al., 1982), even though they used a high dose (20 mg/100 kg body weight) This was interpreted as no effect of bambermycins on ruminal fermentation. In situ rate of digestion was not affected by addition of bambenrycins or ionophores in steers on alfalfa-hay diets (Early et al., 1996). On the other hand, bambermycins decreased OM, CP and CF digestibility in the rumen (Poppe et al., 1993). Bacterial microbial protein production was also reduced. However, 30 g/d more amino acid, apparently of dietary origin, reached the gut in bulls supplemented with bambermycins

PAGE 38

31 Total tract digestion Bambermycins did not affect total tract digestibility of DM, CP, fat, CF, ash and N-free extract in wethers offered a diet of 50% beet pulp and 50% concentrate at maintenance level (De Schrijver et al., 1991) Flachowsky and Richter (1991) also reported no effect of 5 or 10 mg of bambermycins /d on apparent OM digestibility in wethers. In an experiment with fattening Friesian bulls, 50 mg of bambermycins/d increased apparent DM, CF and N-free extract digestibility (Alert et al., 1993). Increased total tract OM digestibility was also reported by Poppe et al. (1993) in cattle. In young cattle, addition of flavomrycin increased total tract CP digestibility (Fallon et al., 1986) Total tract digestibility tended to increase in steers supplemented with bambermycins or ionophores on alfalfa-hay diets (Barley et al., 1996). However, no effect of feed additive was observed in 90% concentrate diets (DelCurto et al., 1996). There is no consistent effect of bambermycins on ruminal or total tract digestibility. Post-ruminal Fffpct.q Rowe et al. (1982) measured the post-ruminal antibacterial activity of bambermycins by inhibition bioassay using Bacinus subtil is. There appeared to be no loss of antibacterial activity of bambermycins in the sheep digestive tract. The presence of active antibiotic in the intestine prompted suggestions that bambermycins may act at

PAGE 39

32 the post-ruminal level, similar to the mechanism of action proposed in monogastrics (MacRae, 1989; Rowe et al., 1991). Bambern^cins increased whole body protein accretion (Table 2-10) when given alone or in combination with clenbuterol. These findings support the notion that antibiotics active in the gastrointestinal tract reduce the mucosal cell turnover by reducing microbial invasion, thereby allowing a greater net partitioning of amino acids towards other body tissues (MacRae, 1989; MacRae and Lobley, 1991) Post-ruminal effects have also been proposed for avoparcin. MacGregor and Armstrong (1984) used mature sheep fitted with ruminal cannulas and re-entrant cannulas at the proximal duodenum and the terminal ileum. Avoparcin and (or) saline solution were continuously infused into the proximal duodenum and subsequently into the rumen of four sheep. Results of their study and those of a previous one where avoparcin was incorporated in the diet (MacGregor and Armstrong, 1982) are presented in Table 2-11. Table 2-10. Protein accretion (g/d) in sheep given clenbuterol (1.5 mg/d) and (or) bambermycins (20 mg/d) Treatment^ Control Clenbuterol'' Control 20.6 35.0 Bambernr/cins'' a — = — — 25.0 39.4 From MacRae and Lobley (1991). Sheep (n = 12) were fed pelleted dried ^rass at twice maintenance level of energy intake. Effect of clenbuterol, P <.001. C Effect of bambermycins, P <.05.

PAGE 40

33 Table 2-11. Effect of avoparcin on the net uptake of amino acids from the small intestine (g amino acid N/g amino acid N entering the small intestine) Duodenal Ruminal Included in infusion infusion feed Amino acid^ Con Avop Con Avop Con Avop Total .648 697 634 .692 .598'' 687^^ Essential .613 679 .664 .734 .553 .682 Non-Essential .628 689 673 .747 .580 .718 ^ MacGregor and Armstrong (1982). P <.05, trends for individual amino acids were in the same direction. Con = control; Avop = avoparcin. Avoparcin increased net uptake of amino acids when included in the diet but not when ruminally or duodenally infused. Nevertheless, the authors felt that the trends were consistent with avoparcin enhancing net uptake of amino acids from the small intestine and that this effect was independent of any effects the antibiotic may have on digestion occurring in the rumen. Further work in rats showed that increasing levels of avoparcin in the diet increased intestinal dipeptidase activity (units/g fresh weight of mucosa) and specific activity (units/mg protein) If the hydrolysis of dipeptides to amino acids by the action of dipeptidases was the rate limiting step in the transfer of amino-N from the intestinal lumen to the portal blood then an increase in dipeptidase activity stimulated by avoparcin could account for at least part of its growth-promoting effect (Parker et al., 1984),

PAGE 41

34 Predominant bacteria in the small intestine are grampositive which are sensitive to many antibiotic additives, whereas those in the large intestine are gram-negative. Therefore it is expected that the small intestine is the more likely site for antimicrobial effects (Parker, 1990) Avoparcin was associated with increased N retention, probably due to lower turnover of gut mucosa (MacRae, 1989; (Parker, 1990) To test the hypothesis that avoparcin will affect gut tissue metabolism of ruminants, Parker (1990) conducted a trial in which weaned lambs were fed a pelleted diet containing either 0, 19 or 28 ppm avoparcin for 6 wk. At the end of the trial, five sheep from each group were anesthetized and injected with vincristine, which causes any cell entering into mitosis to be arrested at metaphase. Rate of cell division in the crypts of duodenal tissue after 90 min was significantly lower with avoparcin treatment, providing evidence for a nutrient-sparing effect in the small intestine. Thus, it appears that the effect of avoparcin in the small intestine of ruminants could be similar to those observed in monogastric species consuming diets supplemented with antibiotics. Other effects Bambermycins reduced the duration and prevalence of Salmonella shedding in calves and reduced the number of Salmonella resistant to other antibiotics (Dealy and Moeller, 1977a) Reduction of Escherichia ml i resistant to other antibiotics has also been reported (Dealy and

PAGE 42

35 Moeller, 1977b) Flavomycin exhibited a preferential inhibition of E. coli and S typhiimirimn bearing plasmids (Huber, 1979). Suinmary of Effects of Bambermycins The limited information available suggests that bambermycins improves animal performance, but in some cases, this effect depends upon the nature of the basal diets. Effects on ruminal fermentation are not consistent. Some researchers have reported increased propionate, while most others reported no difference in VFA concentrations or molar proportions. Total tract digestibility has been increased in several reports, but not in others. The presence of active antibiotic in the small intestine suggests possible postruminal effects, and modes of action may be similar to those reported for avoparcin. Bambermycins improved gain in several experiments, but it was seldom associated with changes in ruminal fermentation that would suffice to explain this effect. Few ruminal effects support the hypothesis of post-ruminal effects. Research is needed to test this hypothesis in cattle. Techniques described by Jin et al. (1994) to evaluate intestinal growth, cell proliferation, and morphology may be useful for such purposes. However, this type of research is beyond the scope of this project. The effect of bambermycins on ruminal function and nutrient digestibility is not completely elucidated, and there is no information with high roughage

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36 diets supplemented with molasses. Therefore this research will address the effect of bambermycins on digestive function in cattle fed these diets. Tnnnphore Antih-iotics lonophores have been defined as substances capable of interacting with metal ions, thereby serving as a carrier by which these ions can be transported across a bimolecular lipid membrane. Monensin can be described as a cation-proton antiporter while lasalocid does not display an obligatory cation-proton antiporter mechanism. lonophores do not display the same affinity for all cations. Monensin mediates primarily Na exchange, because the affinity of monensin for Na"" is ten times higher than that for K*. Lasalocid displays a higher affinity for (Bergen and Bates, 1984) Accepted mechanisms by which ionophores negatively affect bacteria include nonphysiological ion leak caused by ionophores and consequently ATP depletion. This effect is greater in gram-positive bacteria. Gram-negative bacteria have a cellular envelope (outer membrane) that serves as a protective barrier, excluding ionophore complexes (Bergen and Bates, 1984; Russell and Strobel, 1989) Bergen and Bates (1984) summarized the effects of monensin as follows: the ionophore acts on the flux of ions through membranes dissipating cation and proton gradients and interfering with the uptake of solutes and the primary

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37 transport system in the cells. The organisms try to maintain primary transport by expending metabolic energy. Because gram-negative bacteria are able to produce ATP through electron transport, they can survive better, so there is a shift to these organisms in the rumen. It is this shift that is responsible for the final effect of an ionophore on ruminal metabolism. lonophores are generally bacteriostatic and not bactericidal (Nagaraja and Taylor, 1987) Bergen and Bates (1984) suggested that monensin would cause entry of protons into ruminal bacteria in exchange for Na*. However, Russell (1987) using S. bovis as a model, showed that direction of Na'' was opposite to this. Monensin produced a decrease in intracellular K* concentration and influx of protons, resulting in lower intracellular pH. Once intracellular pH was acidic, monensin produced an efflux of protons in exchange for Na*. The inhibition of S. bovis was attributed to futile cycling of ions across the cell membrane resulting in loss of intracellular K*, accumulation of intracellular Na^ and depletion of ATP (Russell, 1987; Strobel et al., 1989) The postulated mechanism of inhibition may be affected by high mineral concentration, as will be discussed later. A list of ionophores used or under investigation is given in Table 2-12, taken from Nagaraja (1995).

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38 Table 2-12, lonophores used or under investigation for use in ruminant diets Molecular lonophore^ weight Cation Selectivity Sequence Monensin 671 Na%K% Li%Rb'>Cs^ Lasalocid 591 Ba^\ K%Rb%Na%Cs*>Li* Laidloirr/cin 721 ND'' Lysocellin 660 Na%K\ Ca'", Mg** Narasin 765 Na">K\ Rh\ Cs\ Li" Salinomycin 751 Rb*, Na%rCs^ Sr\ Ca'% Mg"* Tetronasin 62 8 Ca"%Mg"%Na\ r>Rb' ^ From Nagaraja (1995). ND = Not determined. Effects on Performance A review by Goodrich et al. (1984) summarized the results of 228 feedlot trials and 28 pasture studies (Table 2-13) In feedlot diets, the most significant effect of monensin is an improvement of feed efficiency, as a result of little effect on gain and a reduced feed intake. A more recent review (Owens et al. 1991) showed the same trend with feedlot cattle fed monensin. Effects of other ionophores in concentrate diets are summarized in Table 2-14, adapted from Owens et al. (1991). The effect on feed intake is dependent on the ionophore used in feedlot diets. Monensin and laidlomycin appear to represent the extreme effects of ionophores on intake. Intake decreased with increasing levels of monensin. Lasalocid showed similar trends with less depression of intake, while laidlomycin increased feed intake (Owens et al., 1991).

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39 Table 2-13. Suininary of effect of monensin on intake and performance Diet type Variable Control Monensin Chanae % Ref Concentrate ADG, kg 1.09 1.10 1.6 1 Intake, kg 8.27 7.73 -6.4 Feed/gain 8.09 7.43 -7.5 Concentrate ADG, kg 1.26 1.27 .6 2 Intake, kg 8.97 8 47 -b D Feed/gain 7.29 6.83 -7.5 Pasture ADG, kg 609 .691 13 5 3 Pasture ADG, kg .786 .893 13 .7 4 Pasture ADG, kg .540 .63 0 17 5 Pasture ADG, kg .560 .650 16.3 r D Pasture ADG, kg .590 680 15.5 6 Harvested ADG, kg biz byy 14.1 b Forage Intake, kg n on / J y J 1 Feed/gain IZ 4 ID b -lb J Small Grain ADG, kg 540 620 15 7 Pasture ADG, kg !540 .605 12 ADG, kg 1.04 1.12 7.8 ADG, kg 1.15 1.24 7.8 Pasture ADG, kg .600 .640 6.7 8 ADG, kg .860 .880 2.3 ADG, kg 1.16 1.25 7.8 ADG, kg .970 1.02 5.2 ADG, kg 610 .690 13.1 1 = Goodrich et al (1984), summary of 228 trials. 2 = Owens et al (1991), summary of 137 trials. 3 = Goodrich et al (1984), summary of 24 trials. 4 = Wilkinson et al. (1980), 12 trials. 5 = Potter et al (1976), 4 trials. 6 = Potter et al. (1986), 24, 11, and 12 trials respectively. 7 = Ellis et al. (1984), each mean is one trial. 8 = Parrot et al (1990), 8, 8, 4, 4, and 4 trials respectively. Monensin delivered by ruminal bolus.

PAGE 47

d H rH CTl rH ro rH CS rH Q) rH CO [~~ U 0 CO >1 a 0 •H CTl o CO CN ro (d ro rH CO O r1 OJ 0) Eh d iw Xl •H 00 <* o u cn rH GO 0 •rl rH (T3 W (1) d -H o u U ID 00 G ro O 00 1 loi rH C7> O X} IW •H U (C3 Q) a on Ti J3 XI •H rH o U m [> phores .lo ro rH CO Lasa o .on a •H LT) rH og rH CTl 00 og [> 1 Cn C -H (N CO kg M ga Q) rH rH (T3 '6 ,Q 0) •H a Q) 0) (0 4J ^ Q Q) Q) Eh H Eh < b 4-1 -O (0 — 4J u 0) 0) 4-1 o 14-1 (V 01 0) o u 0 01 w o 0) -rH >-l 4J 4J O (C (0 duo O (0 >4-l a ( 0) 0) i-l o XI ^ ^ Oi 0< 0< coo c c c o o o M-l <4-l MH 0 0 0 U J2 (0 0) M o 14-1 0) U 4J 4J 4J u o 0) 0) 14-1 M-l M-l 144 14-1 4-1 0) 0) (U 4J u 0) o 4J 4J c 01 „ g-H 0) P c w o 01 T) S 01 w -H -H g T3 U ^ m MH ^ 3 14-1 0) 0 u (0 0) 0) c > •H 4-) (0 rH 0) M w 4J c 01 M u (0 (8 0) 0) c a 0) > o 01 01 > > ••00m w XI J3 0) 0) (0 (0 (8 )-l O O 0) 0) 0) j:: 4:: X j3 a a 4J 4J 4J 0 0 c c o o o O O 4J 4J 4J u o >4-( H rH 01 0) a > rr< T3 rH 0) 4J to 3 O O -H -H 4J 4J H -rl -H 0) TJ Ti "O > TITI TI 0) nj (0 (0 0) > 0) w ^ rH rH 5^ ^ ^ -H -H ^ 4J 4J (B 0) 5 m n •* "5 0 oi^„ o o V V V 01 S rH in IT) 000 c (0 u 01 -H g 4J (0 01 ^ Xi TS 4J (0 4-1 & (0 01 c Di 10 (0 a< Oi V V -'~-' — a< Oi 4-1 4J 4J — — u u c; 0) 0) 0) 01 01 '4H 14H >4-l Dl O) MH MH 14H C G 01 0) 0) m m ^ j3 U U o u 4J 4-1 4-1 (0 (0 (IS M >-l M 01 di T3 T3 73 c! c: (0 US (0 3 3 3 u u (TS (0 u (0 0) 2 J o< 01 01 O J3 u 01
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41 Effect of monensin on high roughage diets is also presented in Table 2-13. In general, the effect is translated into increased ADG and feed efficiency. The effect on feed intake appears variable. Under grazing conditions, ionophores usually improve ADG. Feed efficiency data are rarely available because inherent difficulty in measurement of feed intake in grazing animals. A more subtle effect such as no change in ADG with decreased pasture intake may occur. In this case, the benefit of feeding an ionophore will be realized only if stocking rate is increased. However, this effect is difficult to measure and may not have a real economic value (Rowe et al., 1991). Horton et al. (1992) reported an increased ADG in yearlings supplemented with ionophores (lasalocid or monensin) while grazing subtropical grass forages, but responses were inconsistent and appeared to be associated with forage quality and environmental conditions. This variable effect of ionophores (monensin) in grazing animals was related to the interaction of monensin with pasture digestibility and digestive function (Ellis et al. 1984). In grazing animals monensin generally reduces the turnover rate of undigested forage residues and thereby increases the digestibility of fiber. Intake of forages (45 to 65% OMD) was increased with monensin apparently as a result of increased undigested fill. Intake of poor quality forages (< 45% OMD) is decreased by monensin caused by a reduced turnover of

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42 undigested dry matter combined with the animals' inability to accommodate further increases in fill of undigested dry matter. Intake of higher quality forages (> 65% OMD) appears to be decreased by monensin perhaps through a metabolic intake regulation, analogous to the lower intake observed with high concentrate diets. Thus the expected gain response to monensin decreases as the quality of forage consumed increases (Ellis et al 1984). Rowe et al. (1991) suggested that in animals grazing low quality pastures, maintaining or losing weight, there is less chance of a positive response to ionophores. This opinion would agree with the depressed intake observed when ionophores are fed with low quality forage (Ellis et al., 1984), Pond and Ellis (1979) reported three trials where the response to monensin was evaluated in cattle grazing bermudagrass pastures. Monensin increased intake (3.4%) in one trial, but it decreased intake in the other two trials (4.6 and 19.4%). Although intake was decreased, monensin increased ADG. This effect was explained by a reduction in rate of passage of digesta. The resulting increased residence time in the rumen increased digestibility of forage. Gains were .460, .565, and .780 kg for cattle on bermudagrass pasture alone, pasture plus .9 kg corn, and pasture plus corn and 100 mg of monensin, respectively (Oliver, 1975) Monensin increased ADG by 38%. In a similar

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43 experiment, cattle on bermudagrass pasture gained .42 and .52 kg for pasture plus .9 kg corn and pasture plus corn and 200 mg of monensin, respectively. Forage to gain ratios estimated for control and monensin were 19:1 and 15:1, respectively (Rouquette et al., 1980). In a different trial, ADG were .45, .47, and .68 for steers on pasture alone, pasture plus .9 kg corn, and pasture plus corn and 2 00 mg of monensin, respectively. Estimates of forage to gain ratios were 20.5:1, 19:1, and 13:1, respectively (Rouquette et al., 1980). Monensin increased gain by 24 to 45% in this trial. Byers and Schelling (1984) used an isotope dilution technique to measure body composition. They reported that digestive tract fill in cattle grazing high-quality pastures was decreased by monensin or lasalocid. When cattle grazed more mature, low-quality forage, lasalocid reduced fill, while monensin had no effect on fill. Thus, fill may be different not only under the conditions as discussed by Ellis et al. (1984), but also with different ionophores. lonophores also can increase the response to supplements. This effect is particularly important because in high roughage diets ionophores are generally fed daily in .5 to 1 kg of a carrier supplement. Pasture plus supplement (no monensin) supported gains of cattle ranging from .24 to .96 kg/d (Potter et al 1986). The addition of 200 mg monensin/d to the supplement increased gain by .09 kg/d (16.3%) across the 24 trials. In a different series of 11

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44 trials, cattle on pasture alone gained .50 kg. Supplementation with .9 kg/d of energy supplement increased ADG by .09 kg, and the addition of monensin to supplement further increased ADG by .09 kg/d (Potter et al., 1986). In a series of 12 trials, monensin supplementation of harvested forage fed in confinement reduced feed intake by 3.1%, improved ADG by .09 kg (14.4%) and improved feed efficiency by 15.3%. Efficiencies of supplemental feed to extra gain (kg supplement : kg gain) were 10.1:1 and 5.0:1 for the supplemented only and the supplemented plus monensin groups, respectively (Potter et al., 1986). Monensin can be administered through intrariaminal devices, avoiding the need for a carrier supplement. Parrot et al. (1990) reported increased ADG in steers and heifers under different environmental conditions (Table 2-13). Response to monensin may be related to stocking rate and pasture quality (Cochran et al., 1990). lonophore feeding has also been shown to benefit cowcalf production systems. Monensin feeding has been shown to increase ADG and reduce age at puberty in beef heifers (Sprott et al., 1988). This effect of monensin appears to be independent of growth rate (Lalman et al., 1993). lonophore Modes of Action The mechanisms by which ionophores improve performance or feed efficiency have been attributed principally to alterations in ruminal fermentation. However, because

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45 ionophores have activity in both prokaryotic and eukaryotic cells, part of the performance may be due to effects outside the rumen (Nagaraja, 1995) According to Bergen and Bates (1984) ionophore feeding affects ruminal fermentation in three major areas: a) Increased production of propionate and decreased production of methane, b) decreased protein degradation and deamination of amino acids, c) decreased lactic acid production and froth formation in the rumen. A summary of known effects of bambermycins avoparcin, monensin, and lasalocid is presented in Table 2-15. Ruminal effects Ruminal fermentation of carbohydrates, protein and glycerol result in anaerobic oxidation to acetate, carbon dioxide and ammonia. Methane, propionate, and butyrate are produced mainly as a result of electron and proton transfer reactions (hydrogen sinks) Methanogenesis keeps the partial pressure of hydrogen very low avoiding the formation of lactate or ethanol as major end products and allowing more acetate to be produced (Van Nevel and Demeyer, 1995) Volatile fatty acids The most consistent fermentation alteration when ionophores are fed is the increased molar proportion of propionic acid produced in the rumen (Table 216) Increased propionate production results in improved fermentation efficiency because of a greater recovery of metabolic hydrogen (Chalupa, 1984). Furthermore, Armstrong

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46 Table 2-15 Effect of antibiotic feed additives on digestive function and disorders Item" Monen Lasal Avop Bamb Ruminal NH3 Total VFA Acetic Propionic Butyric A: P Methane Rumen fill Liquid turnover Solid turnover nc/+ nc^ nc nc/nc -/nc + nc/-/+ nc/ + nc/+V'-/nc nc/+/-/nc +/nc -/nc nc/Rumen bacteria Yield nc -/nc Gram positive _ Lactate utilizers nc nc Rumen protozoa nc Rumen fungi Rumen digestibility DM nc/+ -/nc Fiber nc nc/+ nc/Protein +/_ Starch nc/Total tract digestibility DM nc/+ + + nc/ + Fiber nc/+ + nc/+ Starch nc nc Protein + + nc Other Bloat Coccidia nc Lactate 3 -Methyl indol ^ Chalupa, (1984); Van Nevel and Demeyer, (1988, 1992); Bergen and Bates, (1984); Schelling, (1984); Faulkner et al (1985); Spears, (1990); Owens et al (1991), De Schrijver et al (1990); Murray et al., (1990); Murray et al., (1992); Aitchison et al. (1989a, 1989b); Galbraith et al., (1983); Alert et al (1983); Poppe et al., (1993); MacGregor and Armstrong, (1982); Chalupa et al (1981); Froetschel et al., (1983); Nagaraja, (1995); Nagaraja et al (1987); Early et al (1996)j^ DelCurto et al (1996). Monen = monensin, ^ Lasal = lasalocid, Avop = avoparcin, and Bamb = bambermycinp References: = decrease; ^ nc = no change; ^ + = increase; V = or.

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47 Table 2-16. Effect of monensin of ruminal volatile fatty acids in cattle Diet^ Variable Control Monensin % change /U : J U Concentration, mM Acetate l\J bl lo Propionate +z 1 Butyrate J.U Q o -z U Proportion, % Acetate / 1 U c c o D D O b Propionate 1 Q 1 j_ O Q +z y cucyrace y y O D 14 Production, M/d Propionate / /4 11 Z + 4b 50:50= Concentration, mM Acetate 65.8 55.9 -15 Propionate 41.1 41 y +z Butyrate lo b y 1 -J J Proportion, % Acetate 53.5 51.3" -4 Propionate 33.4 38.4" +15 Butyrate 11. 0 8.3 -25 Production, M/d Acetate 7.32 8. 68 + 19 Propionate 4.82 7.30 + 52 Butyrate 2.12 1.76 -17 Production, g/d 70:30^ Propionate 441 659" + 49 20:80"^ Propionate 510 899" + 76 Source: Nagaraja (1995) ^ Roughage : concentrate Prange et al (1978). = Rogers and Davis (1982). Van Maanen et al (1978). Different from control, P <.05. and Blaxter (1957) showed that the efficiency of utilization of acetate was low when infused into the rumen as the only energy source, but was increased by the addition of propionate. The heat increment of acetate is reduced by addition of glucose precursors to the diet (Tyrrell et al., 1979) Acetate clearance rate is increased by an incresed

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48 ruminal propionate : acetate ratio (Cronje et al., 1991). Propionate can be used in gluconeogenesis or oxidation, while acetate can not be used for gluconeogenesis. The increased molar proportion of propionate in ruminal fluid may represent a conservative estimate of the amount available for subsequent metabolism. It has been shown in steers fed a high roughage diet with added monensin that propionate production rate was increased by 49% while the molar proportion of propionate in the ruminal liquor only increased by 15%; comparable figures for steers fed low roughage diets were 76% and 25% (Van Maanen et al., 1978). Methane Methane production can be as great as 12 L/h in beef cattle (Thorton and Owens, 1981) Methane production can represent as much as 12% loss of feed energy. lonophores can decrease methane loses by 30% (Schelling, 1984) The effect of monensin on methane production in vivo is variable. Reduction of 16 to 31% in methane production has been reported according to research reviewed by Van Nevel and Demeyer (1995) About half of the decrease in methane production when monensin was fed was associated with the reduced feed intake. When cattle were fed hourly no effect of monensin on methane production was observed (0' Kelly and Spiers, 1992) However, depression of methane production may be transitory, with methane production returning to normal within two weeks (Rumpler et al., 1986). Monensin is not directly toxic to ruminal methanogens, but inhibits

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49 organisms converting formate to carbon dioxide and hydrogen. Thus, monensin reduces the supply of substrate for methanogenesis (Van Nevel and Demeyer, 1995) Ruminal nitrogen metabolism Ruminal ammonia production often exceeds the needs of ammonia-utilizing species. Excess ammonia in the rumen is absorbed and converted to urea in the liver. Although some urea is recycled back to the rumen, much of it is lost in the urine (Russell and Strobel, 1989). Monensin decreases ammonia production in vitro (Van Nevel and Demeyer, 1977) and in vivo (Dinius et al., 1976; Hanson and Klopf enstein, 1979; Poos et al 1979). lonophores appear to affect ruminal degradation of peptides and deamination of amino acids to a greater extent than proteolysis. An apparent contradiction in the nitrogen sparing effect of ionophores was that the most active ammonia-producing bacteria (gram-negative) were resistant to ionophores. Three new gram-positive species with high activity of ammonia production have been isolated. These new isolates were sensitive to ionophores and had a 20-fold greater ammonia production than previously identified ruminal bacteria species (Russell, 1991; Russell et al 1991) Decreased peptide degradation and amino acid deamination caused by ionophores is attributed to inhibition of these ammonia-producing bacteria (Yang and Russell, 1993b) Monensin decreased by 10-fold the number of highly active amino acidfermenting ruminal bacteria in vivo. These

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50 bacteria utilize peptides and amino acids, but not carbohydrates for growth. As a result, there was less amino acid deamination and less ammonia production. In this study monensin did not increase soluble protein, peptides or amino acids in ruminal fluid. Monensin, however, increased the concentration of bacterial protein in ruminal fluid, which would provide additional protein for the animal. They suggested that monensin-resistant bacteria utilized the peptides and amino acids from dietary protein (soybean meal) that were spared from deamination and converted these to microbial protein. Some ruminal bacteria, such as Prevotella ruminicola showed increased growth efficiency when peptides and amino acids were their source of N (Russell, 1984) In another experiment Yang and Russell (1993a) added protein hydrolysates directly to the rumen. Peptide and amino acid concentrations in ruminal fluid decreased at a logarithmic rate after addition. When cows were fed 350 mg monensin/d the rate of peptide and amino acid disappearance and ammonia concentration were decreased. Monensin increased the ruminal outflow of peptides and amino acids from infused hydrolysates. The effect was dependent on the source: greater ruminal outflow for gelatin than soybean hydrolysates. These reports suggest that monensin increased passage of amino-N out of the rumen, although by a different mechanism (increased bacterial-N and dietary amino-N flow, respectively) It is noteworthy that the experimental model,

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51 cows fed at frequent intervals to achieve steady state conditions, was the same in both experiments. Monensin decreased ruminal urease activity by 66% (Starnes et al., 1984). This effect would have a beneficial effect on urea utilization in ruminants, because the rate of urea hydrolysis is faster than the rate of ammonia assimilation by ruminal bacteria (Starnes et al., 1984). The net effect of ionophores on the nitrogen economy of the animal will depend upon specific dietary situations. Increased flow of amino acids to the small intestine could improve production when diets are: a) marginal in crude protein, b) the crude protein is high quality true protein, c) the rate of proteolysis is rapid and d) the rate of carbohydrate fermentation is slow (Russell, 1991) Thus, it is conceivable that the effect of ionophores on metabolizable protein for the host animal will depend upon the net result of all these processes. Other effects Other effects of ionophores on rumen function include reduction of turnover rate of solids and liquids, modification in ruminal fill and retention time, and depression of ruminal motility (Lemenager et al., 1978a; Ellis et al., 1984; Deswysen et al., 1987). These changes may explain changes in feed intake, especially in high roughage diets.

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52 Total tractdigpsti bi 1 i Spears (1990) suggested that the higher energydigestion could be explained by increased fiber digestion (Table 2-17) This higher fiber digestibility may result from longer solid retention time in the rumen, thus allowing greater time for microbial digestion of fiber. Total tract digestibility of starch was not affected by ionophores. However, lasalocid and monensin reduced ruminal digestibility of starch and increased the quantity of starch digested in the intestine. This shift in site of digestion should have resulted in more energy absorbed from starch as glucose rather than as VFA and improved energy utilization (Muntifering et al 1981; Spears, 1990). Higher apparent nitrogen digestibility could be explained by: a) a higher ratio of dietary to microbial protein entering the small intestine because feed protein is usually more digestible than microbial protein, b) fecal endogenous nitrogen losses may be reduced, by decreased microbial protein synthesis in the large intestine and cecum or by decreased sloughing of intestinal cells (Spears, 1990) Ionophores improve absorption of several minerals. In his review. Spears (1990) concluded that ionophore supplementation increased apparent absorption of Mg, P, Zn and Se, whereas absorption of Ca, K and Na were affected inconsistently by ionophore feeding.

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53 Table 2-17. Apparent digestibility of energy and nitrogen in cattle fed ionophores Nutrient/ lonophore^ Digestibility, % Control lonophore Percent change Number of trials Energy Monensin 70.3 72 .4 -.9 to 9.2 17 Lasalocid 75.7 77.7^ 1.9 to 2.2 8 Nitrogen Monensin 62.2 65.7^ .3 to 8.0 15 Lasalocid 70.8 76.4'' .2 to 7.2 3 Adapted from Spears (1990) Means for control and treated animals differ (P<.05) when analyzed by analysis of variance using experiment as replicate. Chalupa (1984) summarized the increased retention of energy and protein produced by addition of monensin in the diet (Table 2-18) In those experiments the increase in energy retention was related to an increase in the amount of metabolizable energy by decreasing fecal and methane energy losses Metabolism of the host an-imal Inhibition of methane production in monensin-treated animals is responsible for about one-third of the improvement in energy utilization (Van Nevel and Demeyer, 1988) There are several studies where significant increases in propionate molar proportions were measured without any improvement in feed conversion efficiency, and there are also studies in which the increase in propionate was too small to explain the magnitude of the improvement observed (Rowe et al. 1991)

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54 Table 2-18. Energy and nitrogen partitioning in animals supplemented with monensin X cem Trial oneep oneep o Leex Energy, % of control Feces 98 93 90 Digested 101 103 104 Urine 92 84 99 Methane C Q "7 A 1 'i Metabolized 105 108 107 Heat 102 105 104 Retained 111 115 119 Nitrogen, % of control Feces 97 98 88 Digested 102 101 107 Urine 92 87 99 Retained 127 138 120 Data siiinmarized by Chalupa, 1984 Monensin at 10 ppm in 50% grain diet. Monensin at 20 ppm in 50% grain diet. Monensin at 3 mg/kg BW'^, 80% grain diet. In cattle and sheep, about half the dose of monensin is absorbed, metabolized, excreted in the bile, and eliminated in feces (Donoho, 1984) This suggests that monensin may have systemic effects. Plasma concentration of minerals (Mg, Na, K) has been altered with ionophores (Spears, 1990; Owens et al., 1991). Depression of heat increment and amino acid sparing effects have also been cited (Bergen and Bates, 1984) A second "protein sparing effect" can occur in the host. Increased propionate production in the rumen and its subsequent absorption may reduce catabolism of amino acids for gluconeogenesis (Van Nevel and Demeyer, 1988) These

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55 investigators also reported effects of monensin beyond the rumen, including changes in blood concentrations of several metabolites, hormones, and minerals. Evidence of an effect of monensin on metabolism in ruminants independent of alterations in ruminal microbial metabolism have been provided (Armstrong and Spears, 1988) Intravenous administration of monensin depressed plasma concentrations of K, P, and Mg, and increased glucose and free fatty acids concentrations. Changes in plasma mineral concentrations were suggested as indices of the cellular effect of monensin. Other indications that effects of monensin on animal performance may not be totally explained by changes in ruminal fermentation are provided by measurement of net nutrient flux (Harmon and Avery, 1987; Harmon et al., 1993). These investigators suggested that changes in the products of ruminal fermentation may not be translated into the products appearing in the portal circulation. Urea recycling was reduced in both concentrateand foragefed cattle. Changes in VFA net absorption from feeding monensin in forage fed animals were not consistent with its role in increasing ruminal propionate production, because total net energy flux did not change. They questioned the role of monensin in improving feed efficiency solely through increased ruminal propionate production.

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56 Animal hf^alt.h Altered ruminal fermentation associated with ionophore feeding reduces the incidence of acidosis, bloat, and acute bovine pulmonary edema and emphysema (Nagaraja, 1995) Probably the more important effect is the reduction in lactate production, resulting in reduced incidence of lactic acidosis in high concentrate diets. Reduction of lactic acid production resulted from a direct antibiotic effect on the gram-positive bacteria which are the more important lactate producers bovis Lactobacillus s pp ) Lactate utilizers are not sensitive to ionophores, providing an additional way of lowering lactic acid concentration (Bergen and Bates, 1984; Nagaraja, 1995). Ionophores in the diet had positive effects on blood glucose levels, reduced blood 3-hydroxybutyrate concentrations in late pregnancy and eliminated signs of pregnancy toxemia in ewes (Parker and Armstrong, 1987) Subclinical coccidiosis in lambs has been reduced when an ionophore was added to the diet. The problem appears to be particularly severe in lambs between 3 and 10 wk of age after which natural immunity builds up. In intensive lamb production, the use of ionophores could give a distinctive advantage over other feed additives that have no effect on coccidia (Armstrong, 1986) Growth promotion has also been observed in young calves. Monensin fed to young calves (7 to 10 d of age)

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57 resulted in 10 to 47% higher gains during the suckling period (30 d) and of 6 to 17% higher gains during the next 90 d. Feed intake was increased during suckling period and decreased thereafter. These effects were independent of coccidia control because no coccidia were found (Ilan et al., 1981). In a separate trial they found increased drymatter digestibility when monensin was administered either in the milk replacer or directly into the rumen. Intera ction with Minerals It has been shown that elevated dietary concentrations of Na and K may decrease the response of cattle to lasalocid and monensin (Rumpler et al., 1986; Russell, 1987; Russell and Strobel, 1989; Schwingel et al., 1989). High dietary K appears to inhibit the antibacterial effect of lasalocid more than that of monensin. Research at the University of Florida evaluating monensin and lasalocid in molasses slurries consumed at 2 to 3 kg/d has not shown improvement in gains of grazing cattle (Kunkle and Bates, personal communication) It was suspected that the high concentration of minerals in molasses, especially K (3 to 4%) and the high consumption of the molasses contributed to the lack of efficacy of ionophores in these studies. The interaction with minerals has not been completely clarified. Greene et al. (1986) has suggested that monensin appears more effective in decreasing the acetate :propionate ratio in lambs when fed with high dietary K. High dietary Na

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58 may also decrease the effectiveness of ionophore supplementation of cattle (Bergen and Bates, 1984; Rogers and Davis, 1982) High dietary Na reversed the depression of microbial synthesis (urinary allantoin) induced by monensin in sheep (Dewhurst et al., 1992). Increasing K concentration in the growth medium of pure cultures of ruminal bacteria increased the resistance of these organisms to ionophore. High extracellular K increased the minimum inhibitory concentration of lasalocid in several species. The effect of K on minimum inhibitory concentrations of monensin was similar to, but not as great as, the effect on lasalocid (Dawson and Boling, 1987). Funk et al. (1986) noted an interaction of lasalocid and K (1 and 2.5% K in the diet) for plasma urea N, acetate:propionate ratio, and NDF digestion in lambs. These interactions, however, were not reflected in lamb gains or feed intake. Research conducted at the University of Florida has shown that changes in the concentration of K and Na resulted in altered in vitro VFA production (Schwingel et al 1989). Important findings were: a) high K concentration increased acetaterpropionate ratio when lasalocid was fed, b) high Na concentration reduced VFA production when either lasalocid or monensin were fed. This research suggested potential problems associated with high dietary K and lasalocid, and high dietary Na and either lasalocid or monensin. The nature of the interaction appears complex, as Bates and Schwingel

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59 (unpublished results) have shown different effects of lasalocid and monensin on S. bovis and R. albus Monensin was less toxic to S. bovis than lasalocid, but more toxic to R. albus Increasing Na in the growth medium allowed 2^ bovis to proliferate in the presence of ionophores, especially monensin. High Na concentration, however, increased toxicity of ionophores to R. albus Increasing K permitted R. albus to survive in the presence of lasalocid, whereas no appreciable effect was observed with monensin. Because high dietary K potentially decreases the efficacy of lasalocid more than that of monensin, it has been hypothesized that monensin will be more effective in supplementation programs utilizing molasses slurries. However, there are reports of improved ADG in cattle on wheat pasture supplemented with monensin (Horn et al., 1981; Davenport et al., 1989) and lasalocid (Andersen and Horn, 1987) Potassium concentration in wheat pasture is usually high, between 2 to 4% of DM (Grunes et al., 1984). Response to monensin feeding in cattle grazing wheat pasture may also be related with reduction of bloat (Horn et al., 1981). It was suggested that monensin may be useful in neutralizing Krelated depression of Mg absorption in ruminants consuming diets high in K (Greene et al, 1986) Hypomagnesemic tetany is a metabolic disorder common in cattle grazing small-grain pastures (Grunes et al., 1984).

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60 Garret et al. (1989) reported improved gain in feedlot cattle consuming a diet containing 3 0% sugar beet molasses and with monensin added. Feed efficiency was improved by monensin in cattle consuming 3 0 and 60% molasses in the diet. Increasing levels of molasses in the control diets depressed animal performance. Apparently monensin was effective in overcoming bloat in supplemented cattle. Frequency of Feeding Molasses supplements are usually delivered 2 to 3 d/wk in most production situations. It is therefore relevant to address the issue of frequency of feeding on the efficacy of ionophores. Efficacy of monensin administration on alternate days compared to daily feeding was evaluated in five trials involving 342 cattle in 32 pastures (Muller et al., 1986). Pooled ADG were .544, .621, and .626 kg for control supplement fed daily, monensin supplement fed daily, and monensin supplement fed on alternate days, respectively. This response was similar to other trials where control and monensin supplements were fed daily (Potter et al., 1976). These results demonstrate that monensin can be effectively administered to pasture cattle in dry supplements that are fed on alternate days. Research conducted at Ona (Horton et al., 1992) with lasalocid fed daily or three times a week in dry supplement showed higher ADG in cattle fed three times a week ( 64 vs .56 kg). Monensin was administrated in a dry supplement (average .7 kg/d) fed every other day to stocker

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61 cattle on wheat pasture (Andrae et al., 1994). Improvement in gain response to supplements containing monensin were similar to previous trials conducted under similar pasture conditions, but with daily feeding of supplement monensin. Soybean meal or corn gluten meal, with or without monensin, was fed on a daily or alternate day schedule to cattle consuming ground corn stalk basal diets (Collin and Pritchard, 1992) Interactions of feeding interval by monensin, and protein source by monensin were observed for ADG and feed intake. Monensin fed at 48-h intervals reduced feed intake in steers. Monensin added to diets supplemented with corn gluten meal reduced ADG by .21 kg but monensin increased ADG in diets supplemented with soybean meal. Monensin fed on alternate days in a high ruminal escape protein supplement was not recommended based on this study. It is noteworthy that protein sources with low ruminal degradability are recommended for molasses slurries (Stateler et al., 1995; Kunkle et al., 1994; Pate et al 1995) Therefore, it is relevant to test the effect of monensin included in molasses supplements formulated with protein sources of low ruminal degradability. Based on the literature reviewed, it appears that frequency of feeding is unlikely to be involved in observed lack of response of ionophore fed in molasses supplements. Inherent differences in the energy substrate in the supplement carrier do exist (starch vs sugar) in addition to

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62 the mineral composition. Interactions of energy substrate and ionophore can not be ruled out. Summary of Eff ects of lonophores Monensin is perhaps the most researched antibiotic feed additive for ruminants. Yet, the mechanism by which it improves animal performance has not been completely elucidated. Effects of monensin on feed intake and gain in cattle fed high roughage diets appears variable. Research has shown that interactions of ionophores with different dietary mineral concentrations are complex. Limited information suggests that monensin is not efficacious to improve gain when fed in molasses supplements. High molasses mineral concentration, especially K, has been suggested as the probable cause of this lack of efficacy. However, monensin has increased gain in diets high in K, such as small grain pastures. More research is needed to clarify this issue. Energy substrate (sugars) protein level and degradability may also play a role and future research may need to consider these factors. The present research was undertaken to evaluate the efficacy of monensin to improve gain in cattle fed a high roughage diet and supplemented with different sources of energy (molasses or corn)

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63 Considerations in Feedin ? Molasses. Animal responses to feeding molasses have been extensively reviewed. Effects of molasses on rumen function, feed intake, digestibility, animal performance, and metabolic disorders were summarized from trials that covered many different dietary situations (Pate, 1983). More recently, the value of liquid supplements for animals on low quality forage has been addressed (Bowman et al., 1994). Kunkle et al. (1994) and Kunkle et al. (1996) summarized research conducted at the University of Florida comparing sources of N (urea and natural protein of different degradability in molasses slurries). Moore et al. (1995) gathered a data base from different dietary situations and analyzed feed intake and animal performance response to liquid supplements. Factors that affect liquid supplement consumption by grazing ruminants were also reviewed (Bowman and Sowell, 1995b) Effects of low levels of molasses on rumen function of high producing animals, especially dairy cows, have also been summarized (Emanuele, 1996) Because recent reviews have summarized many aspects of molasses-based supplements these topics will not be discussed. Rather, the effect of molasses on rumen function will be emphasized and highlights on intake and animal performance will be siammarized.

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64 Digestive Fimchion Ruminal microorganisms Most of the ruminal bacteria that degrade complex carbohydrates are also capable of fermenting some of the simple sugars. In addition, Treponema bryantii, T. saccharophilum Lactobacillus vitulinus and ruminus have been identified as sugar fermenters in the rumen (Stewart and Bryant, 1988) The utilization of soluble sugars is thought to be the major role of the large bacteria Quins 's Oval, which has been found to proliferate in the rumen when sugar-rich diets are fed (Stewart and Bryant, 1988) When molasses is fed in high amounts, methanol can be produced from the breakdown of pectin by pectinesterase (Russell, 1984) Eubacte rium limosum which is capable of using ethanol, was found in rumen of sheep fed a molassesbased diet. Secondary fermentations in the rumen of cattle and sheep on high-molasses diets have been reported (Rowe et al., 1979b). The bacteria Methanosarcina bakerii is capable of transforming acetate to methane and carbon dioxide. They suggested that this finding may explain the low acetate concentration found in ruminal fluid when high molasses diets are fed. This bacteria is found in mud and sludge, and because it has a slow growth rate, its survival would be possible only under low dilution rate, a condition in the rumen which is known to occur in high-molasses diets (Rowe et al., 1979a). Pate (1983) concluded that a somewhat different microbial population would be expected in the

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65 rumen of cattle fed molasses diets in view of fermentation patterns (see below) and the substrate requirements of different microorganisms. He also suggested that more research is needed to identify the microbial population in molasses-fed cattle. Protozoal densities in molassesand sugar cane-based diets are similar (1 to 5 x 10^/mL ruminal fluid) but their species population differs. Protozoal biomass is larger with sugar cane diets because large holotrichs predominate. With molasses based diets the smaller entodinia predominate. It appears that at feeding, protozoa are distributed through the rumen more uniformly. After feeding, the large isotrichs quickly store carbohydrate and through increased density they settle in the ruminal fluid and congregate. This results in selective retention of protozoa in the rumen. In slaughtered cattle on sugar cane diets, large isotrichs were not found in omasal fluid. On molasses diets, approximately 2 0% of the small entodinia left the rumen (Preston and Leng, 1980) Because considerable engulfment and breakdown of bacteria by protozoa took place in the rumen, a reduction in bacterial protein available to the animal occurred. Estimations suggest that the hourly turnover rate due to protozoal predation is higher than that of ruminal fluid turnover in most cases (Ushida et al., 1991). In a summary of 11 experiments (Ushida et al., 1991), defaunation under different dietary conditions resulted in increased flow of

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66 microbial CP and efficiency of bacterial CP synthesis (3.17 vs 4.78 g N/100 g OM digested). Defaunation consistently resulted in higher animal performance in animals on high energy (molasses or sugars) and low protein (urea) diets (Bird and Leng, 1978; Bird et al., 1979; Bird et al 1984). Defaunation appeared to have greater effects on wool production than on growth, reflecting perhaps a specific sulfur amino acid requirement for wool production. Increased protozoal numbers with inclusion of sugar in the diet was not always shown. Chamberlain et al (1985) reported an increased protozoal population in starchsupplemented rather than in sucrose-supplemented diets. Khalili and Huhtanen (1991a) could not find differences in protozoal populations between a grass silage basal diet and basal diet plus 1 kg (16% of DM) of sucrose, infused either continuously or two times a day. Bird (1989) cited by Leng (1990) suggested that response to defaunation may not be related to protozoal population densities, a low density being as detrimental as a relatively high density. Defaunation improved animal performance on molasses-urea diets but the mechanism was not clear. Improved microbial protein supply to the host, changed protein : energy ratios in absorbed products of digestion, and improved efficiency of feed utilization have been offered as explanations (Leng, 1990) On the other hand, defaunation did not result in improved animal performance on higher quality diets high in

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67 true protein. In a review, Veira (1986) stated that the major nutritional effect of protozoa is to change the ratio of protein to energy in the nutrients absorbed, with faunated animals having lower protein and higher energy availabilities compared with defaunated ruminants. Ryle and Orskov (1987) suggested that the positive response to defaunation in molasses-fed animals may be related with the particular population of protozoa. Because holotrics are more sensitive to pH fluctuations, entodinia predominate and they are more active predators. They suggested that increasing dietary fiber (long hay) may create favorable conditions for holotrics. They also noted that holotrics were associated with high propionate concentrations, while entodinia where associated with higher butyrate concentrations. Defaunation under Florida conditions, where the basal diet (medium to low quality hay or stockpiled pasture) is supplemented with molasses slurries (often containing true protein) may not be beneficial. If urea is used as the major source of N, then the protozoal population may become relevant Ruminal volati le fatty acids Feeding molasses to cattle increases the molar proportion of butyric acid in the rumen. This increase appeared to be at the expense of propionic acid when molasses is substituted for grain, or at the expense of acetic acid when molasses is fed as a

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68 supplement in forage-based diets (Pate 1983). Feeding of molasses did not appear to have a consistent effect on the total VFA concentration or ruminal pH, Beever (1993) summarized three dietary scenarios (Table 2-19) These suggest that an acetate-inducing fermentation is more efficient with respect to both VFA production and ATP yield than high propionate or butyrate fermentation. However, the yield of methane is higher in acetate fermentation. With the high-cereal diet, more energy is recovered in the end products of fermentation (VFA and VFA plus ATP) Net ATP production is important because it will be used for microbial growth and maintenance. Russell and Wallace (1988) suggested, from the pathways of VFA production, that the net ATP production is 4, 4, and 3 mol/mol of hexose fermented for acetate, propionate and butyrate, respectively. Only 2 mol of ATP will be produced if propionate is synthesized by the acrylate pathway. Because VFA absorption rates may change with pH or VFA concentrations, VFA molar proportions in ruminal fluid may not reflect the actual VFA proportions in which they are produced (Dijkstra, 1994) Protozoal contribution to VFA production in the rumen varied between 16 and 37%. End products of protozoal fermentation are mainly acetic and butyric acids, while only trace amounts of propionic acids are produced. Thus, starch and sugars fermented by bacteria would yield more propionic

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69 acid and less acetic and butyric acids than would fermentation of the same substrate by protozoa (Dijkstra, 1994) This investigator stressed that VFA produced is not only related to type of substrate, but also the characteristics of the diets. Stoichiometric yield parameters for VFA production from soluble carbohydrate, derived from a large data set, were 1.38, .41, and .10 for acetate, propionate and butyrate in high roughage diets. For a high concentrate diet, the estimated yields were .90, .42, and .30, for acetate, propionate and butyrate, respectively. Table 2-19. Fermentation of 1 mol of contrasting carbohydrate sources High High High Item^ forage cereal molasses VFA produced, mol 1.90 1.80 1. 67 Acetate 1.34 .90 .94 Propionate .45 .70 .40 Butyrate .11 .20 .33 Methane produced, mol .61 .38 .54 ATP produced, mol 4.62 4.38 4.54 Energy from original substrate : VFA energy, % 73 80 75 VFA + ATP energy, % 85 92 87 VFA, mol/100 mol Acetate 70.5 50.0 56.2 Propionate 23 .7 38.9 24. 0 Butyrate 5.8 11.1 19.8 Based on estimations of Beever (1993).

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70 Table 2-20. Effects of molasses level on volatile fattyacid concentrations, airanonia and pH No Low Medium High Item molasses molasses molasses molasses Molasses level^, kg DM 0 1.5 3.0 4.5 % of total DM 0 13.9 26.0 35 2 Total VFA, mM 103 2 102 .4 89 4 100.6 Molar proportion Acetate 72 6 67.7 64 8 57 3 Propionate 16.0 18 4 17 4 20.5 Butyrate 10. 6 12 8 16 4 20.5 Ammonia, mM 9 .45 7.71 6.29 5 75 pH 6 62 6 57 6 57 6 24 Molasses level kg DM 0 1.0 2.0 3 0 % of total DM 0 12.9 24.5 36.5 Total VFA, mM 101.3 117.8 117.7 109.6 Molar proportion Acetate 70.0 69.2 67.7 63.8 Propionate 16.2 16.9 18.3 21.7 Butyrate 11.4 12.0 12.5 12.8 Ammonia, mM 7.65 7.50 7.14 9.71 pH 6.43 6.31 6.32 6.34 Khalili (1993). Basal diet: grass hay ad libitum and 2 kg of cottonseed cake. Linear contrast (P < .05) for individual VFA, ammonia and pH Osuji and Khalili (1994). Basal diet: grass hay ad libitum and 4 kg DM of wheat bran. In the other diets wheat bran was replaced with molasses. Linear contrast (P < .05) for individual VFA. The increase in butyrate concentration in ruminal fluid appeared to be related to the level of molasses in the diet. In fattening systems using 77% molasses in the diet, molar percents were 31, 19 and 41 for acetic, propionic and butyric acids respectively (Marty and Preston, 1970) Two experiments where molasses was added to a basal diet (or substituted for other ingredient) are summarized in Table 220. Increasing molasses from none to 4.5 kg doubled the proportion of butyrate in the first experiment. In the

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71 second, however, increasing molasses from none to 3 kg had small effects on butyrate molar percent. It is noteworthy that 2 to 3 kg molasses (representing 30 to 40% of diet DM) have beeb supplemented in Florida. Data summarized by Pate (1983) showed that sugars, not the ash, in molasses are responsible for the increase in butyrate. Inclusion of molasses or sugars in the diet almost always increased the proportion of butyrate in ruminal fluid. The magnitude of the increase, however, was variable and not always related to level of inclusion of molasses or sugars in the diet. Nitrogen utilization Much of the N in molasses is nonprotein. Stateler (1993) estimated from an in vitro semicontinuous fermentation trial that between 75 and 85% of total N was available for bacterial growth. Urea is usually added to molasses to increase the CP content. Because molasses is low in P, phosphoric acid is usually added. When urea and phosphoric acid are combined, a urea-phosphate salt is formed. Urea-phosphate given to sheep resulted in lower ruminal pH and blood ammonia than when urea alone was given (Perez et al., 1967). Addition of 3% phosphoric acid to a 10% urea liquid supplement prevented ammonia toxicity, apparently due to decreased ruminal pH caused by the phosphoric acid addition, reducing absorption of free ammonia (Davidovich et al., 1977). Increasing urea levels in molasses depressed molasses intake. Urea and monensin.

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72 independently or combined, have been used to regulate intake of molasses. Intake was regulated effectively with 30 g urea per kg of molasses or with 12 0 mg of monensin per kg of molasses (Gulbransen and Elliot, 1990) The addition of sugar-based or starch-based supplements to a basal diet almost always resulted in a decrease in ammonia concentration in ruminal fluid. The lowered ruminal ammonia levels in energy-supplemented animals is associated with an increased rate of fermentation. Intake of energy supplements are often associated with an increased influx of urea into the riimen, but ruminal ammonia levels are decreased because of increased uptake of ammonia by microbes (Obara et al., 1991). In sheep fed a lucerne hay basal diet, infusion of 200 g sucrose (17% of the DM intake) improved N balance, reduced ruminal ammonia and plasma urea N concentrations, increased transfer of urea to the gut and rumen and increased ammonia capture into microbial N. In a similar experiment (sucrose infusion, 20% of DM intake) using sheep fed fresh lucerne, the results were similar (Obara et al. 1991) The use of molasses has been proposed in diets with a high concentration of non-protein N such as silage. Increased nominal microbial protein synthesis has been reported when silage was supplemented with sugar (Khalili and Huhtanen, 1991a) or molasses (Huhtanen, 1988) Supplementing silage with a source of readily available

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73 energy has been found to reduce ruminal artimonia concentration and increase the flow of microbial protein to the small intestine (Rooke et al., 1987; Huhtanen, 1988). These studies were conducted with restricted feeding. When Petit and Veira (1994) fed ad libitum timothy silage mixed with 7.5 or 15% molasses, they found that molasses decreased ruminal ammonia concentrations. Nitrogen retention or plasma urea concentration, however, were not affected by molasses addition to silage diet (Petit et al., 1994). They suggested that sugar supplementation in animals fed ad libitum would decrease ruminal ammonia N concentration as a result of decreased degradability of silage CP, whereas sugar supplementation in feed-restricted animals would reduce ruminal ammonia N concentration as a result of increased microbial CP synthesis in the rumen. Supplementation of silage diets reviewed by Emanuele (1996) suggested that molasses and sugar can be used to replace corn or barley without detrimental effects at low levels of inclusion in the diet. He concluded that molasses fed with protein sources that supply amino acids and peptides to ruminal bacteria supports a higher level of performance than molasses alone or molasses and urea combinations. According to data summarized by Pate (1983), ureanitrogen was less efficiently utilized in forage diets supplemented with molasses than those supplemented with starch or corn. Bates et al. (1988) found that N retention

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74 was lower with molasses-urea than with aeschynomene hay or alfalfa meal supplementation of a basal diet low in CP and digestibility. The inefficient use of supplemental nonprotein N occurred because much of the N absorbed from the gastrointestinal tract was excreted in the urine. They suggested that efficient N recycling in ruminants may limit the effectiveness of supplements which contribute primarily to the ruminally available N pool. Pate (1983) suggested that the feeding of moderate to high levels of molasses reduced the apparent digestibility of CP by 5 to 15%. Practical implications would be an increase in CP requirement above the levels recommended at that time, with the old CP system (NRC, 1976) The fact that young bulls gaining 1 to 1.1 kg/d needed 3 0 to 60% more CP (as fish meal supplement) than recommended by NRC (1976) may indeed reflect high ruminal N losses as ammonia and (or) low microbial yield. The finding that 15 to 25% of N from molasses may be unavailable (Stateler, 1993) may provide a partial explanation to lower CP digestibility. Molasses may also depress protein digestibility of the basal diet. It was shown that sucrose or molasses supplementation depressed ruminal degradability of silage CP (Huhtanen, 1988; Petit and Veira, 1994) Efficiency of microbial synthesis was low on molassesbased diets, and efficiency was increased with the addition

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75 of a starch source (Rowe et al., 1980). They suggested that addition of starch provided a more uniform supply of fermentable energy for the ruminal bacteria. Barley-urea increased duodenal N flow more than molasses-urea when sheep were given cereal straw, suggesting better efficiency of ammonia capture in microbial protein when starch was the energy source (Oldham et el., 1977). Obara et al (1991) infused sucrose (20% of the DM intake) in the rumen of sheep fed fresh alfalfa. Nitrogen balance was improved and ruminal ammonia concentration was reduced by sucrose infusion. An unexpected result was that there was no increase in ammonia incorporation into microbial N. Calculation from data presented shows that microbial efficiency was 4.03 and 2.75 g N/100 g OM digested, with basal and basal plus sucrose infusion, respectively. No difference was observed in the protozoal population. In his review. Pate (1983) found evidence that sugars, and particularly sucrose, were less effective than starch in promoting microbial synthesis from urea. Nitrogen retention was also lower for molasses-urea than for corn-urea diets. He inferred that if biological value of all microbial protein is similar, then the higher urinary-N losses observed in animals fed molasses-urea indicate that urea-N was less efficiently synthesized into microbial protein. Pulse of glucose added to glucose-limited cultures of S. ruminant -i urn and B. ruminicola caused an immediate

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76 doubling of heat (energy spilling) production and little increase in cell protein (Russell, 1986) Van Kessel and Russell (1996) reported that when ammonia was the growth limiting nutrient of predominant ruminal bacteria, the impact of energy spilling was very great, and additional ammonia caused a large increase in yield. However, when energy-excess batch cultures were provided with amino N, the growth rate increased and less energy was spilled (Van Kessel and Russell, 1996). Rixminal conditions created by feeding molasses and non-protein N with low quality forage may be similar to those described for energy-excess cultures. This may explain, at least partially, the low efficiency of N utilization. Digestive asso ciative effects Pate (1983) concluded that molasses increased the digestibility of the total diet, but depressed forage DM and fiber digestibility, particularly low quality forages. The degree of depression was dependent upon the level of molasses in the diet and the crude protein balance. With properly balanced forage-based diets, molasses increased DM digestibility and did not appear to severely depress the digestibility of fiber. Brown et al (1987) found no effect on OM digestibility and depression of NDF digestibility when limpograss hay was supplemented with 25% DM molasses. The same effect was seen when the basal diet was rice straw. Kalmbacher et al (1995) found that molasses-based supplements increased the apparent

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77 OM digestibility of total diet (creeping bluestem basal diet) but decreased NDF digestibility. Similar results were obtained by Brown (1993) using ammoniated stargrass as the basal diet. Mould et al. (1983) reported that reduction of fiber digestion by molasses supplementation appeared related to the presence of highly fermentable carbohydrate (carbohydrate effect) rather than to low ruminal pH. Ruminal infusion of sugars depressed fiber digestion although pH was not affected (Huhtanen, 1988; Rooke et al 1987). Increasing levels of molasses supplements (1.5 to 4.5 kg molasses) caused a linear increase in DM and OM apparent digestibility and a decrease in NDF digestibility with increasing level of molasses (Khalili, 1993) Addition of bicarbonate with the higher level of molasses (37% of the diet) did not affect DM, OM or NDF ruminal digestibility. He suggested that the depressed fiber digestibility may have been associated with a preference by ruminal microbes for soluble carbohydrates, as previously observed in vitro (Russell, 1984). Intake and Performance Feed intakp. Moore et al (1995) analyzed the effect of liquid supplements on forage intake based on 151 comparisons of voluntary forage intake when fed alone and with supplement. When the forage was balanced (DOM: CP < 7) supplements almost always decreased forage intake. When the

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78 forage was very unbalanced (DOM: CP > 12) all types and levels of supplements increased forage intake. When forage DOM: CP was between 7 and 12, forage intake was both increased and decreased by supplements. When intake of forage fed alone was >1.75% of BW, supplements decreased forage intake; when forage intake was <1.75% BW, supplements increased forage intake. The level of supplement was also important: forage intakes were depressed by liquid supplements when supplement intake was >.8% BW. Supplement CP concentration also affected forage intake. Forage intake was increased when liquid supplement CP was >25% of OM. Animal performance Moore et al (1995) analyzed the effect of liquid supplements on animal performance based on 148 comparisons of non-supplemented control (grazed or fed forage) and supplemented with molasses. They concluded that daily gains were generally, but not always, increased by feeding liquid supplements. When a source of N was added, gains were greater than when molasses alone was fed. When supplement CP concentrations were above 15% of OM, gains were almost always increased. When supplemental CP intake was greater than .1% of BW, gains were always increased. Forage quality was also important. When forage intake was low and DOM: CP was unbalanced, liquid supplements increased both intake and gain, but gain was still low or even negative. When forage intake was high and the DOM: CP

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79 was balanced, liquid supplements decreased forage intake generally, but increased gains if the supplement contained meal or combination of meal and non-protein N. Pate (1983) concluded that a source of N should be provided in molasses when supplemented to low quality forage diets. He also recognized that natural protein was superior to non-protein N sources. Kunkle et al (1994) reviewed experiments where molasses slurries were fed as supplements on basal diets of subtropical pastures or hays. They found that supplemental ruminal undegraded protein (feather, blood and/or corn gluten meal) increased gains in growing cattle from .08 to .30 kg/day and averaged .15 kg/day. They recommended that a source of protein of low ruminal degradability be included after the requirements for ruminal degraded protein are met. Pate et al. (1995), and Stateler et al. (1995) obtained a good response of ADG in growing cattle when the molasses slurries contained part of the total CP as ruminally undegradable protein. Summary of Feeding Molasses When high levels of molasses are fed, ruminal fermentation is characterized by high butyrate molar proportion, increased population of entodinia protozoa, and lower ammonia concentration. Secondary fermentation (sludgetype fermentation) and low ruminal motility has been reported with high molasses diets. Molasses supplementation

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80 at about 3 0% of the diet, as recoininended in Florida, is not expected to produce dramatic changes in ruminal fermentation. In silage diets, a low level of molasses supplementation appeared to improve N utilization. However, provision of ruminal degradable protein appears necessary. Addition of non-protein N was better than molasses alone when the basal diet was low in CP. Research with molasses slurries showed that natural protein sources improved performance over non-protein N. Provision of additional protein sources with low ruminal degradability in molasses slurries increased gain in growing cattle fed forage diets. Several reports suggested that cattle used N (especially non-protein N) less efficiently with molasses than with grain. Research conducted with silage diets suggested that nominal feed N degradability may be depressed by molasses supplementation. Furthermore, between 15 to 25% of N in molasses may be unavailable. There is no direct measurement of provision of non ammonia N (an estimator of metabolizable protein supply) in cattle fed high roughage diets supplemented with molasses. More information is needed to understand the supply and utilization of nutrients, especially protein, when molasses is fed.

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The present research will evaluate the effects of moderate levels of molasses and corn supplementation on characteristics of ruminal fermentation, efficiency of microbial growth, and nutrient supply in cattle fed bermudagrass hay.

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CHAPTER III EFFECT OF BAMBERMYCINS AND MONENSIN IN CORN OR MOLASSES SUPPLEMENTS ON PERFORMANCE OF GROWING CATTLE Int-rodurf-ion Florida has approximately 1 million beef cows and some of the weaned calves are stockered after weaning. Molasses is usually the lowest cost energy source available for supplementing grazing beef cattle in Central and South Florida. Liquid feeds require less labor to feed than grainbased supplements which reduces supplementation costs. Researchers at University of Florida developed molasses slurries by adding 10 to 25% dry ingredients such as cottonseed meal, feather meal, blood meal and (or) wheat midds. Molasses slurries are consumed at higher levels than traditional liquid supplements. These higher levels of intake are usually needed to reach the desired performance in growing calves grazing perennial forages in Florida during the fall and winter. Molasses slurries are often limit-fed 3 d/wk in tubs or troughs. Molasses slurries formulated with natural protein that is undegraded in the rumen have been shown to improve the performance of growing cattle (Pate et al., 1995; Stateler et al., 1995). 82

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83 lonophores such as monensin (Rvunensin™) and lasalocid (Bovatec™) have been effective in improving gains of grazing cattle. Limited research at the University of Florida evaluating monensin and lasalocid in molasses slurries has not shown improvements in gains of grazing cattle (Kunkle et al., 1990; Pate, 1995). However, more evidence is needed to corroborate this finding. Bambermycins (Gainpro™) is a feed additive that has improved gains of grazing cattle (Deetz et al., 1990). Its efficacy in molasses-based supplements is not known, at least under Florida conditions. A feed additive that improves gains when delivered in molasses supplements fed at high levels is needed to improve the cost effectiveness of supplementation. The objective of this experiment is to evaluate the efficacy of monensin and bambermycins in corn and molasses slurry supplements. Materials and Methods Performance trials were conducted at the University of Florida Pine Acres Research Unit located in northern Marion County from December 1, 1994 to March 23, 1995 (Year 1, 112 d) and at the Santa Fe Research Unit located in northern Alachua County from December 20, 1995 to April 3, 1996 (Year 2, 106 d)

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84 Year 1 Seventy-six Angus and Brahman x Angus steers and 92 heifers of varying percentages of each breed, weighing from 190 to 320 kg (average 244 kg for all cattle) and 7to 12-months-old at the beginning of the trial were used. Cattle were balanced by sex and breed type in each pen. Each pen (experimental unit) had three heifers and three steers, except for eight pens which had four heifers and two steers. Seven treatments were completely randomized across the 28, .9 -ha paddocks, dormant bahiagrass ( Paspalum notatum ) frosted before the trial. All animals were dewormed and deloused at the beginning of the trial (Ivomec™ pour on) Full weights were taken on d 0, 28, 56, 84 and 112. Shrunk weights were measured on d 1 and 113 after an overnight feed and water withdrawal. Body condition score (BCS) was evaluated by a single evaluator on d 1 and 113 using a 1 to 9 scoring system (Herd and Sprott, 1986) Initial hip height was calculated as the average of measurements made on d 0 and 1, and final hip height was the average of measurements made on d 112 and 113. Blood samples from all animals were collected via jugular venipuncture on d 28, 56, 84 and 112 for plasma urea N (PUN) analysis. Blood was collected with polypropylene syringes containing 1 6 mg potassium EDTA/mL of blood as an anticoagulant (Monovette, Sarstedt Inc., Newton, NC) Riuninal fluid was obtained from two animals per pen on d 28, 56, 84 and 112 using a stomach

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85 tube. The tube was fitted with a ruminal strainer at the end and introduced via the mouth through a Frick speculum. Ruminal fluid was aspirated with an electric vacuum pump. Ruminal fluid samples were filtered trough four layers of cheesecloth and acidified with 5 mL of 20% sulfuric acid/100 mL of ruminal fluid. Samples were obtained between 3 to 5 h after feeding corn supplements. Sampling was conducted the day after new molasses supplements were offered (it was not possible to control sampling time after feeding in this case) Rectal grab samples of feces were collected from all animals on d 28, 56, 84 and 112 for coccidia oocyte and nematoda egg counts. All samples were stored on ice until being translated to Gainesville for processing and storage. Blood samples were centrifuged at 1,500 g the morning after sampling. Riominal fluid was frozen upon arrival. Fecal samples were refrigerated until analysis were conducted within one week. Year 2 The breed types of steers and heifers were similar to those used in Year 1. Initial weight ranged from 245 to 275 kg (average 260 kg for all cattle) Animal allocation to pen and treatment was similar, except that sex was confounded with pen, so that in any given pen only one sex was present. Different animal allocation was decided based on animal availability and the need to gather data useful for modeling work (e.g., intake and performance data by sex)

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86 Animal management and sampling were conducted as described for Year 1, with the following differences: Full weights were taken on d 0, 28, 56, 84 and 106; shrunk weights were measured on d 1 and 107; BCS was evaluated on d 1 and 107; initial hip height was measured on d 0 and 1, and final hip height was taken on d 106 and 107. Blood samples for PUN were obtained from 3 animals per pen on d 28, 56, and 84. Rumen fluid was obtained from 2 animals per pen on d 2 8 and 84. Fecal samples were collected from 2 animals per pen on d 28, 56, 84 and 106. ** Diets Diets consisted of bermudagrass ( Cynodon dactylon ) hay (large round bales harvested during the previous summer) fed alone or with corn or molasses supplements. Treatments were: 1. Hay + corn meal (CC) 2. Hay + corn meal + monensin (200 mg/day) (CM) 3. Hay + corn meal + bambermycins (20 mg/day) (CB) 4. Hay + molasses slurry (MC) 5. Hay + molasses slurry + monensin (2 00 mg/day) (MM) 6. Hay + molasses slurry + bambermycins (20 mg/day) (MB) 7. Hay alone (HAY) Details of supplement formulation (projected intake and ingredients) are described in Table 3-1. The source of bypass protein in both molasses slurry and corn-urea was corn protein. Ruminal degradable protein was balanced using urea. Eighty-five percent of CP in molasses was assumed to be available in the rumen (Stateler, 1993); the rest was considered unavailable. Amount of

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87 supplement delivered as fed was calculated to provide the same quantity of TDN from the supplements. Table 3-1. Supplement formulation and estimated composition Supplement" Item Corn Molasses Ingredient, % as fed: Corn meal 93.8 0 Cane molasses'' 0 89.6 Corn gluten meal 0 10. 0 Urea 2.8 .4 Limestone 1.0 0 Dicalcium phosphate 1.8 0 Dynamate .6 0 Estimated composition" TDN, % DM 84.4 73 .4 CP, % DM 18.4 16.7 DIP, % CP 74 74 UIP, % CP 26 26 Ca, % DM .86 .90 P, % DM 66 .67 Rumensin and Gainpro added in the corresponding treatments to deliveg200 and 20 mg/an/d of monensin and bambermycins respectively. Blackstrap molasses not less than 40% inverted sugars, fortified with phosphoric acid and 25,000 U.S. P. units vit A, 33,000 U.S. P. units vit D, and 22 Int. units vit E per kg, and .0005% Cu, .00001% Co, .02% Fe, .001% Mn, .0025% Zn, and .00007% I. Sulfur concentration no less than 1%, as fed basis (U.S. Sugar Corporation, FL) Calculated from tables (NRC, 1984) and Stateler (1993). Assumes 85% of molasses N available for microbial growth. Feed additives were diluted in a carrier and mixed with the total supplement (corn-urea) or with the dry ingredients (corn gluten meal-urea) for the molasses supplement. A mineral supplement containing 17.2 to 2 0.6% salt, 17.2 to 20.6% Ca, 9% P, 1% Fe, .2% Mn, .01% I, .01% Co, .2% Mg, .12% F, 1,500 ppm Cu, 20 ppm Se, and 4,000 ppm Zn was offered free choice in mineral feeders in all pens.

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88 Feeding procedures Hay bales were weighed, coresampled with a forage sampler 2,5 cm in diameter, and offered free choice in round-bale feeders. Corn supplements were offered daily while molasses supplements were offered on Monday, Wednesday and Friday of each week. Molasses and dry ingredients were weighed and mixed mechanically in open trough feeders in each pen on each feeding day. Uneaten molasses supplements were weighed and recorded the next delivery day, but uneaten supplement was found only during the first 3 wk of the trial. Following the consumption of every third bale (Year 1) hay orts were collected, weighed, and sampled for dry matter determination. A visual estimation of hay waste not collected was recorded each time orts were collected. Weights of hay offered, orts, and waste estimates were used to estimate hay intake. During the last 3 to 4 d of the experiment a fresh bale was delivered to each pen which had less than a third bale of hay left in the feeder, attempting to avoid bias due to fill in the final weights. In Year 2, hay orts were collected after feeding every 5 to 6 bales because hay refusal was small, resulting in lower weigh back expressed as percent of offered hay. Laboratory analysis Hay and dry supplement ingredients DM was determined at 105 C for 18 h in a forced air oven and OM at 550 C for 6 h in a muffle furnace. Nitrogen concentration in hay, corn mix, corn gluten mix, and base molasses samples were determined by the method of Gallaher

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89 et al. (1975) (aluminum-block digestion), and colorimetric analysis (Technicon AutoAnalyzer, Technicon Instruments Corp., Tarrytown, NY; Technicon, 1978). Neutral detergent fiber was determined in hay, corn, and corn gluten meal following the procedures of Goering and Van Soest (197 0) modified by Moore and Foster (1986), with the addition of alpha anylase to concentrate samples only. Hay in vitro OM (IVOMD) digestibility was determined by the procedure of Moore and Mott (1974) Molasses DM was determined by freeze drying. Molasses samples were diluted (weight /volume) in 20 parts of distillate water and pH measured with a portable pH meter (Corning M90, Corning, Inc. NY) Whole ort samples (about 50 to 100 g) were analyzed only for DM at 65 C for 48 h in paper bags. Mineral concentration of supplement was determined following the procedure of Fick et al. (1979). Calcium, Mg, Na, and K were determined by flame atomic absorption spectrophotometry using a Perkin-Elmer AAS 5000 (PerkinElmer, Norvalk, Connecticut) Phosphorus was analyzed by a colorimetric procedure (Harris and Popat, 1954) Plasma urea N was analyzed as described by Hammond et al. (1994), using an automated colorimetric procedure (Technicon AutoAnalyzer II Industrial Method no. 339-01, Technicon Instruments Corp., Tarrytown, NY) based on the diacetyl monoxime method of Marsh et al. (1965). Samples of nominal fluid were thawed, centrifuged, and supernatant filtered through .45 fjm

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90 microcel filters (Gelman Sciences, Ann Arbor, MI) Volatile fatty acids were analyzed by gas chromatography (Perkin Elmer AutoSystem XL, Norwalk, CT) using a packed column (Supelco, 1990b) Fecal nematoda eggs and coccidia oocyte counts were determined using the Wisconsin flotation technique (Benbrook and Sloss, 1948). For Year 2, coccidia oocyte were not counted but given a score from 0 (no coccidia present) to 4 (more than 40 cysts per field) Statistical analysis Statistical analysis was conducted using the GLM procedure of SAS PC (SAS, 1987), as a completely randomized design using the pen as the experimental unit. The model included the following effects: year, treatment, and year x treatment. Repeated measurements (PUN, VFA, parasites) were also analyzed by year using the repeated statement in the GLM procedure. Probability level for time and time by treatment interaction were obtained from F using adjusted degrees of freedom (G-G test, Littel, 1989) When no time by treatment interaction was detected, data were pooled and analyzed with treatment as the only effect in the model. In addition, six single degree of freedom preplanned contrasts were evaluated. Coefficients for all contrasts are presented in Table 3-2. These partition the five degrees of freedom in the 3x2 factorial arrangement of the six supplement treatments (supplements, additives and the interaction of each additive by supplement) the remaining degree of freedom was used to

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91 compare hay alone vs all supplements. All probabilities levels for treatment contrasts are presented in tables grouped for related variables. Table 3-2. Coefficients for preplanned comparisons for treatment effects Treatments Contrasts CC CM CB MC MM MB HAY Cl-Corn vs molasses 1 1 1 -1 -1 -1 0 C2-Monensin vs Ctrl 1 -1 0 1 -1 0 0 C3-Bamberm vs Ctrl 1 0 -1 1 0 -1 0 C4-Monensin x supp 1 -1 0 -1 1 0 0 C5-Bamberm x supp 1 0 -1 -1 0 1 0 C6-A11 supp vs hay 1 1 1 1 1 1 -6 Results and Discussion Composition of supplements used each year is presented in Table 3-3. Average supplement CP concentrations analyzed were 16.6 and 16.3% of DM for cornand molasses-based supplements, respectively. Calculated TDN concentrations were 84.4 and 73.4% of DM for cornand molasses-based supplements, respectively (Table 3-2). The CP concentration of the molasses in this trial was higher than tabular values (NRC 1996), but consistent with values reported by Chapman et al. (1965) for molasses produced from cane grown on organic soils in Florida (7 to 10% CP) Phosphorus concentration in blackstrap molasses is typically .1% of DM.

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92 Phosphoric acid was added to increase the P concentration of the molasses base. Analyzed concentrations of monensin and bamberirrycins in supplements are presented in Appendix Tables A-1 to A-3. Apparently, there was difficulty with the bambermycins analysis. All mix formulations were recalculated and found to be accurate. The original premix was still 100% efficacious 4 months after the experiment was finished. Table 3-3. Composition of supplements by analysis Corn gluten Corn mix meal mix^ Molasses base^ Item Year 1 Year 2 Year 1 Year 2 Year 1 Year 2 DM 86.1 86.1 89.9 91.5 77.5 77.5 As % DM: OM 95.2 94.6 98.3 98.3 83.4 83.7 CP 16.3 16.9 67.0 75.2 9.3 8.5 NDF 9.3 9.3 5.2 5.2 Ca .65 .76 .03 .04 .71 .74 P .67 .71 .44 .46 .88 .74 Mg .25 .29 .07 .09 .49 .49 K .45 .51 .13 .16 4.28 4.26 Na .01 .01 03 .02 .11 08 PH 4.51 4.71 Calculated CP in offered molasses slurries (10.4% corn gluten mix, 89.6% base molasses) were 16.1 and 16.5% of DM for Year 1 and Year 2, respectively. Composition of bermudagrass hay is given in Table 3-4. As expected, there were no differences (P > .3) for any component due to treatment. All bales were kept in a barn and delivered as needed, therefore a random assignment was presumed and composition data were pooled across treatments.

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Table 3-4, Composition of bermudagrass hay 93 Variable Mean Std Dev Minimum Maximum Year 1 (n = 265) Dry matter, % 90.5 32 89 5 91.6 Percent in DM Organic matter 93.8 62 91.8 95.1 Crude protein 9.62 J. / f* 15.96 NDF 81.9 1.88 76.2 87.3 I VOMD % 42 7 2.98 34.1 49 7 Year 2 (n = 218) Dry matter, % 88.8 1.10 82.6 90.4 Percent in DM Organic matter 94.8 .58 92.1 96.3 Crude protein 9 .90 1.56 6.37 14.50 NDF 79.4 2.16 73 .9 87.7 I VOMD, % 46.6 2.59 39.6 53.4 The model level TDN concentration of 1 (NRC, 1996) the hay from was used observed to estimate the gains of animals fed hay alone. Predicted and observed gains were similar when 54% TDN was used in both Year 1 and Year 2. Therefore, a value of 54% TDN (DM basis) for the hay offered was used for estimation of efficiency of feed utilization. Animal Performance There was no interaction of year by treatments for measures of animal performance, therefore means are discussed by year (Table 3-5) and by treatments (Table 3-6) Probability values (F test) for differences due to year, year by treatment interactions, and contrasts are presented in Table 3-7. Results for each year are summarized in Appendix Tables A6 and A9.

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94 Animals used in Year 2 were heavier (P = .0001) and had lower BCS (P = .0007) at the beginning; they gained more (P = .0001), consumed more hay (P = .0001), had lower height change (P = .0001) and higher BCS change (P = .013) than animals used in Year 1. Initial shrink (kg BW loss after feed and water withdrawal/100 kg BW, data not shown) was higher (P = .0001) in Year 1 (7.4%) than in Year 2 (2.6%). Final shrink was also higher (P = .0002) for animals in Year 1 (8.7%) than those in Year 2 (7.4%). Differences in size, hay intake and shrink may explain the higher shrunk ADG for animals in Year 2. The winter was warmer in Year 1, resulting in some pasture regrowth providing additional feed, which may explain the lower hay intake recorded. In contrast, in Year 2 the animals were completely dependent on the hay supply. The lower temperatures in Year 2 may have increased the maintenance energy requirement. The effects of higher maintenance requirements and lower initial fill for Year 2 would tend to cancel each other when estimating efficiency of gain. Animals fed corn gained .047 kg more (P = .005) than those fed molasses, due to greater effect of feed additives in corn than in molasses supplements. Cattle fed CC and MC had similar gains (.621 and .616 kg) and hay intake (1.54 and 1.64% BW) indicating that the TDN from corn and molasses was utilized with similar efficiency when no antibiotics were added. The feeding value of molasses has

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95 U3 in O >^ VD ro >x> in (Ti ro m o ro o cn tH iH VD in ro ro o iH r~o -vf ro VD rm in in tn o 00 in o ro ro in • • • • • • • • • • • • • • • • r-i rH in iH iH iH rH rH iH iH O o cn O o og O O O O O O O O O O o O LD o o iH o o o O O CD o o O ^D o O O o o O o o o fl • o o o o o o C3 n o CO [> vH .H cTi ro ro O o o o in iH IT) o O >X) ro o en o in in 00 in O] in oi >^ 1 ro og ro rsi ro BW >i ^ Cn -H na 12 U H H H PQ (U ( J-) c -H Q IS Q PQ a 0) S m rH Q (d M-l a o :i X Q do w 5 15 -u 2 n3 o E-l MH O Di Q Q (0 (d 4-) 4-) O O Eh Eh (8 CD g 0 "4-1 2 ^ Q 00 Eh Oi U C 06 o s •rl (0 Tl rH 01 3 OT U 3 rH (0 01 o u u (0 w XI 0) ~3 Q > Eh XI ^ rH in ja to 18 6 -iJ 3 c w 6 (0 0) (0 T! tt TI 4J 3 0) (0 0) 3 H U O rH b(0 O • 0) vo (U (71 ? rH >i x: a (8 z 0 t3l II 01 Dl 01 C •H B) J.; (8 -U 4J C C 01 •rl g (8 0) u (8 01 4J C O I rH 18 >i <8 J3 C O

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96 CO rH 00 ID CN ro o 00 o o c~o 00 rH m CTl iH U5 O VD rH o o o O 00 o CTi rH o CN U3 ID (J\ IT) rH CN rH IT) fN O 00 in o rH in r in ^ — 1 CN rH CTl o 00 ro in ro CN] in 00 in rH CN rH U3 __1 r^ 1 1 \^ in 00 in in rH CS rH r~m CN o • in rH in 00 • • in rH CN rH rn m in in cr\ in rH CN rH rH o CN 00 ro IX) rH CN in 00 in rH CN rH u g U CD 0) tn to -H c; S U 0) O (0 OQ CQ ^ Q U r-H 1— 1 rH (0 (t3 (t3 H -H -H JJ 4J 4J •H -H -H •H a C C d) H H H to CO ro CN o 00 CN 00 O VO rH in (N I ^ rH CT> VO VO O O O ro in VO t# rH I CTi ^ VO r~ VO rH I in ro t~VO r# rH I CTl O O O l> rH I ro ^ VO in in rH I tJI c to u to U IS m 12 00 VO in in ro 00 CN 00 ro VO "^i* r~00 VO VO in CTi ro VO in r00 VO rH in rH O rH O ro •'^ Q Eh T3 (0 -U O 4.) U (tj 0) T) OQ O o g -US (US* Q rH Q 14-1 Eh MH a o o O. tn S O) :3 A! Q c>P to r~ ro O rH ro VO rH rH VO VO 00 ro ro rH rH rH rH 00 cn [> CN ro VO CTi a\ ro ro rH P Q Eh E-" (0 (0 O O Eh Eh II X! C o ro 4-> c e (1) 0<<4-l 3 ^ § 4J (1) •H W S 0 3 (U i-i S U' 00 cri >i (0 CO 3 u (0 (U c C O -H nJ 3 m 4J m (V (0 g U. (1) r-1 3 rH 3 3 w 0) 4J c (0 o U I* O II 14-1 0) D) >i C g (0 >H x; 0) o c J (0 o J3 o 01 C •H W 3 4J c g 4J <0 0) (V c o

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97 Table 3-7 Probability values for contrasts, animal performance Contrasts^ Variable CI C2 C3 C4 C5 C6 Initial BW, kg Initial BCS Initial height .8747 .8426 .3706 .9814 .9273 .9196 .2744 .9383 .2879 .1109 .4855 .9889 .4541 5623 6047 .3191 .3637 .3866 ADG kg .0049 .8508 .0004 .1148 .1010 .0001 Height change, cm .6436 .4858 .4507 6171 .9853 0001 BCS change .3442 6672 1221 .3591 .5203 0001 Intake, Hay kg Hay, % BW Hay change, % BW .3658 .2257 .2257 0341 0236 .0236 6145 6431 6431 .6725 9198 .9198 .2281 .3459 .3459 0001 0001 Supplement Kg DM/d DM as % BW % Of total DM Kg TDN/d % of total TDN .0001 .0001 .0057 0001 .2257 .0590 .6086 .0473 0590 .0482 .9265 .8589 .4629 .9265 .4700 0590 .1023 .7447 .0590 .7887 .9265 .1690 .2938 .9265 .2809 Total TDN, kg/d Total TDN, % BW .4703 .2532 .0309 0161 6786 6728 6309 .7297 .2371 .4632 0001 0001 Contrasts: Cl = corn vs molasses C2 = monensin vs control C3 = bambermycins vs control C4 = monensin by supplement type 05 = bambermycins by supplement type 06 = all supplements vs hay alone. produced mixed results in previous research. Earlier work suggested that molasses is utilized with decreasing efficiency as its level in the diet increases (Lofgreen and Otagaki, 1960). Brown and Weigel (1993) presented data suggesting a lower feeding value of molasses when compared with corn and soybean hulls. However, Preston et al. (1969) reported that the efficiencies of metabolizable energy

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98 utilization were 15 to 20% and 26 to 32% for diets with 33 and 72% molasses, respectively. Lofgreen (1965) estimated that the NEg of molasses was .78 and .70 Mcal/kg when fed at 5 to 15 and 20% of the diet, respectively, a difference smaller than previously determined (Logfreen and Otagaki, 1960). Similarly, data presented by Pitzer et al. (1986) suggested that the TDN in molasses and corn was used with similar efficiency (.21 kg of added gain/kg supplemental TDN) In the present experiment, each kg of TDN, either from corn or from molasses slurry, increased gain by .24 kg. Associative effects on intake were similar in animals fed CC and MC (-.56 and -.46% BW lower hay DM intake when compared with animals consuming hay alone) Digestibility may also exhibit associative effects (deviation from additivity) An empirical model was developed from a dry supplement data set to predict, among other effects, the expected change in ME concentration of the total diet when supplements are fed with forages (Brant, 1993; Moore and Kunkle, 1995, 1996). Hay and supplement intake, and hay and supplement OM, CP, and TDN concentration are inputs for this model component. A small negative associative effect on digestibility was predicted (-.08 Meal ME/kg) for both, cornand molasses-supplemented diets. This probably resulted from the moderate level of supplementation (.64 to .73% BW) and a balanced total diet (TDN: CP < 7) when supplements were fed.

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99 Gains observed with CC (.62 kg) were similar to gains reported with bermudagrass plus corn-soybean meal fed at 25% of total TDN intake (Garces-Yepez, 1995), while ADG and intake of the hay alone also was similar (.3 0 vs .25 kg and 1.99 vs 2.10% BW, respectively). Higgins et al., (1991) also reported a similar gain in one of the bermudagrass hays evaluated (.29 and .67 kg/d with hay alone and hay plus .75% BW of corn-soybean meal supplement) Intake of hay alone in the current experiment was similar to intake measured by Stateler et al. (1995) using a similar technique (1.92 to 1.96% BW) Gains reported by Stateler et al (1995) with molasses slurries including soybean meal or blood mealhydrolyzed feather meal were lower (.4 6 kg) and higher (.71 kg), respectively, than gain observed with MC (.62 kg). Animals supplemented with blood-feather meal consumed .14 kg of ruminal UIP from the supplement while in this experiment estimated UIP intake was .09 kg. Quantity rather than quality of UIP was probably more important in explaining the lower gain observed in this experiment because the NRG (1996) model did not predict limiting amino acids at this rate of gain. Moreover, energy and not protein was limiting gain. However, escape protein may provide substrate for gluconeogenesis and improve the efficiency of energy utilization (Preston and Leng, 1980) Inclusion of monensin tended to produce different ADG changes when fed in corn or molasses (monensin by supplement

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100 type interaction, P = .11). Monensin increased ADG .035 kg in corn supplement and depressed ADG .029 kg in molasses supplement. Animals fed CM had .067 kg higher ADG than those fed MM. Effects of monensin have been variable in high roughage diets. Reports involving several trials of grazing cattle or cattle fed forage diets suggest a 14 to 17% increase in ADG when monensin was added to dry supplements fed at 5 to 1 kg/d (Potter et al. 1976; Wilkinson et al. 1980; Potter et al., 1986), Horton et al.(1992) concluded that improvement in ADG to feeding ionophores (lasalocid and monensin) was inconsistent in cattle grazing subtropical grasses, and the variable responses appeared to be associated with forage quality and environmental conditions. Ellis et al. (1984) suggested that the expected gain improvement to monensin decreases as the quality of forage consumed increases, and as the realized gain approaches the genetic potential for gain by the animal. Under the conditions of this experiment, neither of these factors would apply because the gain on hay alone was moderate, indicating low forage quality, and the highest gains were below the animal genetic potential for gain. Improvement in gain when monensin was fed in corn was 21.9% in Year 1 but gain was not improved in Year 2. Reasons for this variability are not evident and can not be explained.

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101 The failure of ionophores to improve gain when fed in molasses supplements was consistent with previous research. Pate (1995) reported that gains were not improved when lasalocid was added to molasses supplements. Kunkle et al. (1990) reported that 200 mg/d of monensin added to molassessoybean meal slurry (2.7 kg/d) in heifers on bahiagrass pasture did not improve gains (ADG responses to monensin were +.02 to -.07 kg). In contrast, Garrett et al. (1989) reported improved gain in feedlot cattle when monensin was added to high concentrate diets with 3 0% of sugar beet molasses, probably because monensin reduced ruminal disorders This experiment and previous research suggests that no change or small depression in ADG should be expected when monensin is added to molasses slurries consumed at about 3 0% of the total DM in warm season grass diets. Cattle fed bamberm/cins in corn or molasses had .074 kg higher (P = .0004) ADG than those fed control supplements. Because of a tendency for bambermycins by supplement type interaction (P = .101) it is risky to generalize. Animals fed CB had .106 kg higher ADG than those fed CC, while animals fed MB had .042 kg higher ADG than those fed MC. Animals fed CB had .064 kg higher ADG than those fed MB. Bambermycins included in dry supplements increased ADG by 15% in pasture fed cattle (Deetz et al., 1990; Deetz et al., 1992). In cattle fed bahiagrass pasture (W.E. Kunkle,

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102 unpublished data), bamberm/cins increased ADG by 22% (.39 vs .47 kg) and 29% (.45 vs .58 kg). Gain increased 28.2% (34.4 in Year 1 and 22.5% in Year 2) with corn supplements, which is similar to values obtained by W.E. Kunkle (unpublished data) and somewhat higher than other reports. Research on the efficacy of bambermycins in molassesbased supplements could not be found. Gain increased 11.3% when bambermycins was fed in molasses supplements (16.7% in Year 1 and 6.3% in Year 2). Because of this variable response, the efficacy of bambermycins in molasses supplements to improve gains is inconclusive and more research is needed. A comparison of responses estimated as the difference between ADG when an additive was included in a supplement minus the ADG obtained with that supplement without additive, was analyzed as a 2 (supplement type) x 2 (feed additive) factorial, with year included in the model. No interactions were found (supplement x additive, P = .95; year x supplement, P = .39; year x additive, P = .74, and year x supp. x add., P .48). Antibiotics added to the supplements improved gain more (P .04) in Year 1 (.062 kg) than in Year 2 (.016 kg), improvements were higher (P .007) in corn (.071 kg) than in molasses (.007 kg), and higher (P = .003) with bambenrr^cins (.074 kg) than with monensin (.004 kg). The order of responses was CB (.106 kg) > MB (.042 kg) and CM (.035 kg) > MM (-.028 kg).

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103 All supplements increased (P = .0001) BCS and height change when compared with hay alone. Similar results were reported by Stateler et al (1995). However, in their experiment cattle in all treatments lost BCS, suggesting that energy retention in BW gain was higher in this experiment. Bambermycins tended (P = .12) to increase BCS and had no effect (P = .45) on height change. Monensin did not affect BCS (P = .67) or height change (P = .49). Corn supplements were completely consumed. Molasses supplements were not consumed completely during the first 3 wk after the start of the trial but were consumed completly thereafter. Monensin included at 120 mg/kg of molasses acted as an effective intake regulator (Gulbransen and Elliot, 1990) which may explain the trend for lower molasses intake when monensin was added at 74 mg/kg in this experiment. All supplements decreased (P = .0001) hay intake from .47% BW (CB) to .70% BW (CM) when compared with hay alone. A similar response was reported by Garces-Yepez (1995) and Higgins et al. (1991) when bermudagrass was supplemented with corn-soybean meal. Stateler et al. (1995) reported .21 to .41% of BW depression of bermudagrass hay intake when about 2 kg of molasses slurries were consumed. Depression of forage intake should be expected when the hay is balanced (TDN:CP < 7, Table 3-4), according to the general conclusion of Moore and Kunkle (1995)

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104 Monensin decreased (P = .024) and bambenttycins did not affect (P = .35) hay intake when included in supplements. However, when analyzed by year, monensin tended (P = .099) to decrease and bambemycins tended (P = .073) to increase hay intake in Year 1. In Year 2, monensin tended to decrease (P = .099) and bambermycins did not affect (P = .40) hay intake Ellis et al. (1984) suggested that the effect of monensin on forage intake depends upon forage quality. It was hypothesized that monensin decreased intake of low quality forage by monensin 's effect on reducing passage rate of undigested forage residue out of the rximen combined with the animal's inability to accommodate further increases in fill of undigested dry matter. Monensin increased intake of mediiim to high quality forages as a result of increased undigested matter fill, and it would decrease intake of high quality forage by a mechanism similar to depression of intake in high concentrate diets (metabolic control of intake) Whether or not this reasoning is applicable to hay based diets supplemented with concentrate at about 3 0% of total DM is not known. In attempting to explain the drop in hay intake, it is relevant to consider the effects of monensin on nomination and ruminal motility. Two-hundred mg of monensin fed daily to 625-kg steers resulted in a 16% decreasing of forage intake of dry winter range (Lemenager et al., 1978b). At the

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105 same time ruminal liquid and solid turnover rates were 31 and 44% slower. In a companion trial, monensin (0, 50, 100, and 200 mg/d) reduced linearly ruminal fluid turnover rate in cattle limited-fed a high concentrate diet. These findings were interpreted as monensin depressing ruminal turnover rate independent of depression in intake. Lemenager et al. (1978a) also reported a 13.6% and 19.6% reduction in forage intake in cows grazing low quality range when 50 and 200 mg of monensin were fed, respectively. Grazing time was also reduced by 14.6% when cows were fed 2 00 mg of monensin. Coombe et al. (1979) reported a reduction in cereal straw intake from 2 to 1.5% BW and from 2.6 to 2.1% BW, depending on the straw treatment, when monensin was fed. Deswysen et al. (1987) reported that 100 mg of monensin given to 290-kg cattle fed corn silage indirectly affected rumination through a lowered ruminal motility. Monensin reduced the daily numbers of normal boli and total boli and increased the mean duration of one rumination bolus cycle. Monensin increased the duration of the main morning meal and decreased total daily ruminal contractions, and tended to depress silage intake. They also found differences among animals and several interactions of monensin by period and monensin by animal, suggesting that the effects of monensin on intake, intake behavior, and ruminal motility were variable. Monensin reduced the strength of contraction of intestinal waves (Job, 1971) Based on this observation.

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106 Deswysen et al. (1987) suggested that strength of ruminal contraction may have been reduced, in addition to reduction in number of contractions. Mastication during ingestion and rumination appears to be the primary mechanism for comminution. Chewing during rumination is more important for the continued comminution of large particles (Ulyatt, et al., 1986). All these findings may explain the slower rate of riominal turnover, increased or no effect on riiminal fill, and variable effects on intake found in other experiments with high forage diets (Lemenager et al., 1978a, 1978b; Pond and Ellis, 1979; Pond et al., 1980; Ellis et al., 1984). The suggested dose of monensin for grazing cattle is 100 mg/d (Delaney and Ellis, 1983). Faulkner et al. (1985) found that 100 mg of monensin was better than 0 or 2 00 mg in a growing trial with 236-kg cattle fed ensiled cornstalks. Monensin linearly decreased intake and quadratically affected ADG. The effects of monensin on rumination and ruminal motility (Deswysen et al. (1987) and depression of feed intake have been found at the 100 mg/d feeding level. The linear effect of increasing monensin levels on ruminal liquid passage rate (Lemenager et al. 1978b) and on intake (Faulkner et al., 1985) suggest that these effects may have been magnified in the current experiment where cattle of similar BW were given twice this dose. High doses were used in an attempt to overcome the presumed inhibitory effect of molasses.

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107 Variable effects of bambennycins on feed intake have been reported in cattle. Some research reported an increased feed intake (Alert et al., 1993), and other research reported no effect (Flachowsky and Richter, 1991; Poppe et al., 1993; Burris and Randolph 1996). Many of the published trials were grazing trials and intake of forage was not available Efficiency of feed utilization was estimated by several variables. Metabolic BW (MBW) was included as a covariate in an attempt to account for maintenance. Gains were predicted by pen with the actual hay and supplement intake, mean BW and sex, using the Level 1 of the NRC (1996) model. For Year 1, predicted gains were obtained for heifers and steers, and averaged. Differences between observed and predicted ADG was analyzed as an another estimator of efficiency. Pen observed and predicted ADG are plotted in Figure 3-1. Points plotted above the line y = x indicate that observed gains were higher than predicted. If the treatments did not affect the efficiency of feed utilization, all points are expected to fall in that line with some variation due to errors of measurements of feed intake and BW gain. Feed to gain and TDN to gain ratios were lower for Year 2, which is consistent with higher gains (Table 3-8). Feed to gain, TDN to gain, and TDN to added gain ratios were lower (P = .0001, .0007, and .037, respectively) in animals fed corn than in those fed molasses supplements, indicating

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108 the better response to feed additives with corn supplements (Tables 3-9 and 3-10) Animals fed monensin had 10% (CM) to 3% (MM) higher efficiency of total gain (P < .03) and similar efficiency of added gain than the ones fed control supplements. Monensin increased (P = .004) by .102 kg (CM) to .041 kg (MM) the difference between observed and predicted ADG. There were trends for interactions for TDN to total gain adjusted for MBW (P = .062) and for difference of observed and predicted ADG (P = .13), indicating that the increased efficiency was higher in corn than in molasses. Animals fed bambermycins had 13% (CB) to 8% (MB) higher efficiency of total (P < .003) and 23% (CB) to 8% (MB) higher efficiency of added gain (P < .007). Bambermycins increased (P = .013) by .063 kg (CB) to .041 kg (MB) the difference between observed and predicted ADG. However, the increased efficiency obtained with bambermycins appeared mostly in Year 2 (Figure 3-2) In Year 1, the increased ADG was explained by increased hay intake. In Year 2, however, there was no effect of bambermycins on hay intake and the increased ADG was explained by increased efficiency of feed utilization. Bambermycins increased feed efficiency in several experiments (Grant et al., 1974; Flachowsky and Richter, 1991; De Schrijver et al., 1991; Alert et al., 1993;

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109 Figure 3-2. Difference between observed and predicted gains, by treatment and year.

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110 CTi 00 in mm ^ in m in U >H -H Is 0) 73 m m Q) O O ^ ^ > Q O Ou rH fN O (N rH moo o o o O (T 00 O CN (N O O 00 in cr\ CTi 00 CN cr\ 00 O U3 LD I> O rH CN rH rO O rH CN rH CN cn o ro O Ln CN CN O CN CO CTi X> LD CN rH ID ID r-co (Tl Cn CN rH ro cTi CO ro O U3 V£> rO ro CN (0 -iH -H IS 'Z Q Q •H (0 (0 (0 tr H Ti T) 0 (0 0) (1) T) t3 t3 (0 T! n3 (d 0) \ T! IS S Ti Q Q o u to 5 o !-> (0 01 4J c: (8 0) g C C I >irH nj (S 4J ja Q) rH 4J (fl ^ o C 4J 4J H -H 01 g (0 0) •l-> rH 0 CU Ci •H nt 01 • T) 0) 01 •H fl} >H -^^ (8 ^ 0 Q 01 4J <0 2 4J c 0) e rH a 0. a 0) u c c a H -H < I 0) Q) ^ OiW 2 ^ > u 0) 01 II 2 2Qaxi H H O + X! 2 g ^ (D Q Q M 4.) Eh Eh -h 10 U S H rH JJ "O (0 0) 4J CUD 0 4H II o 0) 4J U c: c 0)
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Ill Table 3-9. Effect of treatment on efficiency of feed utilization (hay alone not included) Treatments Item CC CM CB MC MM MB SE AVGBW^, kg 285 292 290 290 284 287 2 8 ADG kg Observed 621 656 727 616 589 658 020 Predicted'' 629 554 664 626 563 617 .030 Feed/gain 10. 20 9. 14 9. 13 11. 30 10. 90 10. 40 .30 TDN/gain"" 6. 45 5. 81 5. 71 6. 77 6. 59 6. 26 .18 TDN/gain'^ 6. 51 5. 73 5. 67 6. 73 6. 67 6. 28 .23 Added gain, kg 376 411 482 371 344 413 .020 TDN/add gain^ a'. 24 3! 96 3 28 4. 23 4. 50 3. 91 .22 TDN/add gain' 4. 20 4. 00 3. 31 4. 26 4. 44 3. 90 .23 TDNA/added gain*" 2. 38 1. 76 2. 21 2. 85 2 29 2 40 .23 DiffS kg 008 102 063 010 026 041 .020 ^ Initial + final body weight / 2. Predicted with Level 1 model, NRC (1996), see text. ^ Total TDN / total gain. Total TDN / total gain, Ismeans with MBW as covariate. ^ Gain with supplement gain with hay alone. Supplemental TDN / added gain. ^ Supplemental TDN / added gain, Ismeans with MBW as covariate. Increment of TDN = (change in hay TDN + supplement TDN) / added gain. ^ Difference = observed ADG predicted ADG. Dhuyvetter et al., 1996). The increase in feed efficiency, however, was lower than the efficiency achieved with monensin in cattle fed corn silage diets (Burris and Randolph, 1996). Monensin consistently decreased hay intake by .14% BW in both corn and molasses, and increased by 10% and 3% total efficiency in corn and molasses, respectively. Monensin may be more useful when hay is in short supply or expensive.

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112 Table 3-10 Probability values for preplanned comparisons for efficiency of feed utilization Effect Contrasts" Effect CI C2 C3 C4 C5 Treat Covar AVGBW^, kg ADG kg Observed Predicted"^ 3440 .9492 .7454 .0326 .1789 .3222 0069 .8567 .0007 .1284 5547 .0174 .6478 .8409 1139 .0005 4303 .0636 Feed/gain .0001 0211 0027 .2609 .7157 .0001 TDN/gain^ 0007 .0264 .0013 .2134 .5376 .0006 TDN/gain^ 0002 .0205 .0007 0615 .2894 .0002 .0540 Added gain^ .0069 .8567 0007 .1284 .1139 0005 TDN/add gain"" .0372 .9689 .0062 .2251 .1560 0090 TDN/add gain' .0568 .9764 0073 .4340 .2456 0196 .3013 TDNA/add gain^ .0445 0152 .1888 .8942 .5553 .0664 Diff", kg .0908 0036 .0131 .1261 6659 .0136 Contrasts: 01 = corn vs molasses; C2 = monensin vs control; 03 = bambermycins vs control; 04 = monensin by supplement type; 05 = bamberg>cycins vs supplement type. Treat = treatment; covar = covariate (MWB) ^ Initial + final body weight / 2. ^ Predicted with Level 1 model, NRO (1996), see text. ^ Total TDN / total gain. Total TDN / total gain, Ismeans with MBW as covariate. ^ Gain with supplement gain with hay alone. Supplemental TDN / added gain. ^ Supplemental TDN / added gain, Ismeans with MBW as covariate. Increment of TDN = (change in hay TDN + supplement TDN) / added gain. ^ Difference = observed ADG predicted ADG. Increased ADG in cattle fed bambennycins apparently resulted from increased hay intake and feed efficiency in Year 1 and through increased feed efficiency and no change in feed intake in Year 2 Bambermycins should be used when maximum use of hay is desired.

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113 Volatile Fattv Acids Volatile fatty acid data are presented averaged over sampling times (Table 3-11 and 3-12, P-values in Table 313). Because a few interactions of treatments by sampling date were observed, data are also presented by year and sampling date (Appendix Tables A-7 and A-10, P-values in Appendix Tables A-8 and A-11) Total VFA concentrations were higher in Year 1 (P = .0001) than in Year 2. This variable should be taken with caution because the sampling technique may introduce considerable error due to salivary contamination. It is possible that differences between years reflect different sampling conditions. Cattle were sampled in temporary facilities in Year 2, and therefore greater opportunity for salivary contamination may have occurred. Total VFA concentrations were higher (P = .0001) in animals fed corn than in those fed molasses supplements, possibly reflecting more substrate for fermentation at the time of sampling. Corn supplements were delivered 3 to 5 h before sampling while molasses were delivered the day before sampling. There was a trend (P = .118) for monensin by supplement type interaction. Monensin tended to increase by 3% total VFA in corn and to decrease by 7% total VFA in molasses supplements. Bamberirr/-cins depressed (P = .011) by 13 and 3% total VFA in corn and molasses supplements, respectively.

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0) p > 4J O u >l (-• ^1 (d 0) QJ rH as -H > ro CM cn o o o rH IT) o 00 o oq CN 00 rH m m CTl O (N U) rH ^ ro rH O O X> O O rH rH O O O O ro vx) o oa ro CN CN rH ro (N rH u> 00 o o VO CN rH 00 Ol ^ 00 rH Cri 00 CTl rH VD rH O o o • rH U Q) (0 C O H 5h < O 4-> > Cn <: P3 ffl > Q) -U (0 JJ 0) < u o o o o o in ro 00 ro rH 00 00 o o o as 1^ 0) -U 03 g C O -H a 0 (0 Q) Q) ^ (0 CQ 0) 03 o rH <

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115 c Q) Di O U 4-> -rH c (0 Q) U :3 m rH a a (0 m -d •H o >1 4J 4J fO 4H (U rH -H 4-) (0 rH O > o -U C! 0) e ( 4H o 4J u rH I ro Q) rH ,Q 03 m 0) m m (tJ rH o d u o u w CO 03 d •H m d 0) d o o u jj d o u 03 d •H CO d 0) d o o d o u (D CO o 00 00 in in in CTl O U5 00 < > 03 O m CTl ^ ro CN 00 m o CO 00 rH l> rH VO in >^ rH O CN rH 00 o r-~ CN ro CTl 00 rH V£) rH rH ix> >x) in r~O VO O rH rH rH in CT> >X> td" rH in (N m C-~ O t> O rH rH rH o o 0) 4-> (0 d o •H 0) 03 J3 M < a >ifc O > ^ 13 u [n < 04 OQ OQ > 00 m ro ro 00 00 rH 0) IC a o •H a o u Q^ 0) -IJ 03 4-) 0 u o 00 in 00 O CTl O fO CTl o CN CN 03 J 03 T3 CO O) 03 g rH CT^ 00 og 00 CN in o 00 ro og 00 CO CO in o IX) CN H >H u o 0) J3 0) (V M t-t O 0) 0} m >l u 0) u u It • d) — O -rl 0) 4J & to H 0) c; 0) ro o 0 c:) (0 H u c ai rH Q) •H .C H u (0 C II > 3 1 0 in 0 o w o CN •H JJ 4J c + 0) JJ )~i c u (U 0) •rl IH to ^1 1 •rl M -a a 00 0) 0) 1 V) c 0 3 2 10 •rl u 0) II M C < o •H u — u > (0 >1 <1> •rl u 0) (0 >i o u u to nS 1 -0 Ji 0) 01 0) JJ u II C ITJ U 0) C 01 -i (C 0) U ^ ^4 r^g DQ CQ 0)

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116 Table 3-13. Probability values for contrasts, volatile fatty acids and plasma urea nitrogen Contrasts^ Variable CI C2 C3 C4 C5 C6 Total VFA, inM .0001 .6383 .0106 .1182 .0979 .0773 VFA, M/lOO M Acetate Propionate Butyrate bcvfa"" 0083 .1574 .0001 0001 .0001 .0001 0001 .8234 .3794 .8387 .4535 .0723 .3953 .0006 0031 .7723 .1498 .2183 .1817 .4565 0001 0001 .0001 .5658 Acetic : propionic 0078 .0001 .9018 0054 .0980 .0001 Plasma urea-N .0001 .1721 .9690 .1043 6920 0001 Contrasts: CI = corn vs molasses 02 = monensin vs control C3 = bambermycins vs control C4 = monensin by supplement type C5 = bambermycins by supplement type 06 = all supplements vs hay alone. Branched-chain VFA. Animals fed hay alone tended {P = .08) to have lower VFA concentrations than those fed supplements. Individual VFA molar proportions and ratios should be independent of salivary contamination. Cattle fed corn had a 2% higher (P = ,008) acetate molar proportion than those fed molasses supplements. Monensin decreased (P = .0001) by 5%, while bambermycins did not affect (P = .38) acetate molar proportion in both corn and molasses supplements. All supplements decreased (P = .0001) acetate molar proportion when compared with hay alone. There was a monensin by supplement type interaction (P = .0006) for propionate molar proportion. Monensin increased by 39% propionate molar proportion in corn and by

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117 17% in molasses supplements. BambernYcins did not affect (P = .84) propionate molar proportion. All supplements increased (P = .0001) propionate molar proportion when compared with hay alone. Animals fed molasses had a 16% higher (P = .0001) butyrate molar proportion than those fed corn supplements. Butyrate molar proportion increased in animals fed molasses or sugars (Marty and Preston, 1970; Pate, 1983; Beever, 1993). There was a monensin by supplement type interaction (P = .003) for butyrate proportions. Monensin decreased by 16% butyrate in corn and it did not affect butyrate in molasses supplements. Bambennycins did not affect (P = .45) butyrate molar proportions in any type of supplement. Animals fed supplements (with the exception of CM) had higher (P = .0001) butyrate than those fed hay alone. Animals fed corn had a higher (P < .0001) molar proportion of branched-chain VFA (BCVFA) than those fed molasses supplements (1.35 vs .51 mol/100 mol) Monensin did not affect (P > .8) BCVFA molar proportion and bambermycins tended (P = .07) to increase by 61% molar proportion of BCVFA in both corn and molasses supplements. Branched-chain VFA are considered growth factors for cellulolytic bacteria and they arise from deamination of branched chain amino acids (Russell, 1984) Animals fed molasses had a 6% lower (P = .008) acetate: propionate ratio (C2:C3) than those fed corn

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118 supplements. Moloney et al. (1994) reported lower CjrCj ratios in steers fed a silage diet and supplemented with molasses than in those supplemented with barley (4.28 vs 3.57). There was a monensin by supplement type interaction {P = .005) for CjrCj. Monensin depressed CjtCj by 44% in animals fed corn and by 21% in animals fed molasses. Bambermycins did not affect (P = .90) CjtCj in either supplement type but tended (P = .098) to exhibit an interaction by supplement type. Animals fed supplements had lower (P = .0001) CjiCj than those fed hay alone. Acetate to propionate ratio decreased when changing from high forage to high concentrate diet (Owens and Goetsch, 1988; Van Nevel and Demeyer, 1988; Beever, 1993) Supplementation of forage diets with highly fermentable carbohydrates (sugars, starch) decreased C2:C^ (Owens and Goetsch, 1988; Beever, 1993). The effect of monensin on VFA molar proportions is consistent with the widely known effect of monensin on ruminal fermentation: a reduction in acetate and butyrate, and an increase in propionate molar proportion (Bergen and Bates, 1984; Van Nevel and Demeyer, 1988) The lack of effect of monensin on performance in cattle fed molasses supplements does not appear to be related to the effect on VFA shift. Dawson and Boling (1987) reported an increased resistance of ruminal bacteria to monensin with increasing K concentration in vitro. Greene et al. (1986) and Chirase et al. (1987) reported that monensin was more effective in

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119 decreasing CjiCj ratio in lambs fed high dietary K. Research conducted at the University of Florida has shown that changes in concentration of K and Na resulted in altered in vitro VFA production (Schwingel et al., 1989). With monensin fermentation CjrCj ratios were higher at 48 mM K than at 168 mM K. The decrease of C2:C3 ratio with increasing K concentration was interpreted to be the consequence of a change in the microbial ecology. Total VFA production was severely depressed by monensin, especially at the high Na levels (Schwingel, 1988) The nature of the interaction appears complex, as Bates and Schwingel (unpublished data) have shown different effects of lasalocid and monensin on the growth of S. bovis and R. albus with different K and Na concentrations. Schwingel (1988) pointed out that the effects may be even more complicated in the rumen, where Na and K concentrations affect the concentration of each other. Total VFA production appeared to be negatively affected by the combination of monensin and high dietary K, because monensin tended to reduce total VFA concentration in animals fed molasses supplements. The combined effects of less feed (hay) intake, less VFA, and probably less bacterial protein available to the animal may explain the failure of monensin to increase ADG when fed with molasses. Coombe et al (1979) reported that monensin markedly reduced intake and gain with all diets (pelleted and alkali-treated barley or wheat

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120 straw) and this effect occurred even though monensin had the usual effects on rumen fermentation. A negative association has been reported for corn gluten meal and monensin when fed on alternate days. Steers fed monensin at 48-h intervals had a reduced feed intake. Monensin supplementation of diets that contained corn gluten meal reduced ADG by .21 kg while increasing ADG with diets that contained soybean meal by .13 kg. Monensin feeding in an alternate day supplementation with high riiminal escape protein was not recommended as a result of this study (Collin and Pritchard, 1992) It is unlikely that a similar effect occurred in this trial because even though molasses was delivered three times a week, molasses was consumed for more than 1 d, especially with monensin included in molasses. Furthermore, monensin has also failed to increase gain when soybean meal was the protein source in molasses slurry (Kunkle et al., 1990). Bambermycins appeared to have little effect, if any, on VFA proportions. Van Nevel and Demeyer (1992) reported that in vitro bambermycins was one of the feed additives with little effect on fermentation of different substrates. In that experiment, bambermycins did not depress fiber degradation. Similarly, Rowe et al. (1982) reported no effect on fiber digestion in situ. Several researchers have found no effects of bambermycins on ruminal fermentation (Galbraith et al., 1983; ElJack et al., 1986; Fallon et

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121 al., 1986; Flachowsky and Richter, 1991; Alert et al., 1993) Other research has reported changes in VFA concentrations (DelCurto et al., 1996; Earley et al., 1996). Plasma Urea Nitrogen Plasma urea N data are presented averaged over sampling times (Table 3-12, P-values in Table 3-13). Because an interaction of treatments by sampling date have been observed for Year 1, data are also presented by year and sampling date (Appendix Tables A-7 and A-10) Plasma urea N was 2.1 mg/dL lower in Year 2, perhaps reflecting the total dependency on hay versus some pasture regrowth throughout the experimental period in Year 1 (Table 3-11) In addition, higher gains may have required higher metabolizable protein. Higher hay (energy) intake may have captured more ammonia in the rumen. Major dietary factors shown to affect PUN concentration in cattle include protein level, ruminal protein degradability, energy level, protein to energy ratio, and level of intake (Hammond, 1992; Hammond et al., 1993). Animals fed corn had higher (P = .0001) PUN concentrations than those fed molasses supplements. Whether this is a true difference or an artifact caused by the short time elapsed between feeding corn supplements and ruminal sampling is not known. Hammond and Chase (1996) showed that in beef cows supplemented with cottonseed meal twice a week, PUN ranged from a high of 14 mg/dL in the afternoon after supplementation to a low of 7 mg/dL 2 d after

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122 supplementation. Diets were formulated to provide the same quantities of rumen degradable protein. The rate of degradability of the main DIP source, urea in corn and nonprotein N in molasses supplements, may be different. Different degradation rates of the N and energy source may also result in excess ammonia being absorbed and transformed into urea in the liver. Molasses-based supplements may have captured more N in the rumen, and therefore PUN would be lower. However, Stateler et al. (1995) reported higher PUN concentration in cattle fed similar levels of molasses slurries but with higher CP concentration. It is unlikely that DIP was limiting in either supplement type because the PUN concentration were 9 mg/dL or higher. Hammond et al. (1993) suggested that PUN concentrations between 9 and 12 mg/dL were a transition range below which ADG response to protein supplementation was greater than above this range. Model level 2 of NRC (1996) predicted a negative bacterial N balance (-13% of requirements) in animals fed molasses and 8% surplus in animals fed corn supplements. Predicted metabolizable protein was not limiting for gain in molasses or corn supplements. There was a tendency (P = .10) for monensin by supplement type interaction on PUN and bambermycins did not affect (P = .97) PUN concentrations. Animals fed supplements had higher (P = .0001) PUN concentrations than those fed hay alone. The average PUN concentration for hay alone in Year 1

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123 was 9.41 (7.86 mg/dL was the lowest in February), while the average for Year 2 was 6.77 mg/dL (lowest 6.07 in March) suggesting that at some point, and especially in Year 2, animal performance of cattle fed hay alone may have been limited by DIP. However, the TDN:CP ratio of the hay fed was less than 7, suggesting sufficient protein relative to energy (Moore and Kunkle, 1995) Garces-Yepez (1995) reported mean ruminal ammonia and PUN concentrations of 2.6 and 3.9 mg/dL respectively in steers fed bermudagrass hay containing 5.7% CP, which may be considered more indicative of N deficiency. Parasites Nematoda egg counts were low in both Year 1 and Year 2 and were not affected by treatments (data not shown) Deworming at the beginning of the trial in addition to low grazing activity during the winter probably created little opportunity for infestation. Counts tended to be lower in Year 2 (means from 0 to 2 eggs/5 g of feces) than in Year 1 (means from 8 to 29 eggs/5 g of feces) probably reflecting 'cleaner' pastures in the facilities used in Year 2. The pens used in Year 2 were finished just before the trial started and that area of pasture did not have high animal density during the previous warm season. Monensin consistently suppressed (P < .0004) coccidia counts in both corn and molasses supplements in Year 1 and Year 2 (Table 3-12) This suggests that monensin was

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124 effective at the intestinal level in molasses supplements, and it can be used for prophylaxis for coccidia in either supplement. This effect of monensin and other ionophores has been documented (Bergstronm and Maki, 1974; Stromberg et al., 1982; Watkins et al 1986). It is unlikely that coccidia control accounted for the effect of monensin on animal performance in this experiment, because: a) the counts were low, b) the animals were yearling and likely less susceptible than calves, and c) suppression of coccidia in molasses did not result in higher animal performance. Bambermycins had no effect (P > .2) on coccidia counts, which is in agreement with literature from the manufacturer (Hoechst-Roussel, 1993).

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CHAPTER IV EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES SUPPLEMENTS ON INTAKE, DIGESTIBILITY, AND DIGESTION KINETICS IN HEIFERS Tnfrndnrt-.ion Feed intake is an important determinant of animal production and changes in intake may be associated with changes in digestibility, and both intake and digestibility may be related to digesta kinetics. Some feed additives, such as monensin affect intake, digestibility and digesta kinetics (Ellis et al., 1984). Results from the performance experiment (Chapter III) suggested that bambermycins may affect intake and efficiency of feed utilization. The present research was conducted to verify these effects. The objectives were to: 1) determine the effects of bambermycins included in corn or molasses on diet intake, digestibility and digesta kinetics, and 2) compare the effects of different energy sources (starch and soluble carbohydrates) on these variables. 125

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Material -q and Mf^hhods 126 The experiment was conducted at the University of Florida Nutrition Laboratory in Gainesville, from November 1, 1995 to February 8, 1996. Experimental desig n. A balanced 4x4 Latin square design with a factorial arrangement of treatments was used. Treatments were: Supplement type Bambermycins level QqsIq Corn-urea 0 mg CC Corn-urea 2 0 mg CB Molasses slurry 0 mg MC Molasses slurry 20 mg MB Each experimental period consisted of 25 d, with 17 d for adaptation to the experimental diet, and 8 days for sample collection. Initial assignment of animals to treatments, and pens was at random. Pre-trial Before the experiment was initiated, a 21-d (14-d adaptation to diet and 7-d collection) preexperimental period was conducted with the purpose of characterization of the hay to be used in the experiment. Feed intake, digestibility and digesta kinetics were measured in four animals (23 9 kg BW) fed bermudagrass (Cynodon dactyl on ^ hay alone using the same methodology described below. Data from the pre-trial period were not included in the Latin square analysis and means are reported separately.

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127 Animals Six crossbred Angus x Brahman heifers were weaned in early September 1995 at the Santa Fe Teaching Unit, vaccinated, dewormed, and transported to the Nutrition Laboratory facilities, where they were grazed on bahiagrass ( Paspalum notatum ) pasture and fed bermudagrass hay for 1 wk. Four heifers were selected by temperament (docility) dewormed, and housed in individual pens with concrete floor. They were given ad libitum access to bermudagrass hay and fed 2 kg of molasses slurry for 2 wk. They were given ad libitum access to water and a mineral mixture containing 17.2 to 20.6% salt, 17.2 to 20.6% Ca, 9% P, 1% Fe, .2% Mn, .01% I, .01% Co, .2% Mg, .12% F, 1,500 ppm Cu, 20 ppm Se, and 4,000 ppm Zn. After 2 wk, heifers were offered only bermudagrass hay. Once the voluntary hay intake was about 2% of BW, the pre-trial period was started (October 11) After the end of pre-trial period, one heifer was replaced due to injury. The estimated shrunk BW (defined as the average BW before the morning feeding on d 1 and d 25 x .95) were 238 kg for period 1 and 287 kg for period 4 of the Latin square. The average weight of each heifer in each period was used to express variables as percentage of BW. Diets Bermudagrass hay harvested during the early summer of 1995 from the Pine Acres Research Unit was stored in a barn for use in this experiment, in the performance trial (Year 2, described in Chapter III), and in a r\aminal metabolism experiment (Chapter V) The hay was chopped as

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128 needed to approximately 6 cm and fed ad libitum twice a day (0730 and 1930), as the base diet. The amount of hay offered was determined by adjusting the quantity fed daily to provide a minimum of 8 kg of orts each day. Supplements were formulated as described for the performance trial (Chapter III) designed to provide equivalent amounts of TDN, DIP and UIP. Supplements were fed daily in separate feeders at 0715. All feeds were offered after cleaning the pens. Amount of corn supplement fed was adjusted at the beginning of each period and fed at .8% % BW (as fed basis) Molasses slurry was delivered to provide the same amount of TDN as the corn supplements. Because molasses slurry consumption was not complete, the dry component (corn gluten meal-urea, with or without bambermycins) was delivered separately before the morning feed was offered to assure that the feed additive and protein source were consumed at the designed doses. Consumption of this dry ingredient was completed in about 10 min in all cases. Molasses was top-dressed with 5% corn meal (no additives) and offered within 30 min after dry ingredient consumption. Top dressing with corn appeared to stimulate consumption of molasses. Water and the mineral mix described above was made available at all times. Feed sam pling procediires Procedures for determining voluntary intake and digestibility in sheep, described by Moore (1981), were followed with pertinent adaptations.

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129 Because the amount of hay offered changed, 5% of the total offering was sampled each day to avoid bias. Grab samples of hay were obtained, placed in air-tight plastic bags and sealed. Daily samples from a given heifer in each collection period was placed in the same bag. The total weight of the hay sampled was recorded prior to grinding. During the 8-d experimental period, orts were collected prior to the morning feeding. Feeders were swept and orts collected, and transferred to woven plastic bags. Weight of orts was recorded prior to grinding. Waste was collected by sweeping the floor around feeders and handled as described above The week following the collection period, composited hay samples were ground in a hammer mill, then ground in a Wiley mill to pass 4-mm and 1-mm screens and stored in plastic bags (Whirl-Pak, Nasco, Fort Atkinson, WI) until analyzed. Orts and waste from each heifer were air dried before grinding, and stored as described for samples of hay. Analysis of DM for intake calculation was performed the same day that the hay, orts or waste were ground. Samples of corn mix and corn gluten meal-urea were taken daily during the last 8-d collection period, composited as sampled and stored in separate bags for each animal and period. One molasses sample per period was collected directly from the storage tank and stored frozen until analyzed.

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130 Preparation of Yht:f^rhiTim -mi^rkpd fiber. Bermudagrass hay was ground in a hammer mill and then ground in a Wiley mill to pass a 4-mm screen, and washed in cloth bags in an automatic washer with water heated to 80 to 90 C. The material was washed through two normal cycles with commercial detergent (Tide™) to remove loose material and most of the cell contents (Uden et al.,1980). Two additional washing cycles were used to remove the detergent After drying at 60 C, fiber particles were immersed for 24 h in aqueous solution of ytterbium nitrate (YbN03.5H20) containing 2% of the dry fiber weight as Yb, Acetic acid was used to keep the pH of the solution below 7 (Ellis et al,, 1982; Luginbuhl et al., 1994). After soaking, excess fluid was decanted through a 0.5-mm screen to recover fiber particles. Six successive washes were accomplished by resuspending the material in deionized water (one per hour) and repeating the screening procedure. Marked fiber was dried in a forced air oven at 55 C for 72 h, analyzed for Yb concentration, and stored in air-tight bags until used. Dosing procedure On d 15 of the pre-trial period and on d 19 of each Latin square period prior to the morning feeding, 60 to 90 g of Yb-labeled fiber (11 mg Yb/g fiber) was mixed with 400 g of corn meal (pure) and placed in the clean feeder. Doses were estimated using an equation presented by Ellis et al. (1982). Any uneaten material was recovered for Yb analysis. Effective doses (average 790 mg

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131 Yb/animal) were estimated from the amount delivered minus the amount recovered. The corn plus Yb-labeled fiber mix was made available to animals for a maximum of 3 0 min. Most of the time, animals consumed the marker in a 15-min period. Immediately after dosing, hay and supplement were fed as usual, except that the amount of supplement was adjusted to account for the corn meal eaten with the fiber. This dosing procedure minimized animal handling to reduce possible effects of stress on voluntary intake. Fecal sampling Feces for use in standards and blanks were collected the day before dosing. Samples for Yb analysis were collected every 4 h after dosing up to 56 h, at 8-h intervals from 56 to 72 h, and at 12-h intervals from 72 to 132 h. Fecal grab samples were taken from the rectum only if animals did not defecate within 15 min of designated sampling time. The act of entering into pens often caused the animals to stand up, move around, and defecate. Typically, minimum disturbance of animals resulted and minimum effect on voluntary intake was presumed. Fecal samples were dried in a forced-air oven at 55 C for 72 h and ground in a Wiley mill to pass a 1-mm screen. During the longer sampling intervals, 1to 2-h modifications were made to allow samples to represent every other hour of the day (from 0400 to 2400, 11 samples) for nutrient analysis. After grinding, a 5-g subsample from

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132 these 11 samples were composited for DM, OM, NDF, and N analysis Ytterbium analysis Each individual sample of ground feces (1 to 2 g) was ashed in 50-mL beaker at 500 C for 8 h. Ash was recovered in 1 M DTPA solution, extracted for 12 h and filtered. The final extract was analyzed by atomic absorption spectrophotometry (Perkin Elmer 5000) with nitrous oxide flame. Standard and blank solutions were prepared from pre-dosing fecal samples treated as described above (Ellis et al., 1982). Similar procedures were followed for analysis of Yb in Yb-marked fiber, except that .2 g of sample was used and standards and blanks were prepared with unlabeled hay. Digesta kinetics Excretion curves for Yb in feces were fitted to gamma age-dependent, ageindependent twocompartment models with increasing orders of gamma age dependency (GlGl to G4G1) using the nonlinear procedure of SAS (1987; PROC NLIN, iterative Marquardt method) described by Pond et al. (1988). The SAS program published by Moore et al. (1992) was used. The age-dependent and ageindependent compartments also are described as the fast and slow compartments, respectively, and passage from the slow compartment represents passage from the rumen (Moore et al., 1992) The model was selected using criteria that best fit the data for all animals in all treatments, considering the

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133 lowest residual errors and the most observations hidden in graphs of predicted vs actual data points (Moore et al., 1992) The best fitting model selected to evaluate digesta kinetic estimates was G2G1 for all period by animal combinations The following variables were directly estimated by the G2G1 model or they were calculated from model parameters according to formulas presented by Moore et al., (1992) and Luginbuhl et al., (1994). 1. Passage rate parameter (L) of the marker from the age-dependent compartment (Lambda in the model) The passage rate from this compartment is the product of L X .59635. The number is a constant related to age dependency (constant = .59635 when age dependency = 2) 2. Passage rate (PR) parameters of marker from the ageindependent compartment (k in the model) The PR from this compartment is equal to k, an estimate of passage out of the rumen. 3. Time delay (TD) in h, time elapsed between dosing and first appearance of marker in feces (calculated directly by the model) 4. Total tract mean retention time (TMRT) = 2/L + 1/k + TD. The mean retention time of the fast compartment (FMRT) = 2/L, and the mean retention time for the slow compartment (SMRT) 1/k, or ruminal mean retention time. 5. Fecal DM output (FO) = (DOSE/CJ x k x 24 h, where DOSE is the amount of Yb dosed and C„ represents the initial Yb concentration if instantaneously mixed in the ageindependent compartment; is estimated by the model. 6. Fill of undigested DM in the whole gastrointestinal tract = DOSE/C^ + (DOSE x k) / (L x .59635 X C^) The first term of the sum represents the fill of the slow compartment and the second term represents the fill of the fast compartment. In addition, ruminal PR was calculated according to Grovum and Williams (1973). In this model (designated LN here) ruminal PR is the absolute value of the slope of the

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134 linear, down sloping portion of the natural log transformed excretion curve. Ruminal mean retention time (RMRT) was calculated as 1/PR. Chemica l analysis The hay, orts, waste, and dry supplement, molasses and composited fecal samples were analyzed for DM, OM, NDF, and CP as described in Chapter III. Hay and supplements were also analyzed for minerals (Chapter III) Intake of OM, NDF and CP were estimated based on their concentration in DM of hay, supplements, orts, and waste. Intake of TDN was calculated assuming no associative effects adding the TDN intake from hay (estimated from IVOMD, Moore and Kunkle, personal communication) and TDN intake from supplements (table values, NRC, 1984) Digestibility (D) of each fraction (DM, OM, NDF, and CP) was estimated using their respective dietary and fecal concentrations: D = (I F)/I x 100; where I = intake of each fraction; F = fecal output of each fraction; and fecal output of each fraction = FO x fecal fraction concentration (% in DM) /lOO Statistical analysis Intake of DM was averaged over 7 d in the pre-trial period and 8 d (d 18 to 25) of each period in the Latin square. Data were analyzed as a 4 x 4 Latin square design using the GLM procedure of SAS (1987) Sums of squares were separated into effects of heifer, period, supplement type, feed additive and the interaction

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135 of supplement type by feed additive. When an interaction of supplement type by feed additive was present, treatment means were separated using the LSD with alpha = .05 (SAS, 1987). Results and Discussion Composition of feeds averaged over the four periods is presented in Table 4-1. Molasses had lower DM (72.0 vs 77.5%) and lower phosphorus concentrations (.34 vs .88 to .74%), and had higher pH (5.14 vs 4.51 to 4.71) than molasses used in the performance experiment (Chapter III) This suggests that less phosphoric acid was added to the molasses used in this experiment. Bambermycins concentrations in supplements are presented in Appendix Table A-4. Apparently there was analytical difficulty with bambermycins. Because the drug was active in the original pre-mix 5 months after the end of the experiment and mix formulation was correct, the formulated concentration was presumed. Bermudagrass hay varied among periods, average IVOMD and CP for periods 1 through 4 were: 47 and 9.7; 44 and 10.3; 48 and 9.2 and 44 and 8.8% respectively, which are lower than TDN and CP of the hay used in pre-trial period.

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136 Table 4-1. Composition of feeds fed during feed intake studies Corn Gluten Bermuda Item Corn Mix Molasses^ Mix Hay'' DM, % 86.5 72.0 89.0 87.9 IVOMD — — 45 .7 As % DM, OM 95.0 82.0 98.4 94.5 CP 15.6 9.8 78.7 9.5 TDlf 84.4 72 89 — NDF 9.3 5.2 75.7 ADF — — 40 5 Lignin — — — 4 .90 Ca 66 61 03 33 P .66 .34 .44 .20 Mg .26 .53 .06 40 K .43 3.76 .15 1.19 Na .01 .12 .03 .02 PH 5.14 TDN:CP ratio 5.41 7.35 1.13 5.37 ^ Blackstrap molasses not less than 40% inverted sugars. fortified with phosphoric acid and 25,000 U.S. P. units vit A, 33,000 U.S .P. units vit D, and 22 Int. units vit E per kg, and .0005% Cu, .00001% Co, .02% Fe, .001% Mn, .0025% Zn, and .00007% I. Sulfur content no less than 1%, as fed basis. Hay composition for pre-trial period was: 87.5% DM, 49.1% IVOMD, 94.5% OM, 10.6% CP, 53.0% TDN, 73.3% NDF, 37.1% ADF, 4.77% lignin, and 5.0 TDN: CP ratio. Calculated from table values for supplements (NRC, 1984) Pre-trial Intake, digestibility, and digesta kinetic data for the pre-trial are summaryzed in Table 4-2. Absolute digestibility obtained with FO estimated through marker techniques may be not comparable to digestibility estimated with FO measured by total fecal collection. Underestimation of FO would increase apparent digestibility.

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137 Table 4-2 Intake, digestibility and digestion kinetics for heifers fed hay alone (pre-trial) Item Mean Std Dev Min Max Intake, kg .59 DM 5 25 ox c o • OM 5 .06 .54 4 .37 5. 69 NDF 3 .89 .43 3 .35 4. 40 CP 58 .05 64 Intake DM, % BW Z .30 1 X it • 60 Apparent digestibility, % 61 68. DM 65 .4 3 41 .3 9 OM 66 o o 2.93 0 z 7 69. 5 NDF C Q D O 4 2.62 n 71 1 CP 62 o O 3.31 c; Q D 0 D J Digestible OM intake, kg 3 38 4(J Q Q 3 J Digestible OM intake, % BW 1 41 .24 J z 1 X 7 n Passage rate^, %/h 26. 56 Age-dependant 20 .8 6.41 13 .49 Aaeindependent J Z D .43 o it 7ft ^ • 83 Ruminal passage rate'', %/h A ft U J .42 .66 4 63 Mean retention time^, h Q O o o Fast compartment r D 2 2 1 A 'i c D Slow compartment 31 .0 4 0 26 .1 35. 9 Time delay 12 .5 2 2 10 .4 14. 8 Total 49 .7 3.0 45 .3 51. 5 Ruminal mean retention time^, h 25 .0 2.4 21 .6 27. 3 Fecal output^, % BW .76 .07 .66 82 Fill undigested DM^, % BW .06 .11 23 Fast compartment .15 Slow compartment .89 .15 .79 1. 10 Total 1 .04 .13 .92 1. 22 Estimated with nonlinear model (G2G1) Estimated from linear model (Grovum and Williams, 1973) Moore et al. (1992) suggested that when the digestibility is of interest, other markers with known concentrations such as chromic oxide should be used because of errors in estimating the amount of marker dosed. However, they found that all nonlinear models provided estimates of

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138 FO within 5% of FO measured by total fecal collection in sheep. Good agreement between FO estimated with pulse dosed and continuously infused marker has been reported also (Luginbuhl et al 1994). Even if an overestimation of digestibility occurred, there is no apparent reason to think that a bias existed that would favor one treatment over the other in the Latin square experiment. Estimation of hay intake, digestibility and digesta kinetics obtained in this period will be referred to when discussing the effects of supplements in the next section. Concentration of PUN (10.4 1.3 mg/dL) was consistent with the TDNrCP ratio of the hay, and indicates that N was probably not limiting ruminal fermentation (Hammond et al., 1993). Latin Square Intake and Digestibility Supplement and additive main effects on intake and digestibility are presented in Table 4-3 and treatment means are shown in Table 4-4. Supplement and additive interactions were not observed (P > .2) for any variables. Total diet CP concentration (% DM) was 11.1% CP and 12.4% for cornand molasses-supplemented diets, respectively. Intake of the molasses-based supplement was less than planned due to incomplete consumption. Lower {P = .098) molasses intake than corn (% BW) may result in a confounding of supplement type with supplement level on all variables measured.

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141 0 E (0 c o o 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours after feeding Figure 4 -1. Pattern of corn and molasses supplement consumption in heifers given ad libitum access to bermudagrass hay.

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142 Supplement intake with and without bambermycins was similar (P = .9) and therefore the effect of feed additive should not be confounded with level of supplement intake. Pattern of supplement consumption was different (Figure 4-1) About 60% of corn and 30% of molasses was consiamed by 1 h after feeding. By 3 h after feeding, consumption of corn was almost complete while that for molasses was only 45%, resulting in consumption of molasses more distributed throughout the day. Animals tended to consume most of the corn in one meal. This pattern of consumption is similar to the pattern observed in production situations. Supplement consumption is affected by many factors. There appears to be an optimum level of feeding competition that reduces intake variation (Bowman and Sowell, 1995a) In four studies that measured liquid supplement intake (review by Bowman and Sowell, 1995b), between 1 and 20% of experimental animals refused to consume any molasses-urea supplement. Supplement consumption varied from .002 to 2.54 kg/d and 30% of the beef cows in the experiment consumed only trace amounts of the supplement (Bowman and Sowell, 1995a) Heifers fed molasses had higher (P = .007) total DM intake (% BW) than those fed corn, resulting from higher (P = .001) hay intake. Bambermycins increased (P = .07) total DM intake (% BW) and tended (P = .14) to increase hay DM intake (% BW) when compared to control supplements. This trend is consistent with results from Year 1 of the

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143 performance trial (Chapter III) In other studies, bambermycins did not affect feed intake when compared with control diets (De Schrijver et al., 1990; Flachowsky and Richter, 1991; Poppe et al., 1993; Burris and Randolph 1996; DelCurto et al., 1996; Earley et al., 1996). However, bambermycins increased intake of hay-corn silage and corn silage diets when compared with ionophores (Hoechst-Roussel 1994; Burris and Randolph 1996). Alert et al. (1993) reported that bambermycins increased intake in fattening bulls but the composition of diet was not available. Compared to the pre-trial period, hay intake changes (HIC) were -.34 and -.06% of BW for corn and molasses supplements respectively, while substitution rates (SR) were .51 and .11, for corn and molasses supplements, respectively. These values are close to those reported by Garces-Yepez (1995) for corn supplemented at .83% BW (-.4 and .48 for HIC and SR, respectively). Goetsch et al. (1991) concluded after analyzing several experiments that each kg of corn DM would decrease bermudagrass intake by .46 kg. The molasses supplement consumed had minimal effects on FIC and SR. When intake of molasses supplements was expressed on an OM basis, the supplementation level (.45% BW) was similar to the low corn treatment (.36% BW) reported by Garces-Yepez (1995). Observed HIC (-.06) and SR (.11) for molasses were similar to reported HIC (-.04) and SR (.11) values for corn supplemented at comparables rate. Similar

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144 effects have been observed with small amounts of grain in many experiments (Horn and McCollum, 1987; Moore and Kunkle, 1995). Moore et al. (1995) suggested, after analyzing a large data base, that there were no obvious inherent differences between liquid and dry supplements in their effects on forage intake. Calculated HIC of -.24 and -.16, and SR of .39 and .26 for control and bambenttycins supplements are consistent with the trend of increased hay intake when animals received supplements with bambermycins The apparent effect (no statistics) of bambermycins on HIC in corn (-.41 vs -.28) and in molasses (-.08 and -.05) supplements was similar in relative terms. The biological significance of the effect of bambermycins on hay intake may be greater when the basal diet is supplemented with a moderate level of concentrate rather than with low levels of concentrate. It also may suggest that with a moderate level of supplement, the effect may emerge and increase the total DM intake. Thus, the low amount of molasses intake in this experiment may have precluded statistical detection of an effect of bambermycins on hay intake. Support for this suggestion is given by the observation that bambermycins increased hay DM intake by .13 and .03% BW in corn and molasses, respectively (Table 4-4). Further research, with several levels of supplementation as well as type of supplements will be needed to test this hypothesis. In such research, pattern of consiamption may

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145 need to be controlled to resolve any confounding with type and level of supplement. This last aspect is of academic interest with little practical value. Digestibility of NDF was higher (P = .027) when heifers were fed molasses compared to corn, perhaps a result of a lower associative effect on fiber digestion due to lower molasses intake. Predicted associative effects (change in ME) using the ForsuplO model (Moore and Kunkle, 1995) were .09 and -.02 Meal ME/kg OM for cornand molassessupplemented diets, respectively. This effect may be associated with longer (P = .049, Table 4-5) SMRT in molasses supplements, which would increase the extent of ruminal NDF digestion. Several mechanisms have been proposed to explain the depression of fiber digestion when a source of highly fermentable carbohydrate is added to forage-based diets. For example, lower pH with negative effects on cellulolytic bacteria, retardation of microbial attachment and increase of the lag time, competition of microbial population for essential nutrients, and use of alternative energy sources by cellulolytic bacteria have been suggested (Horn and McCollum, 1987; Orskov and Ryle, 1990) The animal may or may not be able to compensate for ruminal escape of potentially digestible fiber by shifting the site of digestion to the lower gut (Owens and Goetsch, 1986; Galyean and Owens, 1991)

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146 Estimates of NDF digestibility with both corn and molasses supplements were lower (no statistics) than when hay was fed alone in the pre-trial period. Several workers reported depressed NDF digestibility when energy supplements were fed. On bermudagrass hay diets, NDF digestibility (% of DM) for hay alone and hay plus levels of corn supplements comparable to those used in this experiment were: 5 6.7 vs 53.5, and 52.9 vs 47.2 (Galloway et al., 1993a); 56.4 vs 53.9 (Galloway et al., 1993b); 64.3 vs 57.1 (Hardin et al., 1989); 57.6 and 45.8 (Brake et al 1989). Based on results from several experiments, Goetsch et al. (1991) suggested that each kg corn DM would depress NDF digestibility by 3.3 percentage units. Similar reports are available on the effect of molasses on NDF digestibility. For example, molasses included at 25% of the diet depressed NDF digestibility (% DM) in limpograss ( Hema r t hr i a a 1 1 i s s ima ) (57 vs 51), and in rice straw(56 vs 46%) based diets (Brown et al., 1987). A similar response has been observed with ammoniated stargrass ( Cynodon nlemf luensis ) (Brown. 1993), and native grass (Kalmbacher et al., 1995). Khalili (1993) reported NDF digestibilities of 68.4 and 64.8% DM when grass hay was fed alone or supplemented with molasses at .58% BW. These digestibilities are numerically identical to NDF digestibility depression when the pre-trial period is compared to the supplementation periods

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147 Bambenrycins did not affect (P > .8) DM, OM or NDF digestibility. Reports found in the literature are contradictory. Bambermycins did not affect in vitro NDF digestion (Van Nevel and Demeyer, 1992), or in situ cellulose digestion (Rowe et al., 1982), or total tract digestibility of DM, CP, fat, CP, ash and N-free extract in wethers (De Schrijver et al., 1991), or apparent OM digestibility in wethers (Flachowsky and Richter, 1991) In contrast, 50 mg of bamberrrycins/d increased apparent DM, CF and N-free extract digestibility (Alert et al., 1993) and increased total tract OM digestibility in cattle (Poppe et al., 1993). Total tract digestibility tended to increase in steers supplemented with bambermycins or ionophores on alfalfa ( Medicaao sativa ) and grass hay diets (Barley et al., 1996). However, no effect of bambennycins was observed in 90% concentrate diets (DelCurto et al., 1996). Heifers fed molasses had 4 percentage units higher (P = .08) apparent CP digestibility than those fed corn. Lower depression of fiber digestion by molasses would result in less escape of potentially digestible OM from the rumen, with the consequence that less substrate would be available for fermentation in the lower gut. With the corn supplement, more fiber and starch may have escaped rumen fermentation with a shift in site of digestion to the intestines. The passage of potentially digestible carbohydrate to the cecum and colon can increase fecal N losses through increased

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148 fermentation and resulting unabsorbed microbial matter (Orskov et al., 1972). Microbial matter accounts for most organic metabolic matter in feces (Van Soest, 1994), and metabolic fecal N is mostly microbial matter (Mason, 1984) in ruminants. Bambennycins tended to (P = .2) increase by 2.7 percentage units the apparent CP digestibility, in agreement with a report of increased apparent CP digestibility in young cattle (79 vs 74%) on ad libitum concentrate diets (Fallon et al., 1986). This effect would be consistent with the lower microbial activity in lower gut and lower intestinal cell sloughing. It has been reported that bambernr/cins survives the ruminal environment (Rowe et al,, 1982). A similar antibiotic, avoparcin, has been shown to reduce intestinal cell turnover (Parker, 1990) Bambermycins increased N balance (MacRae and Lobley, 1991) and a reduction of gut cell turnover has been suggested as the mechanism of action. Intake of digestible OM tended (P = .098) to be .29% BW higher with molasses resulting from a trend for (P = .18) 2.6 percentage units higher DM digestibility and .15% BW higher (P = .007) total DM intake. Earley et al. (1996) reported no effect of bambernvcins or ionophores on digestible DM intake of alfalfa/grass hay diets, even though DM digestibility tended to increase by feeding feed additives

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149 In short, baitibermycins tended to increase DM intake from hay and had no effect on total tract digestibility. Even though there were no interactions ( P > .2), these effects were apparent when bambermycins was fed with corn but not with molasses supplement. T.^tin .qqnare H i gresta Kinetics Data for the pre-trial period are presented in Table 42, while data for the Latin square experiment are presented in Table 4-5 (main effect means) and Table 4-6 (treatment means) Biological interpretation of the age-dependant, ageindependent, twocompartment model has been described by Ellis et al. (1994). All estimations should be taken as the average over the sample collection period. According to Ellis et al. (1994), the age-dependant flow-paths appear to correspond to the large rumination pool as conceived by Hungate (1966) Residence time in such a nomination pool appears to be of similar magnitude to that resolved for FMRT. This flow-path also appears descriptive of the flow process involving interactions between rumination and fermentation-based buoyancy. The ageindependent flow process appears to be described by mass action competition for escape. The residence time of this compartment appears to be resolved by SMRT of the model. There is not agreement, however, on the adequacy of a particular model to describe

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U 0) W U O IM 0) rH (0 > > 4J a rH D D CO CO CO w CO (0 CQ (U c o :3 CO cn CO (C5 rH o o u rH (d •H > o rH CN rH ro CN O ro CN ID en rH CTl rH rH rH CTl Ln O o O CN CTl CTl ro o CTl in O LD V£) CN ID O 'J X) cr\ ro CO o • 00 cn LD 00 o o rH ro U5 VD CN CO 00 ID CN CTl ID rCN CTl CJ> ro rO m LD og O 1 % 1 MD ro rH O O rH O rH o O rH rH Ln ID ID og LD O rro rH o LD og O • o o o rH rH rH <£> LD LD 00 o a\ o ID 00 ID ro CO 00 ro ro CO rH cn O rH O CN iH rH rH CN rH ^ CN CT\ C nn rH 00 CTl r00 o 00 og rH o CO • rH 00 ID • ro rH O LD o rH rH CN rH CN ID o rH 00 'vf CN ID O rH rH ro 00 OJ 00 rH rH rH CN rH rH rH rH CN 00 00 m ID rH ^ o CTi U3 LD CTl Ln rH CTl CO ID CTl ro o rH rH rH 'J CN SI QJ 4J (0 U 0) Dl (0 J-) CO a t! CO (u a (0 T3 Q) a c a CO aTD Q) QJ C rH T) -H U I I •H Q) Q) 4J tn tn a QJ Pu QJ o e g o u l+H Q) g -H 4J C o •H Jj QJ 4-) QJ U (0 Q) JJ 4J c d QJ QJ g g (0 fO >i a a (0 g g -H O O QJ OUT) rH 4J S QJ (0 CQ O g -U (C rH -H O h CO E-" E-" QJ T3 O g g o u d QJ 12 4-) d QJ g JJ u (0 T5 a Q QJ JJ CO u QJ CD JJ •H CO d fa D JJ d Q) g JJ o u 5 pa JJ a JJ o ^ (0 rH O JJ (0 rH O U CO E-" QJ fa ro o ra\ n 0) rH rC3 o\ Q rH C • 01 g c (0 CO -U -H H g 0 CJ rH (0 0 >1 (4-1 g M-J H Wil Hi (U <1> •H n a g nd d g (0 II HI > >-i o rl 0) u JJ A o 0 •H ^ M (0 o rH < de rH 0 0) g T) 0) -H 0 Ci. JJ M g •rl (0 II 0) Li T) C (0 H rH ne 73 -H W ID g rH 0 m t-i H g Dl <4H 0 (8 >i u rH XI T3 <4H o e JJ rH JJ o C O 0) U 01 JJ 0 g O -H lO 0) JJ g C rH CO •rl M Oi rH 01 JJ O Dl 0) Tl CO U 3 0) 0) W )OUI JJ lO 0) U g U O Li •H a> inea: 0) JJ <4H C Oi MH 0 lO a 0) -H CO 0 JJ rH CO •H C o (0 JJ •H 10 O a a (0 !h (0 g a c 0) jj JJ rH 0) JJ c g 0 u u a M -rl 0) JJ Hi g II Ll (0 0) (0 a rH ft TS a g D. X 01 Q.CO 1 1 rH iH 3 Lmat (0 (0 CO C CO •H •rl II c JJ g •H M la a: CO o >1 lO

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u 0 4-1 LM W u o 4-1 (D 13 rH > Id JJ d 0) e jj (13 Q) ^1 Eh CO CO CO 2 U P3 U U U O rH T}( o ^ in CTi 00 o 00 in T-{ o CN ro CT\ ro in 00 iH CTl o fSl in ro 00 o in o o in in in in (Ti iH CTl ro iH CT\ iH O O U3 CN tn VD rs in o in v£) in o ^ O rH CTl i> in ro O CTl IJD CTl rCN CN ro CN rH fs] i> ro IX) 00 CTl in O] 1X5 00 ^ CTl CTl ro rH v£> ro rH rH in 1X5 O IX) O rH O O in rH 00 ro 00 CN 00 ro 00 CN rro 00 og 00 CN in oj ^ r~rH CTl 00 00 VX) 00 1X> rH 00 0 0) 4J Xi del dp (0 Sh o e mo rH 0) 4J o •H 4J 4-) IS H Di C rH d d J dP 03 ^ (U 0) (U 0 d B g g u g g s -u -u to 0) a o 4J 4J o o (13 T3 0) e •H U U u Q ^ Vh 4J a d a 4-) (t3 n3 >i 4H (0 n3 13 d a a (0 T) a a a to aT) e e -H OJ g g 4J 0) (u (u d 4J 0 O 0) i 4J o o rH rH T! -H e 0) U U to u u o Xi U 1 1 o u rH Q) rH (0 •H (U Q) 4J ^ Q) (t3 d Cn 4-) ^ (tS rH •H d) 01 4H d CO O g 4J 0) •H CO 0 4J (tJ ^H <: <; (0 (t3 rH -H 0 g (13 rH O u (0 (0 Q) fc, CO Eh &H d [JH CO Eh 0) > 0^ a. Pi D m 0) 0) D) nJ rH Q — e M n 0 r-II CTl 0) rH r~ CQ c 2 o rH CO g i-H CO o 4J rl g M u rH (fl 4J 0) rH •H M-l rl rH 0 H-l IS rH u 0) •H IS CO c T3 0) •H D] (0 (0 e g (fl (0 rH r> g g > 0 p E •H u > 4J C5 0 II H )-l T3 C5 o -0 rH <: 0) TJ rH -a 0 Q) 0) 0) > g c 1 ^1 u rH X> 73 MH 0 0 0) u g 4J rH 4-> TJ c o 2 (1) II (U g 4.4 0 g o •rl § CQ 0) 4-) g u c CO •rl u a rH d) 4-) o ft 0) CO rH u 3 Tl (D (1) o CO 0 4-> ^4 g (fl 0) 4-) 4J <-l u g c o o u •rl 0 0) (0 a) 4J u

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152 0 20 40 60 80 100 120 140 Hours post dose Figure 4-2. Natural log transformed fecal ytterbium concentrations in heifers given ad libitum access to bermudagrass hay and fed corn at .67% BW. Each line represents one animal. Control: ; Bambermycins : -.

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153 Hours post dose Figure 4-3. Natural log transformed fecal ytterbium concentrations in heifers given ad libitxam access to bermudagrass hay and fed molasses at .54% BW. Each line represents one animal fed control or bambermycins supplement.

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154 biological processes during digestion and passage (Faichney, 1986; Mertens, 1993; Van Soest, 1994). The slow ageindependent compartment appears confined to preduodenal sites (rumen) while the fast age-dependent compartment seems to reside both in preduodenal (about 60%) and postduodenal segments of the gastrointestinal tract (Pond et al., 1988; Huhtanen and Kukkonen, 1995). However, this partition may depend upon the type of particle and (or) level of intake, as Poore et al. (1991) found that with marked corn in the lactating dairy cow most of the slow and fast compartment occurred in the rumen. The fast compartment also was the compartment that varied the most depending on the model used to fit the data, suggesting that partitioning of total retention time into small compartments may be difficult (Lalles et al., 1991). Even though the G2G1 model was selected, this model was not the best for all animals. If the best model for each animal were selected, all GlGl to G4G1 models would had been selected for a particular animal by period combination. Plots of log transformed Yb fecal concentration vs time after dose are presented in Figures 4-2 and 4-3. Data from corn supplemented heifers appear more variable than that from molasses supplemented heifers. Part of this higher variation was likely due to effects of bambermycins on digestion kinetics in corn but not in molasses.

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155 Marked fiber passed 34% faster (P = .065) out of the riimen in heifers fed corn than those fed molasses supplements (non-linear model) When the PR was estimated by the LN model, there was an interaction of supplement type by bambermycins (P = .075). Passage rate in CC was higher (P < .05) than all other treatments. Use of rare earth as markers has been criticized because of possible marker migration (Faichney, 1986; Combs et al., 1992). Moore et al. (1986) found that 6 to 23% of Dy or Yb dissociated from marked feeds that were incubated in vitro for 24 h, but only .3 to 1.4% of the rate earth markers migrated to unmarked feed, which suggested that dissociated rare earth would pass with the small particles and liquid fraction of the digesta. One possibility is that marker may have partially dissociated from the Yb-marked fiber to enter the more rapidly flowing liquid portion of the digesta. Moore et al. (1992) reported that in sheep given fescue hay or commercial pelleted diets, fluid PR were 8.7 and 8.9%/h, respectively, which were higher than the particulate PR (4.26 and 6.06%/h for Yb-marked hay and pellets) We did not measure fluid PR in this experiment. Predicted fluid PR using the equation reported by Owens and Goetsch (1986) were 8.97 and 9.52%/h for corn and molasses, respectively, which is almost double the PR observed for Ybmarked hay. Another consideration is the marker concentration to avoid saturation of binding capacity of

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feedstuffs (Teeter et al., 1984). Marker concentration in our fiber (1.1% Yb) was similar to 1.7 and 2% reported by Moore et al (1992), but higher than the Yb-marked fiber (masticated stem, .46%) reported by Luginbuhl et al., (1994) A third consideration is the dosing procedure. In experiments conducted by Moore et al. (1992) the marked fiber was dosed in gelatine capsules or placed in closed paper bags in the dorsal cranial sac of the rumen (Luginbuhl et al., 1994). We fed the marker already mixed with corn meal, which would subject the marked fiber to ingestive chewing, salivation and more mixing before arriving at the reticulorumen. The marker mixed with heavy corn particles may have been deposited in close proximity of the reticuloomasal orifice. In this position, it is possible that part of the marked material directly exited the rumen. This possibility would be greater when marked material is dosed at the beginning of the meal. Pond et al. (1989) reported PR of 5.96 and 3.32%/h for Yb-marked NDF from bermudagrass hay when the fiber was dosed at the beginning and at the end of a meal, respectively. Taken all those considerations together, it appears that the Yb-marked fiber may not have dissociated in significant proportion, and the PR observed is consistent with marker administered at the beginning of the meal. The increase in PR observed in corn supplements (5.7%/h) was

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157 higher (no statistics) than PR from pre-trial period (3.26% h) which is consistent with numerical increments observed when a basal hay or pasture diet was supplemented with a small level of grain (Pordomingo et al, 1991; Fredrickson et al., 1993). In pre-trial period (hay alone) the PR estimated by the LN model was 4.03%/h. Reported PR estimated using the LN model for bermudagrass hay and hay plus corn at comparable levels were (%/h) : 3.89 vs 4.26 (Galloway et al., 1993a), 4.18 vs 4.80 (Galloway et al., 1993b), 3.51 vs 3.80 (Hardin et al., 1989). These PR are similar to our estimates for hay alone and hay plus corn using the LN model. With this model heifers fed corn had higher (P = .006) ruminal PR, and consequently lower (P = .003) RMRT than those fed molasses. The interaction (P < .09) observed for PR and RMRT was due to lower effect of bambenrrycins in molasses than in corn supplements (Table 4-6) The discussion on PR of hay particles when hay is fed alone would also apply to PR of hay particles when hay was supplemented with molasses. This suggestion is based on a low associative effect (higher NDF digestibility with molasses) high rate of sugar fermentation, and intake of a small amount of molasses distributed throughout the day. Very little OM from the molasses supplement would exit the rumen, except for a small amount (about 13 0 g, assuming 40% degraded in the rumen) of undegraded corn gluten meal.

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158 There was a tendency (P = .13) for higher FMRT in corn supplements, which would be consistent with more rumination with molasses supplements. If a total ruminal location is accepted for this compartment, then it appears that mixing, rumination and fermentation processes were more efficient in releasing small particles to the ageindependent pool in molasses-supplemented diet. Higher hay intake with molasses would be consistent with more rumination. Lower associative effects on ruminal digestion may also explain lower retention time in this compartment. Another possibility is the existence of other mixing, age-dependent site acting postruminally in corn supplemented diets. Passage of undigested fiber (reduced NDF digestibility with corn) and possibly undigested corn matter may form a mixing pool in the abomasum, cecum or colon. Pond et al. (1988) suggested that about 40% of the fast compartment may be at the postduodenal site. Excretion curves of marker were more variable with corn supplements (Figures 4-2 and 4-3) suggesting perhaps more interactions between hay and corn particles Ruminal retention time was higher (P = .049) in heifers fed molasses, resulting also in higher (P = .028) TMRT. Partitioning of TMRT was also different with corn and molasses supplements. Marked hay particles in corn supplemented diets tended to have longer FMRT, slower SMRT and faster TD. In molasses, particles tended to have shorter

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159 FMRT and faster SMRT, Total MRT (43 to 46 h) was consistent with TMRT reported by Pond et al. (1989) for marked fiber dosed at the beginning of the meal (48 h) The TD, however, was considerably longer (22 h) than the TD in our experiment (10 to 11 h) Luginbuhl et al. (1994) reported longer TMRT, 57 to 80 h, depending on the digesta fraction marked, for steers fed at 99% of ad libitiim. Estimation of ruminal MRT (SMRT) were similar for non-linear and LN models. Total fill was not affected (P = .45) by supplement type. The partitioning of undigested DM fill into fast and slow compartments is related to FMRT and SMRT. There was a trend (P = .11) for .11 % BW lower fill in the fast compartment and numerical (P = .16) .16% BW higher fill in the slow compartment with molasses, probably reflecting the higher hay intake. A large pool of small particle size which would qualify for exiting the rumen exists in forage-fed animals. The rate of comminution does not appear to be the rate-limiting step for exiting the rumen (Kennedy and Doyle, 1993) The tendency for higher fill in the fast compartment with corn supplement may reflect the already discussed less efficient flow of hay particles from the age-dependent to the ageindependent compartment in the riimen (ruminal raft to small particle pool) and the possible existence of postrimiinal mixing pool. Bamberm/cins did not affect (P > .39) digesta kinetics estimated by the non-linear model. There was an interaction

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160 (P = .09) for riominal PR and ruminal MRT estimated by LN model. The ruminal PR was lower (P < .05) in CB than in CC and similar in MC and MB. Numerical differences in the same direction were observed with the non-linear model. The resulting ruminal MRT with the LN model was 11% higher (P < .05) for CB than for CC, while MB was only 3.4% (numerical) higher than MC. The statistically significant differences detected by the LN model may not be biologically significant because they are numerically small and were not translated into effects on digestion (Tables 4-3 and 4-4) Early et al. (1996) reported that indigestible ADF fill and passage were not affected by bambermycins Other reports on the effect of bambermycins on digesta kinetics could not be found. Inspection of Table 4-6 suggests that differences produced by bambermycins appears greater in corn than in molasses and the same trend was observed for intake. Because molasses intake was lower than corn, it is not possible to know if the trends observed were related to supplement type, supplementation level or both. Other feed additives are active in digesta kinetics. Monensin depressed the PR by 14 to 21 % (Ellis et al., 1984) probably through effects on rumination and ruminal motility (Deswysen et al., 1987). Monensin also increased fill and retention time, and these changes were associated with decreased forage intake and increased digestibility (Ellis

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161 et al., 1984). These effects were also dependent on forage quality. Voluntary hay intake and gain were similar in animals receiving CC or MC treatment in the performance experiment (Chapter III) The higher hay intake observed in this experiment can be explained by lower molasses intake. Heifers fed molasses had higher NDF digestibilities and a longer mean retention time, which suggests less associative effect compared to heifers fed corn supplements. Higher hay intake was not associated with higher nominal passage rate of undigested hay DM. Bambermycins increased hay intake in Year 1 (.14% of BW) and had no effect in Year 2 (Chapter III) Bambermycins increased gain in both Year 1 and 2, but apparently through different mechanisms. The effect of bamberitYcins on gain tended to be higher in corn than in molasses supplements. Bambermycins produced small increases (.08% of BW) of total DM intake and tended to increase hay DM intake in heifers given ad libitum access to hay in this experiment. However bambermycins did not affect total tract digestibility. These minor effects of bambermycins on digesta kinetics may not have biological significance and they do not explain the trend for higher intake or the increased gain.

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CHAPTER V EFFECTS OF BAMBERMYCINS FED IN CORN OR MOLASSES SUPPLEMENTS ON RUMEN FUNCTION Tnfrndurhion Antibiotic feed additives may increase animal performance through several mechanisms. Monensin changes the pattern of ruminal fermentation favoring propionate production, decreases deamination, ruminal motility, and feed intake (Bergen and Bates, 1984) The mechanism of action of bambermycins has received less research attention and contradictory information exists on its effects on digestive function (Chapter II) Bambermycins increased gain in the performance experiment (Chapter III) This effects appeared associated to increased hay intake in Year 1 and to increased efficiency of feed utilization in Year 2. This experiment was designed to evaluate the effects of bambermycins on ruminal function when used with molasses and corn supplements, and to document characteristics of digestion of diets fed in the previous experiments. 162

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Mafpria] ^ and Methods 163 This study was conducted at the University of Florida Nutrition Laboratory in Gainesville, from September 27 to December 19, 1995. Ex perimental design. A balanced 4x4 Latin square design with a factorial arrangement of treatments was used. Treatments were: Su pplement typ e Bambermy nins level Code Corn-urea 0 mg CC Corn-urea 3 0 mg CB Molasses slurry 0 mg MC Molasses slurry 3 0 mg MB Each experimental period consisted of 21 d, with 13 d for adaptation to the experimental diet, and 8 d for sample collection. Initial assignment of animals to treatments and pens was at random. Animals Four mature crossbred steers, weighing 630 kg (average shrunk BW) with ruminal and proximal duodenal Tcannulae were used. Surgical fitting of cannulae was performed 20 mo before conduct of this experiment. Steers were dewormed and deloused before the beginning of the experiment and allotted to individual pens, with ad libitum access to water and mineral mix containing 17.2 to 2 0.6% salt, 17.2 to 20.6% Ca, 9% P, 1% Fe, .2% Mn, .01% I, .01% Co, .2% Mg, .12% F, 1,500 ppm Cu, 20 ppm Se, and 4,000 ppm Zn.

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Dipf H anri fppfiina nrnrpdnres Diet formulation was described in Chapter IV. Feed intake and dietary proportions of bermudagrass f rynndon dartvlon ) hay and concentrates are presented in Table 5-1. Feed allocated was restricted to maintenance level and quantities of hay and supplement delivered were designed to match hay to supplement proportions of diets fed in the performance experiment (Chapter III) The molasses slurry supplement was delivered to provide the same amount of TDN as the corn supplement. The 5% lower calculated TDN intake from molasses is based on actual intakes and was due to lower molasses consumption (20%) by one animal and to lower DM concentration of molasses than expected. Except for the noted refusal, all animals ate all the hay and supplement offered. Hay was delivered at 0800 (60% of allowance) and at 2000. Supplements were delivered after complete consumption of the first allotment of hay (at 0830). Feed sampling and analysis. Procedures described in Chapter IV were followed, except that no orts or waste collection was necessary. Hay sample sizes were 200 and 150 g for the morning and evening feedings, respectively. Digesta Kinetics Ruminal fluid and fiber particle kinetics were estimated with cobalt-EDTA and Yb-marked fiber as external markers

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Table 5-1. Steer feed intake for each supplement Item Corn Molasses Intake DM, kg OM, kg 8.00 7.54 8.00 7.26 Intake, % of Hay Supplement total DM 65.6 34.4 64.7 35.3 Intake, % of Hay Supplement total TDN^ 54. 0 46.0 56.5 43.5 Calculated from IVOMD for hay (J.E. Moore and W.E. Kunkle, personal coramunication) and from table values for supplements (NRC, 1984) Ruminal fluid kinetics. Cobalt-EDTA was prepared in two batches according to the technique described by Uden et al.(1980). Recovered Co-EDTA complex was dried at 90 C in a forced air oven and stored until used. On d 14, 60 mL CoEDTA solution containing 2 g Co was placed under the ruminal mat (cranial, middle and caudal locations within the rumen) immediately before hay feeding (0800), using a 100 mL syringe fitted with polypropylene tubing. Ruminal fluid samples (approximately 500 mL in each collection) were collected using a core sampler described by Firkins et al. (1986) from the anterior, middle, and posterior regions of the rumen at 2, 4, 6, 8, 12, 18, 24, and 36 h after dosing. Riominal content samples were strained through four layers of cheesecloth into a plastic collection bucket. Riiminal fluid was acidified (5 mL of 20% H2SO4 per 100 mL of

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166 fluid; Streeter et al., 1990), transferred to bottles, and frozen at -20 C until analysis. Prior to analysis, ruminal fluid samples were thawed, centrifuged (10,000 x g, 15 min) and filtered (Whatman no. 541) Cobalt concentration of the filtrate was determined by atomic absorption spectrophotometry (Model 5000 series X03, Perkin-Elmer, Norwalk, CT) Liquid turnover was estimated by regressing the natural log of ruminal cobalt concentration vs time (Grovum and Williams, 1973). Ruminal fluid passage rate (FPR, %/h) was the absolute value of the slope, and riominal fluid volume (L) was estimated by dividing the amount of cobalt dosed by the antilog of the intercept (zero time) The product of ruminal volume x FPR x 24 h was the ruminal fluid outflow (L/d) Mean retention time (MRT) expressed in hours was the inverse of FPR. Ruminal particle kinetics Procedures described in Chapter IV for Yb-marked fiber preparation, marker analysis and model fitting for the Yb marker excretion curve were followed, with the following exceptions. Marked fiber was administered on d 14 immediately before Co-EDTA dosage. Average dose was 173 g of marked fiber (1.9 g Yb/an) the total dose was split and enclosed in two paper bags, introduced through the ruminal cannula and mixed with the ruminal raft at the anterior and posterior regions of the rumen. Approximately 250 mL of duodenal digesta were collected through the duodenal cannula, discarding the

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initial plug, in plastic bags (Whirl-Pak, Nasco, Fort Atkinson, WI) at 4, 8, 12, 16, 20, 24, 32, 40, 48, 60, 72 and 84 h after dosing. After collection, bags were closed and stored frozen (-20 C) Before analysis, frozen samples were transfer to flat containers and the whole sample was lyophilized. Marker concentration curves were fitted to agedependent, age-independent, twocompartment model (GlGl to G4G1) Because the ascending part of the excretion curves was slow, the G4G1 model best fit the data and this model was used for all animals in all periods. Charactf ^ri sties nf Rnminal Fluid Ruminal pH Ruminal fluid pH was measured with a portable pH meter (Corning M90, Corning, Inc. NY) in the same samples obtained for Co analysis immediately after filtration in collection buckets. Rnm-inal volatile fat ty acids, lactic acid, and ammonia. Samples obtained for Co analysis (2 to 24 h after dosing) were frozen at -20 C. Samples were thawed, centrifuged, and supernate filtered through .45 //m microcel filters (Gelman Sciences, Ann Arbor, MI) Volatile fatty acids were analyzed by gas chromatography (Perkin Elmer AutoSystem XL, Norwalk, CT) using a capillary column (Supelco, 1990a) lactic acid was analyzed by gas chromatography (Perkin Elmer Sigma 3B, Norwalk, CT) using a packed column (Supelco, 1990b) Ammonia concentration in ruminal fluid, prepared as described for

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168 VFA, was determined by automated colorimetric technique (Technicon, 1978) Rnniinal sodium and potassium In the same samples (2 to 24 h after dosing) prepared for VFA analysis, Na and K were determined by atomic absorption spectrophotometry (Model 5000 series X03, Perkin-Elmer, Norwalk, CT) following procedures outlined by Pick et al. (1979). Riim-inal and Total Tract Digestion Digestion was estimated by in situ degradation and using Cr as an external marker to estimate duodenal and fecal DM output. In situ The nylon bag technique was used to determine lag time, rate and extent of disappearance of DM and CP from corn gluten meal (CGM) in the rumen. Nylon bags of 10 x 21 cm (Ankon Co., NY) with an average pore size of 30 x 70 //m were used. The open side of bags was closed using a rubber stopper (no. 8) which was secured with two rubber bands (no. 18) Five grams of CGM (as fed basis) was weighed directly into one nylon bag. Bags were placed inside a polyester bag (38 cm X 45 cm) that was attached to the ruminal cannulae through a plastic string. Starting on d 16 at 2400, bags were placed in the rumen in reverse order of the following incubation times: 0, 1, 2, 4, 6, 12, 18, 24, 36, 48 and 60 h. All bags were removed at the same time (d 19 at 1200) and rinsed several times with tap water in a 20-L container until the water appeared clear. Bags were then washed

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169 individually under tap water and dried overnight in a forced air oven at 55 C, air equilibrated and weighed. Concentrate remaining inside the bag was analyzed for DM and N. Percent of DM and N remaining at each incubation time was fitted to the model described by Mertens and Loften (1980) In vivo digestion Gelatin capsules containing 10 g of Cr203 were dosed at 0800 and 2000 during d 13 to 21. Duodenal (300 mL) and fecal grab samples (200 g) were collected at 6-h intervals during days 20 and 21. Therefore, 8 individual samples for each site representing 0300, 0600, 0900, 1200, 1500, 1800, 2100 and 2400 were collected. Duodenal samples were collected as described for Yb and stored frozen at -2 0 C until analyzed. At the end of all four periods, duodenal samples were thawed, transfer to 600mL beakers, thoroughly mixed, and 200 mL from each beaker were composited by animal and period. A 300-mL aliquot of the composited duodenal samples for each animal-period was freeze dried, ground in a lab mill to pass 1-mm screen, and stored in Whirl-Pak bags until analyzed. Duodenal digesta and feces were analyzed for DM, OM, N and NDF using the same techniques described in Chapter III. Chromiiam concentration of duodenal digesta and feces was measured as described by Williams et al.(1962) using atomic absorption spectrophotometry. Fecal samples were placed in a plastic pan and dried in a forced air oven at 55 C. The whole sample for each

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170 animal -period was ground through a l-mm screen using a Wiley mill, and a subsample of 30 g was saved for analysis of Cr, DM, OM, N and NDF. Duodenal and fecal DM output were calculated by the marker ratio technique (Schneider and Flatt, 1975) Digestibility of each component was calculated using their respective dietary, duodenal, and fecal concentrations Nitrogen Flow an d Microbial Efficiency Procedures suggested by Broderick and Merchen (1992) were followed. Purine content was used as an internal bacterial marker. Bacterial rich material Riominal contents were sampled as described by Cecava et al. (1990), and Ludden and Cecava (1995). Riominal contents (approximately 1,000 mL) were collected twice a day from each steer at 3 and 9, and 6 and 12 h after the morning feeding on d 20 and 21, respectively. Samples were collected from anterior, middle and posterior sites within the reticulo-rumen using a core sampler described by Firkins et al. (1986). At each collection, 500 mL of contents and an equal volume of .9% saline were homogenized in a Waring blender set at high speed for 1 min to dislodge particulate-associate bacteria. Blended contents were strained through four layers of cheesecloth, 1% (wt/vol) formaldehyde was added, and 250 mL of filtrate was stored at 4C. After collection was completed (d 21), samples (homogenized fluid + small particles) were

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171 composited by animal, centrifuged at 500 x g for 20 min at 5C, pellet discarded, and supernate centrifuged at 500 x g for 10 min. Clarified supernate was centrifuged 27,000 x g for 20 min, washed with saline solution, and final microbial-rich pellet collected, and stored at -20 C until freeze dried. Dry material was ground in a Wiley mill to pass 1-mm screen. Analysis In addition to analysis described above, a portion of the wet duodenal digesta (100 mL) was centrifuged (25,000 X g for 20 min at 5 C) and the supernatant analyzed for ammonia N, as described for rumen fluid. Purine content of the bacterial-rich fraction and duodenal digesta were analyzed according to Zinn and Owens (1982, 1986), as modified by Ushida et al. (1985) using RNA Type IV from torula yeast (Sigma Chemical Co., St Louis, MO) as a standard. The bacteria-rich fraction and dry duodenal digesta were analyzed for N, using the same technique described in Chapter III. Calculations Duodenal flow of DM (g/d) was equal to amount of Cr dosed (g) divided by the Cr (g/g DM) concentration in duodenal digesta. Organic matter, NDF, and N flow were calculated by multiplying DM flow by the concentration of each component in duodenal DM. Microbial N (MBN) flow at the duodenum (g/d) was estimated for each steer and period combination by dividing purine :N ratio of duodenal digesta by the purine :N ratio of the bacterial-rich

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172 fraction and multiplying this quotient by the total duodenal N flow for each individual observation. The remaining N was assumed to contain undegraded N of feed origin, ammonia N, and endogenous N. Duodenal ammonia N flow was estimated by dividing the ammonia concentration in duodenal fluid by duodenal DM and multiplying this quotient by the duodenal DM flow. Duodenal non-ammonia N flow (NAN, g/d) was estimated as the difference between total duodenal N and ammonia N flow. The non-ammonia, non-microbial N flow (NANMN, g/d) represents the feed N plus endogenous N flow and was estimated as the difference between the NAN and MBN flow. Feed N flow was the difference between NANMN and endogenous N flow. A value of 4 g N/kg of DM flow to duodenum was assumed for endogenous N (Taminga et al., 1979) Microbial efficiency was expressed as g of microbial N per 100 g of OM apparently and truly digested in the rumen. True OM digested in the rumen was estimated by the difference between the OM apparently digested and the OM of microbial origin. Bacterial CP was also expressed as g CP/100 g of OM digested in the total tract. Statistical analysis Data were analyzed as a 4 x 4 Latin square design using the GLM procedure of SAS (1987) Sums of squares were separated into effects of steer, period, supplement type, feed additive and the interaction of supplement type by feed additive. Ruminal data sampled

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173 over time were analyzed as a split-plot over time with the Greenhouse-Geiser correction of degrees of freedom for all F-tests involving time effects, using the REPEATED statement of the SAS program (Littel, 1989) If no interaction between treatment and time was present, then means averaged over time are reported. When an interaction of supplement type by feed additive was present, treatment means were separated using the LSD with alpha = .05 (SAS, 1987). Results and Discussion Composition of feeds averaged over the four periods is presented in Table 5-2. Molasses used was the same used in the experiment described in Chapter IV. Molasses had lower DM (72 vs 77.5%) and phosphorus concentrations (.34 vs .88 to .74%), and had higher pH (5.14 vs 4.51 to 4.71) than molasses used in the performance experiment described in Chapter III. Bermudagrass hay composition was similar among periods, except for a lower IVOMD in period 4. Average IVOMD and CP for periods 1 through 4 were: 50 and 10.8; 48 and 10.9; 49 and 9.2 and 44 and 10.3%, respectively. Patterns of supplement consumption were similar for both molasses and corn supplements. By 2 h after offering, 70 to 80% of supplements were consumed. Restricted level of feeding may be responsible for this pattern of consumption because the molasses supplement was consumed at a slower rate than the corn supplement when animals were given ad

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174 libitum access to hay (Chapter IV) In this experiment both supplements were consumed at the desired levels and had similar patterns of consumption. Results from this experiment, therefore, may not be completely applicable to typical production situations where a different pattern of molasses consumption would be expected. Table 5-2. Composition of supplements and hay fed in metabolism trial Corn Bermuda Item Corn Mix Molasses^ Gluten Hay DM, % 86.5 72.0 89.0 88.8 IVOMD 47.7 Concentration in DM, % OM 95.0 82 0 98.4 94.3 CP 15.6 9.8 78.7 10.3 tdn" 84.4 72.0 89.0 52.1 NDF 9.3 5.2 76.4 ADF 39.0 Lignin 4.59 Ca .66 .61 .03 .33 P 66 .34 .44 .20 Mg .26 .53 .06 .40 K .43 3.76 .15 1.19 Na .01 .12 .03 .02 PH 5.14 TDN:CP ratio 5.41 7.35 1.13 5.06 Blackstrap molasses not less than 40% invert sugars, fortified with phosphoric acid and 25,000 U.S. P. units vit A, 33,000 U.S. P. units vit D, and 22 Int. units vit E per kg, and .0005% Cu, .00001% Co, .02% Fe, .001% Mn, .0025% Zn, and .00007% I. Sulfur content no less than 1%, as fed basis. Calculated from table values for supplements (NRC, 1984), and from IVOMD for hay (J.E. Moore and W.E. Kunkle, personal communication).

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175 Diaesta Kinetics Fluid and hay particle digesta kinetics are presented in Table 5-3 (main effect means) and Table 5-4 (treatment means) Fluid kinetics Supplement type and bambermycins did not affect (P > .2) FPR, fluid MRT, and fluid outflow. Observed FPR were consistent with predicted FPR (6.4%/h) from equation presented by Owens and Goetsch (1986) Steers fed bermudagrass hay and corn supplement (1.35% BW total DM intake) had FPR of 6.4%/h (Galloway et al., 1993b). A similar value (6.7%/h) was reported by Galloway et al. (1993a) in steers fed at a higher level (2.17% BW DM total intake) Molasses fed at 1% BW or higher reduced FPR (Rowe et al. 1979a) Steers fed molasses had 8.8% higher ruminal fluid volume expressed in L (P = .049) or as percent of BW (P = .054) than those fed corn supplements, which is consistent with ruminal volume reported in animals on high molasses diets. Rimiinal fluid volume increased from 32 L when .75% BW forage was fed with molasses to 75 L when the forage was removed from the diet (Rowe et al., 1979a). In cattle, ruminal fluid volume averaged 15 to 21% BW (Owens and Goetsch, 1988). Predicted ruminal volume (14.5% BW) from their equation based on feed intake is close to estimated volumes (overall mean 13% BW) They also reported that ruminal fluid volume and FPR are negatively related, tending

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176 to produce consistent estimates of ruminal fluid outflow. This probably reflects the physiological control of outflow or may be due to errors in marker use that bias volume and FPR estimates in opposite directions (Owens and Goetsch, 1986) Animals were observed drinking water (not measured) during and after molasses consumption, which may explain part of the higher volume. Possibly the high osmolarity of molasses induced water consumption to keep riominal fluid tonicity within the physiological range (Carter and Grovum, 1990) Under production situations, the high tonicity and high rate of fermentation of molasses may explain frequent consxamption of small meals. Bamberrt^cins did not affect (P = .17) ruminal fluid volume as percent of BW. Earley et al. (1996) reported lower liquid fill and higher FPR in steers fed alfalfa-grass hay diets supplemented with bambermycins compared to steers supplemented with monensin and lasalocid. Particle kinetics Steers fed corn tended (P = .14) to have increased (8.82 vs 6.2 0 %/h) hay PR from the fast compartment than those fed molasses, which may imply higher mixing, ruminal motility and rumination. Higher fluid volume and lower numerical FPR and hay particle PR would suggest lower ruminal motility with molasses. The opposite trend was observed with ad libitiom intake (Chapter IV) The difference, however, may be explained by higher hay intake

PAGE 184

177 u Q) 4-1 4-1 W i-l o 4-1 0) (0 > 0^ 0) > a CO CO w CO (0 CD o 2: CO 0) CO CO (0 rH o S o u rH ;Q (C3 •H ^H (d > o m o og rH CN CN ro o m rn CO o cr\ r~ m m rH (N rH CO rH o CT\ U) CN ^ x) in c-~ m rH 00 rH 00 CN CO ro in rn o o CN rH ^ 00 rH o ro U3 rH >x> in rH m in CN o r~>^ rH U3 ch ro 00 in in >.o cN in in 00 *x) ro cr\ rH rH CN CTl VX) VO O rH O O V£) o m o m o o m CO CN CN o vo in CN o ^.D Og CN CN in 00 m 00 ro (N ^3 -H rH 0) 4J (0 n3 CO CO 03 04 Pi T! J c>f> a o 4-1 o o > > 4J CJ (0 0) ft (U 0) CU ft-d Q) C rH I I c Q) 0) -H < < 3 Pi rH CTl VO -^Jt VO CN ro rH r~ VO 00 in ro rH ro o^ o en in cn rH rH I> rH in ro 1 ^— r^ CN 00 t~ 00 o ^ CN O O in rH rcn 00 rH ro f — i fO 00 ro 1 — 1 in CT> in in in 00 r~r~• • CTi CTi ro cn CN rg o CN ro ro ro o o cr\ cn ro 00 in CN in t~~ 00 rH CN ^ rH rH ^ ^ o ^ O rH ro rH rH o o o o ro in rH ro 00 ro c~CTl rH CTl rCT\ rin rH VO • • • 00 rH rH rH rH CN ro in ro T — 1 ^ 1 I N IX) o en in U 1 ^ N L ro 00 CN rH rH CN ro rH r~. ^ o ro V£> (N o 00 in CN 00 ro rH cn in rH rH CN LO o r^ rH o cr\ 1 U 1 00 ^ rH VO • • • CN VO ro rH rH rH CN ro VO DM, (U g OJ JJ Tl -U -t-l Q) n JJ g g to g g C -!-> !-> 4-1 CQ (U j-> jj O ^ >H Cn >H >H •H (fl rtJ >i -H 03 03 U ft ft (0 C g g rH c o 4J 5 03 to o g to O 4J C IC5 rH H O O r-i 03 rH O (0 fa CO Eh E-" rH fa CO H 0) Pi Q fa V o Q) t4_| (4_| n Q) rH ams. •H > rH •H rH 4J •rl •H T3 T3 73 (C r~ CTl an 73 IK rH 0) > 0) -rl 01 •H g 0 II 73 <0 u 73 •H o <; (0 rH -73 rH •rl rH m 0) s (V 01 01 fH 73 M >*-l Q> 73 0 V) 73 C g (C >i 0 m rH ja e 0 g J-' 0) c (0 > c H Q) 0) 0 •H 0 e c u rH Q) •H o rH g u 0< 1 0 O Oi c rH u O 3 0 Q) MH m c 73 0) 0 ^ MH g g g O 0 0 •H 0) <4H 4J c 0) 0) •9 6 (B rH 43 a (0 PQ CO CO 4J OT -H c u m S •H nJ m R u CU a05 J3

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178 o ro O CN og X) ^ ^ 00 ro r~ rH rr00 v£) in rH 00 ro rH 00 rH CN ^ U3 rH O ro r~ CN rH ro t> rH in rH o in 00 o r~ ro ro ro rH 00 rH 0) dp c o •H c (U 4J 0) o ro v£) rH VO in rH ro in CN O VJD rH VD (T\ ro 00 in in U5 rg in rin 00 r~ vo ro CTl rH rH -sil CN o ro in (7^ CN l> CN rH rH ro 00 >^ CN rH rH 00 CN CN CN >^ ro rs ro 00 rH 00 CN ro rCN CN O rH 00 VD ^ CTi CN CN 4J 03 >H JJ JJ (0 O T3 S > a ^H \ CQ o (0 C M-i J (x; -d 0) J dp c aoi 0) Q) a< oj 0) 0) 0) a rH -H (0 rH CTl CT\ CN CN ro rH CM x> rH 00 ro oj ro rH rH rH rg ro in rH O in VO 00 00 00 in O in • in o CN 00 rH rH CN ro in ^ a\ en 00 ro in ro rH ro ro rg U5 CTV CN ro in rH rH rH f-l o H JJ c cu JJ 0) JJ c Q) e JJ u g e -H O O QJ U U g o o JJ m (0 t, Q) 2 (0 a •H CQ dP 2 Q T3 Q) JJ IS CO CQ 0) Di dP -H T3 c: JJ d 1=1 JJ o O rH rH JJ 0) (1) g g JJ >H (C (0 o o u u rH JJ ^ ( CO O JJ (t3 rH O CO E-i C! •rt u I* O 0) C O c II 0) 01 > o u JJ C JJ T3 0) < (0 0) -H rH JJ o •-< g II II i< (0 o 0) •M w m a (C •H rH u o a u o -u u o 0) II ^ OQ 0) u U CO CO S (0 -rl G cn fO iH O (0 £ u (0 01 c 01 -H Q C T3 ^ O >-l u *J 10 0) 0) r; JJ -H (tJ rH U U o u 0) "H 0) o nS <4-i 01 01 0) (0 4J 0< (C 0) -H (8 01 W "J ns 3 a< o: o:

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179 by animals fed molasses in that experiment. Estimates of PR from the slow compartment (riimen) by LN and G4G1 models have high standard errors. Possible explanations are that sampling from duodenum may be subjected to error because of unrepresentative sampling (Faichney, 1975), the level of intake was low enough to depress PR, or the time of sampling after dose may have been too short (84 h) Using the same steers and marker, but giving steers ad libitum access to hay, Garces-Yepez (1995) sampled for only 72 h after dosing and detected differences among treatments. He reported PR of Yb-labeled particles of 8.5%/h from the fast and 3.3%/h from the slow compartments for steers fed hay plus corn (35% of diet) which are similar to the PR estimates for corn supplemented diets in this trial. There was a trend for supplement type by feed additive interaction (P = .13) for slow compartment particle MRT and total MRT (P = .11). Steers supplemented with molasses tended (P = .12) to have longer (55.0 vs 44.5 h) ruminal particle MRT compared to steers supplemented with corn estimated with the LN model, which may be biologically significant. However the ruminal NDF digestibility (Table 514) was similar with corn and molasses diets. Ruminal DM output was not affected (P > .5) by supplement or feed additive and numerical values are similar to values reported by Garces-Yepez (1995)

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180 An interaction of supplement type by feed additive was detected for fill of the slow compartment (P = .095). Fill of the slow compartment was consistent with the ruminal fluid volume, with an estimated 8 to 10% of undigested hay DM in rumen content. Total hay DM fill was higher (P = .019) in steers fed molasses than the ones fed corn. The increased fill of hay DM and fluid volume, and the tendency for longer MRT with molasses supplementation may be the result of less ruminal motility. A more sluggish vago-vagal reflex by lower afferent stimulus from a diet producing lower tactile stimulation of mechanoreceptors has been suggested (Rukebusch, 1988) High osmolarity of ruminal fluid may also depress ruminal motility (Grovum, 1986 ; Carter and Groviim, 1990) One or both mechanisms may have operated in animals fed molasses. In the experiment with heifers (Chapter IV), total fill was not affected and the model partitioned total fill differently. Heifers fed corn tended to have higher fill in the fast compartment and lower fill in the slow compartment than heifers fed molasses. However, differences found in that experiment may be explained by higher hay (ad libitum) and lower molasses (20% of the diet) intake. Combined data from this experiment and the one described in Chapter IV do not suggest a biologically significant effect of bambermycins on digesta kinetics. Data also suggest minimal differences between energy sources. The possible confounding of level of supplement intake and

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181 supplement type (Chapter IV) and the restricted level of feeding imposed in this experiment limit extrapolation to production situations. Characteristics of Ruminal Fluid Treatment means averaged over time are presented in Table 5-5. Because there were supplement type by time of sampling interaction for several variables, means are also presented by time in Tables 5-6 and 5-7. Time postfeeding in the following discussion refers to time after the morning feeding. Animals were fed a second meal of hay alone 12 h after the morning feeding. Ruminal p H. Ruminal pH exhibited a supplement by time interaction (P = .0001), therefore treatment means are presented by time in Table 5-6. At 4 and 6 h after feeding, steers fed molasses had lower (P < .02) pH than those fed corn. Immediately before the morning feeding (24 h) at 2 and 12 h postfeeding, pH was lower (P < .08) in steers fed corn. Lowest pH values were at 6 and 12 h postfeeding molasses and corn, respectively. Soluble sugars are fermented at a faster rate than starch, which explains the faster depression of ruminal pH after feeding molasses. Galloway et al. (1993a) reported pH from 5.95 to 6.27 in the ruminal fluid of steers fed bermudagrass hay and ground corn (30% of the diet, 2.17% BW total intake), with lowest pH recorded 8 to 14 h postfeeding. They reported total corn

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182 (0 Q) (0 IT) I in rH (0 E-" u Q) 4-1 W o rH > ID to CO w CO u u u rH X! (0 -H U (0 > 1 1 — 1 o o ro o o o rH • • • 00 o 00 o \J 1 m rH CO rH a\ m >X) •=d< >X) rH rH (0 Pi o o :z: I m rH <^ rH O O in CN O rH rH rH O O in O O i> o o 00 o rH r-~ CN o in rrH rH U) rO CN rH r~CN (N in o in in iTi rH cN rr~ OA 00 O us CTi r~ CTv rH o O rH si' =1' rH 00 m CO rH m o CO CO 00 ro o ro r~ o o ro in 00 O CO <~D 00 O O rH rH O rH O rH rH CO rO r~ CO U3 O O O rH O x> o iH rH U5 >X> O CN Cri rH CN CN CN UJ >X) (T5 o m o U5 o CO CO o 1^ CO 00 •=4< rH 1^ o rCN rH in cn t> vo rH VD o rg o rH in rH o rH rH rH rH U c: -H •rl on o o, 0) 0) H o 0) 4J 4J u a rH 4J^ (0 (0 0 \ 4-1 (0 0) >H dJ 5h 13 Sh rH 0) C JJ >l-U 0) Q) 0^ o 0 (0 4J (d rH ^ e nJ M u :i u (0 u u a 0) > c H 0) O -U 0 rH O (0 < U >H 3 CO (0 CO ^ Oj CQ H > H CQ u > < m c H o Iu •i ^ nS ja 0) c o c II 0) (0 (U > o C 4-) O -H o n T3 M (0 01 m 73 m 0) (0 01 rH Cl4 II l< CJ (1) CO to to c m u c • -H 0) (tJ to o •H JJ o nj u -l o 0) 3 rH (0 > >1 u II "O ^^ ^4 •2 i> TS % m 0) I M-l (1) 14-1 73 0) g O 3 0 • to 5 'C3 H 1^
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183 (0 0) e -H •H c 0 CO d CO -H e u JJ em mer ij (0 (D ^ u 4-1 C n3 0 H Q) -U ft (0 >i Sh JJ 0) 4J a (0 d e 0 QJ •H rH ft ft o ft u :i ft CO 0 4-1 4-) 0 0) 4J 4J U (0 0) 4J 4-1 0) 4-1 CJ W (C3 4-) CJ QJ 4-1 4-J W O 4-1 (D > 4J d g 4J (0 0) Eh CO PQ u o u 0) rH H (CJ > VO (N H IT) o CN m 1J3 rH o >x> in in o x) VX) (.D VX) >^ >X> U> r~00 (T\ in in rm CO ro in in U> VD VO >^ *.D VO 00 00 rH o 00 v£i in in in x> ro o ro r~ oo vD 00 in m U5 >X) X) ft d -HCN'^VOOOCNVD'^ S rH rH CN CTi 00 ^ o 00 cn (N o in in X> O CN rH 00 en VD VD 00 CN CN CN CN O in rH CN O CT\ rH 00 rO rH CN CN CN CO in o o CTi rH in rO O CN CN CO 00 CTl CO 00 en vi) ^ r~ CN >^ CO CO CTi CN CTi CO O O CTl CT\ 00 00 rrH U3 cn in cr\ o cr> o CT\ cn cTi in r~ >-D cTi in o cTi CT\ 00 CTV CT\ rH CT m 00 CN in o r~ O xi XI ^ x: ^ O rH o CN o in ^ in CN cTi rH in CO CTi CTi >X> O CN C^ O rH CO l> rH <~D cr\ rCN >x> cTi CO CTi Cr\ 00 CTi CN rH CN CN 00 ro in CN O O CO 00 CTi 00 rH o in r~CTi t~~ CN O O O O CO CO CTi rin CTi ro rH CN ro CN CO O 00 CTl rH >i) O CTi CN in CO CN CN CO ^ in in in CTi O CN ro CN ro U) CN CN CO rH CO 5j< CO ^ in ^ CN CN ro in o in cr\ ro in en in in tji o r~o rH CTi in in ^ en CO en ro n3rN'^>x)cocN>X)'^ 4-) rH rH CN O in in ^ in (d d o •H Oi o u o, mm ^XXXXXiXX (C3 4JCN'^UDC0CN>>O'^ Q) rH rH og u < CO Hi H U u 0) JQ U e O (S 0) c M O 0) c 01 0) (0 4J rH U O 0) 2 II (1) O 0) ^ > 4J -H C 4J O -H U n m nS d) to 01 (0 0) rH Cl. (0 V) U >i c CO u 0 4-> <4-l u o 0 (1) II c 0 PQ 0) •rl o 4J c (J •H (C rH M 0 % 0) M 4J 4-1 4J C C c H 0 0) u e II c r^ u 0. 0 0. CO o D Ui II D) II C u •H u CO O XI

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184 m 0) 4-1 4-1 0 (d a o u H 0) a 4-1 0 w a 4-) U Q) <4-l 14-1 w o 0) :3 rH (d > 4J a (D e 4J fd Q) >H CO CO W CO U rH (d •H (d > fNj 00 ro ^ CTi ^ C >X) rO 00 O CTi rH o r~>^ 00 00 cj^ vD rn IT) m ro U) ^ 00 <~D ro o 00 in CN rH in ^ vo in rH (N 00 00 00 m rH rO m rH 00 m in o 00 in o in o cNj 00 in CN O rH (N CN in CTi (N O x) rrt~~ r~ in en in 05 00 cTi in rH O rc^ >x> rH in o CTi ro CN (N rH ro o r~ c~rin v£> U3 rH m m m CN rH rH CN m i> i> r~ i> ri> O e o o O 4J (d 4-) (U u ^ ^ ^ ^ j:: ^ CN IX) CO (N >X) rH rH CN vo ro CT> rH O CO o m cj^ CO in CN O rH o vo r~ r~ m r~ m o ro CN CN o in CN in rH rH m ro m <.D in CN o rH m ro ro in cTi 00 00 en CN rH ro r00 CTi O CN O 00 rH 'St O O 00 rO O CTv o o o >^ rH in in in CTi O CX) CT\ 00 CT\ CN "-D C7^ O ro r~^ 00 in m 00 >x) in m ^ rH rg cTi ^ CN cri t>x> CTi r~ in in rH rH rH rH rH rH rH 00 CTi CN in U5 O O ro ro in >X) U3 *X) in vx) r~ o U3 c~ro ro in in ro O e o o o 0) iJ -H aCN ^ O rH rH CN u On ro CN 00 00 in 00 CN o CO 00 ro in cr\ o in rH cri CN in 1^ r~x) in cT\ CN ro ^ ro CN o o o > cTi cTi t-~ cr\ in 00 CN O rH in 1^ CN U3 00 C3^ rH rH O rH in CN O o >X) CTi m m in 'S' r~[--v£) ro in in rH in m ro >J3 cr\ O rH rH rH O CTl rr~ CN rH rH m m rH O rH rH rH O O CTl o CN o CN in >^ cr\ CT> cTi cr> cyi CT\ 00 cri in cy\ in ro CTi O O O rH rH O o • o u >,fN'*>X)OOCN>^'* 4- rH rH CN 01 c •H u u d) •9 M B 0 nJ XI 0) c m 0 0) c V) D) <8 4J u o :fe II 0) CQ •iH i-H 0 u > 4-) •rl c 4-> 0 -H O T! Tl 0) (0 0) 0) OT 0) > (C 0) •H I-H b 4J H i II T3 T3 II (8 o T3 s m 0) d) iii ^ •n rH -Q u o 0 a u 3 m a o •H JJ U ( 4J O 4J u o o 0) II >*H CQ (1) • O C -'^ u o u 4J 4J fl fi c I-" O d) U g II Max O ftCO o II II U -H U CO u ^ 6 (1) (0

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185 consumption in less than 20 min. Similarly, Royes (1996) reported fast corn consumption followed by low ruminal pH. Inclusion of urea in the corn supplement may explain the lower rate of consumption. Feeding urea generally increased ruminal pH (Owens and Zinn, 1988) Both rate of consumption and inclusion of urea (2.8% as fed) in corn supplements may explain higher ruminal pH in this experiment. The magnitude and timing of the postprandial drop in ruminal pH in steers fed molasses agrees with other reports (Khalili, 1993; Khalili et al 1993; Osu j i and Khalili, 1994) They observed a somewhat faster increase in pH after the lowest value, probably due to higher forage intake. Animals given ad libitum access to hay may have increased salivary secretion, dilution rate and buffering capacity in the rumen There were no time by feed additive (P = .8) or supplement type by feed additive (P = .5) interactions for ruminal pH. Bambermycins increased (P = .046) ruminal pH (6.63 vs 6.52) in steers fed both corn and molasses supplements. This effect may be partially caused by a lower rate of fermentation because the total VFA concentration was slightly lower with bambermycins. Ruminal pH apparently was measured with less error than total VFA, hence statistical difference was detected only for pH. Reported effects of bambermycins on ruminal pH are variable. Bambennycins increased ruminal pH at the end of wk

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186 4, however by the end of wk 9 there was no effect (Murray et al., 1992). Aitchison et al. (1989a) reported that bambentiycins increased rumen pH (6.94 vs 6.57) in sheep fed at maintenance. However when animals were given ad libitum access to the same diets, the tendency was the same but not statistically significant (Aitchison et al., 1989b). No effect on pH was found in diets supplemented with bambermycins and different sources of sulfur (Murray et al., 1991), or different concentrations of bambermycins (Murray et al. 1990) Under these experimental conditions the increase in pH may not be biologically relevant. Mould and Orskow (1983) suggested that bacterial fermentation of fiber may be depressed when pH is below 6.2, but the pH was always above 6.2 in this experiment. Ruminal volatile fatty acids Total VFA concentrations exhibited a supplement by time interaction (P = .067), therefore treatment means are presented by time in Table 56. The time trends of total rxaminal VFA concentration seems to reflect the different rates of degradability of sugar and starch. The peaks of total VFA in nominal fluid of steers fed corn did not necessarily result in a low pH, probably reflecting salivary buffering with consumption of hay and (or) rumination. Total VFA tended (P = .12) to be higher at 4 h postfeeding in steers fed molasses and it was lower (P = .002) at 16 h postfeeding compared to those fed corn.

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187 There was not a feed additive by time interaction (P = .76) and bamberm/cins did not affect (P = .41) total VFA concentration. Small trends due to supplement type and feed additive were consistent with results from the performance experiment (Chapter III) Previously reported effects of bambermycins on total VFA have been variable. Bambermycins decreased the total VFA concentration (65.3 vs 78.5 mM/L) by the end of wk 4, but there was no effect by the end of wk 9 (Murray et al., 1992) Bambenir/-cins increased total VFA when the diet was supplemented with methionine, but it had no effect on total VFA when the diet was supplemented with other sulfur sources (Murray et al., 1991). Bambermycins did not affect total VFA in lucerne ( Medicago sativa ) -luoin and hay-fishmeal based diets (Murray et al., 1990) or in high quality diet fed at maintenance level (Aitchison et al., 1989a). When sheep where given ad libitum access to these diets, bambermycins decreased total VFA concentration (Aitchison et al,, 1989b). In fattening cattle bambermycins did not affect total VFA (Flachowsky and Richter, 1991; Alert et al., 1993). However, higher total VFA concentration were reported in steers fed a 90% concentrate diet with bambermycins (DelCurto et al., 1996) Acetate molar proportion exhibited a supplement by time interaction (P = .026), therefore treatment means are presented by time in Table 5-7, At 2, 4, and 6 h postfeeding

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188 steers fed corn had higher (P < .025) acetate molar percent than those fed molasses. In the performance experiment (Chapter III), animals fed corn had higher acetate proportion than the those fed molasses. Results are difficult to compare, however, because this experiment demonstrates that individual VFA molar proportions change with time after feeding and feeding time was not controlled with molasses supplements in that experiment. There was not a feed additive by time interaction (P = .5) and bambermycins tended (P = .12) to increase acetate proportion at 2 h postfeeding. Propionate molar proportion also exhibited a supplement by time interaction (P = .003), therefore treatment means are presented by time in Table 5-7. Propionate was higher in steers fed molasses at 2 h (P = .009), 4 h (P = .0004), and 6 h (P = .08) postfeeding than in those fed corn supplements. By 12 h postfeeding, the trend was reversed and animals fed corn tended (P = .11) to have a higher ruminal propionate molar proportion. Propionate molar proportion increased shortly after molasses consumption followed by lower propionate proportion 8 h or more postfeeding. In animals fed corn, propionate showed less variation over sampling times. Because the rates of supplement consumption were similar, this trend reflects the faster rate of sugar fermentation. A rapid fermentation rate usually results in more production of Hj. Excess reducing equivalents combined

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189 with low pH has been reported to increase reduction of pyruvate to lactate and propionate (Owens and Goetsch, 1988) Lactic acid (see below) was present shortly after feeding only in animals fed molasses. There was not a feed additive by time interaction (P = .7) and bambentiycins did not affect (P = .26) propionate molar proportion. Butyrate molar proportion also exhibited a supplement by time interaction (P = .012), therefore treatment means are presented by time in Table 5-7. Steers fed molasses tended to have higher butyrate proportions at 2 h (P = .15), 4 h (P = .14), 6 h (P = .065), and 8 h (P = .12) postfeeding than the steers fed corn. Pre-feeding butyrate proportion, however, was higher (P = .097) in steers fed corn. Molasses fermentation has been reported to have a high butyrate proportion (Marty and Preston, 1970; Pate, 1983). Butyrate was also higher in ruminal fluid of cattle fed molasses in the performance experiment (Chapter III) There was no feed additive by time interaction (P = .49) and bambermycins tended (P = .12) to depress butyrate proportion (averaged across time. Table 5-5). Bambermycins depressed (P < .05) butyrate molar proportion from 12 to 24 h and from 16 to 24 h postfeeding in animals fed corn and molasses, respectively (Table 5-7) Van Nevel and Demeyer (1992) reported that bambermycins decreased butyrate and increased acetate production when the substrate was NDF in

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190 vitro. Murray et al. (1990) also reported a lower butyrate molar proportion in sheep fed banibennycins There was no supplement or feed additive by time interaction (P > .26) for the minor VFA (Table 5-5). Valerate molar proportion exhibited a supplement by feed additive interaction (P = ,074). Bambermycins decreased valerate proportion in animals fed molasses but it did not affect valerate proportion in animals fed corn supplements. Valerate can be formed by carbohydrate or amino acid fermentation. Isobutyrate and isovalerate are derived from the fermentation of the amino acids valine and leucine respectively (Russell, 1984) Isobutyrate and isovalerate molar proportion were affected (P < .06) by time and they will be discussed together. Branched chain VFA (BCVFA) were affected by time (P = .01) and animals fed molasses tended (P = .12) to have lower BCVFA molar proportions. When analyzed by time (data not shown), BCVFA (mol/100 mol) was lower (P < .06) in steers fed molasses at 2 h (1.3 vs 1.9), 6 h (.65 vs 1.65), and 16 h (1.19 vs 1.53) post feeding compared to steers fed corn. Isovalerate molar proportion decreased with sucrose (Huhtanen, 1988; Khalili and Huhtanen, 1991a) and molasses supplementation (Petit and Veira, 1994) They suggested increased utilization of isovalerate for microbial growth. However, BCVFA molar proportion and ruminal ammonia concentration were increased with increasing levels of

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191 soybean meal supplementation (Yang and Russell, 1993b), suggesting increased BCVFA production resulting from ruminal proteolysis and deamination. In the performance experiment (Chapter III), cattle fed molasses had lower BCVFA concentrations in ruminal fluid compared to cattle fed corn supplements. Lower BCVFA may suggest lower feed protein degradation in animals fed molasses. Fiber digesting bacteria may require BCVFA which would be supplied by amino acid degradation (NRC, 1996). Russell et al. (1992) suggested that BCVFA deficiency may occur if highforage diets are low in true protein and non-protein N is used as supplement Bambermycins also affected BCVFA, but the effect was not consistent: it decreased (P = .05) BCVFA proportion at 6 h (.86 vs 1.11 mol/100 mol) and increased BCVFA (P = .03) at 16 h (1.2 vs 1.6 mol/100 mol) after feeding. Bamberwcins tended to increase BCVFA molar proportions in the performance experiment (Chapter III) Bambermycins increased extent of in situ CGM CP digestion after 18 h of incubation (see discussion below. Tables 5-10 and 5-11) Leucine and valine comprise 28% of CP in CGM (Cozzi et al., 1994). The effects of bambermycins on CGM digestion appears consistent with increased BCVFA proportion. Acetate: propionate ratio (C2:C3) also exhibited a supplement by time interaction (P = .003), therefore treatment means are presented by time in Table 5-6. At 2 h

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192 (P = .01), 4 h (P = .0001), and 6 h (P = .05) postfeeding, C2:C3 was lower in ruminal fluid of steers fed molasses; at 12 h after feeding, however, C2:C^ tended (P = .099) to be lower in steers fed corn. In steers fed molasses, acetate and propionate had their lowest and highest values, respectively, at 4 h postfeeding resulting in the lowest CjrCj at this time. This was also coincident with the lowest ruminal pH. There was no feed additive by time interaction (P = .8). Bambermycins did not affect (P > .36) C2:Cj, which is consistent with results from the performance experiment (Chapter III) and other reports (Aitchison et al., 1989b; Murray et al., 1991; ElJack et al., 1986; Fallon et al 1986). However, Earley et al. (1996) reported that bambermycins depressed C2:C3 in cattle fed alfalfa and grass hay. Lactic acid With the exception of one sample, lactic acid was detected only in animals consuming molasses (Table 5-8). Lactic acid concentration was not affected (P = .8) by bambermycins. Lactic acid was only detected in 13% of the samples and it was present as a transient peak shortly after feeding, probably related to high rate of intake of molasses. Whether this peak of lactic acid occurs in production situations is not known because the pattern of molasses consumption may be different. As occurred in the experiment with heifers (Chapter IV) molasses intake tended

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193 to be distributed throughout the day. Some animals, however, tended to eat at faster rates shortly after molasses was delivered, consuming considerable amounts that may have caused accumulation of lactic acid. Table 5-8. Lactic acid concentration (mM) in ruminal fluid of animals fed molasses^ Time after feeding Number of Samples Mean Std Dev Min Max 2 6 14.79 11.50 .89 33.46 4 5 3.66 2.70 1.31 7.94 6 1 .17 8 2 .54 .49 .19 .89 ^ Except for one sample from corn (4 h, .32 mM) lactic acid was not detected in all other samples from corn or molasses fed animals. Presence of lactic acid in molasses, but not in corn supplemented cattle, is consistent with a fast rate of sugar fermentation and has been observed in grass silage diets supplemented with sucrose (Khalili and Huhtanen, 1991a) and with molasses (Moloney et al., 1994). Lactic acid concentration may have been underestimated with our sampling schedule because the highest concentrations have been observed at .5 and 1 h postfeeding (Khalili and Huhtanen, 1991a; Moloney et al., 1994). Lactic acid was higher in ruminal fluid of cattle fed sucrose twice daily than in those given a continuous intraruminal sucrose infusion (Khalili and Huhtanen, 1991a) suggesting that the pattern of molasses consumption may dictate the prevalence of lactic acid production.

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194 Ruminal ammonia Riominal ammonia N concentration was affected (P = .0001) by time after feeding in all treatments (Figure 5-1) In steers fed corn, ammonia N concentration decreased from 26.5 mg/dL at 2 h to 4.4 mg/dL at 12 h post feeding. In animals fed molasses, the maximum value (21.3 mg/dL) was observed at 4 h postfeeding and decreased to 3.3 mg/dL at 12 h postfeeding. The small increase after 12 h postfeeding, observed with both supplements, was associated with hay feeding at 12 h, which should also have increased salivary urea input. Because there was a tendency (P = .17) for a supplement type by time interaction, data were also analyzed by time (data not shown) Ammonia N concentrations were higher (P < .07) at 6 h (14.0 vs 10.8 mg/dL), and lower at 12 h (3.3 vs 4.4 mg/dL) and 16 h (5.5 vs 7.0 mg/dL) postfeeding, and pre-feeding (5.1 vs 8.0 mg/dL) in steers fed molasses than in those fed corn. At 2 h postfeeding ammonia concentration tended (P = .11) to be higher in steers fed corn (27 vs 18 mg/dL) suggesting that the ammonia peak may have been greater with corn. The ammonia peak in steers fed corn may have been underestimated because the first sampling was obtained 2 h postfeeding and after that time only a decreasing trend was observed while in those fed molasses ammonia concentration continued to increase up to 4 h postfeeding (Figure 5-1) There was no feed additive by time interaction (P = .5) Ammonia N concentration averaged over time tended to be ^ I

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195 Figure 5-1. Effect of supplement type on ruminal ammonia nitrogen concentrations, means by time after feeding.

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196 lower in animals fed molasses (P = .18) and in those feed bambermycins (P = .19) (Table 5-5). Intake of N estimated from chemical analysis was 154 and 17 0 g/d for corn and molasses diets, respectively. About 22% of dietary N was provided as non-protein N (urea) in corn, which likely explains the increase of ruminal ammonia N concentration shortly after feeding. Urea N will be hydrolyzed at a fast rate and enter into the ruminal ammonia pool (Owens and Zinn, 1988). Stateler (1993) in his literature review stated that about 60 to 70% of N in molasses is present in relatively simple form. These include amides, albuminoids, amino acids and simple N compounds. Protein and amino acids usually comprise less than 25% of total N and complex N in molasses and they are present mainly as Maillard reaction products. It is conceivable that most of the available N in molasses would be degraded at a fast rate. Rate of degradation of the soluble N fraction in molasses is listed as 350%/h (NRC, 1996) Salivary contribution of urea will also increase during feed ingestion but the relative contribution in corn and molasses diets is not known. Concentration of ammonia in ruminal fluid is the result of several processes. Degradation of feed N, urea recycling and intraruminal microbial turnover contribute to the ammonia pool. Ammonia is removed from this pool by microbial ammonia N utilization for protein synthesis, ruminal ammonia absorption into the blood, and ammonia outflow to the lower

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197 tract (Obara et al., 1991). Sucrose and molasses supplementation decreased ruminal ammonia concentration, probably reflecting increased microbial ammonia capture when microbial growth was rapid (Rooke et al., 1987; Huhtanen, 1988; Rooke et al., 1989; Khalili and Huhtanen, 1991a; Obara et al., 1991). R\iminal clearance of urea (rate of urea degradation per plasma urea concentration) was increased bydietary sucrose and negatively related to ruminal ammonia concentration (Kennedy, 1980) Ammonia is absorbed though the rumen wall by passive diffusion and the quantity absorbed is positively related to ruminal ammonia concentration and to rumen pH. Ammonia absorption is depressed at low pH because proportion of NH4* increases and the charged form is absorbed at a lower rate (Merchen, 1988) This suggests that more ammonia may have been absorbed in animals fed corn supplements because the ammonia peak was higher and the pH during the time of highest ruminal ammonia concentration was also higher than in those fed molasses. Sampling time is an important consideration for plasma urea N (PUN) interpretation (Hammond, 1992; Hammond and Chase, 1996). In the performance experiment (Chapter III), PUN was lower in steers fed the molasses supplement. However, ruminal sampling was conducted 3 to 5 h after feeding corn supplements, which would coincide with high ruminal ammonia concentration. Plasma urea N peaks about an

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198 hour after the rximinal ammonia peaks (Van Soest, 1994) Ruminal ammonia concentration in animals fed molasses was probably lower before sampling because fresh molasses was delivered the day before sampling. Therefore part of the lower PUN concentration may be due to sampling time. Energy intake (1.4% TDN as percent BW) and gain (.6 kg) was similar in animals fed CC and MC, suggesting that supply of ruminal degradable N was not different. Furthermore, PUN concentration in animals fed molasses suggested that a response to additional DIP was not likely (Hammond et al., 1993; Hammond et al 1994). It is unlikely that ammonia N availability may have limited microbial growth and ruminal fermentation because the levels were almost always above the suggested 3 to 5 mg/dL levels for microbial growth (Satter and Slyter, 1974) Mehrez et al. (1977) suggested a higher level (23.5 mg/dL) for optimal ruminal fermentation, but Ortega et al. (1979) did not find a benefit in rate of fermentation by increasing ammonia concentration from 6.3 to 27.5 mg/dL. Bambenrr/cins did not affect (P = .19) ruminal ammonia N concentration, which is consistent with the lack of effect on PUN observed in the performance experiment (Chapter III) The effects of bambermycins on ruminal ammonia have been contradictory in previous research. Bambermycins increased ruminal ammonia concentrations in lambs and adult sheep (Murray et al., 1992). Other reports by the same researchers

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199 showed no effect of bainberrnycins on ruminal ammonia with diets of different qualities that were supplemented with several sources of sulfur (Murray et al., 1991). When alfalfa-lupin and hay-fishmeal diets were supplemented with bambermycins, ruminal ammonia concentration was decreased only in the hay-fishmeal diet (Murray et al. 1990) Bambermycins added to high or low quality diets fed at maintenance levels increased ruminal ammonia in the high quality diet only (Aitchison et al., 1989a). However, when those diets were fed ad libitum bambentYcins reduced ruminal ammonia concentration in the high quality diet (Aitchison et al., 1989b). Bamberirycins inclusion in concentrate fed to young calves had no effect on ruminal ammonia concentrations (El-Jack et al., 1986; Fallon et al., 1986). Rowe et al. (1982) also did not find an effect of bambermycins on ruminal ammonia in cattle. Van Nevel and Demeyer (1990) reported that bamberitr/cins did not affect proteolysis or deamination in vitro. The overall effect of bambermycins on ruminal fermentation evaluated through pH, VFA and ammonia concentration showed only minor changes which would not suffice to explain the increased performance when bambermycins was added to supplements (Chapter III) Effects of corn and molasses supplements on ruminal fermentation appear more related to timing of events rather than great differences in end products of ruminal

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200 fermentation. This is consistent with similar improvements in performance observed in growing cattle fed CC and MC supplements (Chapter III) Sodium and potassium Ruminal fluid Na concentration exhibited a supplement type by time interaction (P = .038). Ruminal Na concentration increased from 8 to 24 h postfeeding in steers fed molasses (Figure 5-2 and Table 59) Increased salivary Na input during rumination may be partially responsible for this trend. At all sampling times steers fed corn had higher (P = .0003) riominal Na concentration than those fed molasses. There was no supplement type by time (P = .2) or feed additive by time (P = .27) interaction for ruminal K. Ruminal K was affected by time (P = .009), with a decreasing K concentration after feeding for steers fed the molasses supplement (Figure 5-2) At all times K concentration was higher (P = .003) in ruminal fluid of steers fed molasses compared to corn. Thus an inverse relationship between these electrolytes was observed, which is reflected in the Na:K ratios. The sum of Na plus K was not affected by time or diet, suggesting that these electrolytes are regulated together in ruminal fluid. The sum of cations and anions tend to remain constant in saliva. The main cations are Na* and K'', which are inversely related. With normal Na intake, salivary Na:K ratio is about 20:1 and drops below 10:1 in animals with Na deficiency (Morris, 1980) Salivary

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u 4-1 4-1 W M O rH > 0^ Q) e (0 0) Sh Eh CO < CO u U U U rH Xi H > 1 CO o (Ti rH o 1 1 1 1 1 1 1 O Ln 1 1 o • O rH • o • o 00 rH ID O <^ CTl O IT) r~ LD CN O CT> LD rH 00 00 CO ^ O CO CN rH O 00 CN LD ro 00 LD 00 LD XI o o 00 ^ (N G\ CN X — 1 ^ rH CN CN LD OA LI ) Ll I cn 00 >^ m rH ro CN cr\ rH rH o m O O O O rH O CN r~O O o o o o o o o LD o o o o • o o o o o o o o o o o • • o 0\ \ — 1 00 00 O CT\ rH ID O U3 ro LD O 00 ro LD LD ^ ro CN rID ID rH rH I> 00 CN 00 ID Vi) CN CO ID CN O CN rH o o O O O rH O rH LD X) "i* rH rH CN fee; + (0 (6 H 01 c -H O Iu (0 to 0) CO w (0 -u rH o O 0) S t-l II 0) S -H g rH O 0) M > 4J -rl C 4J 0 -rl U Tl •a to (0 (V M "O to 0) (tJ 0) rH fc, o S M II <; o •S to (U — to to to d (C •H rH u o u 0) II <4H CQ 0) o c 0) >H (0 to a o •H 4J u ns u 0) JJ c to 4J c 0) g •H o 0) rH > ^^ ^ T> -2 Q) T5 S to (fl -9 to 0) I ^ J (1) u a) >1 -a J O) -H 2 a f_ 6 3 c o 73 o u c o 00 rtH o ro u 0) 4J (0 c (0 c e 0) rH a Ll (11 m rH ^ -H (t! JJ ft) W m >H o U 05 4J T3 S 3 (1) to 0) u
PAGE 209

202 Figure 5-2. Effect of supplement type (corn or molasses) on ruminal sodium and potassium concentrations, means by time after feeding.

PAGE 210

203 concentration of Na and K are regulated by aldosterone (Carter and Grovijm, 1990) Dietary Na could not be assessed because the intake of mineral mix (8% Na) was not measured and water could be an important source of Na. From hay and supplement compositions, it is evident that K and Na intake was higher in animals fed molasses. Corn diets were Na-deficient (.02% Na in DM) Intake of 4 0 g of mineral supplement would increase Na concentration to .06% DM, which is the minimum requirement (NRC, 1996) Mineral supplement was available at all time and was offered at a rate equivalent of at least 100 g/d. Sodium is absorbed from the rumen by the Na-K ATPase system. Sodium absorption is increased by increasing ruminal K or Na concentration, and by increasing the osmotic pressure of ruminal fluid (Carter and Growiam, 1990) These electrolytes were measured in an attempt to relate to the performance experiment (Chapter III) because of the interaction of monensin with mineral concentrations. Direct comparison may, however, not be possible because monensin may change the concentration through changes in absorption. Starnes et al. (1984) reported Na:K ratios of 3.7 and 5 in ruminal fluid of sheep fed corn-soybean mealcottonseed hull diets without and with monensin, respectively. They reported 111 and 122 mM of Na, and 29.5 and 24.3 mM of K for control and monensin diets.

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204 respectively. Monensin increased Na, Mg and P absorption and reduced K concentration and osmolality in ruminal fluid. Magnesiiom absorption may be depressed in molasses diets because it has been shown that increasing Na:K ratio from .5 to 5 linearly increased Mg absorption (Martens and Rayssiguier, 1980) Bambermycins did not affect (P > .28) Na or K concentrations, which was expected because this antibiotic has not been shown to act by altering membrane permeability to ions. Ruminal and Total Tract Digestion In situ degradability of CGM is presented in Tables 510 and 5-11, digesta flow in Tables 5-12 and 5-13, and ruminal and total tract digestibility in Tables 5-14 and 515 by main effect and treatment, respectively. In situ In situ rate of digestion (K^) of CGM DM was increased by molasses (P = .034) and bambermycins (P = .013), and bambermycins tended (P = .13) to increase of CGM CP, Extent of CGM CP digestion was higher (P < .08) at 18 and 24, and 48 h ruminal incubation with molasses supplemented diets, Bambermycins increased (P < .07) CGM CP extent of digestion at 18, 24, 36, and 48 h incubation times. Higher rate and extent of digestion with molasses diets may be related to the presence of CGM as an ingredient of this diet. Corn protein should be similar in both, corn

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205 4J U 0) M-l IW w o (U rH > 0) > H 4J •H a e rH a a CO CO w CO (TJ PQ a o w Q) CO CO (C5 rH o Sh o u 0) fH (0 H > (N CTl rH >X> LT) CO (N ^ CX) CT> V£> O CN IT) n >X) (T> VO rH 00 O m rH o in m CO o m v£) rH in in t~~ in o rH in cn CN o 00 00 < 'Jl rH rO rH rH m iH O rH a\ cn m cn cn CN 00 00 tH rH in rH in vo CO 00 ro O CN rH 00 O (N CN m in CN o o ix> m CN rH 00 00 m o cri rH ro o in CN rH 00 i> CN og CN O O CN CN O rH 00 00 o 00 CO 00 O >X) in 00 CN O o ro o o 1 ri; m u ffl o •H u >H 4H dp a o -H -U CO Q) &) -H 4H o >H [li b pLj Q 4J o ^ 00 "-D CTl CTi CTi rH rH rji CN r~rH 'J' 00 CN m >^ o vD CO rH Cri CT\ CTi I> 00 CTi in in o o o o rrH CN 00 t> in CN o •^a' in in t~~ CN in rH <^ >^ ro rH o o in CN 00 CN rH CO O CN I> ro O rH rH 00 ro o o o ro "X) ro rH CN O 00 CT\ CN oj ro rH ^ CJ^ cri r~i> ro CN UD 00 og rH C~~ CTl rH Cri ro CTi t> 00 00 00 og ro ro [> o rH ro 00 rH Cr\ 00 rH rH og ro in r~rH O O r00 CO ro CTi og t~ ro 00 00 00 ro r-iH in rH rH CN CN o o cr> in CN in 00 rH Tl< CN to <£> og in in CTi cr 00 rH ro 00 rH rH CN ro in r-~ 00 cTi og cTi CN r~ 00 cr\ 00 og CN rH rrin rH rH CN CN dP dP dP <)(> (u < m o c u o a-H -U 0) u ^ u o H u (d u PQ C O H 4-) U rc5 Sh dP O H 4J CO 0) D) -H T3 UH o o ^ d1 4-) ^ CN -O (0 X rH fcic; J w 00 Vi3 00 rH CN ro 0) 4J M 0) Dl -H Tl C D 0) CQ 4J (C c M 0 •H 0) 4J ^ nj 10 M U n-t 0) •H b. 4J •H II T3 < CJ m 0) 0) 0) D) MH m & rH o e 4J C u 0) 0 g (V rH 0. 0 0, u to 4J <*-i u 0 0) M-l C 0 0) •H 4-) c: U •H (0 (8 e (1) 4J 4-) m (0 -H .2 CO Q)J3 3 •H to (U

PAGE 213

206 4-3 U Q) 4-1 14-1 W u o 4-1 :3 rH > 0^ 4J (U e (0 0) CO C/3 u S U U U 0) rH (C3 •H ^H > CM 'J' CTi rH ID 00 CN >^ 00 CTv O (N LD ro <^ cri u> rH 00 O rO rH X> o r~in ro 00 o ro vo rH in in c^ in o rH CN in CTi (N o 00 00 'd* 00 rH ^ m iH rH m rH O rH rH [> t~~ cn 00 rH rH rH rH in cNj ro CXi CTi O <^ o in n ro rH 00 o x) O rrH CN o rH 00 o 00 ^ OJ rH O rH 00 00 o 00 I> ro CO O V£> in 00 CN O o n o o in rH rH o # CO 1^ o in in 00 ro in ro o in P 4-) 4J s >1 .< PQ U (0 (0 m o •H 4J u 03 u O •H -U CO 0) Cn •H ^ Plj [n p ,C! 4-" o ^ Di 4J >X) •o 03 X CN ro J W o rH O rH U3 00 CN in CN ^ o CO rH rH CT^ CN 'd' ro rH 00 O 5^ CTl CN CTl O X> rH rH ro CO CT\ • ••••• o CN CN O ro 43 00 CO in CN rrH vo o in CTl CN ro CN r~ in o o in ro CTl CO 00 CO CN ro 00 CN CT^ CO rH V£) ^ CO 00 in CO CN o in rH ro in in rc~CN ro dP dp dp H ~ 0) <; CQ u 4J o u an u u dP 03 ii! O •H 4J U 03 u dp o •H 4-) m 0) O) •H 4H •o 03 4J 0) 4J VD W ^ ^ ^ CN ro 00 d) w 4J CO •H d) u >1 •H & T3 D S c o nj 0) O ja d C Dl 0 O 0) a •H ) 4J to U (0 44 (C rH u ^4 0 Q> b e >44 <4-l CQ II 0) (U 4J CQ fl c •r4 i-l (0 o (l* r. ^ o a) o JJ -rl C 44 T3 E '-' (1) JJ -rl 0) 0) > 0) D) g II 2 (0 II < 0) o 2; 0) 0) C3 C (I) 01 c S 2 O TO c <" ?i .^i 44 -H rH ja (0 y O e 44 (1) n ,5 c o "J rl £ 0) 0) >H 3 -fl u O 44 MH u u o 0) II <4-l fl ^ U rn 3 •0 44 c (U e 11 rH ft >< ftCO 3

PAGE 214

207 and molasses diets. However, processing (in CGM) and organic matrix (in corn) may produce differences in the microbial population that digest the protein from CGM and corn. Loerch et al. (1983) suggested that in situ N disappearance of protein sources may vary with different dietary protein supplements. Overall mean for the soluble CP fraction was 7.9% in agreement with 7.2% reported for CGM solubilized in sterilized ruminal fluid (Waldo and Goering, 1979) Cozzi et al., 1994) reported 12.3% water soluble CP in CGM. Overall mean for the undegraded fraction at 60 h (used as fraction C) was 4% of CP in CGM, which is similar to the reported 3.7% of CP remaining after 72 h in situ incubation (Cozzi et al., 1994). Van Soest (1994) reported 2% of CP from CGM insoluble in acid detergent, which is an estimate of indigestible CP. Overall mean for was 2.7%/h which compares well with 2.87%/h reported by Cozzi et al. (1994). Bacterial contamination is one potential problem with estimation of CP degradability in situ (Nocek, 1988) Microbial CP contamination of CGM was 4.5, 6.7, 9.3, 2.3, .2, 0, and 0% for 8, 12, 16, 24, 48, 72 and 120 h of in situ incubation (Cozzi et al., 1994). Underestimation of extent of digestion may be expected in our data because correction for bacterial contamination was not done. Bias in treatment comparisons could result if treatments affect differentially bacterial attachment to CGM. The in situ method probably

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208 underestimates CP degradability of CGM due to hydrophobic properties of CGM protein (Stern and Satter, 1982; Cozzi et al. 1993) The practical significance of changes in degradability may be assessed using the NRC (1985) equations. Using PR from Table 5-3 (G4G1 model) degraded CGM crude protein concentrations were 46 and 52% for corn and molasses, and 45 and 53% for control and bamberrt^cins, respectively. When PR was fixed at 4%/h, corresponding values were 42 and 44%, and 38 and 48%, respectively, which would suggest that supplement type had less impact than feed additive. These values agree with estimated 57% 11 escape value from in vivo estimation by regression technique (Stern et al., 1983) Degradability measured with nylon bags really measures disappearance from the bag. Degradation of insoluble protein to ammonia by ruminal microbes include attachment of bacteria to protein, proteolysis to polypeptides, further hydrolysis to oligopeptides, dipeptides and amino acids, and deamination with production of ammonia (Broderick et al., 1991) Protein may disappear from bags as soluble peptides and amino acids, which could be further degraded or pass out of the rumen with the fluid phase contributing to metabolizable protein. The trend for decreased NANMN flow (Table 5-17) for MB compared to MC tends to support that more dietary protein was degraded in the rumen in steers fed

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MB. The lack of effect of bambermycins on ruminal ammonia concentration does not support increased deamination. Comparison between in situ and in vivo estimates of CP degradability is difficult because the treatment effect on hay CP is unknown and changes in particle PR dramatically alter extent of rumen degradability. Tn vivo ruminal digestion. Dry matter intake was similar for corn and molasses diets (Table 5-12) because molasses had less DM than expected, resulting in higher OM intake from corn diets. Total N intake was different between supplements by design, as the diets were balanced for DIP and UIP. Because of these differences, digesta flow data presented in Tables 5-12 and 5-13 will not be emphasized. Instead, digestibility will be discussed (Tables 5-14 and 515) Apparent ruminal DM (P = .13) and OM (P = .1) digestibility tended to be higher in steers fed the molasses supplement. Riominal NDF digestibility was not affected by supplement type or feed additive. Steers fed corn had 10.2 percentage units higher (P = .015) ruminal feed CP digestibility. Ammonia has been considered as the only protein degradation product absorbed through the forestomach epithelium. However according to recent research the stomach may be capable of amino acid and peptide absorption (Webb et al 1992). In vitro studies with ruminal and omasal epithelial tissue have shown that both tissues have the

PAGE 217

o o iH LD U5 CTi CN U) >XI rH n m rO rH O O rH 00 cr\ 00 LT) rro ro ro ro x> rH ro x> m ro ^ cr\ >x> O O rH 00 CT\ r-o rH rO 00 rH Vi) >X) OJ rH ro 00 <^ CTi rH rro O O CN o r00 00 00 in CTl rO CTi CT\ o o in ro *x) iH in rH rH I> O rH CTl 00 rH in rH m rrH CO in ro CTi •rjl rH rH in rH in m o U5 00 (N cr\ in CO 00 CO o 00 in ix> o o o 'J ro in o O O 00 o o rH r~ in cN cTi CO a\ a\ in x) rjt t-U5 CTi in >J3 CN (N H 00 in in rcN CN o ro rH in CN "X" ro rrH o in og rH in rH 00 in rH CN CN Xi \ \ g T3 rH \ UH Ot D) rH (0 o (U a rH X (0 rH (d fa fa (0 4J 2 S 0 s S p o o 2 2 a Q o IS QJ Q O H Q fa 00 ro in fa 0) (D e II -H XI a. ^ II CO CO i II a •H CO CO u

PAGE 218

211 T3 C o rH 4H (0 c Q) O 0) (0 c; iH a o m d •H u >H 0) -i a a (0 (0 0) 0) g 4-) C 4J 0 (0 aa 13 4J 4-1 o ^3 a o 4J U rH 4-) e 4J (0 (U Sh C/3 U CQ U U U 0) rH (d -H ^ ; (d > r-o o rH in U3 cn CN U) VO rH ro ro CTi O O rH Ln ^ CTl Cn V£) rH U3 ro oj VD 00 rH rH O in o o cy> o o o O O N ro rH Ln ro o o 00 o r~ 00 vo CTl rH 00 rH r~ rro in 00 CO (N cn c~o ro CTl rH 00 ro o in in in in rH in O ro (N rH 00 'd' in in in ^ >JD ro rH in CTl in CN rH t> r73 0) 03 -u S S G Q O ro rH O O rH CO OA 00 in r~ro rH ro 'I' ro O U3 00 CO ro cr\ (T> 00 Og O in rH in ^ rH rH 'i* x> rH in rH in 00 in CN in r~ [~ in r~ rH in rH o CTl o en ro in rH t~~ t~~ in r~ rH in rH ro CN o o rH ro ro o rg ro CN CN rH 00 vo KD ^ CT in VD CN CN rH rH rH ro o in in oo ^ CN CN rH rH CN "^Ji ro 00 CN in ro CN CN rH 73 \ 0) i o JJ rH 4H a 4J rH (0 0 a rH Q) 03 rH 73 [l, 4J 03 o u 0) Q O m C •H •g 0 (0 01 C! to 0 d) a m m nj 4J rH O 0 0) g "4-1 UH II (1> C •rl 1 1— 1 0 u > 4J •H c 4J 0 -H u TJ T3 w (0 (1) to T3 to 0) (0 0) rH b i II II < 73 (0 O 73 S 01 <" 0) 0) to to CO C (0 >i •rl rH XI O O >1 e 4J g K HMO) < aw 3 cn II V) II c CJ -H O CO u >1 S XI g II (1>
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212 4J U 0) IW W S-l o 14-1 0) <-\ (0 > a, > •H •H 4J a rH a a CO CO <: CO W CO (0 0) G O CO Q) CO CO (0 rH O a u o o rH (0 > rH 00 00 cr\ r~ in O O o [~rH 00 ro ro CN r^ l IX) IX) ro CN 00 ro CN \ — \JJ r^ in 00 in CX) LD LO ro rH ro VO 00 00 o • • • 00 LO CI> 00 in ^ CTi ro oq O 00 CN r^ m ro CN O rH o CTl in in in oq rH rH O ro in rro r^ vL^ rH *x> 00 cri CTi 00 u> 00 cn rH ro in rH U5 CTl >X) rH rH rH o 00 00 VX> 00 CN in o in cs o ^ en ^ rH rH rH rH CN rH rH CN rH rH ro c ^ -r:^ ^ ^ in ^ O 00 00 rH CTl ro o rH VD 00 og in 00 ro rH V£> in a\ ro ro ^ in in CX) CTl in IX) fO o c^ rH 00 ro CTl >X> >X5 00 *X) cn O U) 00 ro iH in CO CN ro in in 00 CT> 14H M-l o O 4-) jj dp rH U C (0 D^ (1) a Sh rH ^ U rH -H 4J (0 (0 (U (C5
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2 u (U 41 w 5l o 4-1 Q) rH (0 > Jj a (d 0) CO W S pa u u u Q) rH to H (0 > IT) 00 00 00 ro ro ro o ro 00 fO CO CN 0) (0 JJ d •H O rH o og 00 rrH O <* in JJ U u JJ JJ o 4J o 00 00 CTl in o o O rH ro r^g rH ro CN 00 CN o 00 rH in ^ CO in CN 00 rH ro i> 00 00 o 00 • • CO 00 in cn ro CN o 00 CN ro (N o rH o cn r~in in in CN o CN ro in rro rH U3 rH o cr\ 00 00 a\ rH ro CTl rH in ro in • • • • • r~ in rH U3 CTl >x> rH rH rH o CO U3 00 ^ CN in o in GO rH rO CO rH rH rH CN rH o ro o rH o CD ro CO o o • o ro O o ro o O • O O • o • rH ro og 00 in CN rH 00 CTl 00 o X> 00 CN rH rH Tjl VO rH ro 00 rH in CTl o ro 1 U) IX) in O iH CN r00 in in 00 a\ 00 1 r~ ro 00 CN in in 00 CJ\ ro o in in in CTl U3 vo IX> f^ Tji vo rH rrH rH (X) 00 in 00 rH in in 00 in IX) >X) IX) •H Sh Q) JJ 0) 4H o d) a H o dp JJ U Id u as S S P O Q O 2 U • m c C •H H u O g e )H 0) g e )H CO 0 (U 0) 0) 0) c: ItJ o r-t 0 £ JJ II u 0) OQ 14-1 t-l iH 0 H 2 J-) g c 0 (1) V > •H tn JJ 0) H 10 "d D) •d m nj rH O T3 g 0) (1) II II o S V) m (1) c to •H 01 u (0 >. rH g 0 g 0) -i 0 (0 c u c 0 0 0 u JJ II u a) 03 <4H o >4H (V rH C 0 •rl u JJ g C! 0 JJ U c 0) c: g u a) II 0 rH U Q.ft 3 W U II CJ V) US x>

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214 ability to absorb and transfer amino acids and dipeptides (Matthews and Webb, 1995) The importance of this absorption in the diets used in the present experiment is unknown. For this discussion, feed CP disappearance in the rumen can be interpreted as passage of feed N to ammonia pool and absorption through the rumen wall, which is the fate of excess ruminal ammonia. Excess ammonia may result from nonprotein N and highly degradable feed protein, especially if readily available energy is limiting. Feed CP can be captured by ruminal microbes as peptides, amino acids or ammonia for microbial protein synthesis. Ruminal ammonia N concentration and microbial N flow were higher in steers fed corn supplements. Higher disappearance of feed N from the rumen with corn diets may be explained by increased ruminal ammonia pool, increased absorption to blood, and increased capture in microbial protein. Ruminal apparent (66.4 vs 57.7%) and true OM (89.6 vs 80.9%) digestibility expressed as percent of the total tract were higher (P < .02) in steers fed the molasses supplement. Alternative expression of same effect is the increased (P = .015) duodenal OM (4.56 vs 4.16 kg/d) flow in steers fed the corn supplement (Table 5-12) Eighty to 90% of the digested OM disappeared in the rumen. Intestinal digestion would be proportionally lower at restricted intake because of long ruminal retention time. Increasing the level of intake would shift the site of

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215 digestion to the intestine and that effect would be greater with high concentrate and mixed diets as opposed to long hay (Galyean and Owens, 1991). Firkins et al. (1986) reported small but significant increases in ruminal NDF digestibility (as percent of intake and as percent of total tract) when intake of chopped and ground prairie hay was reduced from 1.6 to 1.1% BW. With bermudagrass and corn diets, true rumen OM digestibility (as percent of intake) was 53.4 (Brake et al., 1989), 50.7 (Galloway et al., 1993a), 57.8 (Galloway et al., 1993b), and 52.9 (Galloway et al., 1992). Ruminal true OM digestibility expressed as percent of total tract digestion calculated from data presented in these reports ranged from 80 to 98%. Estimation of feed CP ruminal digestion by the difference method should be taken with caution because errors could be great (Stern and Satter, 1980) Estimates of feed protein entering the duodenum were 8 and 47% higher than predicted by the NRC Model Level 2 (NRC, 1996) for CC and MC treatments, respectively. Only 20% of the difference can be explained by assuming that 15% of N in molasses is indigestible (Stateler, 1993) Other possible explanations are that protein from CGM passed out of the rumen faster than corn protein and (or) that molasses depressed hay protein degradability Passage rate of CGM was higher than soybean meal and distiller grains (Stern and Satter, 1980)

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216 and molasses has been shown to depress ruminal CP degradability in silage (Huhtanen, 1988; Petit and Veira, 1994) Molasses increased in situ extent of digestion of CGM CP. However this effect was expressed after 18 h of incubation. If PR of CGM was high, the effect of molasses would probably disappear. Bambermycins apparently affected dietary CP degradability in opposite directions in each supplement (interaction, P = .12), but differences within each diet were only numerical. Reports on the effect of bambermycins on ruminal digestion are limited. The few reports available show that bambermycins did not affect in situ cellulose degradation in cattle (Rowe et al., 1982), or NDF, starch, and casein degradation in vitro (Van Nevel and Demeyer, 1990; Van Nevel and Demeyer, 1992) In situ rate of digestion was not affected by addition of bambermycins or ionophores to alfalfa-hay diets fed to steers (Barley et al., 1996). In contrast, Poppe et al. (1993) reported that bambermycins decreased OM, CP and CF digestibility in the rumen. Intestinal digestion Steers fed corn had higher intestinal digestion (as % entering) of DM (P = .013) and OM (P = .005), suggesting that starch was not completely digested in the rumen. Intestinal total N digestibility exhibited an interaction (P = ,08) of supplement by feed additive, due to no effect of bambentr/cins in corn but

PAGE 224

217 lowering (P < .05) total intestinal N digestion in molasses supplements. This effect could be related to a numerical increase in feed N digestion in the riomen resulting in less available feed N entering the small intestine of steers fed MB. Intestinal digestibility of NDF was not affected bysupplement type (P = .33) or feed additive (P = ,9). Total tract digestion Steers fed corn had higher total tract DM (P = .018), OM (P = .001), NDF (P = .002), and CP (P = .035) digestibility than those fed molasses (Table 514) Although steers fed corn supplements had lower ruminal true OM digestibility (55.1 vs 57.9 %) than steers fed molasses supplements, the intestinal OM digestibility (46.0 vs 37.4% of OM entering the duodenum), and total tract OM digestibility (68.0 vs 64.5%) were higher. Riominal NDF digestibility was similar (58.7 vs 58.4%) between corn and molasses supplemented steers but intestinal NDF digestibility was niomerically higher in corn supplemented steers (5.0 vs -.9%) with a high SE. Total tract NDF digestibility had a low SE and steers supplemented with corn had a higher total tract NDF digestibility (61.0 vs 58.1%) than steers supplemented with molasses. Hsu et al. (1987) reported that 71 and 9% of NDF was digested preand postduodenum, respectively, in sheep fed corn fiber. Molasses or sucrose supplementation depressed NDF digestibility in several studies (Brown et al., 1987; Brown, 1993; Khalili,

PAGE 225

218 1993; Khalili and Huhtanen, 1991b; Kalmbacher et al., 1995). Royes (1996) compared the effect of corn and molasses supplements fed at 15 or 3 0% of the diet on total tract digestibility in steers fed ammoniated stargrass hay. Digestibility of OM and NDF was lower with molasses than with corn when supplement comprised 15% of the diet (50.9 vs 54.7 % OM, and 50.7 vs 56.9% NDF), but OM and NDF digestibility was similar when supplements were 30% of the diet When the same supplements were fed with ad libitum hay intake (Chapter IV) NDF digestibility was lower with corn diets. Apparently there was more depression in fiber digestibility because of higher corn than molasses intake. Royes (1996) reported that steers fed .43% BW of molasses had a ruminal pH similar to those fed aitimoniated stargrass hay. However, steers fed .52% BW of corn had a ruminal pH significantly lower than steers fed ammoniated stargrass hay. Apparently this pH reflected the feeding habits of steers (corn consumed in 3 0 min and molasses consumed in 12 to 24 h) which were similar to patterns of supplement consumption described in Chapter IV. Hay intake was also higher in heifers fed molasses. More fermentable OM entering the large intestine with corn may explain the tendency for lower CP digestibility observed in heifers given ad libitum access to hay. High intake of mixed diets has been shown to

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219 shift the site of digestion, increasing intestinal digestion (Galyean and Owens, 1991) Differences in total tract CP digestibility were numerically small between supplements (Table 5-15) suggesting that animals fed molasses could partially compensate for lower ruminal protein degradability Pate (1983) suggested that feeding moderate to high levels of molasses reduces the apparent digestibility of crude protein in the range of 5 to 15%. Depression of intestinal microbial or dietary protein digestibility and (or) increased metabolic fecal N excretion have been suggested as possible explanations. In this experiment, this difference can be accounted for by the estimated 15% (7 g N) indigestible N from molasses (Stateler, 1993). However, consideration of fecal N is relevant. Cattle do not have intestinal sucrase (Merchen, 1988) It is generally assumed that soluble sugars are completely fermented in the rumen. At medium to high levels of molasses supplementation, part of the sucrose may exit the rumen dissolved in the ruminal fluid. Oldham et al. (1977) estimated that only 6 to 9% of sucrose ingested with molasses reached the duodenum in sheep fed 60% molasses. Using the upper limit, it can be estimated that up to 90 g of sucrose may reach the duodenum in cattle fed molasses in this experiment. The fate of this sucrose would likely be to support microbial fermentation in the large intestine. An

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220 increase of fermentable substrate at this site would result in increased airanonia capture for microbial growth with the net result of increased fecal N (Orskov et al. 1972). Sucrose supplementation increased fecal N in grass silage diets. Fecal output of N was closely related to the excretion of RNA in feces indicating increased hindgut fermentation (Khalili and Huhtanen, 1991a) Bambermycins did not affect DM, OM, NDF or CP total tract digestibility. In contrast, bambermycins tended to increase N digestibility in heifers (Chapter IV) One of the mechanisms, lower intestinal cell turnover, suggested as an explanation may be not as important here. Animals fed at maintenance have lower visceral metabolism and tissue mass (Ferrell and Jenkins, 1985) Reported effects of bambermycins on total tract digestion have been variable as discussed in Chapter IV. Nitrogen Flow and Microbial Efficiency Total N intake, duodenal total N, duodenal ammonia N, and duodenal NAN flows (g/d) were higher (P < .07) in steers fed molasses compared to those fed corn (Table 5-16) Steers fed bambermycins had 10.9 g/d lower (P = .077) NAN flow than those fed control supplements. Flow of NAN expressed as percent of N intake was not affected by supplement type (P = .4) or feed additive (P = .35). Total duodenal N flow was 22.6 and 20.9 g/d higher than N intake in steers fed corn and molasses supplements, respectively. Recycled N causes

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daily duodenal N flow to exceed N intake when diets contain below 13 to 15% CP (Owens and Zinn, 1988) Total diet CP was 12 and 13.3% in corn and molasses, respectively. Duodenal flows (g/d) of NANMN and undegraded feed N exhibited a supplement type by feed additive interaction (P < .08). Steers fed MC had higher (P < .05) NANMN and undegraded feed N than those fed CC or CB. Steers fed MB had higher (P < .05) flows of these N fractions than steers fed CC but similar to the those fed CB (Table 5-17). When expressed as percent of intake, steers fed molasses had higher (P < .051) NANMN and undegraded feed N flow than the ones fed corn. Urea was used to provide the same amount of DIP in both diets, which resulted in 34 and 5 g of dietary N contributed from urea in corn and molasses diets, respectively. Urea and most of the CP from molasses has a similar rate of fermentation (400 and 350%/h, NRC, 1996). However the rate of fermentation of starch and sugar differ greatly (15 to 35 vs 300%/h, NRC, 1986) It would be expected that the N entering the ruminal ammonia pool would be utilized more efficiently in molasses diets, through incorporation into microbial protein. Ruminal ammonia concentration was higher in corn, which may imply that part of accumulated ammonia was absorbed through the rumen wall. Partition of NAN flow was different between corn and molasses because bacterial N flow was higher (P = .053) in steers fed corn. The lower

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222 (0 O C 0) O u OJ O m a •H u I (0 (0 0) a 4-) >1 u OJ JJ -H d u e M-i Q) 4-1 rH 0) a a d m Di o UH u o -u •H -u d u 0) rH llH -H 0) ^ OJ d 4-) H U (0 (0 S 42 LD 0) rH 43 4J u Q) HH <4H W o (U rH > > 4-) d rH a a CO CO CO (0 d o m 0) m m rH o d o u rH 43 •H > cn rH o o o o (N LD rHrH'*x)Lnooo r~-ro crioocorHr-oooi^oq u>ro moom*^cNOrHOrH co cri'<*(Nooo^rHocn'* r-oo Lnmr--ooro>x)00{Nr~ r^cN O'*U5'J'O000q OOrH iHOOrotHrocomoo vovo rH(Nr~-oro C--(NU>VDV^OOCNrHrH n (N (N m fN m CN ooooor-fNOOcrij3ir( Crit>rHrHOOrOOO>X> 00 oOrHcr\oom>^ro rHOOmr-rHCNrOOrH CTi ooocrio^inr-^ >x>c^cr^oo(TiLnu50 ru)rHO^D'^-<*ro 0) (U ^ ^ O o CTiocrimu3'*^^>X) rHO 00 CO IT) O OrHoOrHinrominro r~-Ln 00 in o rHOrHUDrHOCr^rHrH inr~ <^ o corHr~-coor~-oooooo rocrv m o in o (0 T3 U QJ Q) (0 O 4.) CO 0) Di -H 00 in vo OJ *x) cr\ 00 in o 00 00 rH CTi O cr\ 00 00 U3 CJ^ CN CS iH rH O (N ID ID O tH O rH rH O 00 rH rH m 00 in cn rH CN <* d o H CO tJ Q) \ cn D) H >1 u d 0) •H u o d 0) Pi 4J 0) (0 rH -H (13 43 d O -H U 13 -H Oi S ro rH ro 1>D iH CO 00 CN m m in CN 00 O CO 00 00 tn CN r^j 00 ro •^1' ro in o o in CO ro ro \ — 1 rH n ro ro o o o cn in rH O ro (T> 00 rH ro CO OJ rH rH CO ro m 00 ro OJ CN rH rH CTl rH rH • • CN CN ro CN rH rH in • CN • • ro CN in CN rH rH o § ^ O O cn 01 di o o M o o rH o o O rH rH a, Q u 4J u cn (0 CD tn Di ^ o 4J o rH QJ 0) rH \ a :3 (d a V4 ^ 4J U Eh o s Eh JJ 0) Q) D) H tn c • •rl u >1 m -H 4J U (fl M 01 JJ (DO) ^ e (0 rH < II p e 5 (0

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223 rH CTi O O O 00 U3 CN LT) CN in rHrH'^ogooujinooD r-m OAOOOOrHr^ooovocN >^ro rOOOrO^(NOrHOrH 00'^ CTl'^CNOOCrirHOCn';!' c^oo inror-ooroujoocNO c^cn OOAC^^UJ-^OOOCN CrH rHOOrorHrooorooo vdvo rHCNrHoofOir)x>ooir) rjtoo UDOoocNromOrH-^ old rn>x>^roLnoLnOrH ooo ooo^ooooo oo (nrHrHLnr~cylma^r^ CN'^rHr-r~r-romo in ro m ro m roc'*-^0'*>x)moo 00 c^o[^>X)-<* CTirHcricriinr^'^ '*t^c^cri'^cN]oo CJ H -H o o I S (0 iw fd o -d o \ 4H rH \ CJldP TJ O (d CJ)<*P (d o O E IS s 0) Q) !^ !2: fa b4 rH CN 00 CN rO'^CTiC^'^int^roo mc^ VX) o cTivocNinr-inojcorH r-vD m 00 vo o >x>inooor^'^(NtnfN cr^ro CN rH in o rHr~-'^c^'^rH'^cNin c^in n o IT) o 00 in VO CN VD cr\ 00 vo in o rcr\ CN 00 00 rH CT\ O C7^ 00 00 00 CN r~ 00 CN O rH CN n o O rH o o o C3^ rH CN <* 1^ rrH cn CN CN ^ m m m ro rH 00 00 in in in ^ ^ ^ O 00 00 in CN CN m ^ in o o 00 m m rH m ro ooo CTl rH rH rH <^ in rCTv rH CN CM ro O O O CN 00 m CN ro ro o o ro CN in rin cTi ro CN in CN CN 00 00 00 ro in ro in CN CN in CN 0 4J <4H u 0 0 (U II u-l c: >u 0 QQ 01 H U 4-> c 0 •H <0 rH (0 u 0 g 0) u 4-) 4-) c a a M 0 0) 0 e II 0) rH u a 0 aw o 3 CO II ta II c o •H CJ w u XI o 0) (0

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224 bacterial N flow in steers fed molasses is not consistent with benefits of synchronization of protein and carbohydrate fermentation in the rumen. Apparent and true OM fermented in the rumen (g/d) was not affected by supplement type (P > .5) or feed additive (P > .8). Efficiency of microbial N synthesis was higher in steers fed corn, expressed as apparently (3.59 vs 2.99 g N/100 g OM, P = .019) or as truly (2.47 vs 2.19 g N/100 g OM, P = .033) fermented OM. Bacterial efficiency expressed as percent of OM digested in the total tract was not affected by supplement type (P > .5) or feed additive (P > .26). The NRC (1996) committee used this form of expression of microbial efficiency and a value of 13% of TDN is recommended for use with most diets. The total tract efficiency of MCP was similar for both diets because increased postruminal digestion decreases efficiency with corn supplement. Apparent microbial N efficiency found in this trial was similar to mean value (2.7 .99 g N/100 g OM) reported by Stern and Hoover (1979) True microbial N efficiencies were higher than reported values (1.38 to 1.63 g N/100 g OM) measured with bermudagrass and corn diets consumed at comparable intake levels (Galloway et al., 1992, 1993b; Brake et al 1989), and similar to values (2.7% g N/100 g OM) measured in steers fed prairie hay at 1.1 to 1.6% BW (Firkins et al., 1986).

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225 Microbial protein synthesis may be affected by dilution rate, ruminal N concentration, N source, carbohydrate source, sulfur to N ratio, and feeding frequency (Stern and Hoover, 1979) The last two factors were not likely factors because S was added to the corn supplement and molasses is high in S (.47% DM, NRC, 1996), and frequency of feeding was similar between supplements. Dilution rate was numerically lower, but not significantly different, in steers fed molasses than in those fed corn. Mixed ruminal bacteria that were incubated in vitro produced 50% less protein at pH 5.7 than at 6.7 (Strobel and Russell, 1986) Because pH was similar in corn and molasses diets, pH per se can not explain the lower efficiency observed with molasses diets. The transient presence of lactic acid in all steers fed molasses suggests that bacteria may have had increased use of the acrylate pathway. Propionate synthesized through the acrylate pathway yields less ATP than when it is synthesized through the randomized pathway (Russell and Wallace, 1988) Although ruminal ammonia N concentration was lower in molassesfed animals, it was above the minimum of 3 to 5 mg/dL required for maximum bacterial growth (Satter and Slyter, 1974) The NRC (1996) Model Level 2 predicted +14 and -18 g bacterial N balance for corn and molasses diets, respectively. However these estimates are driven by predicted bacterial CP synthesis. Calculated recycled N

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226 (NRC, 1985) was 37 and 33 g/d for corn and molasses diets, respectively. Flow of NAN was higher than N intake in all diets suggesting that recycled N was captured into microbial protein. Most of the DIP should have originated from readily available non-protein N (urea and simple N compounds from molasses) with true feed protein contributing at later post feeding times because of lower rates of degradation. Recycled N can supply rinninal ammonia, but not ruminal peptides. Steers fed molasses had higher duodenal NANMN flow and lower BCVFA molar proportion in ruminal fluid, suggesting a decreased feed protein degradation. Amino acids and peptides are needed for optimum bacterial protein synthesis (Russell et al., 1992), although according to NRC (1996) a lack of amino acids or peptides is unlikely to be a problem in typical diets for beef cattle. However, this generalization may not be applicable to the molassessupplemented diets in this experiment. Molasses provided 44% of the total TDN with a high degradation rate of the energy substrate. The main N source for bacterial growth when soluble sugars were fermented should have been derived from non-protein N rather than from true feed protein, which may have implication in the efficiency of microbial growth. Bates and Denham (1990) emphasized that different sources of CP degraded in the rumen are not necessarily equivalent in capacity to support efficient microbial protein synthesis.

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Energy source deserves careful consideration because it differs between diets. In his review, Pate (1983) found evidence that sugars, and particularly sucrose, were less effective than starch in promoting microbial synthesis from urea. Nitrogen retention was also lower for molasses-urea than for corn-urea diets. He inferred that if the biological value of all microbial protein is similar, then the higher urinary-N losses observed in animals fed molasses-urea indicate that urea-N was less efficiently synthesized into microbial protein. Khalili and Huhtanen (1991a) reported increased duodenal NAN flow and dilution rate in cattle fed grass silage supplemented with sucrose. However, the efficiency of microbial N synthesis was not significantly increased by sucrose supplementation. Obara et al. (1991) reported increased N balance and a reduced nominal ammonia concentration in sheep infused with sucrose intrarruminally There was no increase in ammonia incorporation into microbial N suggesting that less feed N was degraded and calculation from data presented shows that efficiency of microbial N synthesis was 4.03 and 2.75 g N/100 g OM digested, with basal diet and basal plus sucrose infusion, respectively. No difference was observed in the protozoal population. Rowe et al. (1980) also reported low efficiency of microbial synthesis with molasses-based diets, and efficiency was increased with the addition of starch. They

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228 suggested that addition of starch provided a more uniform supply of fermentable energy for the rumen bacteria. Combination of barley-urea increased duodenal amino acid flow more than the combination of molasses-urea when sheep were given cereal straw, suggesting a better efficiency of ammonia capture in microbial protein when starch was the energy source (Oldham et el., 1977). In vitro experiments indicated that bacteria use energy for purposes other than growth (energy spilling) and a variety of pathways for energy spilling have been proposed (Russell and Cook, 1995) Fermentation of energy-rich substrate can continue with VFA and ATP production, but without the increase in microbial mass. A pulse of glucose added to glucose-limited cultures of S. rumi nant ium and ruminicola (formely B. rumin icola) caused an immediate doubling of heat production and little increase in cell protein (Russell, 1986) Energy spilling appears to be a phenomenon that can occur any time bacteria have excess energy (Russell and Cook, 1995). Van Kessel and Russell (1996) reported that when growth rate of the predominant ruminal bacteria was being regulated by the limited ammonia concentration, the impact of energy spilling was very great, and additional ammonia caused a large increase in yield. This probably was not the situation observed in the present experiment because ammonia appears to be in excess during molasses fermentation. However, when energy-excess batch

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229 cultures were provided with amino N, the growth rate was increased and less energy was spilled (Van Kessel and Russell, 1996) It is therefore likely that during sugar fermentation the supply of amino N (peptides, amino acids) may have been insufficient to balance the anabolic and catabolic rates of fast growing bacteria. Consequently, part of the energy available from fermentation may have been spilled. Rooke et al. (1987) reported that the efficiency of microbial N synthesis was unchanged by infusion of casein, urea or glucose syrup in cattle fed a grass silage diet. However, when glucose syrup and casein were infused together the efficiency increased from 2.6 to 3.8 g N/100 g OM digested. Inclusion of a protein source of high ruminal degradability in the molasses slurry may increase microbial protein flow to duodenum through availability of growth factors, amino acids, and peptides. Good responses to escape protein may result from lower than expected microbial protein contribution. More information about effects of different N sources on microbial growth in diets supplemented with molasses would allow evaluation of different feeding strategies to meet metabolizable protein (MP) requirements. The NRC (1996) Model Level 2 predicted 534 and 728 g bacterial protein for CC and MC treatments, respectively. Estimated bacterial CP flows in this experiment were 659 and

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230 569 g for CC and MC treatments. It should be noted that model estimates are for steady state conditions, which may not be applicable to restriction of intake imposed in this experiment. Animals fed molasses, for example, showed ruminal characteristics associated with active ruminal fermentation for only 6 to 8 h postfeeding and the bulk of the nutrients were consiamed in two hours or less after feeding. Metabolizable protein is the composite of bacterial and feed protein. Steers fed molasses had higher NAN (g/d) flow than the ones fed corn. However, if the calculated unavailable N originating from molasses is subtracted, the NAN flows in both, corn and molasses are close (17 0 vs 17 6 g/d) Predicted MP (NRC, 1996) was 548 and 612 g/d for CC and MC in the performance experiment (Chapter III) This MP protein would allow .92 kg of gain in animals fed corn, which indicates that energy and not MP was limiting. Under these circumstances, the lower efficiency of microbial growth may not be relevant because after discounting for lower microbial N efficiency, an estimated 570 g MP would be available in animals fed molasses. Amount of N digested in the total tract tended (P = .086) to be higher in steers fed molasses. Fecal N was also higher (P = .0004) which resulted in the lower apparent CP digestibility discussed above.

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231 Bamberrnycins tended to (P = .16) decrease microbial N flow and did not affect (P = .4) microbial efficiency. In vitro, bambermycins did not affect microbial yield (Van Nevel and Demeyer 1992) Report of effects of bambermycins on microbial growth in vivo could not be found. In summary, bambermycins had minor biologically relevant effects on ruminal or total tract digestibility. The effect on pH was small but consistent and may be more important at higher feed intakes. The increased gain observed in the performance experiment (Chapter III) can not be explained by digestibility alone. Bambermycins had a variable effect on hay intake in that experiment. Bambermycins increased total DM intake and tended to increase hay DM intake when heifers where given ad libitum access to hay. However, these effects could not be explained by changes in digesta kinetics or digestibility (Chapter IV) The effect of bambent^cins on animal performance may be also mediated postruminally This possible mode of action, however, is difficult to reconcile with variability of response, not only in these experiments but also in the available literature. If in fact bambermycins lowered maintenance energy and protein requirements through lower intestinal tissue turnover, a dietary effect would not be expected. Experiments with different levels of feeding to estimate maintenance requirement and efficiency of energy utilization may be one way to test effects on energy

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utilization. Measurement of variables associated with gut metabolism (cell turnover, oxygen consumption) and N balance would be indicated also. Molasses and corn supplements appear to elicit a slightly different response in digestibility, and this effect appears dependent on level of intake. Rate of supplement consumption may be relevant in some of the responses. This aspect has rarely been addressed in the literature and may warrant further research. Diets including corn and molasses apparently provided excess of MP, therefore energy may have been the nutrient limiting gain (Chapter III) Animal performance was similar in CC and MC indicating that the energy from corn and molasses supplements was used with similar efficiency. Apparent lower riominal digestibility of feed CP with molasses and the apparent contradiction with in situ degradation may warrant further research. The lower efficiency of microbial growth detected with molasses merits additional research. Effects of non-protein N and amino N on microbial growth efficiency should be evaluated with molasses supplements.

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CHAPTER VI SUMMARY AND CONCLUSIONS Exppr-imRnf 1 Animal Per formance (Chapter III) Yearling cattle fed bermudagrass hay during two winters and supplemented with corn DM at .65% of BW gained .047 kg/d more (P = .005) than those supplemented with molasses DM at .74% of BW, due to greater efficacy of feed additives in corn than in molasses supplements. Cattle fed corn (CC) and molasses (MC) without antibiotics gained .621 and .616 kg/d, increased height 6.18 and 5.9 cm, increased BCS .28 and .30, and consumed 1.54 and 1.64% BW of hay DM, respectively. This indicates that the energy (1.57 and 1.56 kg TDN/d) provided by corn and molasses based supplements was used with similar efficiency when no antibiotic was added. Addition of 200 mg of monensin tended to produce different ADG response when fed with corn and molasses (monensin by supplement interaction, P = .11). Monensin increased ADG .035 kg in corn and decreased ADG .029 kg in molasses supplements. Animals fed CM had .067 kg higher ADG than those fed MM. Addition of 20 mg of bambermycins tended to produce different ADG when fed with corn and molasses (bambermycins 233

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234 by supplement interaction, P = .10). Bambennycins increased ADG .106 and .042 kg in corn and molasses supplements, respectively. Cattle fed CB gained .063 kg/d more than those fed MB. Cattle fed monensin consumed .14% of BW less (P = .024) hay DM than those fed control supplements. Cattle fed bambermycins consumed 1,62% of BW hay DM, similar (P = .64) to 1.59% of BW hay DM consumed by those fed control supplements. Bambermycins increased (P = .07) hay intake in Year 1 and had no effect (P = .4) in Year 2. Monensin did not affect (P = .49) height change or BCS change (P = .67) while bambermycins did not affect (P = .49) height change but tended (P = .12) to increase BCS change (.14), when compared to control supplements. Monensin tended (P = .13) to interact with supplement type for efficiency of feed utilization. Monensin increased (P = .004) by .102 and .02 6 kg the difference between observed and predicted gain in corn and molasses supplements, respectively. Bambermycins increased (P = .013) by .063 and .041 kg the difference between observed and predicted gain in corn and molasses supplements, respectively. However, the increased efficiency with bambermycins was most apparent in Year 2 Animals fed supplements gained .4 kg/d more (P = .0001), had 1.9 cm more growth in height (P = .0001), and .72 more increase in BCS (P = .0001) than those fed hay

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235 alone. Cattle fed supplements consumed less (P = .0001) hay, [.47% BW (CB) to .70% BW (CM)], than those fed hay alone. Animals fed corn had higher (P = .0001) ruminal VFA concentrations (70.1 vs 58.7 mM) higher (P < .0001) molar proportion of branched-chain VFA (BCVFA) (1.35 vs .51 mol/100 mol), lower (P = .0001) butyrate molar proportion (9.7 vs 11.3 mol/100 mol), and a 6% higher (P = .008) acetate:propionate ratio (CjiCs) than those fed molasses supplements There was a trend (P = .118) toward a monensin by supplement type interaction for total VFA. Monensin tended to increase by 3% total VFA in corn and to decrease by 7% total VFA in molasses supplements. Bambermycins decreased (P = .011) by 13 and 3% total VFA in corn and molasses supplements, respectively. Monensin decreased (P = .0001) by 5%, while bambermycins did not affect (P = .38) acetate molar proportion in both corn and molasses supplements. There was a monensin by supplement type interaction (P = .0006) for propionate molar proportion. Monensin increased propionate molar proportion by 39% in corn and 17% in molasses supplements. Bambermycins did not affect (P = .84) propionate proportions. There was a monensin by supplement type interaction (P = .003) for butyrate proportions. Monensin decreased butyrate in corn by 16% and it did not affect butyrate in molasses supplements. Bambermycins did not affect (P = .45)

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236 butyrate molar proportions in any type of supplement. Monensin did not affect (P > .8) BCVFA molar proportion and bambermycins tended (P = .07) to increase by 61% molar proportion of BCVFA in both corn and molasses supplements. There was a monensin by supplement interaction (P = .005) for C2:C3. Monensin decreased C2:C^ by 44% in animals fed corn and by 21% in those fed molasses. Bambermycins did not affect (P = .9) C2:C^ but tended (P = .098) to interact with supplement type. Acetate: propionate (treatment) were: 4.14 (CO, 2.88 (CM), 4.33 (CB) 3.81 (MC) 3.17 (MM), 3.64 (MB), and 4.41 (hay alone). All supplements decreased (P = .0001) C2:C3 when compared with hay alone. Cattle fed corn had 2.13 mg/dL higher (P = .0001) plasma urea N (PUN) concentration than those fed molasses supplements but part of this difference may be related to sampling time after feeding. There was a trend (P = .10) toward a monensin by supplement interaction and bambermycins had no effect (P = .9) on PUN concentrations. Animals fed supplements had higher (P = .0001) PUN concentration than those fed hay alone (10.9 vs 8.0 mg/dL). Minimum mean values of 7.86 (Year 1, February) and 6.07 (Year 2, March) suggest that performance may have been limited by ruminal degradable CP in animals fed hay alone. Monensin consistently suppressed (P < .0004) coccidia counts in both corn and molasses supplements and bambermycins had no effect (P > .2) on coccidia counts.

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237 Rypf^rimf^nh 2 Intake and Diaest.ibilitv fChaPt.er IV) Heifers fed molasses consumed .15% of BW more (P = .007) total DM and .28% of BW more (P = .001) hay DM, and .13% of BW less (P = .098) supplement DM than those fed corn. Heifers fed bambermycins consumed .08% of BW more (P = .073) total DM and tended to consume .08% of BW more (P = .14) hay DM than those fed control supplements. Effects of bambermycins on hay intake resembled those observed in Year 1. Pattern of corn and molasses consumption was different, with molasses consumption distributed throughout the day while 80% of the corn was consumed in 2 h after feeding. Apparent digestibility of DM and OM was not affected by supplement type (P > .18) or feed additive (P > .8). Heifers fed molasses had 4.6 percentage units higher (P = .027) NDF and 3.9 percentage units higher (P = .084) CP digestibility than those fed corn. Heifers fed molasses had .29% of BW higher (P = .098) intake of digestible OM than those fed corn, reflecting the higher total DM intake and the lower negative associative effect on fiber digestion when molasses was fed. Bambermycins did not affect NDF (P = .9) and CP (P = .2) digestibility or digestible OM intake (P = .35). Increased efficiency of feed utilization due to bambermycins (Chapter III) can not be explained by changes in total tract digestibility. Heifers fed corn had 34% faster (P = .065) marked-hay

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238 particle passage rate out of the rumen, tended to have 32% longer (P = .13) fast compartment MRT, had 20% shorter (P = .049) slow compartment and 6% shorter (P = .028) total tract MRT, and tended to have .11% of BW higher (P = .11) hay DM fill of the fast compartment than heifers fed molasses. Higher hay intake in animals fed molasses may be related to a trend (P = .13) for shorter hay particles MRT in the agedependent pool (mixing, rumination, comminution, fermentation), a trend for .16% BW higher ruminal fill (P = .17), and longer ruminal (P = .049) and total tract MRT (P = .028). Higher intake was not associated with higher ruminal passage rate of undigested hay DM. Bambennycins depressed (P = .018) ruminal passage rate by 11% and increased (P = .006; 1.5 h) ruminal MRT by 7% in heifers fed the corn supplement (supplement by bambemycins interaction, P < .09). This effect on digesta kinetics was small, it was detected only with the linear model, and is probably biologically non relevant. Increased intake in growing cattle fed bamberrrr/cins can not be explained by digesta kinetics or digestion. In general, trends in digesta kinetics due to bambennycins were greater in heifers fed corn compared to those fed molasses. Experiment 3-Riimen Function an d P-i aestibilitv (Chapter V) Pattern of corn and molasses consumption was similar, probably due to feed restriction.

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239 Steers fed molasses had 1.1% of BW higher (P = .055) ruminal volume and .19% of BW higher (P = .019) total hay DM fill than those fed corn, suggesting less ruminal motility. There was no effect (P > .17) of bambermycins on fluid or hay particles kinetics, except that it tended (P = .095, supplement by bambermycins interaction) to decrease ruminal DM fill .29% of BW in steers fed corn and to increase riominal DM fill .21% of BW in those fed molasses. Ruminal pH, total VFA, acetate, propionate, butyrate molar proportions and acetate to propionate ratios exhibited a supplement type by time of sampling interaction (P < .07). Postprandial changes reflected the different rates of digestion of sugars (faster) and starch (slower). Steers fed molasses had lower (P < .025) acetate molar proportions at 2, 4, and 6 h post feeding, higher (P < .08) propionate molar proportions at 2, 4, and 6 h post feeding, lower (P < .05) acetate to propionate ratios at 2, 4, and 6 h post feeding, compared to those fed corn. Butyrate molar proportion was higher (P = .065) in steers fed molasses at 6 h postfeeding but lower (P = .096) at 24 h postfeeding compared to those fed corn. Averaged across time, steers fed molasses tended (P = .11) to have lower acetate (71.3 vs 72.9 mol/100 mol) had higher (P = .08) propionate (16.1 vs 14.8 mol/100 mol), had lower (P =.097) acetate to propionate ratios (4.57 vs 4.99), and tended (P = .12) to have lower branched-chain VFA (1.31 vs 1.61 mol/100 mol) than those fed corn supplements.

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240 Lactic acid was detected only in animals consuming molasses and it was present as a transient peak shortly after feeding, probably related to high rate of intake of molasses and high rate of sugar fermentation. Steers fed bambermycins had higher (P = .046) pH (6.63 vs 6.52) averaged across time and lower (P < .05) butyrate molar proportions at 12, 16, and 24 h postfeeding compared to those fed control supplements. Averaged across time steers fed bambermycins tended (P = .12) to have lower butyrate (9.8 vs 10.6 mol/100 mol) than those fed control supplements. Bambermycins did not affect (P = .88) acetate to propionate ratio. Ruminal ammonia N decreased with time after feeding. Postprandial peak of ammonia N was earlier and tended to be higher (P = .12) in steers fed corn compared to those fed molasses (27 mg/dL at 2 h vs 21 mg/dL at 4 h postfeeding) The lowest ammonia N concentrations were observed at 12 h postfeeding with both corn and molasses, and were lower (P = .08) in animals fed molasses (3,3 vs 4.4 mg/dL) than in those fed corn. Ammonia N concentration was also lower (P < .05) at 20 h (5.5 vs 7.0 mg/dL) and 24 h (5.1 vs 8.0 mg/dL) postfeeding in animals fed molasses. Effect of supplemental energy source on ruminal fermentation appears more related to timing of events rather than great differences in total VFA concentration or VFA molar proportions. This is consistent with similar gains

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241 observed in animals fed CC and MC (Exp. 1) Overall, effects of bambentiycins on ruminal fermentation evaluated though pH, VFA and ammonia concentration showed only minor changes which would not suffice to explain the increased performance observed when bambermycins was added to supplements in Exp. 1. Animals fed molasses had lower (P = .0003) ruminal Na (78 vs 105 mM) and higher (P = .003) ruminal K (71 vs 42 mM) concentrations than those fed corn. Ruminal Na increased and ruminal K decreased with time after feeding in steers fed molasses Rate of DM digestion of CGM was higher when incubated in situ in animals fed molasses (P = .034) or bambermycins (P = .013) than when incubated in steers fed corn or control supplements. Bambentiycins tend (P = .13) to increase CP rate of degradation compared to control supplements (2.17 vs 3.22). Extent of CP degradation was higher (P < .08) after 18, 24, and 48 h of ruminal incubation in animals fed molasses compared to those fed corn. Bambermycins increased (P < .07) the extent of CP degradation after 18, 24, 36, and 48 h of ruminal incubation compared to control supplements. Apparent ruminal OM digestibility was higher (P = .098) in steers fed molasses than in those fed the corn supplement (42.9 vs 39.4%). Ruminal NDF digestibility was not affected by supplement type (P = .89) or bambermycins (P = .9). Steers fed corn had higher (P = .015) ruminal feed CP

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242 degradability (69.2 vs 58.9%). Pre-duodenal apparent and true OM digestion (% of total tract) was higher (P < .02) in steers fed molasses than in those fed corn. Steers fed corn had higher (P < .013) intestinal digestion (% entering) of DM and OM than those fed molasses. Steers fed corn had higher (P < .035) total tract DM (65.4 vs 63.2%), OM (68.0 vs 64.5%), NDF (61.0 vs 58.1%), and CP (64.8 vs 62.1%) digestibility than those fed molasses. Bambermycins did not affect (P > .2) DM, OM, NDF or CP ruminal or total tract digestibility. However, bambermycins tended to increase ruminal feed CP degradability (62.0 vs 55.9 %) and decreased (P < .05) intestinal CP digestibility (64.7 vs 67.1%) in molasses but not in corn. Non-ammonia N flow was higher than N intake in all treatments indicating that recycled N was captured in microbial protein. Flow of NAN expressed as percent of N intake was not affected by supplement type (P .43) or feed additive (P = .35) Steers fed molasses had higher (P = .05) NANMN (53.9 vs 45.8%) and higher (P = .015) undegraded feed N flow (41.1 vs 30.8%) than those fed corn, expressed as percent of intake. Bacterial N flow was higher (P = .053) in steers fed corn than in those fed molasses (100 vs 91.1 g/d) Lower bacterial N flow in steers fed molasses is not consistent with benefits of synchronization of N availability and carbohydrate fermentation in the rumen.

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243 Bamberitr/cins tended (P = .11) to decrease total duodenal N flow (179 vs 189 g/d) increased (P = .093) ammonia N flow (8.0 vs 7.1 g/d), and decreased (P = .077) NAN flow (171 vs 182 g/d) compared to control supplements. Steers fed corn had higher (P = .019) apparent (3.59 vs 2.99 g N/100 OM) and higher (P = .033) true (2.47 vs 2.19 g N/100 OM) microbial N efficiency (g N/100 OM fermented in the rumen) Microbial N efficiency expressed as percent of OM digested in the total tract or as percent of calculated TDN intake was not affected by supplement type (P > .5) or bambermycins (P > .26). It is likely that during sugar fermentation the supply of amino N (peptides, amino acids) may have been insufficient to balance the anabolic and catabolic rates of fast growing bacteria under excess energyconditions. Consequently, part of the energy available from fermentation may have been spilled. Prediction of MP supply from diets used in Exp. 1 indicates that energy, not protein was limiting gain. Under these circumstances, the lower efficiency of microbial growth may not be relevant Bambermycins tended (P = .16) to decrease microbial N flow and did not affect microbial N efficiency. Bambermycins had minor biologically significant effects on ruminal or total tract digestibility. The effect on pH was small but consistent and may be important at higher feed intake. The effect of bambermycins of increasing situ extent of

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244 digestion at long incubation times, higher ruminal feed N digestion, and lower bacterial N flow may be relevant in diets that include molasses. The increased gain due to bambermycins in Exp. 1 can not be explained bydigestibility. Effect of bambermycins on animal performance may be mediated postruminally More research is needed to elucidate the mechanism of action of bambermycins. rnnclusions Supplemental energy supplied by corn and molasses without feed additives was used with similar efficiency for gain. Monensin was not effective for improving gain in growing cattle fed bermudagrass hay and supplemented with molasses slurry DM at .7% of BW. Bambermycins improved gain by 17% and 7% when included in corn and molasses supplements, respectively. Because of a trend toward a supplement by bamberm/cins interaction, definitive conclusions on the efficacy of bambermycins to improve gain in molasses supplemented-diets can not be made and further research is needed. Monensin depressed hay intake while bambermycins either had no effect or tended to increase hay intake, which indicates that bambermycins should be used when maximum use of forage resource is the objective. Monensin and bambermycins improved feed efficiency.

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245 However, bamberrrtycins increased gain by increasing hay intake and feed efficiency in Year 1, and by increasing feed efficiency in Year 2. Effect of bambemrycins on gain does not appear mediated through notable changes in ruminal fermentation, digesta kinetics or digestibility. Increased ruminal pH and extent of CP degradation in situ, and decreased butyrate molar proportion were the only significant effects of feeding bambennycins Steers fed molasses had higher ruminal propionate and butyrate, lower acetate and branchedchain VFA molar proportions, lower acetate to propionate ratio, and lower ruminal ammonia concentrations than those fed corn supplements Ruminal feed CP digestibility, duodenal microbial N flow, and microbial N efficiency were lower in cattle fed molasses-based supplements compared to cattle fed corn-based supplements. Lactic acid was present only in animals fed molasses. Riaminal fluid volume and DM fill were higher in animals fed molasses. More research is needed to evaluate these changes in animals fed at production levels of intake. Ruminal Na concentration was lower and K was higher in animals fed molasses than in those fed corn. At restricted intake, total tract nutrient digestibility was higher in animals fed corn than those fed molasses. At ad libitum hay intake, NDF digestibility was

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higher in animals fed molasses, hindered by lower molasses than were given ad libitum access to 246 However, this conclusion is corn intakes when animals hay.

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APPENDIX A TABLES Table A-1. Monensin concentration (g/ton) in supplements Exp. 1 Date sampled Corn^ Corn gluten meal^ Expected Analysis Expected Analysis Year 1 Dec-14-94 84 91 640 645 Dec-29-94 84 80 640 639 Jan-16-95 84 105 640 575 Jan-26-95 84 74 640 685 Feb-07-95 84 83 640 636 Feb-19-95 84 83 640 555 Mar-07-95 84 84 640 549 Mar-20-95 84 88 640 629 Year 2 Jan-04-96 84 85 640 623 Jan-18-96 84 83 Feb-01-96 84 86 640 570 Feb-15-96 84 82 640 586 Mar-05-96 84 82 640 611 Mar-14-96 84 91 640 606 Apr-01-96 84 77 Year 1: Corn mixed on 11-30-94, 12-13-94,1-4-95, 1-19-95, 1-3195, 2-21-95, and 3-14-95. Corn gluten meal mixed on 11-30-94, 15-15-94, and 1-12-95. Corn gluten meal was 10.4% of molasses slurry. Year 2: Corn mixed on 12-14-95, 1-4-96, 1-24-96, 2-14-96, 3-7-96, and 328-96. Corn gluten meal mixed on 12-14-95 and 2-1-96. Corn gluten meal was 10.4% of molasses slurry. 247

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248 Table A-2. Bambennycins concentration (g/ton) in supplements Exp. 1, Year 1. Corn^ Corn gluten meal^ Date sampled Expected Analysis Expected T3*"i Y*Qh r J. X. o analysis cpapnnd analysis Dec-14-94 8.4 6.7 64 7.1 53.4 Dec-29-94 8.4 6.7 64 7 5 Jan-16-95 8.4 7.5 64 7.2 52.3 Jan-26-95 8.4 6.9 64 7.6 51.7 Feb-07-95 8.4 7.0 64 7.2 52.8 Feb-19-95 8.4 7.3 64 7.3 52 .3 Mar-07-95 8.4 6.8 64 7.5 51.7 Mar-20-95 8.4 7.5 64 7.6 52.3 Corm mixed on 11-30-94, 12 -13-94, 1-4-95, 1-19-95, 1-31-95, 221-95, and 3-14-95. Corn gluten meal mixed on 11-30-94, 15-15-94, and 112-95. Corn gluten meal was 10.4% of molasses slurry. Original drug premix analyzed in June/96 tested 100% effective. Table A-3. Bambermycins concentration (g/ton) in supplements Exp. 1, Year 2. Corn^ Corn Gluten meal^ First and Date First Second second sampled Expected analysis analysis Expected analysis Jan-04-96 8.4 < 2.5 7.9 64 < 25 Jan-18-96 8.4 < 2.5 8.0 64 < 25 Feb-01-96 8.4 < 2.5 7.7 64 < 25 Feb-15-96 8.4 < 2.5 < 2.5 64 < 25 Mar-05-96 8.4 < 2.5 < 2.5 64 < 25 Mar-14-96 8.4 < 2.5 < 2.5 64 < 25 Apr-01-96 8.4 < 2.5 < 2.5 64 < 25 Corn 28-96. Corn mixed on 12 gluten meal -14-95, 1-4mixed on 12 -96, -14195 24-96, and 22-14-96, 3 1-96. Corn -7-96, and 3gluten meal was 10.4% of molasses slurry. Original drug premix analyzed in June/96 tested 100% effective.

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249 Table A-4. Bambermycins concentration (g/ton) in supplements Exp. 2 Corn^ Corn gluten meal^ First OCCOIIU. First Second Period Expec analysis analysis Expec analysis analysis 1 old 9 8.3 71 < 25 1 new 9 < 2.5 71 < 25 2 9 < 2.5 8.5 71 < 25 < 25 3 9 < 2.5 71 < 25 4 9 < 2.5 71 < 25 a Corn mixed on 9-30-95 and 11-14-95. Corn gluten meal mixed on 10-1-95, 11-20-95, and 12-27-95. Expec = expected concentration. Original drug premix analyzed in June/96 tested 100% effective. Table A-5. Bambermycins concentration (g/ton) in supplements Exp. 3 Corn^ Corn gluten meal^ First Second First Second Period Expec analysis analysis Expec analysis analysis 1 9 8.3 71 62.9 2 9 8.4 8.9 71 64.2 3 old 9 8.3 71 62.9 3 new 9 < 2.5 71 < 25 4 9 < 2.5 8.5 71 < 25 64 ^ Corn mixed on 9-30-95 and 11-14-95. Corn gluten meal mixed on 10-1-95, 11-20-95, and 12-27-95. Expec = expected concentration. Original drug premix analyzed in June/96 tested 100% effective.

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252 4J U Q) m (d jj a o u Eh X Eh U in U o o o o o o I I I I I I I I og cr\ CTl 00 U3 rg o rH rg o • I I o o o I I I iH iH O O O O O O O O O I I I I I I I I O O O O O O 00 o 00 o H O 00 o o I I I I O VO (N UD rH 00 O Og I rn ^ o o o m o o en o 00 CN CTl 00 in 00 og ro o in in rH in ro CN CN ro 00 ro in CN in o CO a\ og CN cr> in in CN cr\ ro CN CTl in o CN r~1 CTl o o 00 in m in in CN CTl ro ro r~ro 00 in o CN og VD (Tl rH vo a\ CN ro cr\ 00 rH ro rH r~CN 00 00 a\ in 00 cr\ in rH o o ro o ro rH og 00 in <>D >X> CTl CO in rH o 1 CN o o o cn rH • 00 00 CN O CN ro 00 CN o CN • rH ro >x> CTl rH rH 00 rH O 00 in o ro CTl 00 ro ro rH o o rH ro ro ro in ro CTl l> [> in rH CN o rg a\ t> I> CN rH in CN in 00 o in 1 00 CN o rH rH o o o CTl 00 o X) CN in cr> 00 rin rH CN rH in o a\ 00 CN 00 00 CTl o CTl in ro o CN o in U) rH 00 CTl CTl CTl ro > CTl in o o 1 ro in o CN rH 00 l> o o o CN O CN O o rH rH o in CN o ro rH n rH o in in rH rH rH rH O o a\ rH rH rH VD ^ o o cr\ m m o o ro o in O ro o rH 00 o in 00 o o o o o ro in O CN 1 o O o ro o m CN CTl cri o O O O 00 ro en O o O rH o • g 0) u ak U •H 0) Q \ PQ C M P JJ (d (d o td a x: d T) 4-) < u T5 Q) < U a (d 73 m O ^ o Q Q rH (d e \ 4J Eh Eh > rH X! x: QJ 2 2 Q u rH (0 u -rH Q a rH Q Q MH Eh 4H rH rH rH •H (d -H (d 0 0 (d (d (0 4-) U u -H w >< 012 ^ Dj tJ) O) U) 4-) Q) (0 (U u (d ^ Q u ID M dP 0 o O U > CO pq tH Eh Eh <: a* (d 0) (d fa 2

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255 u 0) 4-1 m (0 a o u Eh •H U5 U ID U u U O U rH (d •H > O P g u a u ^: -H 0) I I I I I I o in I I I I I I I I I I I I o ID O O I I G) C U CO u CQ 4-) \ H Q 5 CQ 0) (0 Q 0) (T3 o JJ a Q) ^ s § .H P Q M-l a o a O) di :3 CO p E-" o 4-1 o D1 IS p p Eh Eh 03 (0 o o Eh Eh O •H o > •• u n3 O Eh o d s-i o 00 o o o rH ro CN CN og LD LD ro og O ro o ro o o CN O 1X> CN 1 CD ( — i 1 1 1 1 1 1 1 O LD o o tH o o o o OJ O rH o ro 00 rH 1 rH ro 00 CN o o 00 >X) ro ro O ID oq o ro 00 ro 00 00 o t> o o L f — 00 ro CTi ro o ro ro ID rH O rH V£) rH rH CN 00 rH rH o ID rH o oq CN rH ro '3' ID cr> CN CN ID ro rH ID o o 00 O rro cn O cr> CN CN O 00 00 O vo LD 00 rog ID 00 CTl 00 CO O 00 O 00 cn ro LD ro ro og rrH rH o O o LD rH 00 00 00 00 CO 00 o IX> ro ro O ro OJ crv O rH cn o CTl ro ID O ID O • ID ID ro cn 00 rH rH ro vo ro CN ro ro O VD vo O rH rH CN rrH rH cn (N VX) rCTi o\ ID LD cn rH ro ro o ro ro ID CTi a\ O CN O ID U) CN o ro o '^f ^ og iH o o O CN O CN rH O o o (N cn ro U) o o rH rH ro ro ro CN 00 CN CN CO CO r00 rH CT\ "^f O O LD CN a\ ro CTi ro >x) og 00 'J' O O 00 r00 cn og rH rH o eg 00 iH rH rH o o OJ ro rH rH rH o O o o rH h (0 (d Q) 5 i (0 u ac T3 CJ e OQ 0) o J-' M 0) • iJ rH — g Q) O • TJ 0 0) 4J O to O C • > rH 01 II nj e aB) (1) 0) o •rl G O Eh -H m > X. 0) 0) (0 (S 0) Eh cn rH a\ 00 CN o • m 0) 0) rH 4J rH ja (C (0 •rl >H rH m rH > ns 2^ 8^' 0) 00 01'-' f8 <^ X! • U C -H m a < 11H ^ l*J 4-) C X 0) g 0) U to ^ g u 0 0) X D cn O C Z m II g II OJ u m <" (U JJ (0 73 X rH in 0< CO < 4J >1 (1) i< rH O) 0) > ftb01 > 0) u u O -iJHffl 0) rH (fl 0) JJ T5 H g u

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APPENDIX B RAW DATA riRtrt Cnrips fnr P prf nrmanrPi Experiment ID YEAR TRT BRD SWTIKG SWT2KG HTl HT2 CSl CS2 ADG AVGWT HAYDM SUPPDM PRDADG ACET PROP BUTY RATIO TOTAL = cattle ear tag number 1 = Dec-1994 to Mar-1995, at Pine Acres 2 = Dec-1995 to Apr-1996, at Santa Fe = treatment 1 = corn control 2 = corn monensin 3 = corn bambermycins 4 = molasses control 5 = molasses monensin 6 = molasses bambermycins 7 = hay alone = breed = initial shrunk body weight in kg = final shrunk body weight in kg = initial hip height in cm = final hip height in cm = initial body condition score (1 to 9 scale) = final body condition score (1 to 9 scale) = average daily gain in kg = average shrunk body weight (initial + final) /2, kg = hay dry matter intake in kg = supplement dry matter intake in kg predicted average daily gain from NRC (1996), kg = acetate molar proportion in rumen fluid = propionate molar proportion in riimen fluid = butyrate molar proportion in rumen fluid = acetate to propionate molar ratio = total volatile fatty acids in rumen fluid, mM PUN = plasma urea nitrogen in mg/dL 256

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257 naf.a Codps for In fakp and ni gpst ihi 1 i tv Heifers PER = period of the Latin Square AN = animal number SUPP = supplement type: c = corn m = molasses ADD = bambermycins c = control, no bambermycins b = 20 mg bambermycins TDMI = total dry matter intake, kg TOMI = total organic matter intake, kg TNDFI = total neutral detergent fiber intake, kg TCPI = total crude protein intake, kg TTDNI = total digestible nutrients intake, kg SDMI = supplement dry matter intake, kg SOMI = supplement organic matter intake, kg SNDFI = supplement neutral detergent fiber intake, kg SCPI = supplement crude protein intake, kg STDNI = supplement digestible nutrients intake, kg FDMOUT = fecal dry matter output, kg FOMOUT = fecal organic matter output, kg FNDFOUT = fecal neutral detergent fiber output, kg FCPOUT = fecal crude protein output, kg CZERO = ytterbium concentration at time = 0 LAMBDA = passage rate parameter of marker from the age-dependent compartment Ks = passage rate of marker from the ageindependent compartment DOSE = ytterbium dosed, mg TD = time delay for first appearance of marker in feces, hours K-LN = passage rate of marker from the rumen estimated with linear model (slope of regression)

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258 naf a CndPH fnr Rni n pn Fnnr^^nn and Pi aestibi] 1 Steers PER = period of the Latin Square AN = animal niunber SUPP = supplement type: c = corn m = molasses ADD = bambermycins c = control, no bambermycins b = 30 mg bambermycins DMI = total dry matter intake, kg OMI = total organic matter intake, kg NDFI = total neutral detergent fiber intake, kg NI = total nitrogen intake, g DMFW = dry matter flow at duodenum, g OMFW = organic matter flow at duodenum, g NDFFW = neutral detergent fiber flow at duodenum, g TNFW = total nitrogen flow at duodenum, g NH3FW = ammonia nitrogen flow at duodenum, g MNFW = microbial nitrogen flow at duodenum, g DMOUT = fecal dry matter output, kg OMOUT = fecal organic matter output, kg NDFOUT = fecal neutral detergent fiber output, kg NOUT = fecal nitrogen output, kg CZERO = ytterbium concentration at time = 0 LAMBDA = passage rate parameter of marker from the age-dependent compartment Ks = passage rate of marker from the ageindependent compartment DOSE = ytterbiiam dosed, mg TD = time delay for first appearance of marker in feces, hours

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259 Raw Data Performance Experiment ID YEAR TRT PEN BRD SEX SWTIKG SWT 2 KG HTl HT2 CSl CS2 J J ^ Kf \J ^ \J 1 1 2 2 5 1 282 358 124.5 133.4 5.8 5.0 3940093 1 1 2 4.0 1 225 278 118.1 124.8 5.5 5.5 3940103 1 1 2 4.0 2 249 292 125.4 131.1 5.2 5.8 3940115 1 1 2 1.0 2 220 284 111.5 119.1 5.2 4.8 3940174 1 1 2 3.0 1 241 307 116.5 124. 5 5.4 5.5 3940906 1 1 2 2.0 2 215 272 111.5 115.9 5.2 6.0 3940008 1 1 6 2.0 2 305 363 128.0 132.7 5.0 6.2 3940025 1 1 6 1.0 1 224 314 113.0 122.3 5.8 5.5 3940027 1 1 6 4.0 2 266 308 121.3 126.4 6.2 6.2 3940069 1 1 6 4.0 1 288 317 122.6 130.2 6.8 5.8 3940142 1 1 6 2 5 1 218 268 118.1 125.1 5.0 5.2 3940909 1 1 6 2 0 2 236 305 122.6 126.1 5.6 5.5 3940004 1 1 20 2 0 1 301 372 129.5 136.9 4.8 5.5 3940137 1 1 20 3.0 1 248 307 128.0 133 .7 5.8 5.2 3940169 1 1 20 2.0 2 190 258 108.9 118.1 5.0 5.2 3940180 1 1 20 5.0 1 222 266 121.3 129.2 5.0 5.5 3940908 1 1 20 5.0 2 213 264 115.9 123 .2 6.2 6.0 3940910 1 1 20 2.0 2 322 373 127 6 128.9 6.2 6.5 3940012 1 1 28 1.0 2 247 333 114.6 122.3 5.2 6.2 3940014 1 1 28 4 0 1 300 356 128.9 134.0 4.8 5.8 3940088 1 1 28 2 5 2 222 298 115.6 122 .3 5.0 6.0 3940106 1 1 28 4 0 2 208 268 115.3 127.0 5.5 6.2 3940131 1 1 28 1.0 1 223 298 114.0 122.9 4.8 5.2 3940903 1 1 28 2.0 2 193 227 110.8 115.6 5.2 5.2 3940047 1 2 10 2.0 2 296 373 120.7 128.0 6.0 4.8 3940092 1 2 10 1.0 1 236 313 118.1 124.8 5.0 3940096 1 2 10 3 0 2 277 341 121.9 127.0 6.5 6.0 3940108 1 2 10 3.0 1 227 298 122.6 129.9 5.0 5.0 3940117 1 2 10 5.0 1 241 300 118.7 124.8 6.0 6.2 3940907 1 2 10 5.0 2 260 319 118.7 126.7 5.5 5.8 3940016 1 2 14 3.0 1 283 353 123.2 130.8 5.2 6.2 3940024 1 2 14 4.0 2 266 322 127.0 133.0 6.0 6.2 3940028 1 2 14 3.0 2 282 345 117.5 123.5 6.5 6.5 3940046 1 2 14 1.0 1 234 307 113.0 120.0 4.8 5.2 3940189 1 2 14 5.0 1 184 232 117.8 125.1 5.2 5.2 3940901 1 2 14 2.0 2 265 313 114.3 120.0 5.3 5.5 3940098 1 2 22 2.5 2 259 342 122.3 126.4 5.4 6.5 3940099 1 2 22 5.0 2 203 251 119.1 122.3 4.8 5.8 3940104 1 2 22 4.0 1 253 317 120.7 127.0 5.7 5.5 3940107 1 2 22 1.0 2 249 335 116.2 122.3 4.8 6.0 3940114 1 2 22 3.0 2 278 346 116.5 127.3 5.8 6.5 3940126 1 2 22 2 5 1 221 283 113.7 123 .2 5.7 5.5 3940026 1 2 24 1.0 2 237 324 114.9 121.9 5.0 5.8 3940065 1 2 24 2.5 2 251 318 117.2 122 .9 6.2 6.5 3940078 1 2 24 2 5 1 290 412 130.2 137.5 5.5 6.5 3940171 1 2 24 3 0 2 200 272 113.0 121.9 4.8 6.0 3940188 1 2 24 4.0 1 205 282 116.2 124.2 5.2 5.8 3940911 1 2 24 5.0 2 228 273 116.5 123.5 6.5 5.8 3940017 1 3 12 3.0 2 298 354 127.0 131.8 6.2 6.2 3940037 1 3 12 4.0 1 286 350 124.2 128.9 5.7 6.0 3940038 1 3 12 1.0 2 248 334 119.4 122.9 5.5 6.5 3940147 1 3 12 4.0 2 222 283 117 .2 125.1 5.5 6.2 3940152 1 3 12 2.5 1 257 331 130.8 136.9 5.2 4.8 3940160 1 3 12 2.5 1 209 275 114. 6 123.8 5.2 5.2 3940018 1 3 17 1.0 1 249 337 114.9 123 .2 5.8 5.8 3940035 1 3 17 1.0 2 233 312 114.9 121.9 5.1 6.2 3940120 1 3 17 5.0 2 208 261 116.8 120.7 5.8 5.5

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260 Raw Data Performance Experiment lU TRT PPM BRD SEX SWTIKG SWT2KG HTl HT2 CSl CS2 J y 4U Iz 0 1 J 1 7 2 231 313 117.8 124.8 6.2 6.2 1 X 3 J 1 7 5 0 1 297 373 123 .2 131.8 6.0 6.5 3 y 4U J. 4 D 1 X 3 1 7 3 0 1 232 307 126.4 135.3 4.8 4.2 J y 4UU / D X -J 1 9 1 0 2 198 289 108.3 117.5 4.8 5.2 J y 4UU y 1 X 3 -J 1 9 X J 4 0 1 298 385 128.9 136.2 5.6 6.5 J y 4U 1 lu 1 3 J 1 Q X ^ 2 228 285 108 6 111.5 5.8 6.0 J y 4U 11 b 1 3 J 1 9 X 7 1 244 327 119.4 129.5 5.0 5.5 J y 4U1 b D 1 •3 J 1 Q X J7 4 n 2 210 276 113 .0 120.3 5.5 6.5 J y 4U 1 0 ^ 1 3 J 1 9 X J 1 220 286 110.2 120.0 5.6 6.0 1 Q >l A 1 "7 A ly 4U 1 / u X 3 J £t f 1 0 2 213 297 110.8 117.8 5.2 6.2 O Q /I A A A C: J y 4 U U U D X 3 J 27 2 5 2 288 349 123.5 130.8 5.2 5.8 "2 Q >1 A A "7 n o y 4 u u / u 1 X 3 J 77 5 0 1 276 355 124.5 133.7 5.4 6.2 J Q >i A A T tf; J y 4u u / D X 3 ?7 3 0 2 232 311 120.7 126.4 4.3 6.5 "5 Q 4 A m "7 J y ft u u / / 1 X 3 27 1 0 1 198 295 112.4 125.1 4.7 5.5 1 Q 4 m in J J ft U 1 / u 1 X -J J 27 4 0 2 233 295 116.8 126.7 6.2 6.2 •3 Q y1 A A 4 Q J y ft U U fi O 1 X A 4 3 0 2 305 357 128.9 135.0 6.2 6.5 J J ft U U D Z X A 4 2 5 1 272 351 123.5 132.7 5.2 5.5 O Q i1 A A C Q o y ftu u D o 1 X A 4 3 0 1 258 331 119.7 127.0 6.0 5.8 O Q ^ A AT /I o y 4 u u / ft 1 X A *4 • 1 0 2 211 243 110.5 117.5 5.2 5.2 J y 4U 1 D D X A ** 4 0 1 229 290 120.7 129.5 5.6 6.2 O Q y1 A Q 1 J y 4 u J 1 o X A ** A 5 0 2 240 280 120.3 126.1 5.8 5.8 O Q j1 A A 0 n J y 4 u uz u 1 X A 7 2 0 2 260 319 117 8 123.8 6.2 6.0 J ^ ft U U ft 3 1 X A ** 7 1 0 1 280 346 121 6 130.5 5.0 5.5 1 X 4 7 3 0 1 266 320 118.4 126.7 5.2 5.8 3 Q 4 A1 "5 1; J _7 *4 U J. J 3 1 X 4 7 4 0 2 238 307 119.7 128.6 5.5 5.8 O -7 ft w i -J O 1 4 7 5 0 1 214 268 118.1 121.9 5.8 5.8 ^ -7 U ^ X *S 1 4 7 2 0 2 214 279 110.2 117.5 5.2 5.2 O Q 4 A A1 A J 17 V W i W 1 4 9 3 0 2 299 360 124.8 129.2 6.5 6.5 ibJ W \/ ^ ^ 1 4 9 1 0 2 210 268 110.2 118.7 4.5 4.8 3940041 1 4 9 5 0 1 242 282 122.6 129.2 4.5 5.2 ^940061 1 4 9 2 5 1 281 344 127.3 133.0 5.2 5.5 3940175 1 4 9 5 0 2 208 245 121.0 126.1 5.2 5.2 3940185 1 4 9 3 0 1 220 284 120.7 130.0 4.7 4.8 3940011 1 4 26 2 5 2 259 330 119.1 123.8 4.8 5.8 3940081 1 4 26 2 0 2 271 311 120.3 126.7 4.5 5.5 3940100 J* ^ V/ i v 1 4 26 1 0 1 240 321 117 .2 123.5 5.6 5.5 3940121 1 4 26 2 0 2 252 308 112.1 123.2 5.8 5.8 3940195 1 4 26 5 0 1 223 268 118.1 126.7 5.7 6.0 J J 'i\J J v J 1 4 26 5 0 2 255 315 124.8 131.1 5.2 5.8 X ? *X VJ ^ V,/ 1 5 5 1 0 2 256 313 113 4 119.4 5.2 5.8 J J •* VJ U J o 1 5 5 3 0 1 254 319 125.1 132.7 5.5 5.2 3940084 1 5 5 5 0 2 210 245 113.0 120.3 5.2 5.5 J J *X VJ X ^ o 1 5 5 2 5 1 237 298 119.1 129.7 5.5 4.8 J J 'X^J A. ^ -7 1 5 5 3 0 2 221 261 116.2 122.3 5.7 5.8 9401 99 1 5 5 5 0 2 208 277 118.1 126.7 5.0 5.8 1 9401 7 1 X 5 g 1 0 1 230 288 116 8 124.5 5.8 4.8 3940051 1 5 8 4 0 2 284 313 123 .2 129.5 6.0 5.5 3940066 1 5 8 3.0 2 225 288 117.8 122.9 5.7 5.8 3940113 1 5 8 2.0 2 256 326 121.3 127.6 5.8 6.2 3940179 1 5 8 3.0 1 224 282 122.9 132.1 4.2 4.2 3940183 1 5 8 4.0 1 236 277 113.4 127.3 5.8 5.8 3940039 1 5 13 2.5 1 240 310 121.3 128.6 5.2 5.2 3940054 1 5 13 2.0 2 298 363 123.5 129.9 5.0 5.5 3940094 1 5 13 5.0 2 233 262 122.6 128.0 5.4 5.2 3940097 1 5 13 3.0 2 221 261 104.8 108.0 6.5 6.2 3940141 1 5 13 3.0 1 225 307 117 .5 126.1 4.5 5.2 3940145 1 5 13 4.0 1 259 337 125.1 130.5 4.2 5.0

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261 Raw Data Performance Experiment TD YEAR TRT PEN BRD SEX SWT 1 KG SWT2KG HTl HT2 CSl CS2 1940037 1 5 21 1 0 2 210 271 111.1 119.7 4.8 5.0 J J w u w u 1 5 21 3 0 2 316 356 127.0 131.8 5.7 6.2 O J \J -J ~J 1 5 21 1 0 1 217 271 114.0 121.3 5.5 5.0 J 17 ri U w O O 1 5 21 3 0 1 222 294 116.8 122 6 5.8 5.2 O _7 fi U J. O 1 J. 5 21 4 0 2 215 268 111.5 118.1 6.2 6.0 O U J. J 1 5 21 5 0 1 225 272 120.7 127.6 5.4 5.8 X J r I./ ^ o 1 6 3 1 0 2 258 316 117.5 122.9 5.7 5.8 J y *\j \j ^ £t 1 6 3 1 0 1 252 309 112.1 121.6 6.5 6.5 J ^ T U U J o 1 6 3 3 0 2 244 312 121.0 128.0 5.4 5.2 ^ J7 'A V J. V/ ^ ]_ 6 3 4 0 1 212 280 109.2 118.1 5.8 6.2 J _7 f4 V J. V/ J 6 3 5 0 2 209 282 117.5 123.5 5.5 6.5 J ^ W X / ^ 1 6 3 3 0 1 240 325 123.5 133.4 5.2 5.8 X J *• V V/ W J 1 6 11 1 0 2 251 321 112.4 124.2 5.0 5.5 J ^ w V ** 1 6 11 3 0 2 264 317 119.4 128. 6 6.5 6.2 "940060 J J ^ \J \J \J \J 1 6 11 2 0 1 305 366 126.7 131.4 6.2 6.2 3940130 1 6 11 5 0 1 236 285 120.0 128.3 5.0 5.2 3940143 1 6 11 4.0 2 221 258 119.4 128. 6 5.8 5.2 3940149 1 6 11 3 0 1 251 321 114.0 122.6 6.2 6.5 3940089 J J ** U V/ t> J 1 6 15 1 0 2 260 323 118.1 122.9 5.0 6.0 3940112 1 6 15 4 0 2 238 302 121.3 130.5 5.8 6.2 3940127 6 15 3 0 2 188 273 108.6 119.7 4.2 5.5 3940153 1 6 15 2 5 1 238 313 120.7 129.5 5.2 5.5 3940164 1 6 15 5 0 1 229 280 123.2 126.4 5.5 6.2 3940177 1 6 15 3 0 1 229 298 121.0 129.5 5.2 5.2 3940003 1 6 25 2 0 2 306 377 121.6 127 .3 5.8 6.8 3940033 1 6 25 1 0 2 186 256 110.2 115.9 4.5 5.5 3940042 1 6 25 3.0 2 298 351 122.9 126.7 6.5 6.5 3940059 1 6 25 2.5 1 254 317 120.7 124.8 5.5 6.0 3940111 1 6 25 4.0 2 221 278 116.5 124.8 5.8 5.8 3940132 1 6 25 4.0 1 221 288 113 .0 124.2 4.8 5.5 3940013 1 7 1 1.0 1 238 259 121.0 127.0 5.2 4.5 3940015 1 7 1 4.0 2 269 295 121.6 128.6 6.2 5.8 3940022 1 7 1 2.0 2 281 295 115.9 121.0 6.5 6.2 3940124 1 7 1 5.0 1 250 262 126.1 132.9 5.5 5.5 3940187 1 7 1 3.0 1 201 218 118.1 123.2 5.2 4.2 3940902 1 7 1 2.0 2 239 231 118.7 121.0 5.2 4.5 3940031 1 7 16 4 0 1 276 286 119.4 123.2 5.8 5.8 3940072 1 7 16 3 0 2 261 277 121.9 123.8 6.8 5.8 3940073 1 7 16 2.0 2 249 265 119.4 127.6 5.0 5.0 3940159 1 7 16 2 0 1 208 226 113.4 117.5 5.8 4.5 3940163 1 7 16 5.0 2 201 215 113 .7 119.4 6.2 5.0 3940167 1 7 16 3 0 1 211 234 111.1 117.5 5.2 4.5 3940021 1 7 18 1 0 1 269 287 121 0 124.8 5.0 4.8 3940043 1 7 18 2 0 2 281 294 120.0 126.4 5.8 5.8 3940083 1 7 18 4.0 2 239 263 129.2 132.4 4.5 4.5 3940151 1 7 18 5.0 1 244 259 124.8 127.3 6.5 5.5 3940166 1 7 18 3.0 2 203 235 110.5 114.9 5.7 5.5 3940191 1 7 18 3.0 1 267 290 127 .0 131.8 4.8 4.2 3940002 1 7 23 2.0 2 302 330 121.3 126.7 6.2 6.0 3940009 1 7 23 3.0 2 255 284 111.8 115.6 6.2 5.5 3940063 1 7 23 2.5 2 238 258 113.4 116.5 5.5 5.0 3940082 1 7 23 1.0 1 250 272 111.8 118.1 6.2 4.5 3940123 1 7 23 5.0 2 233 254 123 .2 125.7 6.2 5.5 3940190 1 7 23 4.0 1 173 199 110.5 113.4 4.8 4.2 908 2 1 14 2.0 2 308 406 121.6 127.6 5.5 6.0 910 2 1 14 2.0 2 231 302 118.1 125.4 4.8 5.2

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262 Raw Data Performance Experiment ID YEAR TRT dKU GT7V 3 W J. X 1\VJ SWT 2 KG HTl HT2 CSl CS2 922 2 1 1 1 yi 1 4 1 u 9 ^ *s u 313 113.0 114.3 5.5 6.2 3950030 2 1 1 4 R n 3 u 9 2 65 329 121 3 125.7 6.2 6.5 3950033 2 1 1 4 9 n 9 295 349 121 0 123.5 5.2 5.8 3950081 2 1 1 4 0 u 9 it J ^ 329 114 0 118.7 5.5 5.8 917 2 -1 1 1 Q 1 n 1 u 9 it 209 277 109 6 115.3 5.0 5.5 3950042 2 1 1 Q 9 n 9 it ^ ^ 340 112 4 116.5 5.5 6.2 3950080 2 1 1 1 Q 9 n 9 it ^ .3 X 283 115.3 116 8 4.8 5.5 3950136 2 -1 1 i y 9 9 it 977 it 1 1 356 114 0 119 4 5.8 6.0 3950139 2 1 1 1 0 j.y 4 9 it 283 333 122 6 126 4 6.0 6.5 3950170 2 1 1 Q 1 y 9 9 it ^ ^ ^ 317 122 6 128.3 4.8 5.8 3950044 2 1 1 0 9 2 2 c D 3 97 9 it 1 it 345 121 6 130.2 5.8 5.6 3950115 2 -I 1 9 9 22 4 5 J 974 349 117 8 127 6 4.5 4.6 3950132 2 1 22 0 3 94R 308 112 1 120.7 5.5 5.8 3950134 2 -1 1 9 9 22 9 3 979 it 1 it 367 121 6 128 0 4.8 5.4 3950177 2 1 0 9 2 2 9 •3 961 ^ Q X 342 119.7 125.4 5.2 5.4 3950203 2 1 2 2 9 J 3 9 "? 1 ^ J X 308 113 0 123 .2 5.2 5.0 3950041 2 1 1 2 1) 9 J 3 9 Q7 itji 37 6 115.6 121 0 5.8 5.5 3950059 2 1 2 5 3 •3 J 9 c: 9 Z 0 z 47 117.2 121 6 5 5 5.5 3950148 2 1 2 b 2 •3 9 97 itit 1 "^97 0 it 1 116 5 121 6 5 0 5.5 3950166 2 1 1 2 I) 4 3 J 9 ftft 351 124 8 130 8 5.3 5.6 3950173 2 -1 i 2 D 1 3 9 8 ^ ^ 0 315 109 9 117 .2 5.0 5.2 r c 3950205 2 -1 1 2 D c D 3 J ^ ^ ^ 340 126 .7 131 4 5.2 5.5 3950016 /. -5 0 9 it 9 T 1 ^ J X 297 111 5 118.4 5.3 5.4 3950020 2 A 9 0 9 9 it itv 0 374 122 3 127.3 5.0 5.8 f\ c r\ r\ Q 395003 8 2 9 J 9 it 9^4 297 120 7 120.7 5.8 5.8 3 9500// 2 Z 9 9 9 it 243 288 119 1 144 5 5.0 5.5 ^ Q c "1 (\o 2 9 9 9 it 268 340 114.3 117 5 5.5 6.2 3 7 D U 0 0 2 2 286 354 118 .7 122.9 6.2 6.2 Q m J V / 9 £t it 5 2 2 274 347 116.2 120.7 4.8 5.8 Q 1 1 J J. £t o 2 2 274 354 122 .3 125.1 5.2 5.8 J y D U U X £1 5 4 2 288 354 120 0 124. 5 5.8 6.0 9 2 5 1 2 249 351 120.0 126.4 4.8 5.5 0 y D U U D X 9 2 5 3 2 209 277 117 .8 119.7 4.5 5.0 J 7 D U X / J 9 2 5 3 2 249 345 115.9 125.1 5.2 6.0 J -7 D W U D / 9 2 7 2 3 238 329 115.9 124.2 5.5 5.6 0 Q c n rt Q 0 J D U U D -7 9 0 it 7 3 3 256 317 117.8 124.8 5.2 5.4 9 9 7 9 3 272 356 116.5 120.0 5.8 5.8 9 2 9 £t / c: •3 ^ 299 374 122 3 128.3 6.2 6.2 3 9 D0iO4 2 9 7 / 9 •3 97 9 it 1 it 3 67 121.6 130 2 5 0 6.0 3 9 DUIDD 2 9 i. 7 / *i •3 286 363 117 5 121 9 5.8 6.0 3 9D00ib 2 9 1 9 9 0 9 n J it\t 3 67 121 6 124 2 5 5 5 8 1 C A A 0 A 3950034 2 Z 12 D 3 J 97 Q ^4 113.0 117 5 5 8 5 8 *5 0 c r\ c "5 2 9 1 9 1 Z 9 3 979 it I it 349 113 0 118 4 5 0 5 8 0 ft C A A ^ ^ 3950066 2 1 9 12 A 4 3 9'?4 Z 0 ri ^94 0 it** 118.1 123 8 4 5 5 5 5 q c n n Q fl J J J U U -7 0 9 2 12 4 3 277 345 119.7 123.2 5.0 5.8 3950140 2 2 12 3 3 259 311 122 .3 125.7 5.0 5.4 3950027 2 3 9 2 2 304 410 122.9 124.2 6.2 6.2 3950047 2 3 9 6 2 229 306 113.4 121.0 5.0 5.3 3950078 2 3 9 2 2 277 363 116.5 122.9 5.8 6.0 3950117 2 3 9 3 2 306 374 121.9 127.0 5.4 6.0 3950182 2 3 9 3 2 238 299 112 .7 117.5 5.5 5.5 3950193 2 3 9 5 2 231 295 123.2 127 .0 4.8 5.5 909 2 3 18 2 2 299 399 123.2 127.6 5.0 6.2 3950024 2 3 18 2 2 231 308 111.1 116.5 5.2 6.2 3950092 2 3 18 2 2 259 354 119.1 121.9 4.8 5.3 3950120 2 3 18 3 2 290 374 121.9 127.6 4.8 6.0

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263 Raw Data Performance Experiment TD YEAR TRT PEN BRD SEX SWT 1 KG SWT2KG HTl HT2 CSl CS2 O J X J 2 3 18 6 2 249 351 118.7 120.7 4.8 6.2 3950171 2 3 18 4 2 249 333 121.6 125.4 4.5 5.4 ^ J J \j \J J yj 2 3 23 6 3 283 397 119.4 128.0 5.2 6.0 3950107 2 3 23 3 3 272 356 118.7 123 .8 5.5 5.5 3950127 2 3 23 1 3 240 329 112.7 119.7 5.2 5.4 J J J Kl i. J ~^ 2 3 23 4 3 290 363 122.6 126.4 5.8 5.8 3950165 2 3 23 5 3 268 342 124.2 128.9 6.0 6.0 3950179 2 3 23 3 3 231 308 111.8 129.5 5.5 5.6 J J .J \J \J •J \f 2 3 27 4 3 279 358 119.7 120.3 5.5 5.4 3950110 2 3 27 6 3 288 381 118.4 123.5 4.8 5.8 3950141 2 3 27 1 3 204 306 104.8 115.3 4.8 5.6 J J J W i J 2 3 27 3 3 243 311 117.2 118.7 5.5 5.4 3950218 J J ^ \J £t ^ \J 2 3 27 4 3 215 283 113.0 123.5 4.8 5.2 3950013 2 4 2 2 2 277 388 123.8 132.4 4.8 5.4 3950055 2 4 2 3 2 279 356 115.3 119.4 6.2 6.2 3950147 2 4 2 2 2 268 354 115.9 120.7 5.4 5.8 3950162 2 4 2 4 2 249 324 119.4 125.1 4.8 5.2 3950176 2 4 2 6 2 254 290 110.8 112.1 5.8 5.8 3950187 2 4 2 1 2 202 265 105.4 108.3 5.2 5.2 3950019 2 4 20 2 2 317 413 123.8 126.7 5.8 6.0 3950083 2 4 20 1 2 238 311 107.6 111.1 5.5 5.8 3950104 2 4 20 3 2 288 329 118.1 118.7 5.6 5.2 3950119 2 4 20 2 2 268 345 117.8 122 .9 4.8 5.6 3950161 2 4 20 5 2 259 327 123.8 127.6 5.2 5.5 3950201 2 4 20 3 2 206 270 110.8 114.3 5.2 5.8 3950031 2 4 24 4 3 274 358 123 .2 129.9 4.8 5.8 3950065 2 4 24 3 3 299 397 121.3 127.3 6.0 5.8 3950076 2 4 24 6 3 249 333 113.7 118.4 5.8 6.2 3950129 2 4 24 1 3 259 313 110.8 115.3 5.2 5.5 3950157 2 4 24 5 3 252 324 121.6 126.4 5.2 5.6 3950167 2 4 24 4 3 297 367 119.7 125.4 5.2 5.5 3950039 2 4 28 6 3 288 365 121.3 125.7 5.0 5.8 3950064 2 4 28 4 3 279 361 120.3 129.9 4.8 5.6 3950091 2 4 28 3 3 293 381 115.3 118.7 5.2 5.8 3950124 2 4 28 1 3 204 286 106.0 114.9 5.2 5.5 3950130 2 4 28 4 3 327 413 124.5 126.4 6.0 6.0 3950172 2 4 28 4 3 238 306 114.6 119.7 5.5 5.8 912 2 5 4 2 2 259 320 120.7 123 .8 5.3 5.5 920 2 5 4 1 2 220 268 112.1 115.3 5.5 3950040 2 5 4 2 2 281 374 115.6 119.1 5.5 6.2 3950068 2 5 4 3 2 215 308 115.3 125.1 4.8 5.2 3950086 2 5 4 3 2 324 372 125.7 126.4 5.8 5.2 3950198 2 5 4 4 2 234 311 119.7 126.4 4.8 5.5 913 2 5 13 2 2 277 358 126.7 132.1 4.8 5.4 3950026 2 5 13 4 2 297 349 110.5 124.2 5.8 6.2 3950057 2 5 13 3 2 231 297 114.6 115.9 4.8 5.2 3950058 2 5 13 6 2 224 317 112.7 120.0 4.8 5.5 3950084 2 5 13 2 2 283 345 112.4 117.8 5.5 5.5 3950163 2 5 13 3 2 274 338 123 .8 126.4 5.2 5.8 3950029 2 5 17 3 3 327 401 117 5 117 .8 5.8 6.2 3950056 2 5 17 3 3 247 333 126.7 134.0 4.8 5.5 3950122 2 5 17 1 3 277 356 113.4 119.1 5.2 6.0 3950169 2 5 17 6 3 279 370 120.0 128.6 5.5 6.1 3950183 2 5 17 5 3 220 290 120.7 126.4 5.2 6.2 3950184 2 5 17 4 3 252 311 115.9 120.3 5.5 5.8 3950087 2 5 26 6 3 311 390 127.0 131.1 4.8 5.8

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264 Raw Data Performance Experiment Tr VT7aR 1 r\ TRT PEN BRD SEX SWTIKG SWT2KG HTl HT2 CSl CS2 O Q C A n Q "5 o A 26 3 3 279 342 116.2 119.7 5.2 6.2 0 D 9 fi ^ D 2 3 288 356 118.1 121.0 5.0 5.6 1 A c A T on z £1 0 4 3 286 349 120.7 126.7 5.2 5.2 O Q C A 1 "a Q ^ y D u X J o p 5 26 3 3 243 327 116.2 134.6 5.5 5.6 ^ J? D V X O J. p 5 26 4 3 238 297 118.1 124.2 4.8 5.4 O Q C A A A Q J y D U U U J •) £1 g 1 2 2 299 365 117.5 118.4 6.2 6.2 o Q c A n 1 n o y D u u X / p 6 1 1 2 229 290 107 6 115.3 5.5 5.5 •> Q c A n 0 0 p D 1 3 2 286 356 124.2 128.3 5.2 5.3 O J J U w -? J P 6 1 g 2 306 358 122.3 124.5 5.8 5.8 O Q C A AQQ J 7 J v U -7 -7 2 6 1 2 2 236 302 106.7 110.2 5.5 6.0 J J 3 U X J 3 2 6 1 4 2 227 283 116.5 124.2 5.5 5.8 Q 1 A X 44 p g g 2 2 299 395 122.9 126.7 4.8 5.5 Q 0 y z D p g g 2 2 268 351 111.8 116.5 5.2 6.2 •J Q C A n 0 P c D g 3 2 243 320 119.7 121.3 4.8 5.7 O Q C A 1 O 7 ^ y D u X J J p g 3 2 213 295 103.5 106.7 5.8 6.0 J y D u X 3 J p 6 g 1 2 234 295 105.4 108.3 5.5 5.5 O y D U X D ft p g g 4 2 249 342 122.3 127.6 5.0 6.0 J ? D U U f -7 p g 10 2 3 247 349 113.0 117.5 5.0 5.4 J y J u u 0 0 P g 10 4 3 263 324 118.7 121.3 5.2 5.4 0 Q c A 1 1 0 J y X X J p <: u 1 0 4 3 331 429 125.4 134.3 5.6 J J J U X J z p g 10 3 3 288 383 120.7 124.2 5.2 5.8 oqcAi CO 0 7 3 u X 0 V 2 g 10 5 3 222 274 118.1 122.6 4.5 5.1 7 Q C A 0 A Q J ? D \J Z y 0 p g 10 g 3 231 320 120.3 127.0 5.5 5.2 0 _7 J U U J 0 2 g 16 g 3 327 417 122 6 128.6 6.0 6.2 •3 Q R A-| Al J J _) U J. VJ J. 2 g 16 4 3 297 356 125.4 131.4 5.8 5.6 J ^ D U X Z 3 p g 16 1 3 213 304 109.6 115.6 5.0 5.5 J ^ J U X ** J 2 g 16 4 3 313 424 118.7 127.0 5.5 6.5 3950189 2 g 16 2 3 229 297 109.2 114.0 5.0 5.5 3950221 2 6 16 4 3 197 293 109.6 114.9 4.5 5.5 925 2 7 8 1 2 222 265 108.0 109.9 5.0 5.2 3950006 2 7 8 3 2 272 304 114.0 116.2 5.2 5.4 3950008 2 7 8 6 2 272 299 120.0 125.7 5.0 4.8 3950014 2 7 8 2 2 299 333 120.3 120.7 6.0 5.7 3950074 2 7 8 5 2 254 277 124.2 128.0 5.2 4.8 3950144 2 7 8 2 2 254 274 112.7 139.7 5.5 5.5 919 2 7 11 2 2 254 306 117.8 118.1 4.8 5.2 3950005 2 7 11 1 2 197 218 107 6 109.6 4.5 4.8 3950018 2 7 11 2 2 286 340 119.4 127.3 5.0 5.2 395005? J ^ \J J £t 2 7 11 g 2 322 340 120.3 124.2 6.0 5.2 39Rnn97 J ^ ^ \j \j J 1 2 7 11 3 2 293 306 125.4 128.9 4.5 4.7 3950142 2 7 11 5 2 238 277 119.7 121.3 5.2 5.4 3950037 2 7 15 g 3 311 342 126.1 128.0 6.0 4.8 3950089 2 7 15 2 3 261 299 116.5 117 .2 5.0 4.8 3950103 2 7 15 4 3 295 333 125.7 128.3 5.5 5.2 3950109 2 7 15 3 3 254 295 118.1 120.3 5.2 5.5 3950159 2 7 15 3 3 286 336 121.3 123 .2 5.2 4.5 3950210 2 7 15 5 3 229 268 116.8 122.9 5.2 4.8 3950021 2 7 21 2 3 302 331 114. 6 117 .8 5.8 4.8 3950046 2 7 21 6 3 240 283 119.4 123 .2 5.0 4.8 3950112 2 7 21 4 3 283 322 120.3 120.7 5.5 5.5 3950114 2 7 21 5 3 322 345 128.9 138.4 5.0 4.5 3950123 2 7 21 3 3 268 295 119.7 122 6 5.2 5.0 3950145 2 7 21 3 3 238 279 116.8 121.6 4.5 5.0

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265 Performance Experiment: Intake, observed and predicted gain OBS YEAR TRT PEN VjW i. HAYDM SUPPDM PRDADG 1 1 1 Z J J o ^ o ^ 3 56 1.86 .545 2 1 1 D c; n • DUO ^ O 3 .72 1.86 520 3 1 1 o r Z U t^l 9 • D X Z ?7ft it 1 V 4 .32 1.86 640 4 1 1 Z O 2 64 3 52 1 86 .555 5 1 2 n QUO 290 3 .30 1.86 .425 6 1 2 14 • D ^ J ^ o ^ 3 63 1 .86 .515 7 1 2 22 Oil 97ft it 1 o 3 81 1.86 555 8 1 2 2 4 0 J 3 it 1 3 54 1.86 525 9 1 3 12 0 U 0 9ft7 4.19 1.86 590 10 1 3 1 / 97 9 it 1 J 4.69 1.86 .700 11 1 3 ly D / U it I Kl 4 27 1 86 660 12 1 3 2 / ft R • 0 O D 97fi 4.19 1 86 620 13 1 4 4 • 3U J 9 ftl Z O J. 4.25 2 12 565 14 1 4 / 1^ 4 ft D 4o 97fi z / o 4 37 2 .05 590 15 1 4 ft y 4 ft 970 3.06 2 .04 .385 16 1 4 2 b ^ 9 D Z D 9ftn ^ o w 4 07 2 15 560 17 1 c D Aft 7 9S8 it U o 3.10 2.06 435 18 1 5 o o ^ / 0 9 KQ ^ Q Z7 3.24 2 07 430 19 1 b U ^ "3 Q D J J? 97 fi it 1 3 44 2 04 435 20 1 b 2 1 AQ n 9 fil ^ O X. 3 92 2 08 580 21 1 c D J n Q 97 0 4.38 2 07 620 22 -I 1 c D 1 1 11 283 3 89 2 10 505 23 -I 1 c D 1 D fin7 D u / 9 fi4 ^ o 4 17 2 07 610 24 1 0 2 D • ^ Q O 280 4 12 2 13 560 2 b 1 / J. 1 90 • J. ^ u 253 5 13 0 .00 .170 26 1 1 1 O • X o 242 4 .18 0 .00 .030 2 / X. I 1 ft J. o 1 8A • X O *m 261 5 62 0 00 .255 -1 i. 1 217 254 5 19 0.00 .195 O Q o 1 J. 1 A J. rt 9 .680 302 5 67 1 86 .740 J U o 1 Q J. ^ o 633 285 3 99 1 86 .540 O 1 0 £t 1 X 3 .738 298 5 .21 1 86 .730 •5 0 it 1 ^ -J 3 .788 301 5 46 1 86 .760 -) £t it •3 .J 2 612 293 4.16 1 86 .540 J O £i 5 2 .767 298 4.30 1.86 550 O C J 0 Z •) it 7 3 .767 311 5 09 1.86 .670 o 9 £t 3 651 307 4 .86 1 86 .650 O 1 •3 J Q _? 0 .734 303 5 60 1 86 .730 J o 3 J 1 ft o O Q u 308 6 .23 1.86 .800 3 Q "3 J £t -J fil f) Q X V/ 307 4 92 1.86 650 4U "3 97 782 287 3 90 1.86 .560 A 1 z 9 71 292 5 90 2 15 .780 42 2 4 2 u £, o oz 9 97 it J I 4 60 2.15 570 43 2 A 4 2 4 J 7 ^3 4 Tin 5 82 2 15 .750 44 2 4 2 o •3 7 fi7 T 1 9 O l.it 6.26 2 .15 810 z c 9 .666 290 5.16 2 11 660 46 2 5 13 2 662 299 4.97 2.15 620 47 2 5 17 3 .731 305 5.56 2.13 .720 48 2 5 26 3 .662 309 4.99 2.11 620 49 2 6 1 2 .590 295 4.33 2.15 .530 50 2 6 6 2 .781 292 4.98 2.15 640 51 2 6 10 3 .788 305 5.57 2.15 .730 52 2 6 16 3 .817 306 5.66 2.15 .740 53 2 7 8 2 .284 277 6.97 0.00 .420 54 2 7 11 2 .313 281 5.53 0.00 .180 55 2 7 15 3 .378 292 6.55 0.00 .330 56 2 7 21 3 .320 292 6.26 0.00 .290

PAGE 273

266 Performance Experiment: Volatile Fatty Acids OBS YEAR TIME TRT RTITY X X RATIO TOTAL 1 1 1 1 2 ID./ ft 9 4.32 91 5 2 1 1 1 c D b 3 U X 0 <3 fi R 0 -/ 4.11 88 5 3 1 1 1 zU c C 9 1 R "X 7 3 .77 79 4 4 1 1 1 1 0 Z 0 C yl ft !>4 • 0 X 4 X q 9 3 99 79 4 5 1 1 2 1 A lU C 9 1 dZ • X •71 1 Z X X 5 1 2 49 80 2 6 1 1 2 14 4 D • D X D fs fi 9 3 12 69 8 7 1 1 2 zz yl C X ^ 4 5 1 3 03 68 3 8 1 1 Z 4 4"^ 9 4 J Z 4 7 2 82 65.5 9 1 1 3 1 0 X Z c;7 1 O / X X Z V 7 7 4 64 78.7 10 1 1 3 J. / yl c: C in 7 5 7 4 .24 63.3 11 1 1 3 X y n DU 0 19 R X z ^ 6 2 4 07 70.8 12 1 1 3 Z / fC9 A OZ • ft 1 R R 8 9 4 .10 88 7 13 1 1 4 4 • X 1 fi 7 1 3 .72 71.4 14 1 1 4 1 49 n 4Z U in 1 X U X 6 0 4 14 58 9 15 1 1 4 Q "5 ft r 7 • ^ 5 9 4 13 54 0 16 1 1 4 z 0 J y D 11 7 XX./ 6 9 3 .39 58 7 17 1 1 c D c D T Q 1 J y X in Q X U 7 6 0 3 60 56.7 18 1 1 5 0 0 3 D D 19 n X z u fi R 0 -/ 2 .96 54 9 19 1 1 c 5 13 4U U in Q X U 7 fi fi 3 .77 58 1 20 1 1 z X 3 0.4 IT <5 X J 7 4 R 4 -/ 2 90 55 6 21 1 1 3 O 3 U 11 7 XX./ fi fi 4 52 72 5 22 1 1 1 1 X X *^ 4 Q n 5 8 4.06 51 9 23 1 -1 1 0 X D 7 7 J D / Q 1 •7 X 5 5 3 93 50 9 24 1 -1 1 c D Z D 77 ft in 1 X w X 5 6 3 84 54.1 25 1 1 X •7 / -1 X 4Q Q fty ^ 11 n X X V/ 5 0 4 55 67 0 26 1 X •7 X D 41 1 ft X • X <} 9 4.7 4 50 56 3 Z 1 -1 1 X I 1 ft X 0 11.0 5 3 4.96 72.4 z o •y 1 X 7 55.0 12 3 6 0 4.45 74.6 1 z z fi4 9 14.7 9 4 4.37 90.0 O A JU 1 1 X D 13.7 8 9 3.16 67 .7 •3 1 J 1 -1 1 1 z u 4R R 10.3 6 9 4. 43 64. 6 1 1 1 X 9 ft 40 8 10.8 9 1 3.79 62 .3 J ^ X •J 9 47 7 23 6 6 9 2 03 80.1 J 4 1 1 z £ X 4Q 9 19.6 5 9 2 54 76.3 J 0 1 z £t 47 24.2 5 8 1 98 78. 6 i D 1 z 0 Z 4 28.6 5 9 1 94 91 3 J / 1 z 1 9 X z 7 7 4 3 93 56 0 -Jo 1 z 0 1 7 X / 0 X • 11 R 4 9 3.25 49 1 1 1 z -5 X y 41 S ft X > o 11 X X 6 6 3 68 61 4 4U -1 1 z J 9 7 Z / 47 ft fi / • 0 1 ? 7 X / 6 6 3 58 70 5 yl 1 41 1 z 4 A f* R4 S D ft J 9n <) Z w 7 11 2 2 62 88 1 4z X z fi 7 / 4ft ft 0 ^ 14 4 9 3 3 .35 74.3 43 -1 1 z 4 Q y J y u 1 fi X 0 • 0 7 Q 2 82 61 8 44 1 z 4 9 c z 0 11 fi X X • 0 7 R / -y 2 97 54 2 4 D -I X z c -J 15.1 7 8 2 .86 67 0 46 1 2 5 8 32.0 12.4 7.2 2.59 52.5 47 1 2 5 13 28.1 9.9 5.3 2.84 43.8 48 1 2 5 21 32 .2 15.5 7.3 2.14 56.2 49 1 2 6 3 46.1 17.6 9.0 2.73 73.5 50 1 2 6 11 32.0 9.8 6.1 3.26 48.7 51 1 2 6 15 31.3 10.6 7.0 2.97 49.6 52 1 2 6 25 33.1 13.7 1.1 2.41 55.2 53 1 2 7 1 45.3 11.6 6.4 3.90 65.2 54 1 2 7 16 39.5 10.3 6.3 3.82 58.1 55 1 2 7 18 34.0 8.5 4.7 4.00 48.7 56 1 2 7 23 41.5 11.3 5.4 3.63 59.7

PAGE 274

267 Performance Experiment: OBS YEIAR TIME TRl 57 1 3 1 1 z / 58 1 3 1 c D oo 4 59 1 3 1 z 0 /I C T 60 1 3 1 28 54 2. 61 1 3 2 10 3 / Z 62 1 3 2 14 62 2 63 1 3 2 22 55.5 64 1 3 2 24 59 1 65 1 3 3 12 50 o 66 1 3 3 17 55.1 67 1 3 3 1 A 19 CO o 68 1 3 3 27 4o J 69 1 3 4 4 CI ft 70 1 3 4 / CO A dZ 4 71 1 3 4 y AC O 4 o z 72 1 3 A 4 2 6 4Z D 73 1 3 5 c D c i 1 51 / 74 1 3 5 8 1 O A 2 9.4 75 1 3 5 13 3 i J 7 6 1 3 5 21 AC ft 45 U 77 1 3 6 3 ^ O A JO 4 78 1 3 6 11 44.5 79 1 3 6 1 b >l ft o 4U o 80 1 J r D Z 3 6 6,1 81 1 3 / -1 i AC T 4 5.3 82 -I 1 •3 J / 1 0 >1 Q Q 4y 5 O J -I J / 1 Q J. o 40 0 O A 1 1 o •7 Z O 4 J J o D 1 A 4 1 1 Z 7 A R o c O D i 4 1 D O / Z a 1 1 /I 4 1 1 0 A Z U O 1 J o o oo 1 1 4 1 J. Z O O J. U o y J. A 4 1 A / O *4 -1 J. 4 Z 4 0 D O -I 1 4 Z ZZ D 4 Z y z 1 4 Z Z 4 D D • 4 y J 1 4 •J J Iz >1 "7 "7 % 1 1 94 1 A 3 1 / An c 4 / 5 y D 1 4 J iy 55 Z 9 6 1 4 3 2 1 C A ft 64.9 97 1 4 4 A 4 c ^ o 53.2 98 1 4 4 1 C ft Q 5U 9 99 1 4 4 9 C T 1 57 3 100 1 4 4 26 48 9 101 1 4 5 5 48.1 102 1 4 5 8 51.3 103 1 4 5 13 54.7 104 1 4 5 21 43.8 105 1 4 6 3 59.2 106 1 4 6 11 55.1 107 1 4 6 15 50.1 108 1 4 6 25 60.1 109 1 4 7 1 50.7 110 1 4 7 16 60.5 111 1 4 7 18 70.9 Volatile Fatty Acids ct\XJc RATIO TOTAL J. 4 • O fi 4.38 85 9 J. / • O ft 7 3 85 96.9 Q Q A 57 f* / 63 4 in c J. U • 0 7 4 R 06 73 6 ZZ J 1 O X 5 Rfi 87 5 J. t> D 7 n T fi5 ^ o z 87 6 17 Q R R J • X 7 80.1 Z U D R fi 7 fit) z • o ^ 86.5 11 K fi 7 4 64 70.4 7 n 4 17 77 2 10 Q J. Z • 7 fi n 4.79 83 1 10 7 J.Z / c c ^ 68.0 11 1 J. J. i. fi fi 4 7? fB / ^ 70.0 7 7 3.78 75.0 ID 1 fi 4 4 55 63 6 in Q fi 4 4 05 60 5 14 R 6 4 3 56 74 0 R R D ^ 0 fi 5.35 38 4 fi 0 o z 4 4 4. 1 4 46.1 10 0 X z z R 70 63 9 fi 1 u X 4.16 55 0 in A X U • ft fi fi 4.30 62 4 11 7 X X / 6 8 3 50 60 4 7 9 4 8 4 .35 46 9 4 7 4 80 60 6 10.0 6 0 4 97 66.9 9 2 4 8 4.75 58 9 10 7 5 6 4 67 67 .7 13 8 9 1 5 43 98 4 14 7 8 7 4 57 92 0 11 8 8 7 5 17 82 8 14 7 12 4 5 47 109 9 28.6 7 9 2.75 116.2 19 1 7 3 3 43 93 9 14 3 5 9 3 .78 75 6 15 0 6 1 3 69 78.0 9 3 6 8 5 .11 77.5 9 3 5 8 5.11 63 2 7 2 5.36 73 8 11 fi X X o 7 7 5 53 86 3 11 R X X 8 3 4.70 74.1 J m ^ 7 0 5 50 67 4 X z o 7 <3 4.66 77 9 11 X X J fi n 68.9 10.1 6.2 4.80 65.1 15.5 9.6 3.32 77.0 12.3 7.7 4.44 75.5 10.3 6.2 4.23 61.0 12.4 9.3 4.78 81.1 10.8 7.8 5.12 74.7 10.8 8.1 4.72 69.4 12.1 8.1 4.98 80.3 9.2 5.4 5.52 65.5 10.9 7.4 5.54 79.7 13.2 7.9 5.36 94.1

PAGE 275

268 Performance Experiment: OBS YEAR TIME TRT FhN 112 1 4 7 2 J D 1 0 113 2 1 1 14 DU 0 114 2 1 1 1 ft 19 41 0 115 2 1 1 22 116 2 1 1 2 b yl Q 1 4o • X. 117 2 1 2 3 A Q 0 118 2 1 2 c D 3U 0 119 2 1 2 1 DU J 120 2 1 2 12 4U / 121 2 1 3 ft 00./ 122 2 1 3 IS >i r '7 4U / 123 2 1 3 2 3 A f\ n 124 2 1 3 27 /II Q 4 1 • 0 125 2 1 4 2 4U / 126 2 1 4 20 127 2 1 4 24 41 D 128 2 1 4 28 25.7 129 2 1 5 4 43 1 130 2 1 5 13 40 y 131 2 1 5 17 36.1 132 2 1 5 26 39 / 133 2 1 6 1 4o 4 134 2 1 6 6 /IT "3 4 / 3 135 2 1 6 10 44 Z 136 2 1 6 i b 3 3.1 137 2 1 7 0 0 3 D 3 138 2 1 7 11 34.0 139 2 1 7 1 b Al 1 4 / 1 140 2 1 7 21 A A 44 D 141 2 2 1 14 3 4.3 142 2 2 1 1 y D U D 143 2 2 1 2 2 4Z U 144 2 2 1 2 b 145 2 2 2 ACS 1 4U 1 146 2 2 2 b ACi 1 4y 1 147 2 2 2 / "3 R 148 2 2 <^ 1 0 i. ^ &.C\ &. ^ U 149 2 2 "3 J Q ^ ft A 150 2 2 i 1 0 151 2 2 •5 J 2 J J J 4 152 2 2 3 2 / 4U 1 153 2 2 4 2 3 / 1 154 2 2 4 20 38 b 155 2 2 4 24 32 y 156 2 2 4 28 39.3 157 2 2 5 4 29.9 158 2 2 5 13 32 7 159 2 2 5 17 25.1 160 2 2 5 26 31.2 161 2 2 6 1 30.4 162 2 2 6 6 34.3 163 2 2 6 10 27.7 164 2 2 6 16 27.6 165 2 2 7 8 51.0 166 2 2 7 11 31.4 167 2 2 7 15 30.3 168 2 2 7 21 41.4 Volatile Fatty Acids PROP BUTY RATIO TOTAL ft ft 5 4 5 .84 66.1 8 6 3 53 76.2 6 9 4. 52 59.9 ft 0 5 4 4 64 51.6 in d 7 1 4 64 67.2 6 4 2.43 77.7 1 ^ ft 8 0 3 .21 76.6 17 4 5 6 2 .96 75.6 14 4 5.7 2 84 62.2 7 4 6 2 4 .96 51.5 ft 4 6 9 4.85 57.6 ft ft 5 3 4. 65 55.8 Q 1 ^ X 5 9 4 61 57.8 11 X X • Q 6 4 3 51 59.6 in 4 X U 6 6 3 91 58.8 in 7 2 4 03 60.2 ^ 4 3 4 4.81 34.7 X 0 • ^ 9 3 2 .73 69.5 14 0 8 1 2 89 63.7 19 n 5 9 3 01 54.8 14 Q 7 8 2 69 63 0 17 '\ X / >> 10 3 2 82 77.5 19 6 X ^ 0 8 1 3.75 69.7 19 1 X A • X 8 9 3 65 66.5 7 4 6 1 4.48 48.4 7 1 4 1 4.91 47.3 7 1 4.0 4.90 47.0 10 8 6 5 4.39 65.9 9 8 5 6 4.59 61.1 10 2 4.4 3.35 49.1 14 1 9.0 3.58 73.9 11 6 7.4 3.62 60.9 12 2 7.0 3.10 56.9 17 0 4.3 2.37 61.4 18 5 8.0 2.73 75.6 15.0 4.3 2.35 54.6 11.3 3.9 4.16 55.7 9.7 6.6 3.94 54.7 10.9 6.8 3.66 57.8 9 1 6.4 3.64 49.2 10 2 6.6 3 94 56.9 13 9 8 9 2 82 59.9 11 7 7 2 3 .28 57 4 8 7 2 7 3 .79 44.3 13.0 8 0 3 05 60 4 4 0 2.74 44 8 11 1 5 1 2 85 48.8 10.4 4.4 2.42 39.9 11.0 4.5 2.82 46.7 11.5 6.8 2.65 48.7 13.6 8.5 2.52 56.6 9.3 6.7 2.96 43.7 7.3 2.5 3.75 37.4 12.4 2.6 4.11 66.0 9.5 2.8 3.23 43.8 9.2 4.9 3.30 44.4 11.9 4.8 3.51 58.1

PAGE 276

Performance Experiment: Plasma Urea Nitrogen OBS YEAR MONTH 1 DEC 2 DEC 3 DEC 4 DEC 5 DEC 6 7 DEC 8 DEC 9 Dbv10 11 DEC 12 Dh.(_ 13 DEC 14 15 16 UfcL 17 18 DEC 19 DEC 20 DEC 21 T~, T-1 f~l DEC 22 JDEC 23 • 24 Ut.C 25 ~ UEiC 26 De.(27 28 UiLL 29 r r
PAGE 277

Performance Experiment: Plasma Urea Nitrogen OBS YEAR 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 o3 O J o4 J} 86 87 88 89 90 Al 91 ?• 92 J93 94 95 96 97 98 99 100 101 102 103 104 105 106 1 107 1 108 1 109 1 110 1 111 1 112 1 MONTH TRT T TV XT JAW 1 X JAJN JA^J JAN X T7VKT JArl z uAW z JAM z J Ar J Ar '3 J JAM 3 J JAW "3 JAW XT JAW A 4 JAW i1 TA XT JAW A *k T7V XT JAW c D TAXI JAW c T7VKT JAW c J JAW c JAW 0 T AKT JAW c. D TAXI O AIM g U /UN 7 7 7 u AIM 7 MAP 1 MAP 1 MAP MAP 1 MAP 2 MAP i'ir\r\ 2 MAP 0 MAP o MAP MAP •5 MAP o MAD •3 MAD MAD ft MAD MAK MAR 4 MAR 5 MAR 5 MAR 5 MAR 5 MAR 6 MAR 6 MAR 6 MAR 6 MAR 7 MAR 7 MAR 7 MAR 7 PPM PUN O z 13.02 D 11 60 Z
PAGE 278

Performance Experiment: Plasma Urea Nitrogen OBS YEAR 113 2 114 2 115 2 116 2 117 2 118 2 119 2 120 2 121 2 122 2 123 2 124 2 125 2 126 2 127 2 128 2 129 2 130 2 131 2 132 2 133 2 134 2 135 2 136 2 137 2 138 2 139 2 140 2 141 142 Z 143 t A A 144 t A C 145 t A ^ 146 z 147 2 148 2 149 2 150 2 151 2 152 2 153 2 154 2 155 2 156 2 157 2 158 2 159 2 160 2 161 2 162 2 163 2 164 2 165 2 166 2 167 2 168 2 MONTH TRT T"! T"! n FEB 1 1 FEB T JL FEB 1 con r bo 1 CCD FEB Z FEB FEB FEB Z FEB •J FEB -J FEB •3 O FEB FEB /I FEB ii 4 FEB A 4 FEB ji 4 FEB c D FEB c D r EB c FEB c r ho D r Ed 0 r Eo D CCD r Ed 0 r Ed 7 r ILD 7 r CiD 7 r cjD 7 U ATi 1 1 .TAM .TAM 1 X TAM TAM TAM TAM 9 £ TAM -5 JAW J T A XT •3 J T7V XT uAN 4 TX XT JAN 4 JAN 4 JAN 4 JAN 5 JAN 5 JAN 5 JAN 5 JAN 6 JAN 6 JAN 6 JAN 6 JAN 7 JAN 7 JAN 7 JAN 7 D17M PUN 1 A X 0 1 CI X J J w 8.68 X -J • ** V/ J 19 fiO c J n 1 19 R3 X ^ 0 J 1 1 Sfl X X 0 0 Q 1 fl S'^ X 0 .J J X 0 in 0 97 8 97 z Q i^fi 7.28 z 0 ft 9 0 A % 11 S6 X X ^ 0 1 -i X J 19 R7 1 7 X / 19 "59 Z D fl ftfi 0.00 1 X 6.98 e V 7.26 X u 8.11 X V 14.57 fi V 7 49 1 1 X X 9 13 15 4.36 21 9.95 14 12 52 19 7.26 22 8 42 25 10.31 3 8.25 5 12 09 7 8 94 12 9 .23 9 10 92 X 0 8.39 11 IT X X X -J 97 11 6"? X X V J 9 7.31 9 n fl 49 9 ^ 19 9 fl ft 09 Q nft 13 8.44 17 9.32 26 7.98 1 1 .SI 6 6.93 10 10.89 16 8.37 8 6.10 11 8.76 15 5.92 21 5.19

PAGE 279

Performance Experiment: Plasma Urea Nitrogen OBS YEAR 169 2 170 2 171 2 172 2 173 2 174 2 175 2 176 2 177 2 178 2 179 2 180 2 181 2 182 2 183 2 184 2 185 2 186 2 187 2 188 2 189 2 190 2 191 2 192 2 193 2 194 2 195 2 196 2 MONTH TRT MAR 1 MAR 1 MAR 1 MAR 1 MAR 2 MAR 2 MAR 2 MAR 2 MAR 3 MAR 3 MAR 3 MAR 3 MAR 4 MAR 4 MAR 4 MAR 4 MAR 5 MAR 5 MAR 5 MAR 5 MAR 6 MAR 6 MAR 6 MAR 6 MAR 7 MAR 7 MAR 7 MAR 7 PEN T5TTKT 14 1 A Q'i 19 1 11 22 Q A'X 2 b J / z o 5 11 / o • 12 lU ou U • Dl 1 O 18 1,11 1 c nA 27 11 1 Q 1 1 1 o *> 11 ASl 1 1 • 40 20 O A 24 ^ Q Q R 14 D 1 4 4 / Z 7 13 8.62 17 13.43 26 7.44 1 5.55 6 6.25 10 6.05 16 10.31 8 4.30 11 5. 60 15 6.81 21 7.59

PAGE 280

273 Heifers: Total Intake OBS PER AN SUPP ADD TNDFT TCPI TTDNI 1 0 1 0 0 o ^ 0.520 2 440 2 0 2 0 0 ^ con ft *i u 0.640 3 190 3 0 3 0 0 D 330 D U / U fi <3 0 J O J L/ 0.580 2.820 4 0 4 0 0 c r\ c f\ D 1 U U 7 Q T 0 0.580 2 680 5 1 1 m c 6.136 D D J U J J J J 0.714 3 439 6 1 2 m b / jlZ C C Q 1 b DoJ. 4 J. J. O 0 877 4.158 7 1 3 c c C COT J J. ^ -7 0.656 3 404 8 1 4 c b 6.848 6.433 4.071 0.766 4.031 9 2 1 m b 5.776 5.390 3.597 0.742 3.220 10 2 2 c D 0 / J. / u O -1 4.082 0.788 3 .966 11 2 3 m C 5.355 5.088 3.876 0.692 2.827 12 2 4 c C 6.197 5.889 3 .750 0.734 3 675 13 3 1 c C 6.465 6.096 3.570 0.713 3.937 14 3 2 m C 8.403 7 .738 4. 668 0.948 4. 520 15 3 3 c b 6.471 6.157 3.624 0.718 3 .940 16 3 4 m b 8.504 7 .832 4.728 0.941 4.888 17 4 1 c b 6.750 6.419 3.995 0.750 4.046 18 4 2 c c 7.631 7.255 4.556 0.807 4 548 19 4 3 m b 6.613 6.251 4.703 0 757 3 514 20 4 4 m c 8.633 7.999 5.114 0.970 4 881 Heifers : Supplement Intake OBS FllK AT* SUPP ADD SDMI SOMI SNDFI SCPI STDNI 1 A U J. 0 0 0.000 0.000 0.000 0 000 0.000 n U 0 0 0.000 0.000 0.000 0 000 0 000 A U J 0 0 0.000 0.000 0.000 0 000 0 000 4 A U ft 0 0 0.000 0.000 0.000 0 000 0 000 D 1 1 m c 1.285 1.080 0.008 0.234 0 947 c 0 1 z m b 1.795 1.509 0.011 0 .327 1.323 7 1 J c c 1.553 1.476 0.144 0 242 1 311 o o 1 X c b 1.553 1.476 0.144 0 242 1 311 9 2 1 m b 1.229 1.064 0.022 0.273 0.936 10 2 2 c b 1.734 1.647 0.161 0.270 1.463 11 2 3 it> c 0.455 0.417 0.015 0.198 0.366 12 2 4 c c 1.647 1.565 0.153 0.256 1.390 13 3 1 c c 1.734 1.647 0.161 0.270 1.463 14 3 2 m c 1.990 1.701 0.028 0.358 1.167 15 3 3 c b 1.734 1.647 0.161 0.270 1.463 16 3 4 m b 1.918 1.641 0.028 0.346 1.445 17 4 1 c b 1.907 1.812 0.177 0.297 1.610 18 4 2 c c 2.081 1.977 0.194 0.324 1.756 19 4 3 m b 0.671 0.599 0.016 0.238 0.525 20 4 4 m c 2,165 1.850 0.030 0.395 1.628

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274 Heifers: Fecal Outputs OBS PER AN SUPP ADD FDMOUT r UMU U i PC POUT 1 0 1 0 0 1.500 1 "J o Q J. U X o 0.180 2 0 2 0 0 1.874 1 1 "i Si X ^ / J. 0.226 3 0 3 0 0 rt c A 2 0d4 1 Q Q "5 J, O J J 1 fil X J O X 0 .243 4 0 4 0 0 1 Old 1 81b 1 CQ A 1 9 fi9 X > ^ w ^ 0.214 5 1 1 m c 1 O A 1 oU4 X D 0 X 1 Oil X ^ X X 0 .205 6 1 2 m b o o c c 2 2dd o n c:'7 Z U D / 1 Aft Q X O ^ 0.266 7 1 3 c c 2.046 1 Q il T 1 o 4 / X J fi J 0.259 8 1 4 c b 2.969 o c cr c Z b D D 0 0 91 0.328 9 2 1 in U D J. o ^ ^ 1.704 1 200 0.256 10 2 2 c b 2.134 1.968 1.414 0.287 11 2 3 m c 1.889 1.734 1.260 0.250 12 2 4 c c 2.338 2.114 1.444 0.316 13 3 1 c c 2.166 2.025 1.515 0.252 14 3 2 m c 2.819 2.596 1.840 0.377 15 3 3 c b 2.370 2.157 1.562 0.271 16 3 4 m b 2.774 2.550 1.834 0.338 17 4 1 c b 1.921 1.771 1.268 0.238 18 4 2 c c 2 664 2.441 1.703 0.379 19 4 3 m b 2.385 2.172 1.597 0.283 20 4 4 m c 2.786 2.559 1.811 0.382 Heifers: Parameter from G2G1 and Log models OBS PER AN SUPP ADD CZERO LAMBDA KS DOSE TD K-LN 1 1 1 m c 361. 1 0 .21879 0. 039228 688 13. 0 0 .043624 2 1 2 m b 274. 1 0 .14299 0. 037868 680 9. 6 0 .038138 3 1 3 c c 426. 1 0 .09062 0. 090558 401 4. 7 0 .059858 4 1 4 c b 270. 7 0 .24736 0. 049136 681 12. 6 0 .047758 5 2 1 m b 372. 8 0 .25555 0. 038045 747 12. 2 0 .041925 6 2 2 c b 359. 2 0 .14050 0. 042876 745 10. 0 0 .039801 7 2 3 m c 339. 4 0 .26937 0. 035678 747 10. 6 0 .047902 8 2 4 c c 470. 0 0 .14489 0. 061949 739 11. 5 0 .052897 9 3 1 c c 462. 2 0 .14396 0. 052313 797 10. 2 0 .054026 10 3 2 m c 269. 0 0 .15638 0. 039459 799 10. 5 0 .042670 11 3 3 c b 590. 6 0 .10101 0. 077357 754 8. 7 0 .053254 12 3 4 m b 398. 9 0 .17379 0. 057870 793 12. 2 0 .050056 13 4 1 c b 573. 6 0 .18110 0. 044373 1035 11. 4 0 .045289 14 4 2 c c 330. 1 0 .15561 0. 035578 1030 9. 7 0 .041431 15 4 3 m b 417. 3 0 .17421 0. 040853 1015 9. 9 0 .047559 16 4 4 m c 430. 5 0 .14647 0. 050709 985 10. 7 0 .047898

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275 Fistulated: Parameter from G4G1 model AJN SUPP CZERO LAMBDA KS TD DOSE 1 1 1 1 m C 283 0. 10824 0 037480 1.23 2200 z •1 J. o m u 246 0. 12879 0 023867 0.00 2200 J -I 1 J c *^ 289 0. 14926 0 030032 0.00 2200 4 1 4 c b 282 0. 18904 0 .023300 0.00 2200 5 2 1 m b ion A U o Z Zj 0 .013652 1.96 1800 6 2 2 c b 651 0. 08993 0 .088586 3.13 1800 7 2 3 m c 249 0. 16577 0 020055 5 47 1800 8 2 4 c c 254 0. 16681 0 .023322 0.00 1800 9 3 1 c c 158 0. 39839 0 .015257 12.15 1800 10 3 2 m c 236 0. 12543 0 .030940 0.00 1800 11 3 3 c b 193 0. 32114 0 .026416 7 .31 1800 12 3 4 m b 219 0. 16423 0 .025055 1.00 1800 13 4 1 c b 243 0. 16623 0 .032718 0.01 1800 14 4 2 c c 203 0. 24695 0 035246 1 .05 1800 15 4 3 m b 198 0. 14900 0 .030196 0 00 1800 16 4 4 m c 222 0. 20987 0 039950 3 .04 1800 Fistulated; ; Nutrient intake OBS PER AN SUPP ADD DMI OMI NDFI NI 1 1 m c 7 .96 7.21 3 .87 173.09 2 2 m b 7 .96 7.21 3.87 173.09 3 1 3 c c 7 .75 7.32 4.10 154.73 4 1 4 c b 7 .75 7.32 4.10 154.73 5 2 1 m b 7 .70 6.94 3.69 155.32 6 2 2 c b 7 .75 7.05 3.92 136.97 7 2 3 m c 7 .41 6.71 3 69 150.81 8 2 4 c c 7 .48 7.05 3.92 136.97 9 3 1 c c 8 .28 7.93 4. 61 161.81 10 3 2 m c 8 .54 7.78 4.36 181.91 11 3 3 c b 8 .35 7.93 4.61 161.81 12 3 4 m b 8 .54 7.78 4.36 181.91 13 4 1 c b 8 .35 7.84 4.23 163.20 14 4 2 c c 8 .35 7.84 4.23 163.20 15 4 3 m b 7 .41 6.75 3.60 161.99 16 4 4 m c 8 .47 7 69 3.99 183.30 I 1

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276 Fistulated: Duodenal flow OBS PER AN SUPP ADD DMFW OMrW ViUV V W 1 IN r rV NH3FW MNFW 1 1 1 m c 5652 1 "J fi Q X O O <3 1 ftQ J, O _7 7.09 77 0 2 1 2 m b 5519 1 T t; "3 X -? ^ 6.32 86.9 3 1 3 c c 413 5 O O T 1 c; Q 1 Do D X J. o 5 02 71.6 4 1 4 c b 5917 48 i / ? J X O w 7.24 81 6 5 2 1 m b 6007 44DO J. 4 J D 91 0 ^ X w 10 93 91 2 c D 2. c V, 6374 4974 1630 212 7.40 95.9 7 2 3 m c 4275 3326 1637 159 6.54 86.1 8 2 4 c c 5700 4457 1745 172 7.37 102.9 9 3 1 c c 6363 5157 1857 195 7.81 113.2 10 3 2 m c 6474 4907 1835 234 7.38 97.4 11 3 3 c b 4378 3480 1629 130 7 .23 96.6 12 3 4 m b 5464 4217 1708 168 9.45 90.4 13 4 1 c b 6225 4988 1775 190 8.28 104. 6 14 4 2 c c 6723 5251 1652 233 6.44 133.8 15 4 3 m b 4189 3274 1498 157 6.80 93.3 16 4 4 m c 6284 4745 1837 214 9.50 106.3 Fistulated: Fecal outputs OBS PER AN SUPP ADD DMOUT OMOUT NDFOUT NOUT 1 1 1 m c 2799 2426 1554 62.1 2 1 2 m b 3020 2610 1719 65.8 3 1 3 c c 2415 2086 1477 45.4 4 1 4 c b 2707 2362 1659 52.0 5 2 1 m b 2868 2507 1566 66.5 6 2 2 c b 2940 2507 1628 58.8 7 2 3 m c 2665 2334 1492 61.0 8 2 4 c c 2735 2356 1594 56.9 9 3 1 c c 2678 2378 1622 48.2 10 3 2 m c 3094 2730 1835 57.2 11 3 3 c b 2850 2510 1720 45.0 12 3 4 m b 3053 2718 1782 55.9 13 4 1 c b 2707 2386 1604 58.5 14 4 2 c c 3095 2707 1805 67.2 15 4 3 m b 2725 2387 1434 67.9 16 4 4 m c 3313 2884 1785 76.2

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In situ digestion: Percent left OBS PER AN TIME 1 1 1 U 2 1 1 1 1 3 1 1 4 1 1 X 4 5 1 1 D 6 1 1 7 1 1 1 1 Q lo 8 1 1 z 4 9 1 1 1 J o 10 1 1 A O 4o 11 -I 1 1 12 1 A u 13 -1 1 J. 14 1 1 15 1 4 1 6 1 o z b 17 1 2, Iz 18 1 1 Q i O 19 1 O A Z 4 20 1 2 J D 21 1 A Q 4o 22 -1 1 Z 2 3 -1 J. J A 2 4 i J. 2 5 1 2 D 1 4 2 / 1 1 o J D 2o 1 o 1 0 2y 1 O 1 R J. O J u 1 i 0 A. £t 4 J i 1 -5 J 7 fy J D 3z 5 ft o 3 3 1 X O -3 o u 3 4 1 1 3 D 1 A *k 3 D X A 3 / X A 4 3 8 -1 X A 0 3 9 -1 1 4 IZ 40 X A 4 1 Q 41 1 4 z 4 42 1 4 J b 43 1 4 A 0 4o 44 1 4 ^ A bU 45 2 1 0 46 2 1 1 47 2 1 2 48 2 1 4 49 2 1 6 50 2 1 12 51 2 1 18 52 2 1 24 53 2 1 36 54 2 1 48 55 2 1 60 56 2 2 0 ^TTPP Ann DM CP m c 92.22 93 66 m c 86.83 85 97 in v.* 84.12 86.98 m 83.36 87 27 m 80.80 84.31 ICl 74.56 82 56 in 64.26 78.01 in 63 43 78.26 ill 47 85 59 58 Tift ill 37 62 45.24 ill 12 13 14.79 ill 92 10 94 27 m iJ 86.22 87 92 m b 85.20 87 30 ill b 80 27 86 90 n\ b 79 63 85 89 m b 68.58 81 .71 m V, u 59.78 73 44 in 59.29 73.15 m 39.75 49 50 m b 31.73 39.60 in b 9 11 10 63 V91.85 95.76 \_> 86 69 92 62 84 68 89 51 77 03 84.66 74. 53 85.74 66 12 79.16 60.21 75.56 55.02 69.71 38.28 47 48 c 18.16 22 10 c 0.67 0.81 b 91.74 92.32 b 84.97 89.92 b 82 54 88 13 b 76 98 83 91 b 72 64 83 .86 b 67 89 83 60 b 59.41 76.13 b 55.51 69 .70 b 40 .79 51 63 \^ b 19 49 22 85 C b 0 05 0 06 TTl ill b 87 91 91 .25 in b 85 89 91 90 m b 83 .70 89.64 m b 80.38 90.57 m b 78.84 88.20 m b 75.49 89.39 m b 61.32 78.04 m b 62.05 77.49 m b 44.02 54.89 m b 24.48 29.63 m b 5.93 6.33 c b 89.18 91.73

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In situ digestion: Percent left OBS PER AN 57 2 2 58 2 2 59 2 2 60 2 2 61 2 2 62 2 2 63 2 2 64 2 2 65 2 2 66 2 2 67 2 3 68 2 3 69 2 3 70 2 3 71 2 3 72 2 3 73 2 3 74 2 3 75 2 3 76 2 3 77 2 3 78 2 4 79 2 4 80 2 4 81 2 4 82 2 4 83 2 4 84 2 4 85 2 4 86 2 4 87 2 4 88 2 4 89 3 1 90 3 1 91 3 1 92 3 1 93 3 1 94 3 1 95 3 1 96 3 1 97 3 1 98 3 1 99 3 1 100 3 2 101 3 2 102 3 2 103 3 2 104 3 2 105 3 2 106 3 2 107 3 2 108 3 2 109 3 2 110 3 2 111 3 3 112 3 3 TIME SUPP ADD 1 c D 2 c y. 4 c -u D 6 c 12 c V. 18 c 24 c Jj 36 c D 48 c O 60 c D 0 m C 1 m C 2 m C 4 m C 6 m C 12 m C 18 m C 24 m C 3 6 m C 48 m C 60 m C 0 c C 1 c C 2 c C 4 c C c 0 c C c C 1 o lo c C z4 c c J 0 c c 4o c D u c r\ \j c J. c \^ c 4 c C 0 c C 12 c C 18 c C 24 c C 3 6 c C 48 c c 60 c c 0 m c 1 m c 2 m c 4 m c 6 m c 12 m c 18 m c 24 m c 36 m c 48 m c 60 m c 0 c b 1 c b CP o 4 Z 0 87 48 / O O o o 95 / 0 0 o 43 / -5 0 A S7 d4 • D u 77 D / 7 1 80 no 41 05 0 1 07 Z / ^ -J 62 c D 1 Q c; 91 Q O OO 0 o y fi fi o 4 Q O J "3 C J O ft7 7 n o U n 7 ft7 / D R O DZ 4Q / u ST 97 D O A 1 4 J. D O fin Dz O D n7 A o 0 0 • z z fin 1 0 • xz 38 n7 0 09 O J fi7 09 ft A O 4 7 1 • / X O J 7 6 Q1 O X 7ft o o 80 HQ R9 9 4 / / S7 34 70 83 .71 V O • ?B X 85 .20 7 77 94 • V o 7 6 53 ft J fin J -J 25 0 • o o 2 97 o o 91 82 87 60 96 64 84 73 93 .70 7Q 17 89 97 77 • OX 86 99 D O 7 4. fid 09 D ^ 1 A • X ^ 86 04 D J. ft ^ 7 8 28 RO a ^ X fi R 60 "^0 40 46 c fiO • oz 89 Q1 .ox Q7 A ft Q 7 1 fi X D oD 7 fi7 83 .40 90 .03 80 .82 87 .44 72 .18 86 .72 61 .13 75 .99 56 .53 70 60 51 .55 64 95 11 .60 13 62 3 .26 3 .83 89 .67 90 .75 85 .76 92 .03

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In situ OBS PER AN TIME TIT lis o o 2 1 1 A 11% •3 5 4 1 1 R 1 J. D J -l 1 1 <^ J. J. O •3 J 12 1 1 T 3 18 X X o •J 5 J 24 1 1 Q X X 7 -i 36 ion X z u 48 XZ X a 60 10 0 X z z 4 0 10 7 X Z J 4 1 1 0 A X Z 4 0 J 4 2 1 0 R XZ D o J fi IOC XZ D *k XZ / J A 4 Xzo J 4 1 S 1 o o xzy A 4 ion X JU 4 J o X J X 4 1 1 o X JZ A 4 111 X J o 1 X 0 1 "5 A X J 4 A ** 1 X 1 "5 R X-3 3 A ft 1 X 0 1 "5 C X J D A 1 X 4 1 X J / A 4 1 X <; V X ^ O 4 1 X 12 1 "^Q X J 7 4 1 X 18 1 Afl X 41 fk 0 z rt O 1 R >l X D4 ft 0 z fin ICC XDO 4 •a ICC XDD A 4 1 157 4 3 2 158 4 3 4 159 4 3 6 160 4 3 12 161 4 3 18 162 4 3 24 163 4 3 36 164 4 3 48 165 4 3 60 166 4 4 0 167 4 4 1 168 4 4 2 digestion: Percent left SUPP ADD DM CP b 82 48 89. 67 b 74.24 84.33 b 70 68 85.33 b 61 51 77 60 c b 57.45 73 .49 b 47.11 57.92 b 16 52 19. 68 b 5.93 6.98 b 0.59 0.69 in b 90.17 96.39 iti b 85.65 92.99 in b 83.38 90.87 in b 79.70 84.39 ill b 77 84 86.20 m b 60 34 74.37 in b 53 52 67.99 m b 49 94 63 .26 in b 19 93 24.38 in b 2 00 2.44 in b 0 .21 0.26 b 86.96 89.00 b 80. 60 86.78 b 77 .77 85.20 b 75.06 83.70 b 72.41 82.68 b 68.11 80.55 c b 62 .74 78.71 c b 58.59 74.88 c b 44.58 56.59 c b 32.93 41.44 c b 1.13 1.42 c c 87.04 92.15 c c 81.68 89.40 c c 78.86 89.08 c c 75.20 85.99 c c 74.16 79.88 c c 66.35 81.00 c c 65.04 77.90 c c 60.68 76.22 c c 50.53 63 52 c c 47.41 59.35 c 1.42 1.77 in b 88.60 90.84 in b 81.81 86.34 m b 81 .06 84.32 m b 74.97 82.05 m b 72.73 81.02 m b 61.32 74.01 m b 48.32 57.87 m b 45.97 56.73 in b 23 .15 27.91 m b 0.52 0.63 m b 0.13 0.16 m c 88.73 91.32 m c 81.81 86.79 m c 79.45 87.56

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280 In situ digestion: Percent left OBS PER AN TIME SUPP ADD DM CP 169 4 4 4 m c 78.19 83.57 170 4 4 6 m c 77.78 85.82 171 4 4 12 m c 61.34 74.89 172 4 4 18 m c 57 .01 70.64 173 4 4 24 m c 50.64 62.46 174 4 4 36 m c 26.28 32.58 175 4 4 48 m c 11.96 14.77 176 4 4 60 m c 0.17 0.21

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281 Rumen Fluid: OBS PER AN SUPP 1 1 1 m 2 1 1 3 1 1 m 4 1 1 m 5 1 1 m 6 1 1 m 7 1 1 m 8 1 2 m 9 1 2 in 10 1 2 m 11 1 2 m 12 1 2 m 13 1 2 m 14 1 2 m 15 1 3 c 16 1 3 c 17 1 3 c 18 1 3 c 19 1 3 c 20 1 3 c 21 1 3 c 22 1 4 c 23 1 4 c 24 1 4 c 25 1 4 c 26 1 4 c 27 1 4 c 28 1 4 c 29 2 1 m 30 2 1 m 31 2 1 m 32 2 1 m 33 2 1 m 34 2 1 m 35 2 1 m 36 2 2 c 37 2 2 c 38 2 2 c 39 2 2 c 40 2 2 c 41 2 2 c 42 2 2 c 43 2 3 m 44 2 3 m 45 2 3 m 46 2 3 m 47 2 3 m 48 2 3 m 49 2 3 m 50 2 4 c 51 2 4 c 52 2 4 c 53 2 4 c 54 2 4 c 55 2 4 c 56 2 4 c 57 3 1 c pH, airanonia, sodium ADD TIME Fn c 2 6 00 c 4 c o o o z o c 6 D J / c 8 5 b / c 12 O / i c 16 6.61 c 24 6 87 b 2 6.16 b 4 b J o b 6 C CO b 8 D b J b 12 D /O b 16 CO 6.59 b 24 6.68 c 2 C T c 6.7b c 4 6 by c 6 6 by c 8 6.41 c 12 6 b / c 16 6 4z c 24 6.64 b 2 o c 6 o b b 4 b / 1 b 6 ^ CO b bi b 8 C AC b • 4 o b 12 C C Q b by b io b o b b 2. 4 C Q 1 o o X D 0 X o b 4 0 D D b 0 D D 0 b o o a *7 Q o / y D £ Q 1 o o X D 1 0 o D ^ 4 0 y D 7 7 ^,11 b 4 b b y b D D 4o b 8 C O 0 b b 12 b 4b b 1 5 c An b 24 b bl c 2 b bb c 4 b 4 / c 6 6 40 c 8 6 2o c 12 b ziU c 16 6.29 c 24 6.65 c 2 6,88 c 4 6.68 c 6 6.51 c 8 6.46 c 12 6.51 c 16 6.46 c 24 6.72 c 2 6.92 and potassium NH3 NA_roM K_mM 23 .66 56 .5 110.0 17 .52 43 5 81 8 11 .55 47 .8 74.2 5 .62 60 .9 84.4 2 .71 69 6 58 8 4 .12 73 .9 56 3 3 61 91 .3 38.4 22 .20 43 .5 94 6 15 61 34 .8 81 8 7 .40 43 .5 74 2 1 .10 47 .8 61 4 1 .71 47 .8 48 6 3 .95 60 .9 61 4 4 .09 60 .9 56.3 24 .40 113 .1 38.4 18 .93 113 .1 38.4 9 .83 113 .1 35.8 4 .27 108 .7 33 2 5 .41 113 .1 43 5 5 .75 121 .8 38 4 7 66 126 .1 23 0 9 .84 108 .7 35.8 14 .25 113 .1 46 0 2 .39 113 .1 30 7 1 .59 108 .7 25 6 5 .76 117 .4 38.4 4 .95 113 .1 33.2 6 .92 126 .1 28 1 18 68 69 6 84. 4 19 .55 78 .3 71.6 14 .45 87 .0 74.2 8 .06 91 .3 71 6 3 .39 91 .3 51.2 6 .79 100 0 58 8 5 .09 113 .1 40.9 30 .33 87 .0 61.4 18 .92 78 .3 43.5 10 .45 82 .6 51.2 3 .58 87 .0 40 9 3 .77 87 .0 30.7 6 .97 95 .7 53 .7 8 .55 82 6 38.4 10 .39 108 .7 53 7 46 .80 104 .4 66 5 19 .98 91 .3 61 4 14 .17 95 .7 66 5 6 .34 91 .3 58 8 4 .18 100 .0 56.3 8 .80 108 .7 51.2 9 .83 117 .4 35.8 23 .82 113 .1 30.7 16 .43 113 .1 25.6 11 .37 117 .4 23.0 6 .57 121 .8 23.0 9 .12 108 .7 33.2 7 .52 121 .8 28.1 30 .51 108 .7 35.8

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Rumen Fluid: pH, ammonia. OBS PER AN SUPP ADD TIME 58 3 1 c c 4 59 3 1 c c 6 60 3 1 c c 8 61 3 1 c c 12 62 3 1 c c 16 63 3 1 c c 24 64 3 2 m c 2 65 3 2 m c 4 66 3 2 m c 6 67 3 2 m c 8 68 3 2 m c 12 69 3 2 m c 16 70 3 2 in c 24 71 3 3 c b 2 72 3 3 c b 4 73 3 3 c b 6 74 3 3 c b 8 75 3 3 c b 12 76 3 3 c b 16 77 3 3 c b 24 78 3 4 m b 2 79 3 4 m b 4 80 3 4 m b 6 81 3 4 m b 8 82 3 4 m b 12 83 3 4 m b 16 84 3 4 m b 24 85 4 1 c b 2 86 4 1 c b 4 87 4 1 c b c O 88 4 1 c b o o 89 4 1 c b 90 4 1 c b 1 b 91 4 1 c b >i Z4 92 4 2 c c 2 93 4 2 c c 4 94 4 2 c c 6 95 4 2 c c o o 96 4 2 c c 12 97 4 2 c c 16 98 4 2 c c 24 99 4 3 m b 2 100 4 3 m b 4 101 4 3 m b 6 102 4 3 m b 8 103 4 3 m b 12 104 4 3 m b 16 105 4 3 m b 24 106 4 4 m c 2 107 4 4 m c 4 108 4 4 m c 6 109 4 4 m c 8 110 4 4 m c 12 111 4 4 m c 16 112 4 4 m c 24 282 sodium and potassium rn NH3 NA_mM K_mM o o D OO 30 1 0 lo ^ c\ A 1 U4 A % 25 6 C C Q D by 1 C 1 D /2 113 X 23 0 o 4 / 6 21 I 1 T I I / • A *k 25.6 C £ Q 4 A 1 4 1 1 1 J X 23 0 D O D y D2 X u 0 1 I 38.4 D oy 0 / / 121. 0 7 .7 C 1 A 21 4b D2 z 99 7 C 1 >! O 14 17 A "3 43 D 89 5 D J 1 12 29 A "3 43 D 94.6 o 4j> D ob A 1 0 0 99.7 b 4o A 0 42 DZ z 102.3 b D o / 2 J> DZ z 104 9 b / J r; 7 b bu y 102 3 b yy 17 oU Ill 113. X 43 5 c on b y u 2 1 y 2 113. 1 X 40 9 b / 4 14 34 11 / A 120.2 b b 4 1 b .20 1 u • X 40 9 0,0/ 3 y 0 11 / A *k 35.8 b -D b 8 ol 113 X 46 0 b y ^ 6 y 0 121. D 0 35 8 C C A b D 4 22 ol o2 D 89.5 C AC b 4 b 21 b4 o2 b 89.5 b 3 0 12 lo oZ c D 84.4 C C A b b 4 b / 4 / 0 71 6 C 1 A b / 4 3 u b 0 z D 53 7 •7 A 1 ft7 n 81 8 0 y D 4 o2 inn X u u 46 0 7 no 4 / • Do 1 n ft X u 0 7 43 5 0 O X 1 1 .XX 1 1 X X 0 1 X 38 4 n 1 Z X 1 1 X X J 1 X 33 2 X Aft 4 0 11"^ X X -J 1 30.7 41 -1 D -? 1 7 fi 28 1 D rs ? 0 fil OX 11"^ 1 38.4 fi 71 X z 1 7 1 X 30.7 Q / ? A1 4 X 60 9 76.7 U ^ J ft fid Q 76.7 f, in X u • J J 0 J • 0 74.2 0 0 7 D 74.2 fi 19 0 • X ^ X DO D ^ £: 0 69 1 0 X 0 Q 1 y X D -7 <: 0 76.7 0 D U 4 A 0 42 7 ft 3 81 8 0 ^ Z / .ID 1 nA X u 4 ft 56 3 0 • 0 z 14 1 1 11 Q 1 y X •3 J 51 2 b 4 b 22 b b 1 nn X u n u 81 8 0 ft J 14 .10 87 0 74 2 6.71 3 .23 95. 7 66.5 6. 61 5 .33 91. 3 53.7 6.76 4 .48 108. 7 46.0 6.53 20 .06 91. 3 92.1 6.48 17 .91 78. 3 76.7 6.48 11 68 82 6 76.7 6.51 5 .72 82 6 76.7 6.80 2 .78 91. 3 58.8 6.66 4 .87 95. 7 61.4 6.83 3 .80 104. 4 48. 6

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A A r E N S A C R R I T U D E O I M I P D T r 0 A M P I I T X D L E L T c u 1 1 2 MOL CON 69.4 1 / z 1 1 4 MOL CON 59.0 2 4 D 1 1 6 MOL CON 68.6 1 / 2 1 1 8 MOL CON 58.6 2 J 1 1 1 12 MOL CON 65.9 15.9 1 1 16 MOL CON 66.2 19.0 1 1 24 MOL CON 58 9 O 1 "7 21./ 1 2 2 MOL BAM 64.9 1 Q T ly / 1 2 4 MOL BAM 65.9 1 O Q lo y 1 2 6 MOL BAM DO 0 IT 1 2 8 MOL BAM 70.0 1 C O 15 y 1 2 12 MOL BAM 7 5.7 Li i 1 2 16 MOL BAM 71.1 1 C •? lb./ 1 2 24 MOL BAM 77 0 1 "3 Q 1 J y 1 3 2 COR CON 70.3 1 C T lb / 1 3 4 COR CON 73.9 14.0 1 3 6 COR CON 70.8 1 c o 15.9 1 3 8 COR CON 69 1 17 9 1 3 12 COR CON 69 7 17 3 1 3 16 COR CON 69 1 16.7 1 3 24 COR CON 71.7 1 yl ft 14.9 1 4 2 COR BAM 72.3 1 C T 15 / 1 4 4 COR BAM 75.2 •\ A C 14 b 1 4 6 COR BAM 7 5.0 1 c c 1 5 b 1 4 8 COR BAM / J O 1 b ^ 1 4 12 COR tsAM / 4 U 1 D y 1 4 16 COR BAM T 1 O / 1 Z 1 b b 1 4 24 COR BAM / D 1 1 D 4 2 1 2 MOL BAM D D 4 1 Q £ 1 o b 2 1 4 MOL BAM D D 5 1 Q n lo / 2 1 6 MOL BAM / U Z 1 c c 1 3 b 2 1 8 MOL BAM 71.1 1 >1 o 14.2 2 1 12 MOL BAM 73.2 IJ o 2 1 16 MOL BAM 7 6.5 11 1 11 / 2 1 24 MOL BAM / 1 O 1 >1 Q 1 4 y 2 2 2 COR BAM T C A 7 5.4 1 O Q 12 y 2 2 4 COR BAM ^ A O / 4 o 1 "3 A 1 J 4 2 2 6 COR BAM / 4 0 1 c c 1 o b 2 2 8 COR BAM 71.5 1 o t 2 2 12 COR BAM / 0 5 1 Q n lo U 2 2 16 COR BAM 72 / 15 b 2 2 24 COR BAM 70.4 15 D 2 3 2 MOL CON 72 7 14.1 2 3 4 MOL CON 69 2 17 4 2 3 6 MOL CON 68 0 19 2 2 3 8 MOL CON 69.7 18 0 2 3 12 MOL CON 70.9 16.8 2 3 16 MOL CON 71.0 16.2 2 3 24 MOL CON 72 .7 14.7 2 4 2 COR CON 72.8 12.6 2 4 4 COR CON 72.6 12.6 2 4 6 COR CON 72.1 13.5 2 4 8 COR CON 70.7 14.5 2 4 12 COR CON 71.3 14.1 283 o T X I Tl c s T m i r\ w L 0 0 V Z D D E V T I> K TT u A A T L I L L 1 0 • £t 1 T 0 54 0 54 105 c • Z> 1 J. ^ v 1 43 1 25 120 1 0 0 u ^ ^ 0 80 0 40 73 7 1 X J / 1 X 87 1 78 114 lU A 4 1 1 .OX o £i 2 71 94 z n 7 n 1 X V X 0 94 100 1 1 X o o ^ 3 6 3 24 110 X o c. o n a it -J 06 0 58 117 X ^ c 97 1 01 0 50 92 1 0 XZ p 1 1 .XX 0 99 0 23 87 1 1 X X o n 3 5 1 44 0 68 83 Q 7 n 05 0 79 0 45 58 Q A ft 77 0 96 0 95 107 D p n 0 62 1 03 80 Q c • o ^ J 1 20 1 24 142 X u • U r\ / o A \J fio 0 74 101 XU • o o / A \J 9 f, 1 18 108 1 n iU X A u 1 X 1 1 X X 1 X 21 112 XU z A u ft 4 .04 A \J 91 1 10 112 1 1 X X 7 A u .OX 1 X 1 16 126 XU ^ A U Q1 -7 X 0 88 1 34 109 Q o c I? A 9 1 12 1 31 100 7 Q o n 7 0 0 79 0 87 104 7 Q • o 0 16 0 71 0 74 117 7 7 0 50 0 71 0 90 107 7 9 0 55 0 69 0 90 100 3 6 0 99 1 23 1 44 108 5 9 0 61 0 60 1 43 98 1 1 ^ 5 0 89 1 34 1 24 114 1 0 X ^ > o 0 59 0 89 0 67 117 1 X o •I • o 0 25 0 69 0 05 105 1 X o 1 • X 0 37 0 81 0 44 88 1 1 X X T • J 0 44 0 86 0 62 79 ^ 9 0 47 0 73 0 71 90 3 8 1 .24 1 08 2 13 69 9 0 7 6 1 01 0 91 95 9 7 0 69 0 65 0 77 72 Q o p o 0 43 0 44 0 13 89 q 1 X. 0 34 0 26 0 56 92 9 8 0 53 0 44 0 78 72 9 7 0 61 0 68 0 74 99 o A \J 99 0 96 1 42 112 X u £r D A u D D A v Q9 1 X 07 87 X u c D A u 7 n ^ 1 \j 1 X u 1 X 1 5 X -J 124 X U 7 A u A 1 4 X A u 7 Q A \J O 99 X u 7 0 28 0 72 0 67 115 10 .9 0 .15 0. 70 0. 52 114 11 .1 0 .11 0. 88 0. 75 109 10 .0 0 .58 0. 90 1. 05 102 11 .7 0 .77 0. 94 1. 18 85 12 .3 0 .65 0. 82 0. 94 82 12 .3 0 63 0. 71 0. 79 87 12 6 0 66 0. 70 0. 84 93 12 .6 0 .53 0. 60 0. 84 87

PAGE 291

Data of Individual (M/lOO p A A f E N S A C K R I T U D E I M I P D T r 0 A M P I I X D L E L T C u 2 4 16 COR CON 72 9 1 A 1 14 1 2 4 24 COR CON 71.7 13 9 3 1 2 COR CON 74.1 12 8 3 1 4 COR CON 70 9 14 3 3 1 6 COR CON 70 8 15 5 3 1 8 COR CON 70 4 15.0 3 1 12 COR CON 69 2 14 J 3 1 16 COR CON 70.4 13 y 3 1 24 COR CON 72.2 13 3 3 2 2 MOL CON 69.4 1 O 1 18 1 3 2 4 MOL CON 67 2 18.2 3 2 6 MOL CON 70.8 15.0 3 2 8 MOL CON 72.9 14.6 3 2 12 MOL CON 73.7 14 1 3 2 16 MOL CON 75 6 12 7 3 2 24 MOL CON 75.0 13 7 3 3 2 COR BAM 74.9 12 7 3 3 4 COR BAM 74.3 12 4 3 3 6 COR BAM 73.5 13.1 3 3 8 COR BAM 71.7 14.0 3 3 12 COR BAM 70.6 1 ^ A 16.4 3 3 16 COR BAM 71.1 16.1 3 3 24 COR BAM 71.9 14 / 3 4 2 MOL BAM 69 3 ly u 3 4 4 MOL BAM by J 1 y 3 3 4 6 MOL BAM by y 1 / y 3 4 8 MOL BAM / i / 10.3 3 4 12 MOL BAM 72.9 1 4 o 3 4 16 MOL BAM 71.5 lb / 3 4 24 MOL BAM 75.1 14.0 4 1 2 COR BAM 74.9 13 o 4 1 4 COR BAM 74.2 1 C A 10 U 4 1 6 COR BAM 1 b 3 4 1 8 COR BAM 72 9 1 ^ A 15.9 4 1 12 COR BAM 72.7 16.2 4 1 16 COR BAM 72.5 15.6 4 1 24 COR BAM 76.0 13 2 4 2 2 COR CON 77 5 12 4 4 2 4 COR CON 76.7 13.3 4 2 6 COR CON 76.7 13.9 4 2 8 COR CON 76.4 14 2 4 2 12 COR CON 75.3 14 2 4 2 16 COR CON 76.2 13 6 4 2 24 COR CON 77.5 13.0 4 3 2 MOL BAM 77.4 13.5 4 3 4 MOL BAM 74.6 15.1 4 3 6 MOL BAM 73 .2 15.9 4 3 8 MOL BAM 72.8 16.2 4 3 12 MOL BAM 75.8 13.8 4 3 16 MOL BAM 75.9 13.8 4 3 24 MOL BAM 75.9 14.3 4 4 2 MOL CON 74.4 15.0 284 [) and Total V r M. ( M \ T 1 V V I TT U c & s T rfi T vJ T. 0 0 V X T> D E V T R TT u p X\. A I T X L L 12 3 n U C A o 4 n on 0 00 100 11 D A o c O D A u 7 1 1.28 85 10 0 u Q Q OO n Q7 y / 1 X it -J 89 11 4 1 A A U4 1 1 n n 1 49 82 11 3 0 I D n U 7 A / f4 1 (T? X U .J 99 12 4 A D O A u i> 0 n 97 96 14 U n / o n u d7 1 17 81 1 J 1 n 1 1 / 1 n \j 7 Q 1 07 104 11.4 n y / n \j fid 1 55 94 in c lU b n o D n fid 0.66 104 11 o 11 o n C Q D y 1 J. 1 00 91 11 A 11.4 n ^ 4 n V 0 23 91 11 A 11.4 A U o n n ft 9 0.19 91 11 r\ 11 u n O 1 z 1 n 7 ^ 0.30 96 1 A 1 10 1 A 1 1 jl n fi fi .00 fi SO 97 9 0 0 ^ 1 bl n w fiQ 0 y 1 m X u 0 87 9 6 U b4 n U Q7 0 / 1 9 10.7 A OO n fi fi b 0 X U .J> 81 11 A 11.0 n / y n fi n 91 11 c 11.0 n 0 0 n u fi fi 0.96 99 11 1 11.1 n 4d n \j R9 0.91 105 1 A Q lU O n J o n fifi 0 88 127 in c lU D n O 0 n fiS V .J 1.29 95 n A1 n 70 0.64 99 Q Q A u • n V/ 7 fi 0 45 128 1 u o A u 9 Q 0 73 0.35 106 in Q n u n 53 0.32 95 in Q n 7 0 J £t n w fi9 0 45 89 in Q 1 u y n 4 / n \j 77 mil 0 72 85 o o n A U Rft 1.13 86 O.I A \) / D n u fi7 1.18 85 C3 / n R fi n 0 89 93 o o n A R n 45 0 .86 89 o D n J 0 n 4ft 0 91 97 Q 1 y 1 n J y n 4.S *x .J 1 09 102 Q "3 y J n 0 fil OX 1.24 104 fi '3 o o n fi 1 b 1 0 \j 1.44 90 O D n "3 "3 n u J X 0.78 111 O O n "3 A n Q 0 7 0.66 106 O J n 0 A Z 4 n U X 100 O "3 O O n Z D n u 9 Q 108 y z A U •3 "3 n '3 1 J 1 1 m X U X Q n y u A u 1 fi 1 0 n J y n fifi 109 •7 Q / y 0. 38 n \j 'K fi 0 91 98 7.2 0. 68 0 .00 1.27 81 8.4 0. 40 0 .55 1.04 94 9.3 0. 21 0 .51 0.84 99 9.5 0. 19 0 .46 0.77 104 9.1 0. 20 0 .38 0.74 89 8.3 0. 49 0 59 0.91 102 7.1 0. 67 0 61 1.41 97 9.4 0. 26 0 .50 0.48 96

PAGE 292

Data of Individual (M/lOO A P S A p T U u R fii 0 I P D T p M P I I I E L T C 0 4 MOL CON 72 .1 16 .7 6 MOL CON 74 6 14 .8 8 MOL CON 75 .8 13 .8 12 MOL CON 76 .8 13 .0 16 MOL CON 79 .0 11 .8 24 MOL CON 79 .2 12 6 n ) oiiu Total VFA (M) D D I V I IT \J s A S T •P 0 L 0 0 V E V T u R A A I T I L L 10.1 0.23 0. 53 0 .31 94 9.8 0.16 0. 37 0 .23 99 9.6 0.21 0. 43 0 .22 90 9.0 0.31 0. 47 0 .48 78 8.0 0.22 0. 49 0 .50 90 6.3 0.45 0. 49 0 .92 79

PAGE 293

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308 Rouquette, F.M., Jr., J.L. Griffin, R.D. Randel, and L.H. Carroll. 1980. Effect of monensin on gain and forage utilization by calves grazing bermudagrass J. Anim. Sci. 51:521. Rowe, J.B., M. Bobadilla, A. Fernandez, J.C. Encarnacion, and T.R. Preston. 1979a. Molasses toxicity in cattle: rumen fermentation and blood glucose entry rates associated with this condition. Tropical Anim. Prod. 4:78. Rowe, J.B., M.L. Loughan, J.V. Nolan, and R.A. Leng. 1979b. Secondary fermentation in the rumen of a sheep given a diet based on molasses. Br. J. Nutr. 41:393. Rowe, J.B., F. Bordas, and T.R. Preston. 1980. Protein synthesis in the rumen of bulls given different levels of molasses and cassava root. Tropical Anim. Prod. 5:57. Rowe, J.B., J.S.W. Morrel, and A.W. Broome. 1982. Flavomycin as a ruminant growth promoter-investigation of the mode of action. Proc Nutr. Soc. 41:56A. Rowe, J.B., P.J. Murray, and S.I. Godfrey. 1991. Manipulation of fermentation and digestion to optimize the use of forage resources for ruminant production. In: Isotope and Related Techniques in Animal Production and Health, pp. 83-99. International Atomic Energy Agency, Vienna, Austria. Royes, J.B. 1996. Source and Level of Energy Supplementation for Cattle fed Tropical Grass Hay. M.S. Thesis. University of Florida, Gainesville. Ruckebush. Y. 1988. Motility of the gastro-intestinal tract, pp In: D.C. Church (Ed.) The Ruminant Animal: Digestive Physiology and Nutrition, pp. 64-107. Waveland Press, Inc., Prospect Heights, IL. Rumpler, W.V. D.E. Johnson, and D.B. Bates. 1986. The effect of high dietary cation concentration on methanogenesis by steers fed diets with and without ionophores J. Anim. Sci. 62L1737. Rumsey, T.S. 1984. Monensin in cattle: Introduction. J. Anim. Sci, 58:1461. Russell, J.B. 1984. Factors influencing competition and composition of rumen bacterial flora. In: F.M.C. Gilchrist and R.I. Mackie (Ed.) Herbivore Nutrition in

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BIOGRAPHICAL SKETCH Osvaldo Balbuena was born April 18, 1955, in Avellaneda, Buenos Aires Province, Argentina, He grew up in the rural community of Ita Ibate, Corrientes Province, Argentina. He attended high school and college at Corrientes City. He received his veterinary medicine degree from the Universidad Nacional del Nordeste in 1978. The following year he was awarded a scholarship to learn research techniques in ruminant pathology at the Institute Nacional de Tecnologia Agropecuaria (INTA) He has been permanent researcher at INTA since 1983 and has worked in mineral deficiencies of beef cattle in Formosa and Chaco provinces. He later came to the University of Florida and in 1988 he received his M.S. degree under Dr. L.R. McDowell. After his return to Argentina he continued his work with mineral deficiencies and started to conduct on-farm research with protein-energy supplementation of beef cattle grazing rangelands. He has been a research coordinator at Colonia Benitez Experimental Station since 1989. In December 1993 he was awarded a thirty-month scholarship from INTA and came back to Florida, with his family, to pursue the Ph.D. degree under Dr. W.E. Kunkle. 315

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. William E. Kunkle, Chair Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Moore or of Animal Science tohn E. 'rof ess I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doeftor of Philosophy. Dougl^'s Bates Associate Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality,^ a dissertation for the degree of Doctor of P]a|losophy. as Andrew C. Hammond Associate Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. h^hn E. Sollenberger /i Professor of Agronomy*-

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1996 jL/f / Dean, College of Agriculture Dean, Graduate School