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Effects of Supplement Type on Performance and Physiological Responses of Brahman-Crossbred Cattle


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EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED CATTLE By REINALDO FERNANDES COOKE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Reinaldo F. Cooke

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This thesis is dedicated to my wife Flavia, and also to my entire family.

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iv ACKNOWLEDGMENTS Firstly, I would like to thank my wife, Flavia Cooke, and my family for all the love, support and patience during my program. I also would like to thank Dr. John Ar thington for the opportunity, guidance and also for the excellent mentorship. I would like to thank the members of my committee, Dr. Cliff Lamb, Dr. William Thatcher and also Dr. Charles Staples, for the support with my research and class work. Other faculty me mbers not involved in my committee, such as Dr. Chad Chase, Dr. Matthew Hersom, Dr. Joel Yelich, Dr. Richard Miles, Dr. Gary Hansen and Dr. Xuejun Qiu, also were fundame ntal for the accomplis hment of my goals. I would like to thank all the staff that help ed me with my research projects, with special thanks to Mrs. Toni Woods. My fo rmer advisor from Brazil, Dr. Jose Luiz Moraes Vasconcelos, is also very important to my academic and research career, since he is the one that introduc ed me to the scientific world. Special thanks are extended to my fellow graduate students, and also to my Brazilian colleagues Guilherme Marquezini, Everton Pereira, Ocilon S F ilho, Davi Brito de Arajo a nd Dr. Joo Vendramini for the indispensable help with my research projects.

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v TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................2 Supplementation for Forage-Based Diets.....................................................................2 Energy Supplementation...............................................................................................2 Molasses-Based Supplements.......................................................................................6 Supplementing Molasses to Forage-Based Diets.........................................................8 Citrus Pulp-Based Supplements.................................................................................10 Supplementing Citrus Pulp to Forage-Based Diets....................................................13 Energy Metabolism.....................................................................................................16 Energy Metabolism and Beef Cattle Performance..............................................20 Energy and Nitrogen Synchr onization in the Rumen..........................................21 Nutrition and Reproduction in Beef Cattle.................................................................24 Onset of Puberty in Beef Heifers.........................................................................25 Reproductive Performance in Postpartum Beef Cows........................................26 Feed Intake and Reproductive Performance in Cattle.........................................28 3 EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE, REPRODUCTIVE AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED FEMALES..................................................................................................................32 Introduction.................................................................................................................32 Material and Methods.................................................................................................33 Animals................................................................................................................33 Diets.....................................................................................................................35 Sampling..............................................................................................................36 Blood Analysis....................................................................................................37 Statistical Analysis..............................................................................................38 Results and Discussion...............................................................................................41

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viExperiment 1.......................................................................................................41 Experiment 2.......................................................................................................47 Conclusions.................................................................................................................52 4 EFFECTS OF SUPPLEMENT TYPE AND FEEDING FREQUENCY ON PERFORMANCE AND PHYSIOLOGI CAL RESPONSES OF YEARLING BRAHMAN-CROSSBRED STEERS........................................................................74 Introduction.................................................................................................................74 Material and Methods.................................................................................................75 Animals................................................................................................................75 Diets.....................................................................................................................75 Sampling..............................................................................................................76 Blood Analysis....................................................................................................76 Statistical Analysis..............................................................................................77 Results and Discussion...............................................................................................78 Conclusions.................................................................................................................83 LITERATURE CITED......................................................................................................93 BIOGRAPHICAL SKETCH...........................................................................................105

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vii LIST OF TABLES Table page 3-1 Composition of supplements fed to heifers..............................................................53 3-2 Nutrient composition and intake rate of supplements fed to heifers....................... 54 3-3 Composition of supplements fed to cows............................................................... 55 3-4 Nutrient composition and intake ra te of supplements fed to cows .........................56 3-5 Effect of treatments on ADG and repr oductive performance of heifers in experiment 1.............................................................................................................57 3-6 Effects of treatments on plasma me tabolites and hormones of heifers................... 58 3-7 Correlations between plasma measurements and ADG of heifers.......................... 59 3-8 Effects of performance and plasma c oncentration of metabolites and hormones on reproductive performance of heifers...................................................................60 3-9 Effects of treatments on plasma metabolites and hormones of cows ......................61 4-1 Composition of supplem ents fed to steers................................................................84 4-2 Nutrient composition and intake ra te of supplements fed to steers .........................85 4-3 Effects of treatments on plasma metabolites and hormones of steers......................86

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viii LIST OF FIGURES Figure page 3-1 Outline of the follicular and lute al function synchronization protocol....................62 3-2 Blood urea nitrogen (BUN) concentrations of heifers supplemented with citrus pulp(CT) or molasses-based supplement (ML).....................................................63 3-3 Plasma glucose concentrations of heifer s supplemented with ci trus pulp(CT) or molasses-based supplement (ML)............................................................................64 3-4 Plasma insulin concentrations of heifer s supplemented with c itrus pulp(CT) or molasses-based supplement (ML)............................................................................65 3-5 Plasma insulin-like growth factor I (IGF-I) concentrations of heifers supplemented with citrus pulp(CT) or molasses-based supplement (ML)............66 3-6 Plasma growth hormone concentrations of heifers supplemented with citrus pulp(CT) or molasses-based supplement (ML).....................................................67 3-7 Forage DMI of cows supplemented with citrus pulp(CT) or molasses-based supplement (ML)......................................................................................................68 3-8 Blood urea nitrogen (BUN) concentrati ons of cows supplemented with citrus pulp(CT) or molasses-based supplement (ML).....................................................69 3-9 Plasma glucose concentrations of cows supplemented with c itrus pulp(CT) or molasses-based supplement (ML)............................................................................70 3-10 Plasma insulin concentrations of cows supplemented with citrus pulp(CT) or molasses-based supplement (ML)............................................................................71 3-11 Plasma insulin-like growth factor I (IGF-I) concentrations of cows supplemented with citrus pulp(CT) or molasses-based supplement (ML)............72 3-12 Plasma progesterone (P4) concentrati ons of cows with CIDR and supplemented with citrus pulp(CT) or molasses-based supplement (ML)...................................73 4-1 Forage DMI of steers supplemented 3x/wk with citrus pulp(CT) or molassesbased supplement (ML), or daily wi th citrus pulp-based supplement.....................87

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ix4-2 Blood urea nitrogen (BUN) concentrati ons of steers supplemented 3x/wk with citrus pulp(CT) or mola sses-based supplement (ML), or daily with citrus pulpbased supplement.................................................................................................... 88 4-3 Plasma glucose concentrations of stee rs supplemented 3x/wk with citrus pulp(CT) or molasses-based supplement (ML) or daily with citrus pulp-based supplement...............................................................................................................89 4-4 Plasma insulin concentrations of st eers supplemented 3x/wk with citrus pulp(CT) or molasses-based supplement (ML) or daily with citrus pulp-based supplement...............................................................................................................90 4-5 Plasma insulin-like growth factor I (IGF-I) of steers supplemented 3x/wk with citrus pulp(CT) or mola sses-based supplement (ML), or daily with citrus pulpbased supplement.....................................................................................................91 4-6 Plasma growth hormone (GH) concen trations of steers supplemented 3x/wk with citrus pulp(CT) or molasses-base d supplement (ML), or daily with citrus pulp-based supplement.............................................................................................92

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED CATTLE By Reinaldo Fernandes Cooke May 2006 Chair: John D. Arthington Major Department: Animal Sciences Two studies were conducted to comp are the performance and physiological responses of forage-fed beef cattle supplemen ted with either molasses-based or citrus pulp-based supplements. The first study consiste d in two separate experiments. In Exp. 1, we compared BW gain, reproductive efficien cy, and plasma concentration of BUN, glucose, insulin, IGF-I, GH, and progester one (P4) of 60 Brahman x British heifers supplemented 3x/wk with either a molasses-ba sed (ML) or citrus pulp-based supplement (CT). Supplement intakes were formulated to be iso-caloric and iso-nitrogenous. Reproductive performance was not affected by treatments; however, mean BW gain was greater ( P < 0.05) for heifers fed CT (0.30 and 0.40 kg/d for ML and CT groups; SEM = 0.02). Mean plasma concentrations of gluc ose, insulin and IGF-I were greater ( P < 0.05) for heifers fed CT, whereas BUN was greater for heifers fed ML. Mean plasma P4 concentration did not differ between treatments. In Exp. 2, we compared forage DMI and plasma concentration of BUN, glucose, insulin, IGF-I, and P4 of 24 Brahman x British

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xi mature cows supplemented with the same treatments of Exp. 1. Forage DMI did not differ between treatments; however, a da y and treatment x day interaction ( P < 0.05) were detected. No differences were detected in any bl ood metabolite/hormone. For blood P4 analysis, the two treatments were further se parated into one of two sub-treatments, in a 2 x 2 factorial arrangement: A) exogenous source of P4 (CD) or B) natural source of P4 (NCD). Only the results from the CD group were reported because cows from the NCD group were at random stages of the estrous cycle. No differences in plasma P4 concentration were detected between CT and ML-fed cows, but a treatment x period interaction ( P < 0.01) was detected because cows fed CT had greater plasma P4 concentrations during the be ginning of the experiment. The second study compared BW gain, forage DMI, and plasma concentrations of BUN, glucose, insulin, IGF-I, and GH of 24 yearling Brahman x British steers supplemente d 3x/wk with either CT or ML, or every day with a citrus pulp-based supplemen t (CD). Supplement intakes were again formulated to be iso-caloric and iso-n itrogenous. Forage DMI did not differ among treatments; however, a day and treatment x day inter action were detected ( P < 0.05). Mean BW gain was greater ( P < 0.05) for CD vs. ML, tended to be greater for CD vs. CT ( P = 0.13), but did not differ for CT vs. ML (0.30, 0.18 and 0.10 kg/d for CD, CT and ML, respectively; SEM = 0.05). Mean plasma concentration of glucose was lesser ( P < 0.05) for CD vs. ML and CT. Mean insulin was greater ( P < 0.05) for CT vs. CD and ML. We concluded that fora ge-fed cattle offered a citrus pulp-based supplement had increased BW gain compared to cattl e offered a molasses-based supplement. Additionally, BW gain was fu rther increased if citrus pulp-based supplements were offered daily.

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1 CHAPTER 1 INTRODUCTION The beef industry in Florida primarily cons ists of cow-calf enterprises with forage as the main component of cattle diets. The re productive performance of the herd is one of the most important factors for the overall effi ciency of these operations, whereas energy is the first nutritional con cern for cattle reproduction. Fora ges grown in Florida usually are lacking in energy density, and conse quently, energy supplementation is often required. A variety of feedstuffs can be used as ingredients for energy supplements, but price and availability are highly variable. Citrus pulp and molasses are energy concentrated byproducts that originate from citrus a nd sugarcane production, respectively. Both enterprises are important and extensive agri cultural systems in Florida. Despite its moderate DM content (approximately 75 %), molasses is classified as a liquid feed, whereas citrus pulp is commonly processed and fed as dry pellets. The different physical characteristics of these products lead to differences in inta ke behavior, which have an important effect in operations where cattl e are supplemented with low frequency. Molasses and citrus pulp also differ in their carbohydrate profile. Sucrose is the main carbohydrate of molasses, whereas pectin is the main carbohydrate of citrus pulp. Pectin and sucrose are fermented differentl y in the rumen, potentially impacting forage intake, diet digestibility and energy utilization. For these reasons, experiments were conducted to compare the effects of supplement type on performance and physiologic responses of Brahman-crossbred cattle.

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2 CHAPTER 2 LITERATURE REVIEW Supplementation for Forage-Based Diets Forage is the cheapest and most available source of nutrition for beef cattle in the state of Florida. With the purpose of mini mizing costs of production, it is the main dietary component of the cow-calf operations located in the state. However, the forages grown in Florida usually do not have adequa te nutrient density to meet the nutrient requirements of cattle consuming typical amounts of DM (Moore et. al., 1991), particularly animals with greater nutrien t demands. According to the NRC (1996), nutritional requirements of beef cattle increas e with lactation, pregnancy, and growth rates. Consequently, growing cattle and lact ating beef cows require a high plane of nutrition to maintain adequate performance. When their diets are based on low-quality forages, as in the case of Florida, nutr ient supplementation is often required. Energy Supplementation Energy supplementation is required to ma intain optimal animal performance in areas where energy availability from grazed forages is limited (Caton and Dhuyvetter, 1997). In Florida, even when protein supplemen ts are fed and forage intake is potentially increased, energy requirements usually are not met because of the lack in forage quality. Bodine and Purvis (2003) also indicated that alt hough protein is typically considered the major limiting nutrient for cattle, its supplementa tion may not result in adequate energy supply to the animals. Consequently supplemental energy is required.

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3 Contrary to protein supplements, energy supplements often decrease forage intake and digestibility (Caton and Dhuyvetter, 1997; Kunkle et al ., 1999; Bodine and Purvis, 2003). A data base containing 66 publications concluded that fo rage intake was decreased when supplemental TDN intake was greater than 0.7 % of BW (Moore et al., 1999). These effects are even more pronounced when supplements contain high quantities of nonstructural carbohydrates (NSC) such as starch and suga rs (Sanson et al., 1990; Olson et al., 1999). The potential mechanisms by wh ich high NSC supplements depress forage intake and/or digestibility in clude a decrease in ruminal pH decrease in synthesis and activity of cellulolytic enzymes (Martin et. al ., 2001), impaired bact erial attachment to fibrous digesta (Hiltner and Dehority, 1983) and an increase in lag time for fiber digestion (Mertens and Loften, 1980). Bowman and Sanson (2000) compiled 116 publications and observed that supplements with high quantiti es of NSC do not have a negative impact on forage intake when fed less than 0.3 % of BW or when the NSC fraction of the supplement does not exceed 0.1 % of BW. However, restricting energy supplementation to these levels may impair animal performance, particularly when animals are fed low-quality forages and have high nutritional demands. An alternative to prevent de pressed forage intake when energy supplementation is necessary is the utilizat ion of fibrous by-products feedstuffs as energy sources in the diet. These feedstuffs usually have reduced NSC with moderate TDN content, are rapidly digestible in the rumen and also are low-pri ced when compared to the costs of high NSC feedstuffs. Some authors have reported no di fferences in performance when animals were fed energy supplements with high or low NSC content (Sunvold et al., 1991; Horn et al., 1995). These data imply that high NSC supplements can be replaced by highly digestible

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4 fibrous supplements without a depression in an imal performance. Other authors reported a small negative effect on forage intake wh en supplements with low NSC content were fed (Bowman and Sanson, 1996; Caton and Dhuyvetter, 1997; Royes et al., 2001). A review by Bowman and Sanson (2000), how ever, concluded that low NSC energy supplements, contrary to those with high NSC concentration, increased low-quality forage intake and digestibility. According to the NRC (2001), the NSC frac tion of a feedstuff is found inside the plant cells, and is composed of sugars, st arches, organic acids and other carbohydrates such as fructans, serving as the major s ource of energy for cattle. The NSC fraction is highly soluble in the ruminal environment. This fraction is degraded rapidly by the microbial population, and because of this, in creases the rate and amount at which VFAs are formed. Increasing the NSC content or av ailability in diets typically results in elevated VFA production/accumulation in the ru men with a decreased acetate:propionate molar ratio. This subsequently leads to a decr ease in ruminal pH that may be detrimental to fibrolytic bacteria, resulting in impaired forage digestibility (Aldrich et al., 1993; Joy et al., 1997; Knowlton et al., 1998). The energy provided by feedstuffs with a low NSC content usually originates either from NDF or pectin. According to the NRC (2001), NDF is the measurement that best differentiates structural from nonstructural carbohydrates and constitutes most of the structural components in plan t cells: cellulose, hemicellulo se and lignin. On average, NDF is less digestible than NSC and its co ncentration in feedstuffs is negatively associated with energy (NRC, 2001). The chemical profile of NDF (ratio of cellulose:hemicellulose:lignin) affects its digestibility. Cellulose and hemicellulose are

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5 potentially but incompletely digested and uti lized as an energy source by the microbial population in the rumen. In contrast, lignin is poorly or even not digested at all (Jung and Allen, 1995). Therefore, feedstuffs with a si milar content of NDF will not necessarily have similar energy concentration. Depending on the lignin concentration within the NDF fraction, feeds with greater NDF may have more available energy than feeds with lesser NDF content (NRC, 2001). The concentration of NDF in the diet is positively associated with ruminal pH since the microbes gene rally ferment the NDF fraction slower. Therefore, lesser VFA production/accumulati on and a greater aceta te:propionate molar ratio in the rumen may result. Pectins are found within the plant cell wall, but are not linked to lignin. Pectins are well digested by the ruminal microbes, but not by mammalian en zymes (Ariza et al., 2001). Although classified as a dietary fiber, pectins are not represented in the NDF fraction, and are classified as non-fiber carbohydrates (NFC; Arthington et al., 2002). The NFC fraction is similar to the NSC. It in cludes all carbohydrates not included in NDF, but also includes pectins, which are not cla ssified as part of the NSC fraction (NRC, 2001). Compared to NDF, pectins are rapidly an d extensively digested in the rumen, as reported by Ben-Ghedalia et al (1989), where the apparent digestibility was 98.7 % for pectin and 79.4 % for cell wall constituen ts by sheep fed high-pectin based feed. Although the digestibility of pectins and NS C components is similar, there are some major differences between them. When pectin escapes ruminal fermentation, it cannot be digested in the small intestine, whereas NSC can be digested. Further, when pectins are fermented in the rumen, they yield greater acetate:propionate ratios compared to NSC fermentation.

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6 Energy supplementation is used to correct deficiencies in order to maintain or enhance animal performance. A variety of di fferent feedstuffs can be used as energy sources in ruminant diets. Supplements with low NSC concentration are a viable alternative to substitute trad itional and costly supplements with high NSC concentration. When designing and formulating a supplementa tion program, the source of energy should be carefully chosen, since the differences in energy fermentation and digestion among carbohydrate sources may impact animal performance. Molasses-Based Supplements According to Curtin (1983), the term mo lasses referred specifically to the final effluent obtained in the prep aration of sucrose by repeated evaporation, crystallization and centrifugation of juices from sugarcane or sugar beets. Nowadays, current feed nomenclature characterizes molasses as any liquid feedstuff contai ning more than 43 % sugar (Curtin, 1983). Sugarcane molasses is a by-product of the sugarcane refining process to obtain sucrose, and must not c ontain less than 46 % total sugar expressed as invert. Invert is a 50:50 mi xture of glucose and fructose, obtained when sucrose is hydrolyzed. If molasses moisture content ex ceeds 27 %, its density is determined by double dilution and must not be less than 79.5 Brix (Curtin, 1983). The industry uses the term Brix as a measurement of molasses total solid content. This term originally indicated the concentration of sucrose in pure sucrose solutions on a weight basis, but molasses also contains glucose, fructose, raffinos e and numerous non-sugar organic materials (Curtin, 1983). Therefore, the Brix value for molasses often will differ from actual sugar or total solid content and will represent not hing more than a number denoting specific gravity, which cannot be related to either sucrose or DM content (Curtin, 1983).

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7 The chemical composition of sugarcane mo lasses varies widely, similar to that observed in many other industrial by-products. It is influenced by seve ral factors such as soil type, temperature, moisture, season of production, plant variety, production practices at a particular processing plant, and by st orage variables (Curti n, 1983). These factors lead to significant variations in nutrient c ontent, flavor, colorati on, viscosity and total sugar content (Curtin, 1983). Sugarcane molasses contains large qua ntities of simple sugars and other carbohydrates, contributing to its high-energy value. Molasses contains around 88 % of the TDN of corn (72 vs. 82 % on a DM basis, respectively), and th e principal sugar is sucrose, constituting around 30 to 40 % of molasses on an as-fed basis (Austin, 2003). In contrast to energy, sugarcane molasses is low in CP content, and its nitrogenous material mainly consists of non-protein compounds. However, CP content can vary according to soil type as reported by Chapman et al. ( 1965), where molasses produced from sugarcane grown on soils high in organic matter containe d 7 to 10 % of CP compared to 3 % for molasses from sugarcane grown on mineral rich soils. Regard ing mineral content, when compared to other energy sources such as ce real grains, sugarcane molasses is relatively high in calcium, potassium, magnesium, s odium, chlorine and sulfur, but low in phosphorus (Curtin, 1983). According to Kunkle et al. (1995), black strap molasses is the by-product of the sugar industries in south Flor ida and Louisiana. Most Fl orida sugarcane is grown on organic rich soils, resulting in a molasses with greater protein content than molasses derived from sugarcane grown on soils with low organic matter (Kunkle et al., 1995). Molasses production is increased during fall and winter, and its price is determined by the

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8 world market. More than 500,000 tons of molasses are produced annually in south Florida and this makes the state a net exporter of the product (Kunkle et al., 1995). Blackstrap molasses typically contains 22 % moisture, 60 % TDN and 7 % CP on an asfed basis (Kunkle et al., 1995). Molasses slurries are common on many Florida ranches. Slurries are created by blending molasses with dry ingredients, typi cally protein sources such as cottonseed meal or feather meal. Therefore, molasses-based supplements ar e classified as energetic supplements, containing high quantities of NSC deri ved from its high sugar content. Supplementing Molasses to Forage-Based Diets A beneficial use for molasses is its additi on to diets based on low quality forages or roughages, providing a readily available s ource of energy. Howeve r, molasses does not contain adequate protein to meet the CP re quirements of cattle fed low quality forages; consequently a source of supplem ental protein is often required. Feeding molasses has been studied extens ively over the past century. Pate (1983) compiled the results of several studies wher e molasses was supplemented to beef cattle diets based on forage. Supplementation usually resulted in decreased forage intake, but total DMI was increased. Animal performan ce was consequently increased, but not compared to when supplements based on othe r energy sources, such as cereal grains, were fed. However, when plant protein was added as a nitrogen source, molasses-based supplements were similar to corn-based supplem ents in terms of BW gain by beef cattle. More recently, a review reported the effects of liquid supplements on the performance of cattle fed forage-based diets (Moore et al., 1995). Generally, BW gain was increased but responses were variable, a nd no consistent rela tionship between rates of supplementation and gain were observed. When molasses was the only ingredient of

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9 the supplement, gains were sometimes decreas ed and at other times increased. However, when a major protein source was adde d, BW gains increased considerably. Supplementation of molasses only increased forage consumption when intake of forage was less than 1.75 % of BW wit hout supplement; otherwise forage intake was decreased. A recent study compared sources and rates of energy supplementation for yearling cattle fed ammoniated hay (Roye s et al., 2001). The authors compared supplements based on sugarcane molasses, corn or soybean hulls, and reported reduced feed efficiency and consequent less BW gain for animals fed molasses compared to the other supplements, supporting the findings reported by Pate (1983). Several authors reported that feeding cattle diets c ontaining high quantities of sucrose or molasses resulted in a increase in the molar percentage of butyric acid in the rumen (Owen et al., 1967; Kellogg and Owen, 1969; Hatch and Beeson, 1972). In addition, Reyes (1974) and Silves tre et al. (1977) also noted a substantial in crease in the molar percentage of butyric acid in the rumen of cattle fed in creasing quantities of molasses. Olbrich and Wayman (1972) compared the molar percentage of butyric acid in the ruminal fluid of steers fed either molasses or corn, and reported a greater concentration for the animals fed molasses. Based on these reports, feeding molasses to cattle increases the molar percentage of butyric acid in the rumen, which usually is at the expense of propionic acid when molasses substitu tes grain or at the e xpense of acetic acid when molasses is fed as a supplement in forage -based diets (Pate, 1983). This variation in VFA profile may be related to differences in ruminal microbial population. Ruminal protozoa population has been positively correlated with but yrate synthesis in the rumen (Hristov et al., 2001), whereas de faunation decreased the concentr ations of butyrate in the

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10 ruminal environment (Mendoza et al., 1993; F aciola et al., 2004). The inclusion of 35 % of molasses in beef cattle di ets increased the ruminal prot ozoa population (Foreman and Herman, 1953). Therefore, an increased prot ozoa population in the rumen might be one of the causes to the greater butyrate synthe sis observed in cattle fed with molassescontaining diets. Most of the available information a bout the effects of VFA proportion on energy metabolism involves acetate:propi onate ratios, which are nega tively correlated to energy utilization and consequent anim al performance. Given that butyrate, like acetate, is not a glucose precursor, a similar effect can be at tributed to butyrate:propionate ratios. This could explain why animals fed molasses-ba sed supplements typically have reduced performance compared to animals fed gr ain-based supplements (Pate, 1983). In summary, although molasses supplement ation usually has a negative impact on forage DMI, overall performance is often improved by supplementing molasses to cattle fed energy deficient diets. Further, molasses al so can be used as an effective carrier to deliver other nutrients, such as protein, vitamins and minerals. Citrus Pulp-Based Supplements Florida produces more than 80 % of the total United States supply of citrus, and almost 90 % of this production is processed into juice (Hodges et al ., 2001). Citrus pulp is the residue from citrus processing, and cons ists of a mixture of peel, rag and seeds. Annually, the Florida citrus i ndustry produces more than 1 million tons of citrus pulp (Hodges et al., 2001) and most of this tota l has been exported to foreign countries throughout the past years, causing local prices to increase, and resulting in a decrease in domestic usage. However, recent declines in exports have made citrus pulp an affordable by-product feed for ruminants in the Un ited States (Arthington and Pate, 2001).

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11 Citrus pulp has been fed to ru minants since the beginning of 20th century, when it was suggested to have a potential value as a feedstuff. Walker (1917) made the initial observation that cattle readily consumed a mix of fresh citrus pulp a nd cull fruits, but the feed often spoiled before animals consumed it all. In the 1930s, dried citrus pulp began to be produced commercially after Scott (1926) dehydrated grapefruit and fed it to dairy cattle. Since then, the production and availability of dry citrus pulp has increased steadily (Arthington et al., 2002 ). The drying procedure consists of grinding or chopping and then dehydrating the fresh fruit residue. To aid in the dehydration process, calcium oxide or calcium hydroxide is mixed in th e process (Arthington et al., 2002). Citrus pulp can be fed wet, as a dry meal or in dry pellets. The wet product is usually shipped directly from the juice plant, arranged as piles in the pastures and offered to cattle. Arthington and Pate ( 2001) suggested that it is t ypically uneconomical to feed wet pulp because of its high wasting rate (up to 30 %) and shipping cost s associated to its low DM content (15 to 20 %). Cattle consume citrus pulp in either the wet or dry form, but dry citrus pulp is more easily transpor ted and handled (Arthi ngton et al., 2002). Dry citrus pulp is a bulky feedstu ff, having a density of approximately 303 kg/m. For this reason and because of economic and transpor t limitations, pelleting dry citrus pulp is favorable since it increases density, handli ng efficiency, and decreases dustiness and bridging in storage bins (Arthington et al., 2002). Ammerman et al. (1967) compared pe rformance of steers fed nonpelleted and pelleted citrus pulp added at different concentr ations within the supplement. When fed at 66 % of the total supplement, steers fed pelle ted citrus pulp gained more weight (1.38 kg/d) compared to those fed the nonpelleted pulp (1.19 kg/d). The aut hors attributed the

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12 differences to the greater density of the pe lleted form (211 to 374 kg/m for nonpelleted and pelleted form, respectively), increasing its consumption by the steers. A similar study conducted with lambs also indicated that feed ing pelleted citrus pulp increased intake and animal performance (Loggins et al., 1964). La mbs fed pellets had numerical greater BW gain (0.12 kg/d) when compared to lambs fed the nonpelleted form (0.11 kg/d) and also had slightly greater intake (0.06 kg/d). Pi nzon and Wing (1976) reported no differences in the ruminal pH, ruminal ammonia concen tration, blood urea nitrogen concentration, and VFA concentration in the rumen of steer s fed pelleted or nonpelle ted citrus pulp at similar rates. Chemical composition and physical characte ristics of citrus pulp mostly depends on the type of fruit and processing methods used to produce it (Arthington et al., 2002). The same authors compared the nutritional va lue of samples collected in two different studies (Ammerman et al., 1968; Arosemena et al., 1995) and tabular values reported by NRC (1996). The authors concluded that rega rdless of some varia tions among the three sources attributed to producti on site, citrus variety and prop ortion of seeds and peel the nutritional values were similar. According to the NRC (1996), citrus pulp usually contains 82 % TDN and 6 to 7 % CP (DM basis). Calcium content is partic ularly high in citrus pulp because of the addition of calcium salts to aid in the dryi ng process. The carbohydrate fraction of citrus pulp is mainly composed of 25 to 44 % pecti n, 12 to 40 % sugar and 1 % or less starch on a DM basis (Hall, 2000). Compared with common cereal grains such as corn, citrus pulp contains moderate amounts of NSC (sugars a nd starch) and high concentration of pectins. For that reason, citrus pulp contains a different carbohydrate profile compared to

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13 traditional energy feeds. Citrus pulp-based supplements are classified as energy supplements, and despite the modest content of NSC, digestibility is great because of the large concentrati on of pectins. Supplementing Citrus Pulp to Forage-Based Diets Several studies have been conducted to investigate citrus pulp and subsequent pectin fermentation in the ruminal environment. Welch and Smith (1971) compared rumination patterns when sheep and cattle were fed pelletized citr us pulp, beet pulp or alfalfa meal in addition to mixed hay. Ce ll wall content was 27, 50, 48, and 68 % of DM for citrus pulp, beet pulp, alfalfa meal, and mixed hay, respectively. Citrus pulp decreased rumination time for sheep and cattle when compared to mixed hay fed alone (0.34 vs. 0.16 min/g of DM consumed for mixed hay a nd citrus pulp, respectively). However, when data were analyzed per unit of cell wa ll content, no difference in rumination time was found (0.50 vs. 0.56 min/g of cell wall cont ent consumed for mixed hay and citrus pulp, respectively). Alternatively, beet pul p and alfalfa meal reduced rumination time when data were analyzed as total DM or cell wall content consumed. The authors attributed these effects to the lack of physic al form required to stimulate rumination in beet pulp and alfalfa meal, which were greate r in cell wall content compared to citrus pulp. The beef pulp and alfalfa meal were finely ground, and c onsequently did not contribute substantially to the rumination proces s. The authors concluded that addition of citrus pulp to forage-based di ets did not influence rumination process and time, despite its low cell wall content compared to forages. Fi ber digestibility increased when citrus pulp was added to sheep diets in a dose-depe ndent pattern (Sudwee ks, 1977). Strobel and Russell (1986) added different sources of carboh ydrate to cultures of ruminal bacteria and measured the fermentation process and produc ts. The authors reported that carbohydrate

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14 fermentation was decreased when pH became mo re acidic, particularly when pectin was the major carbohydrate source. Pectin utiliz ation decreased by 53 % when culture pH dropped from 6.7 to 6.0, indicating that organisms that degrade pectins are pH sensitive. At neutral culture pH (around 6.7), the aut hors reported that addi tion of starch and sucrose increased lactic acid production, whereas little lac tic acid was produced when pectin was added. When culture pH was lo wered to 5.5, lactate production quadrupled for the starch, doubled for sucrose and did not ch ange for pectin. Ace tic acid production was always doubled for pectin compared to al l other carbohydrates, even when pH dropped below 6.0. Therefore citrus pulp may be le ss likely to cause la ctic acidosis and consequential impaired fiber digestibility when compared to these other feedstuffs high in NSC. Several other researches also have reported that feed ing citrus pulp increased the concentrations of acetic acid in the rumen a nd also increased the acetic to propionic acid ratio when compared to traditional energy sour ces such as cereal gr ains (Loggins et al., 1964; Hentges et al., 1966; Drude et al., 1971). Although gr eater ruminal propionate production usually was associated with high c oncentrate diets and negatively associated with high forage diets, when forage (corn silage) was replaced with increasing amounts of citrus pulp (0 %, 33 % 67 %, and 100 %) in steers diets, only a moderate decrease in the ruminal pH (7.12, 6.97, 6.81, and 6.76, respec tively) was reported (Schaibly and Wing, 1974). Furthermore, acetate synthesis wa s the greatest and propionate was the poorest when diets contained 100 % citrus pulp, even if compar ed to 100 % corn silage diets. The authors concluded that citrus pulp, though not c onsidered a forage in the strictest sense, does contain some forage -like properties which promote less acidic ruminal pH values. All these data imply that citrus pulp, rich in pectin, can have less

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15 detrimental effects on ruminal pH and fermen tation products compared to other energy sources, such as high NSC containing feedstuffs. Research was conducted in the 1950s a nd 1960s to investigate the impact of supplementing citrus pulp on the performance of beef cattle fed forage-based diets. Chapman et al. (1953) reported no significant differences in gain or feed efficiency when steers grazing St. Augustine pasture were s upplemented with 2.3 kg/d of citrus pulp or snapped corn, or had free-choice access to sugarcane molasses. But in a similar study, steers on pasture fed citrus pulp tended to have greater gains (0.78 kg/d) compared to steers fed ground snapped corn (0.72 kg/d), s ugarcane molasses (0.65 kg/d), or a mixture of corn, citrus pulp and cottonseed meal (0.75 kg/d) (Chapman et al., 1961). Research resulting from several studies indicated positive results from feeding citrus pulp as an energy source to feedlot an imals. Kirk and Davis (1954) fed steers for 120 d with either ground snapped corn or citr us pulp and reported similar gains (1.08 and 0.99 kg/d, respectively), but animals fed citrus pulp had greater feed efficiency (5.40 and 4.70 kg of required TDN / kg of BW gain, respectively). Peacock and Kirk (1959) conducted three 140 d trials in wh ich steers were fed either corn or citrus pulp at 70 % of the total diet, and did not detect differences in gain or feed efficiency. Ammerman et al. (1963) replaced ground corn with citrus pulp at 22, 44 and 66 % of the total diet of feedlot steers and did not find differences in gain across treatments, but gains were numerically greater for the 22 and 44 % inclusion rate (1.35 and 1.48 kg of ADG, respectively). Kirk and Roger (1970) published a review that contained 23 separate trials conducted at the University of Florida Range Cattle Research and Education Center in Ona, Florida, which used citrus products in b eef finishing diets. Citrus pulp was the main

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16 energy source in 20 of these studies. The author s concluded that the am ount of citrus pulp fed was positively associated with feedlot performance. Research demonstrates that citrus pu lp is a good energy source for ruminants, particularly for animals fed forage-based diet s, since it does not seem to interfere with fiber digestion and rumen health. With a large citrus industry in Flor ida, citrus pulp may provide an economical advantag e for producers in the southeas tern portion of the U.S. Energy Metabolism According to an extensive review by Bergman (1990), the VFAs, also known as short-chain fatty acids, are mainly produced in the rumen by microbial fermentation of carbohydrates, and contribute appr oximately 70 % to the caloric requirements of ruminants. The major VFAs are acetate, pr opionate and butyrate, produced in a ratio varying from approximately 75:15:10 to 40: 40:20 (acetate:propiona te:butyrate). After synthesis, VFAs are absorbed across the ru minal epithelium, from which they are either carried by ruminal veins to the portal vein a nd hence through the liver or are retained and metabolized for energy by the ruminal epit helial cells (Bergm an, 1990). Acetate and butyrate are oxidized to produce energy whereas propionate is reserved for gluconeogenesis. Approximately 90 % of the but yrate formed in the rumen is retained by epithelial tissue and converted to ketone bodies or carbon dioxide whereas the remainder is removed by the liver (Bergman, 1990). Little acetate approximately 30 % is retained in the rumen compared to butyrate (90 %). Most of the acetate is oxidized throughout body tissues to generate ATP. Acetat e also is the major source of acetyl CoA for lipogenesis (Bergman, 1990). Propionate is 50 % retained in the rumen and the remainder is removed from portal blood by the liv er, where it serves as a major substrate for gluconeogenesis (Bergman, 1990). This proce ss is essential to gl ucose status of the

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17 ruminant animal because almost all dietary glucose is fermented in the rumen and does not reach the small intestine fo r absorption. In summary, propiona te is directed to glucose synthesis whereas acetate and butyrate are oxi dized for energy generation, but butyrate is highly retained in the rumen a nd poorly contributes as an en ergy source for other tissues. Although acetate is the larg est single energy source ab sorbed through the portal system, accounting for more than half of the total energy absorbed as VFA (Bergman et al., 1990; Baird et al., 1975), it is inefficiently utilized as an energy source by tissues when compared to glucose, which is effectiv ely synthesized from propionate in the liver. Glucose is crucial for maintenance and pr oductive functions in ruminants such as growing and lactating cattle (Reynolds, 2005) and propionate accounts for up to 76 % of the glucose synthesized in the liver (Re ynolds et al., 1994). Ruminants eating foragebased diets depend on liver synthesis of gluc ose to meet their metabolic requirements (Huntington, 1997), and increased liver glucose synthesis is positively associated with energy balance and animal performance. Resear ch by Aiello et al. (1989) also reported detrimental effects of butyrate on VFA me tabolism by restraining hepatic propionate utilization, thus inhi biting the conversion of propionate to glucose. In addition, the inhibition is specific and not due to a general competition among VFAs for metabolism. High energy diets and supplements selected to enhance animal performance are typically associated with increased VFA production, pa rticularly propionate, leading to increased glucose synthesis. Even when these are base d on ingredients that are not propiogenic, such as citrus pulp or molasses, overall form ation of propionate is increased in the rumen as a consequence of increased total VFA production.

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18 Insulin is a hormone synthetized by betacells in the Islets of Langerhans in the pancreas, and primarly regulates glucose uptake by cells (Austgen et al., 2003). This hormone also has an important effect on li pogenesis and cellular upt ake of amino acids and some electrolytes (Austgen et al., 2003) Although there always is a low rate of insulin secretion in the pancreas, the amount secreted into the blood increases as blood concentrations of glucose rises, and simila rly decreases as blood concentrations of glucose falls. Therefore, insulin is impor tant for body homeostasis because it maintains blood glucose under constant concentrations a nd also has an anabolic role because it increases cellular uptake of nutrients. The mechanism by whic h insulin promotes glucose uptake by cells consists in the activation of insulin receptors embedded in the plasma membrane, which regulates the number and ope ration of protein molecules also in the plasma membrane that transport glucose into the cell (Austgen et al., 2003). Once inside the cells, glucose enters glycolysis and the re spiratory cycle to gene rate ATP molecules. Insulin-like growth factor I is a protein hormone that resembles insulin on its structure, and is mainly synthetized by th e liver in response to GH (Gluckman et al., 1987). However, several studies in cattle dem onstrated that IGF-I concentrations in blood were positively correlated to feed intake (Bossis et al., 1999; Armstrong et al., 2001; Rausch et al., 2002) and blood insulin concen tration (Keisler and Lucy, 1996; Webb et al., 2004). The IGF-I molecule binds to specif ic receptors present on many cell types in several tissues and has an anabolic func tion promoting cell growth and multiplication (Quin, 1992). Growth hormone, also known as somatotr opin, is a protein hormone synthesized and secreted by cells called somatotrophs in th e anterior pituitary (Austgen et al., 2003).

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19 It assists in the control of se veral physiologic proce sses, particularly st imulating anabolic processes such as cell growth and multiplication. Growth hormone can stimulate tissue growth by two different mechanisms; 1) direct effects by binding with receptors on target cells, or 2) indirect effects by stimulating IGF-I s ecretion by the liver. The majority of the growth-promoting effects of GH are actually due to IGF-I acti ng on target cells, especially in muscle and bone (Austgen et al., 2003). Growth hormone also affects nutrient metabolism since it stimulates prot ein anabolism in tissues, enhances the utilization of fat by stimulating triglyceride oxi dation in adipocytes, a nd also is one of a series of hormones that serves to maintain blood glucose within a normal range (Austgen et al., 2003). Synthesis a nd secretion of GH are regul ated primarily by two wellcharacterized hypothalamic peptides: stimulat ed by GHRH or inhibited by somatostatin. In addition, GH synthesis and secretion also are regulated by IGF-I concentrations in blood. Increased concentrations of IGF-I in blood decrease the secretion of GH by directly suppressing its synthesis in the so matotrophs and by stimulating the release of somatostatin from the hypothalamus (Austgen et al., 2003). Since in sulin concentration and feed intake are positively correlated with IGF-I, they consequently are negatively associated with GH secretion and concentra tions in blood. The integration of all the factors that affect GH synthesis and secretion lead to a pulsatile pattern of release of this hormone (Austgen et al., 2003). Studies with beef cattle demonstrated that frequency of GH pulses is not affected, but pulse height, amplitude and overall GH concentration in blood are inversely correlated with energy in take (Nolan et al., 1990; Hornick et al., 1998; Bossis et al., 1999).

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20 Energy Metabolism and Beef Cattle Performance Feed intake, blood metabolites associated with nutritional status such as the glucose/insulin/IGF-I/GH axis, and conse quent animal performance are usually associated in cattle. Research by Vizcarra et al. (1998) analy zed the influence of BW gain on concentrations of blood glucose and insulin in primiparous beef cows. The authors fed cows in order to attain gains of 0.45 or 0.90 kg/d and reported that animals under a higher plane of nutrition had greater concentrations of glucose and insulin in blood. In a similar study with beef steers, blood gluc ose, insulin, and IGF-I typica lly increased with rate of gain (Hersom et al., 2004). Animals fed to 60, 100 or 160 % of maintenance energy and protein requirements also had increased blood concentrations of gluc ose, insulin and IGFI when feeding level increased (Lapierre et al., 2000). Ellenb erger et al. (1989) compared serum concentration of glucose, IGF-I and GH in steers fed either ad libitum gaining 1.40 kg/d, or restricted amounts gaining 0.37 kg/d. Mean serum c oncentrations of GH were elevated (45.6 vs. 23.4 ng/mL) and serum con centrations of IGF-I were decreased (108 vs. 167 ng/mL) for feed-restricted steers. Bossis et al. (1999) compared blood concentrations of glucose, insulin, IGF-I and also blood concentration, pulse frequency and pulse amplitude of GH from heifers fed either to maintain body condition score or restricted diets to induce negative energy balance. Decr eased blood glucose (58.7 vs. 71.5 mg/dL), insulin (0.95 vs. 1.85 ng/mL), and IGF-I (15.35 vs. 94.1 ng/mL) concentrations but increased GH concentration (38.7 vs. 12.1 ng/mL) and pulse amplitude (36.3 vs. 17.6 ng/mL) for feed-restricted he ifers were noted. Pulse frequency of GH did not change between groups. It can be concluded that animal performa nce is associated with energy intake and metabolism. There is a direct relationship among diet and blood concentrations of

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21 glucose, insulin, IGF-I and GH, and these me tabolites also have direct effects on each other. Additionally, blood concentration of IG F-I is typically associated with BW gain and therefore, should be maximized. Energy and Nitrogen Synchronization in the Rumen Ruminants have a high rate of ammonia absorption from the rumen, which like peptides and amino acids, is a product of di etary protein degrada tion by the microbial population in the rumen (Huntington, 1990). Amm onia is a major precursor for microbial protein synthesis, and its excess is abso rbed by ruminal epithelium and transported mostly to the liver. In liver, ammonia is conve rted into urea, which can be re-utilized as a nitrogen source in the rumen via saliva or ci rculatory system (blood urea nitrogen; BUN), or be excreted in urine by the kidneys. He patic ammonia metabolism and renal excretion of urea require energy. Therefore, elevat ed amounts of ammonia produced in and absorbed by the rumen increase the animals requirement for energy (Huntington, 1990). Microbial protein synthesis is optimal wh en both energy and nitrogen sources are available in the rumen (Hammond, 1997). Diets ri ch in protein conten t but lacking either in energy availability or synchrony between ruminal energy and nitrogen supplies may limit the utilization of available nitrogen by ruminal microorganisms (Huntington, 1997), and also increase BUN concentrations (S antos, 2004). Alternatively, diets with inadequate protein concentrations limit rumi nal microbial growth and compromise fiber digestion. Therefore, to increase efficien cy of nitrogen utilization in the rumen and consequent microbial protein synthesis, energy and nitrogen should be available in the rumen in a coordinated pattern. Ruminal de gradability and diges tibility of dietary carbohydrates and protein differ among feedst uffs (NRC, 2001) and these differences may influence microbial fermentation and pr otein synthesis in the rumen. Diets or

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22 supplements containing highly de gradable energy sources, su ch as high NSC feedstuffs, should also contain protein s ources with high rumen degradab ility in order to grant a synchronized availability of energy and ni trogen to the microbial population in the rumen. Conversely, when fibrous ingredients such as citrus pulp are the main energy source, protein sources with lower de gradation rates should be added. Several studies examined the effects of synchronizing energy and nitrogen supplies in the rumen. When steers were infuse d with starch and casein in their rumen or abomasum, they had reduced ammonia absorp tion and increased nitr ogen retention when both nutrients were delivered in the rumen (Taniguchi et al., 1995) In research with lambs, Matras et al. (1991) fed three diffe rent sources of energy varying in ruminal degradability (barley > steam-flaked corn > dry rolled sorghum) combined with three different sources of protein also varying in ruminal degradation ra tes (urea > 50:25:25 of urea, blood meal and corn gluten meal > 50:50 blood meal and corn gluten meal) in a 3 x 3 Latin square design. Nitrogen utilization was most efficient when rapidly degraded sources (barley and urea) and slowly degraded sources (sorghum and blood + corn gluten meal) of energy and protein were fed toge ther. The authors concluded that it was advantageous to feed animals sources of protein and energy containing similar ruminal degradation rates. Spicer et al. (1986) fed feed ingr edients differing in ruminal degradation rates to beef st eers (barley > corn > sorghum) and reported that microbial protein synthesis and efficiency of nitrogen utilization was positively associated with protein and carbohydrate degradabil ity of the feedstuff. Studies with dairy cattle indicated that synchronizing ruminal fermentability of carbohydrate and protei n sources increased nitrogen utilization and consequent outflow of bacterial protein from the rumen. Herrera-

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23 Saldana et al. (1990) fed lactating dairy cows barley or milo as energy sources (90.5 vs. 70.5 % of ruminal carbohydrate degradability, respectively) and co ttonseed meal or brewers dried grains as protein sources ( 56.6 vs. 38 % of ruminal protein degradability, respectively). The authors reported increased microbial protein s ynthesis when animals were fed synchronized diets, pa rticularly diets with highly de gradable ingredients, which resulted in increased milk production. In a similar study, Aldrich et al. (1993) fed lactating dairy cows four diets formulated for high and low ruminal degradability of carbohydrate and protein. The author s reported that passage of bacterial N to the small intestine was the greatest (262 g/d) when highly ruminally-degradable nonstructural carbohydrate was combined with high rumen-available protein, and was the least when high rumen-available nonstructural car bohydrate was combined with low rumenavailable protein (214 g/d). Monitoring BUN is a technique that can be used for measuring protein and energy status in cattle and is highly correlat ed with the amount of ammonia produced and absorbed in the rumen (Hammond, 1997). Th erefore, in healthy ruminants, BUN concentrations are indicative of the protein to energy ratio in the di et, and furthermore, synchrony between dietary energy and protein degradability in the rumen. Little work has been reported in beef cattle with regard to the influence of ruminal energy and nitrogen synchronization with BUN concentrations. Most of the work analyzed the effects of dietary protein concentration or degradability. Hammond (19 83) fed steers isocaloric diets having three concentrati ons of CP and reported that BUN concentrations increased from 2.6 to 11.1 mg/dL as dietary CP increased from 6 to 18 % (DM basis). Burris et al. (1975) fed steers isocaloric and isonitr ogenous corn-cottonseed hull based diets

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24 supplemented with either urea or soybean meal. Urea increased BUN values (6.7 vs. 4.8 mg/dL). Similar to the studies examining ruminal synchrony of energy and nitrogen, Chase et al. (1993) fed bulls diets formulat ed to provide 75 or 150 % of maintenance energy requirements but equal CP intake. The concentrations of BUN averaged 5.6 mg/dL in bulls fed the highest energy diet and 19.7 mg/dL at the lowest energy intake, demonstrating that increasing dietary ener gy intake, while holding protein intake constant, decreased BUN concentrations. In other studies with beef cattle, BUN concentrations between 11 and 15 mg/dL were associated with maximum rates of gain for growing animals (Byers and Moxon, 1980) and concentrations of BUN of 7 to 8 mg/dL were associated with maximum performa nce of finishing steers (Preston et al., 1978) or mature cows (Hammond, 1997). The vari ance in BUN concentration reported in these studies may be related to the differ ent protein requirements for each animal category. Optimal concentrations of BUN can be associated with maximized microbial protein synthesis, leading to increased perf ormances. Concentrations that are less than these optimal values may indicate a deficiency in dietary protein re lative to the energy intake, while excessive values of BUN may indicate unnecessary dietary protein and/or lack of either energy availa bility or synchrony between ruminal energy and nitrogen supplies, leading to increased ammonia formation and absorption in the rumen. Nutrition and Reproduction in Beef Cattle The primary nutrient consideration for op timum reproductive performance in beef cattle, particularly females, is energy (Mass, 1987). Inadequate energy intake is correlated to decreased reproductive perfor mance, delayed onset of puberty, extended postpartum anestrous and decreased conception and pregnancy rates in cattle (Santos, 2004). Therefore, one important goal for beef cattle operations, particularly cow-calf

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25 enterprises in Florida, is the development of nutrition programs that emphasize energy supplementation to maintain or enhance th e reproductive efficiency of the herd. Onset of Puberty in Beef Heifers Age at puberty is a major determinant of lifetime reproductive performance of beef cows, and is defined as the age at which heifers have their first ovulation and consequential luteal phase (Santos, 2004). Sexua l maturity is defined as the age at which animals reach their maximum reproductive poten tial and are definitely able to conceive and gestate. In cattle, sexual maturity is associated with onset of puberty, and heifers typically attain sexual maturity around three or four normal estrous cycles (around 60 to 90 d) after the onset of puberty (Santos, 2004) One of the most important goals for cowcalf operations is to develop heif ers so that they are able to conceive at 14 to 16 mo of age and calve at approximately 2 yr of age (Schillo et al., 1992). Heifers should also experience two or three estrous cycles before the onset of the breeding season in order to be sexually mature. For that reason, replacem ent heifers should at tain puberty around 12 mo of age to achieve optimal reproductive pe rformance during their first breeding season (Schillo et al., 1992). Heifers that conceive early in th eir first breeding season and therefore calve early in thei r first calving season wean mo re and heavier calves during their productive lives (Lesmeister et al., 1973). Age at puberty is directly associated with BW, body composition, and breed type. Furthermore, heifers with greater energy inta ke and consequent greater BW gain attain puberty earlier (Schillo et al., 1992). The onset of puberty seems to be associated with increased frequency and amplitude of LH pulse s that stimulates development of ovarian follicles to the preovulatory stages. These LH pulses are clearly associated with nutritional status (Sch illo, 1992). As explained previous ly, energy intake increases VFA

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26 production in the rumen, particularly propiona te, which increases glucose synthesis and consequent insulin and IGF-I secretion. Th ese hormones and metabolites appear to increase gonadotropin secretion (LH a nd FSH) by influencing hypothalamic-hypophyseal secretory activity (Butler and Smith, 1989; Sc hillo et al., 1992), a nd also appear to amplify the effects of these hormones in ovarian cells (Spicer and Echternkamp, 1995). The onset of puberty is a gradual process. During the pre-pubertal period, ovarian follicles secrete basal quantities of estrogens which inhibit LH secretion. This negative feedback declines as puberty approaches due to increased pulsatile secretion of LH (Day et al., 1984). An increased nutri tional plane may decrease the sensitivity to the negative feedback from estradiol and enhance concen trations of LH released (Foster, 1988). In summary, the exact mechanism by which a higher plane of nutrition anticipates puberty is not well known, but the increase in the availability of metabolites and hormones (glucose, insulin, and IGF-I) that may enhance gonadotropin secretion/activity and decrease the sensitivity of the hypot halamic-hypophyseal tissues to the negative feedback from estrogens may be potential explanations. Reproductive Performance in Postpartum Beef Cows Postpartum interval, characterized as th e period from parturition to resumption of estrus, largely determines the likelihood of cows becoming pregnant during the breeding season (Wiltbank, 1970). Calves derived from co ws that faced long postpartum intervals tend to be younger and lighter at weani ng (Ferrell, 1991). Therefore, decreased postpartum interval is a major goal to be attained in cow-calf enterprises in order to increase the efficiency of the operation. The mechanisms associated with the ac quisition and maintena nce of reproductive capability in postpartum cows results from functional integration of the hypothalamic-

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27 hypophyseal-ovarian axis (Hess et al., 2005). Du ring late gestation, elevated production of steroids by placenta and maternal ovaries, particularly estradiol and progesterone (P4), exerts intense negative feedback on the hypot halamus, decreasing th e release of GnRH (Short et al., 1990; Lucy, 2003) and theref ore depleting hypophyseal reserves of LH (Wettemann et al., 2003). Despite the relativel y fast restoration of LH supply in the hypophysis after calving, postpartum anestrous is sustained because of the sensitivity to negative feedback effects from estradiol (Shor t et al., 1990). Postpartum anestrous can be even prolonged due to the negative effect s of calf-nursing (Williams, 1990) and severe negative energy balance on the amplitude and fr equency of LH secret ion (Schillo, 1992). During the early postpartum period, when the re quisites for energy increase primarily due to lactation, energy intake often does not satisfy energy requirements. Consequently, decreased blood concentration of glucose, in sulin, IGF-I, P4 and also GnRH and LH pulse frequency are observed (Santos, 2004) The mechanism by which energy affects GnRH and gonadotropins secre tion is yet unknown, but blood c oncentrations of glucose, insulin and IGF-I seem to mediate this proce ss. Glucose is the primary metabolic fuel for the central nervous system, and low gluc ose concentrations appear to reduce hypothalamic secretion of GnRH (Wettemann et al., 2003). Due to its impact in glucose metabolism, insulin may be an important link between nutritional and reproductive processes. Insulin combined with glucose may stimulate GnRH release from the hypothalamus (Arias et al., 1992). Insulin medi ates follicular dynamics (Webb et al., 2004), stimulates cell prolifer ation and steroid synthesis in the ovary (Wettemann and Bossis, 2000) and also facilitates hepatic production of IGF-I (Keisler and Lucy, 1996; Webb et al., 2004). Blood concentrations of IG F-I were increased for beef cows that

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28 resumed estrous cycles early postpartum, but not for those that remained in anestrous (Roberts et al., 1997). Other studies also report ed the presence of IG F-I receptors in the ovaries and dominant follicl es of beef cows (Armstrong and Benoit, 1996; Bao and Garverick, 1998). Therefore, IGF-I may be a positive signal to the hypothalamohypophyseal-ovarian axis to resume norma l functionality afte r parturition. Feed Intake and Reproductive Performance in Cattle The endocrine events that lead to resump tion of luteal activity in postpartum cows and the attainment of puberty in heifers are similar (Looper et al., 2003). Elevations in blood P4 concentrations were detected dur ing the period that immediately preceded puberty in heifers (Gonzalez-Padil la et al., 1975), and this P4 is originated from ovaries (Berardinelli et al., 1979), specifically from compact luteal tissues embedded within the ovary. The same pattern was observed in postp artum beef cows, since first ovulation after parturition was usually followed by a temporary increase (4 to 5 d of duration) in P4 (Williams and Ray, 1980; Perry et al., 1991). The increase in concentrations of P4 appears to be a necessary factor for the resu mption of normal estrous cycles. Looper et al. (2003) indicated that 81 % of cows that had a rise in P4 before first estrus had normal length estrous cycles (19 to 23 d) whereas only 36 % of cows that di d not have increased P4 had normal estrous cycles. The rise in P4 before the onset of puberty in heifers and first estrus in cows may have a role in the endocrinal changes leading to the establishment of gonadotropins and gonada l hormone secretions, preparing the reproductive system to attain normal and fe rtile estrous cycles (Kinder et al., 1987). Circulating concentration of P4 is also critical for the onset and maintenance of pregnancy, especially during the early phase s (Spencer and Bazer, 2002). Progesterone prepares the uterine environmen t for gestation, and also inhi bits the release of hormones

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29 that may disrupt pregnancy. In cattle, blood conc entration of P4 before or after breeding has been associated with conception rates. Conception rates were influenced by plasma concentrations of P4 before artificial insemination in dair y cows (Folman et al., 1990). Cows were estrus synchronized by two injectio ns of PG, and cows with P4 concentration below 5 ng/mL 2 d before second PG admi nistration had conception rates of 36 %, whereas cows with concentration of P4 above 5 ng/mL had conception rates of 75 %. Xu et al. (1997) also synchronized estrous in lactating dairy cows with two treatments of PG 13 d apart, but half of the animals were s upplemented with exogenous P4 for 5 d before second injection. The author s reported improved estrous response (89.6 vs. 82.9 %) and conception rate from first AI (65.1 vs. 59.7 %) for animals that received exogenous P4. Robinson et al. (1989) supplemen ted dairy cows with exogenous P4 between d 5 to 12 or d 10 to 17 after insemination, and detected increased pregnancy rates compared to untreated cows (60 to 30 %). These findings are also supported by several other studies (Wiltbank et al., 1956; Folman et al., 1973; Fonseca et al., 1983), validating the importance of P4 for the onset and maintenan ce of pregnancy, and subsequent conception rates. Several studies with dairy cattle reported that high feed intake may decrease circulating P4 concentrations, and therefore impair reproductive efficiency. Vasconcelos et al. (2003) suggested that f eeding pattern may alter blood P4 concentration in pregnant lactating Holstein cows. In this study, feed was withheld 12 h before the experiment started and then cows were pr ovided with either 100 % of a total mixed ration (TMR) at once, half of TMR every 12 h, a quarter of TMR every 6 h, or leaving animals unfed for an additional 12 h (control). After first feeding, blood was sampled hourly for 24 h. The

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30 authors reported that supply ing either 100 % or 50 % of TMR decreased circulating P4 concentration by 1 h after feeding and concentrations remained depressed until 8-9 h after feeding, whereas feeding 25 % of TMR did not reduce concentrations of P4. In a similar study, Sangsritavong et al. (2002) aspirated follicl es and regressed the corpus luteum of non-lactating Holstein cows in order to sync hronize the follicular wa ve, and then treated animals with no feed, feed intake at 0.5 of maintenance requirements (M) or at 1.5 M. The authors determined that blood P4 concen trations were inversely correlated with feeding regimen at 4 h after feeding ( 4.14, 3.33 and 2.70 ng/mL of P4 for unfed, 0.5 M and 1.5 M, respectively). The same authors co mpared circulating P4 concentrations in lactating cows either unfed or fed 0.5 M, 1.5 M, or 2.2 M. Again, feed intake decreased P4 concentrations between 1 h and 4 h afte r feeding, although no differences were found between cows fed 1.5 or 2.2 M. Research by Rabiee et al. (2001) compared P4 concentration of non-lactating grazing dairy cows with full or restricted access (2 h/d) to pasture. Animals were drenched 2x/d with chromic oxide capsules to allow daily feed intake estimation. Circulati ng P4 was supplied by exogenous sources and initial blood concentrations of P4 were similar among animals, since endogenous production was eliminated prior to the study via GnRH-agoni st implants. The av erage daily DMI of pasture was greater for cows in the full access group (15.9 vs. 6.3 kg of DM) and their plasma P4 concentrations were less ( 1.08 vs. 1.71ng/mL) compared to the restricted group. In research with beef cows, Pescara et al. (2005) synchroni zed the ovulation of 364 cows. After AI, cows were fed daily 2 kg or 6 kg of corn (as-fed basis). Blood concentrations of P4 were greater for anim als fed 2 kg of corn (1.7 vs. 1.3 ng/mL) on d 7 after AI, but conception rates did not differ between the treatments. Arthington et al.

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31 (2004) supplemented beef heifers (3x/wk) with either a molasses/cottonseed meal slurry or range cubes. Heifers supplemented with range cubes readily consumed the entire amount of feed, whereas molasses-fed heifer s had a longer period for total supplement consumption (48 h). Animals had similar rate s of BW gain, but pregnancy rates were different (76.3 vs. 49.2 % for molasses and ra nge cube-fed heifers, respectively). The authors attributed this effect to the different supplement intake pattern, associating faster feed intake with acute decreases in circulat ing P4 and consequently impaired pregnancy rates, although blood concentrations of P4 were not determined. The mechanism by which feed intake decrea ses blood P4 concentration in cattle is well defined by Sangsritavong et al. (2002). After meal c onsumption, liver blood flow increases in order to transpor t digested nutrients from the gut through the liver and on to the rest of the body (Huntington, 1990). Liver is the main site of peripheral steroid inactivation and catabolism by enzymatic pr ocesses, converting biologically active hormones into inactive forms, which are then eliminated primarily in the urine (Steimer, 2003). Blood flow to the liver is substantially increased when large amounts of feed are ingested, therefore increasing the rate by whic h P4 is inactivated and catabolized, causing acute and chronic reductions in circ ulating concentra tions of P4. In summary, high planes of nutrition may be detr imental to reproductive efficiency if not managed correctly. During pe riods where P4 is particularly fundamental, either for priming the hypothalamus-hypophysis axis for the onset/re-attainment of estrous cycles or for the onset/maintenance of pregnancy, feed intake should be carefully controlled in order to avoid sharp decreases in circulating P4.

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32 CHAPTER 3 EFFECTS OF SUPPLEMENT TYPE ON PE RFORMANCE, REPRODUCTIVE AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED FEMALES Introduction The primary nutritional consideration fo r optimum reproductive performance of beef females is energy (Mass, 1987). With greater energy intake and consequent improved energy status, heifers attain puberty earlier (Schillo et al., 1992) whereas cows resume postpartum estrous cycles earlier (Robe rts et al., 1997). Alternatively, inadequate energy intake is correlated highly with delayed onset of puberty, extended postpartum intervals and decreased concep tion and pregnancy rates (Santo s, 2004). In Florida, where the beef cattle industry is primarily constitute d of cow-calf operations with diets based on forages lacking in energy availability (M oore et al., 1991), nutritional programs with emphasis on energy supply are therefore required. Citrus pulp and molasses are energy byproducts originated from orange and sugarcane industries, respectiv ely, two of the most important agricultural operations in Florida. Sucrose is the main carbohydrate of molasses, whereas pectin is the main carbohydrate of citrus pulp. Pectin and sucros e are fermented differently in the rumen, potentially impacting forage intake, di et digestibility, a nd energy utilization. Molasses is classified as a liquid feed, whereas citrus pulp is processed and fed commonly as dry pellets. Previous res earch by our group (Arthington et al., 2004) reported that differences in physical form of the supplement led to variances in intake behavior and reproductive performance. B eef heifers supplemented with dry feed

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33 consumed the entire amount of supplement faster than heifers supplemented with an equivalent amount of CP and energy from a liquid supplement. Although no differences in BW gain were detected, heifers fed the liquid supplement had greater overall pregnancy rates. Supported by st udies that indicated that in frequent and/or acute feed intake is associated negatively with ci rculating progesterone (P4) concentration (Sangsritavong et al., 2002; Vasconcelos, et al., 2003), we hypothesized that heifers supplemented with dry feed had decreased circulating P4 and consequent impaired pregnancy rates. The objective of the present experiment s was to investigate the effects of supplement type on performance and reproduc tive efficiency of Brahman-crossbred cattle, including plasma metabolites and hormones. Material and Methods Two experiments were conducted at the University of Florida IFAS, Range Cattle Research and Education Center, Ona. The first experiment was conducted from September to December 2004 and was divided into a sampling phase (September and October) and a breeding phase (November and December). The second experiment was conducted during the months of October and November 2004. The animals utilized in these experiments were cared for by acceptable practices as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999). Animals Experiment 1 Sixty Brahman x British crossbred heifers (BW SD = 246 23 kg; age SD = 10 1 mo) were utilized in this experiment. For the sampling phase (d 0 to d 45), heifers were stratified by initial BW a nd age, and randomly allocated to 12 pens (5

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34 heifers/pen). Pens were assigned randomly to one of two treatments: 1) molasses-based supplement (ML) or 2) citrus pulp-based supplement (CT). Pen was considered the experimental unit (6 pens/treatment) and each pen was a 1.3 ha pasture of bahiagrass ( Paspalum notatum ). For the breeding phase (d 46 to d 107), heifers were re-allocated by treatment into two bahiagrass pastures and exposed to bulls for a 60-d breeding season. Experiment 2. Initially, 40 non-lactating and non-pregnant multiparous Brahman x British crossbred cows had follicular and lu teal function synchronized (Figure 3-1) by treatment with GnRH (100 u g; Cystorelin; Merial Ltd., Duluth, GA) and insertion of an intra-vaginal P4 releasing device containi ng 1.38 g of P4 (Eazi-Breed CIDR; Pfizer Animal Health, New York, NY) on d 21, followed by treatment with of PGF2 (25 mg; Lutalyse; Pfizer Animal Health, New Yo rk, NY) and CIDR removal on d -14, and a second treatment with GnRH (100 u g) 48 h after the PGF2 injection (d -12). Transrectal ultrasonography was performed immediately and 48 h after the second GnRH injection (d -10). Cows that were detected at the same stage of the follicular wave were then chosen to be enrolled in the experiment (Figure 3-1). Thirty-four cows were selected and stratified by age, parity, BW, and BCS (1 = emaciated, 9 = obese; Wagner et al., 1988) and randomly assigned to one of the two treatm ents: 1) exogenous source of P4 (CD) or 2) natural source of P4 (NCD). In order to ob tain 12 cows at the beginning of the estrous cycle in each group at th e start the experiment (d 0), al l cows were again treated with PGF2 (25 mg) on d -5 (Figure 3-1). At this time two CIDRs were inserted in the vagina of the cows from group CD, and remained throughout the entire experiment. Two days later (d 3), cows from NCD were treated with GnRH (100 u g) (Figure 3-1).

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35 At the beginning of the experiment (d 0), both groups CD and NCD containing twelve cows each (BW SD = 502 55 kg; age SD = 5 2 yr) were stratified by BW and randomly allocated to 12 feedlot pens (2 cows/pen, 6 pens/group). Pens were then randomly assigned to one of the two treatmen ts in a 2 x 2 factorial arrangement of treatments: A) molasses-based supplement (ML) or B) citrus pulp-based supplement (CT). Pen was considered the experimental unit. Diets Experiment 1. Pasture quality was estimated to be 56 % TDN and 7.4 % CP (DM basis) from samples analyzed by a commerci al laboratory (Dairy One Forage Laboratory, Ithaca, NY). A complete mineral mix and wa ter were offered ad libitum throughout the experiment. Stargrass ( Cynodon nlemfuensis ) hay was offered when pasture availability was limited. Hay quality was estimated to be 52 % TDN and 6.5 % CP (DM basis) from samples also analyzed by commercial la boratory (Dairy One Forage Laboratory). Treatments consisted of two energy supplem ents (Table 3-1), fed 3x/wk (Mondays, Wednesdays and Fridays) at a rate of 2.1 a nd 2.3 kg of DM per heifer daily for ML and CT, respectively. Supplement intakes were formulated to be iso-caloric and isonitrogenous (Table 3-2) and also balan ced for calcium content, given the high concentration of calcium in citrus pulp. Experiment 2. Limpograss ( Hemarthria altissima ) hay was offered ad libitum throughout the entire experiment, and hay qual ity was estimated to be 54 % TDN and 9.1 % CP (DM basis) from samples analyzed by commercial laboratory (Dairy One Forage Laboratory). Cows had free access to a complete mineral mix and water. Treatments consisted of two energetic supplements (T able 3-3), fed 3x/wk (Mondays, Wednesdays and Fridays) at a rate of 4.1 and 4.5 kg of DM per cow daily for ML and CT,

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36 respectively. Supplement intakes were formul ated to be iso-caloric and iso-nitrogenous (Table 3-4) and also balanced for calciu m content, given the high concentration of calcium in citrus pulp. Sampling Experiment 1. One week before the start (d -7 and d -6) and at the end of the experiment (d 107 and d 108), heifers were we ighed for 2 consecutive days to determine both full and shrunk (after 16 h of feed restri ction) BW. Heifers also were weighed every other week to monitor averag e daily gain (ADG) rates, bu t overall ADG was calculated using initial and final shrunk BW values. Blood samples were collected weekly (on Wednesdays) throughout the entire experiment to determine onset of puberty using blood P4 concentrations. Heifers were considered pubertal if P4 valu e was greater than 1.5 ng/mL for 2 consecutive weeks. During the sampling phase, in addition to the weekly collections, blood samples were obtained once a day during four consecutiv e days, every other week, starting at 4 h after supplements were offered for determina tion of glucose, blood urea nitrogen (BUN), insulin, IGF-I, GH, and P4 concentrations. Th ese samples were collected from d 0 to d 3, d 14 to d 17, d 28 to d 31 and d 42 to d 45, which were classified as periods (P 1, P 2, P 3 and P 4, respectively) for further statistical analysis. Periods alwa ys started on Mondays and ended on Thursdays. During the breeding season, heifers were exposed to mature Angus bulls from d 46 to d 107. Each group was exposed to 2 bulls at the same time (1:15 bull to heifer ratio), and bulls were rotated weekly between groups to account for potential effect of bull. Heifer pregnancy stat us was verified by presence of fetus using transrectal

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37 ultrasonography (5.0 MHz transducer, Aloka 500V, Wallingford, CT) 70 d after the end of the experiment (d 177). Random samples of feedstuff, pasture, a nd hay were collected during the trial and analyzed for nutrient composition at comme rcial laboratories (Dairy One Forage Laboratory for cottonseed meal and citrus pulp samples, and SDK Laboratories, Hutchinson, KS for molasses samples). Experiment 2. During the first 3 weeks of the experiment (d 1 to d 21), blood samples were collected immediately prio r and 4, 8, 24, 32 and 48 h after the first supplement feeding of the week (d 1, 8 a nd 15) for determination of glucose, BUN, insulin, IGF-I and P4 concentr ations. Weeks were classified as periods (week 1 = P 1; week 2 = P 2; week 3 = P 3) for further statistical analysis. During the second part of the experiment (d 22 to d 37), daily forage DMI was recorded. Hay was offered in ad libitum am ounts, and refusal was collected and weighed daily. A sample of the offered hay was coll ected twice during this period for nutrient analysis, while samples of refusal were collected daily from each pen to determine DM content. Hay samples were dried for 96 h at 50 C in forced-air ovens. Random samples of the feedstuffs also were collected duri ng the trial, and all the samples obtained for nutrient composition were analyzed at comm ercial laboratories (Dairy One Forage Laboratory for cottonseed meal and citrus pulp samples, and SDK Laboratories for molasses samples). Blood Analysis Blood samples were collected via jugul ar venipuncture during Exp. 1 and from coccygeal vein or artery during Exp. 2 into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Fr anklin Lakes, NJ) containing sodium heparin,

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38 placed on ice immediately, and centrifuged at 855 g for 30 min for plasma collection. Plasma was frozen at -20C on the same day of collection. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine both plasma glucose (Coulombe and Favreau, 1963; modified and described by Bran + Luebbe Industria l Method #339-01) and BUN (Gochman and Schmitz, 1972; modified and described by Br an + Luebbe Industrial Method #339-19). A double antibody radioimmunoassay (RIA) was used to determine plasma concentrations of insulin (Malven et al., 1987; Badinga et al., 1991), IGF-I (Badi nga et al., 1991) and GH (Badinga et al., 1991). The extraction procedure used in the IGF-I assay was modified from Badinga et al (1991) by using a ethanol:acet one:acetate ratio of 6:3:1. Concentrations of plasma P4 were determined using Coat-A-Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid phase 125I RIA. The intraand interassay CV for Exp. 1 were, respectively, 9.66 and 10.05 % for insulin, 8.13 and 5.87 % for IGF-I, 5.95 and 18.71 % for GH, and 4.48 and 8.15 % for P4. The intraand inter-assay CV for Exp. 2 were, respectively, 12. 89 and 15.92 % for insulin, 9.90 and 11.00 % for IGF-I, and 2.41 and 11.19 % for P4. Statistical Analysis Experiment 1. Data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001). The statistical model used was: Y = + TRTi + Pj + PENk(i) + HEIFERl(k) + DAYm(j) + TRTPij + TRTDAYim(j) + Eijklm where Y = response variable = mean

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39 TRT = fixed effect of treatment P = fixed effect of period PEN = random effect of pen within treatment HEIFER = random effect of heifer within pen DAY = fixed effect of day within period TRTP = effect due to intera ction of treatment and period TRTDAY = effect due to interaction of treatment and day within period E = residual error Results are reported as least square m eans. Means were separated using LSD. Significance was set at P 0.05, and tendencies we re determined if P > 0.05 and 0.10. Only significant interactions are reported. Experiment 2. Data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001). The statistical model used for forage DMI data analysis was: Y = + TRTi + PENj(i) + DAYk + TRTDAYik + Eijkl where Y = response variable = mean TRT = fixed effect of treatment PEN = random effect of pen within treatment DAY = fixed effect of day TRTDAY = effect due to interaction of treatment and day

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40 E = residual error The statistical model used for plasma hormones and metabolites data analysis was: Y = + TRTi + Pj + PENk(i) + COWl(k) + TIMEm(j) + TRTPij + TRTTIMEim(j) + Eijklm where Y = response variable = mean TRT = fixed effect of treatment P = fixed effect of period PEN = random effect of pen within treatment COW = random effect of cow within pen TIME = fixed effect of time within period TRTP = effect due to intera ction of treatment and period TRTTIME = effect due to interaction of treatment and time within period E = residual error Results are reported as least square m eans. Means were separated using LSD. Significance was set at P 0.05, and tendencies were classified at P > 0.05 and 0.10. Only significant interactions are reported. Al l plasma metabolites and hormones (but P4) data were analyzed comparing CT vs. ML without distinction be tween CD and NCD. Progesterone data compared CT vs ML within CD and NCD groups.

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41 Results and Discussion Experiment 1 Heifers were provided supplement tr eatments on Mondays, Wednesdays and Fridays at 0700 h throughout the entire experiment. Heifers fed ML required approximately a 48 h period to completely c onsume the supplement, whereas heifers fed CT consumed the whole amount of supplement 24 to 36 h after supplements were offered. This intake behavior was unexp ected, since previous research by our group (Arthington et al., 2004) repor ted that heifers in similar conditions of management, consumed entire amounts of dry supplement within 2 to 3 h after feeding, whereas liquid supplement also was entirely consumed in approximately 48 h. Therefore, we concluded that intake behavior did not differ between treatments as expected, and the results obtained for heifer performance and reproductive efficiency in this experiment should be primarily attributed to the nutritiona l differences between CT and ML. For all plasma metabolites and hormone da ta, day(period) and period effects were significant ( P < 0.01). The significance in day(period) effect can be explained by the daily variability in animal behavior and consequent supplement consumption, considerably affected by the environm ent and management activities, although supplement feeding and blood samplings always started at the same time throughout the entire experiment. Regarding the significance in period effect, it can be attributed to animal maturation and also to the adapta tion of the animals to the experimental procedures such as feeding and handling ma nagement, since it was observed that both energy and protein status of the animals im proved with the advance of the experiment without any modification in the treatments.

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42 Heifers fed CT had greater ADG compared to heifers fed ML (0.40 vs. 0.30 kg/d, respectively; P < 0.05, SEM = 0.02; Table 3-5), concur ring with previous data reporting that animals fed molasses-based supplements us ually have inferior BW gain compared to animals fed supplements based on other energy sources, such as corn or soybean hulls (Pate, 1983; Royes et al., 2001). However, th e superiority in performance from CT-fed heifers was not reflected in reproductive effi ciency. There were no treatment effects on pregnancy rate, puberty rate and number of days required to attain puberty (Table 3-5). Heifers fed ML had greater ov erall BUN concentrations ( P < 0.05) compared to heifers fed CT (5.17 vs. 4.17 mg/dL, respectiv ely; SEM = 0.24) (Table 3-6; Figure 3-2). However, this effect was mainly observed dur ing the first two periods of the experiment. During periods 3 and 4, the significance was less apparent ( P = 0.07 and P = 0.60, respectively). Therefore, a treatment x period interaction was detected ( P < 0.01). A treatment x day(period) inter action also was detected ( P < 0.01) and can be mainly attributed to the differences in supplemen t intake behavior and supplement ruminal degradability, leading to differe nces in daily BUN concentra tions between the treatments. Monitoring BUN is a technique used for measuri ng protein status in cattle and is directly correlated with the amount of ammoni a produced and absorbed in the rumen (Huntington, 1990). Ammonia is a product of dietary protein degradation by the microbial population in the rumen and is a major precursor for microbial protein synthesis. Concentrations of BUN should range between 11 and 15 mg/dL for growing animals to ensure maximum rates of gain (Byers and Moxon, 1980). This goal can be achieved by feeding animals with correct qua ntities of protein and energy (Hammond, 1997; Huntington, 1997), and also synchronizing the supply of these nutrients in the

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43 rumen by feeding energy and protein sources wi th similar rates of ruminal degradability (Spicer et al., 1986; Matras et al., 1991; Taniguchi et al., 1995 ). In the current study, both treatments would be deficient in protein a ccording to the BUN concentrations observed, since they were consistently below the optim um (Table 3-6). However, supplements were formulated to supply the protein requiremen ts of developing heifers gaining similar weight as observed in this experiment, using the NRC model (N RC, 1996). Therefore, BUN might not be the most accurate indicator of the protein status in our case, since the BW gain of heifers were in an expected range and the negative effects of protein deficiency were thus not obser ved. The differences between treatments may be attributed to the faster rate of ruminal degradation of sucrose compared to pectin (NRC, 2001), optimizing synchrony of protein and energy supp ly in the rumen of heifers fed ML during the initial periods. Plasma glucose (83.30 vs. 74.68 mg/dL for CT and ML, respectively; SEM = 2.46), insulin (0.89 vs. 0.75 ng/mL for CT a nd ML, respectively; SEM = 0.04) and IGF-I (121.53 vs. 108.91 ng/mL for CT and ML, resp ectively; SEM = 4.10) concentrations were greater for heifers fed CT ( P < 0.05; Table 3-6; Figure 33, Figure 3-4 and Figure 35, respectively). The differences between the tr eatments increased with the advance of the experiment for glucose and insulin concen trations; therefore, treatment x period interactions were detected ( P < 0.01). A treatment x day(period) interaction was only observed for glucose ( P < 0.01), and similar to BUN, may also be attributed to the differences in supplement intake behavior. Seve ral researchers have reported that feeding diets containing high molasses or sucrose inclusion rates resulted in an increase in butyric acid concentration in the rumen of cattle (Owen et al., 1967; Kellogg and Owen, 1969;

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44 Hatch and Beeson, 1972), whereas diets rich in citrus pulp, consequently pectin, produced a greater proportion of acetic acid (Loggins et al ., 1964; Hentges et al., 1966; Drude et al., 1971). Both butyric acid and acetic acid are VFAs that oxidized for energy production (Bergman, 1990). The epithelial tissu e of the rumen retains approximately 90 % of the butyrate produced and converts it to ketone bodies or carbon dioxide, whereas only 30 % of acetate is metabolized by ruminal epithelium, and the remaining is oxidized throughout most of the body tissues to ge nerate ATP (Bergman, 1990). In addition, butyrate also has a detrimental effect on VFA metabolism by restraining hepatic propionate utilization, thus inhibiting gluconeogenesis a nd restraining glucose synthesis and availability (Aiello et al., 1989). Ruminal pH and forage intake and/or digestibility were not measured in the present experime nt. However, several authors reported that feeding supplements based on non-structural carbohydrates (NSC), such as sucrose, depressed forage intake (San son et al., 1990; Olson, et al ., 1999) and digestibility by decreasing ruminal pH and consequent micr obial activity (Merte ns and Loften, 1980; Hiltner and Dehority, 1983; Martin et. al., 2001 ). Alternatively, when supplements based on fibrous by-products such as citrus pulp were fed, forage intake and digestibility was less negatively affected (Bowman and Sa nson, 1996; Caton and Dhuyvetter, 1997; Royes et al., 2001) or even increas ed (Bowman and Sanson, 2000). The positive relationship between glucose, insulin, and IGF-I c oncentrations with ADG, observed in this experiment, is also supported by others (V izcarra et al., 1998; Lapierre et al., 2000; Hersom et al., 2004). Some authors also included analysis for plasma GH concentration and observed a si gnificant inverse re lationship between GH concentrations and animal performance/energy status (Ellenberger et al., 1989; Bossis et

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45 al., 1999). In these studies, blood was sampled in serial collections (every 20 and 10 min, respectively) to accurately measure GH concentrations, which is released in a pulsatile pattern (10 to 15 pulses/d; Hornick et al., 1998; Bossis et al., 1999). This might be an explanation to why no differences were det ected in GH concentrations between CTand ML-fed heifers (41.45 vs. 39.27 ng/mL, respectively; P = 0.62, SEM = 3.20) (Table 3-6; Figure 3-6), given that blood was sampled once a day and we were not able to identify pulses or basal concentrations of this hormone Therefore, GH analysis in this experiment was not accurate enough to be a ssociated directly to the gl ucose/insulin/IGF-I axis. The correlation between all plasma metabolites and hormones with ADG also was determined with Pearson correlation coefficients (Tab le 3-7). The only significant correlation was observed for IGF-I ( P < 0.01). In order to verify if plasma P4 concentr ations would be infl uenced by treatments and consequent intake behavior, only samp les from pubertal heifers with plasma P4 greater than 2 ng/mL during the sampling period were used. In vi ew of the fact that these heifers were at random stages of the estrous cycle and consequently concentrations of P4 varied significantly across heifers, we anal yzed the difference of P4 concentration on a percentage basis, comparing the values from days that supplements were offered to values from days that supplements were not o ffered (set as 100 %). Both treatments had a decrease in P4 concentra tion during feeding days ( P < 0.01; SEM = 2.9); however, no differences were observed between tr eatments (89.5 and 90.3 % for CT and ML, respectively; P = 0.66, SEM = 4.1). Elevated or infre quent feed intake may decrease circulating concentrations of P4, and therefor e impair reproductive e fficiency given that P4 is essential for the onset of puberty (Gonzalez-Padilla et al., 1975) and for the

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46 recognition and maintenance of early pregna ncy (Spencer and Bazer, 2002). Vasconcelos et al. (2003) fed pregnant lact ating Holstein cows with eith er 100 % of the daily diet at once, half of the diet every 12 h, a quarter of diet every 6 h or le ft animals unfed. The authors reported that the first two treatments decreased circulating concentrations of P4 by 1 h after feeding and concentrations remain ed depressed until 8 to 9 h after feeding. An inverse relationship between feed inta ke and blood P4 concentrations also was reported by others (Rabiee et al., 2001; Sangsritavong et al ., 2002; Pescara et al., 2005). In the current experiment, the differences obs erved in feed intake behavior (24 to 36 h and 48 h for total supplement consumption fo r CT and ML, respectively) were not as substantial as expected, and might explai n why no differences were found in blood P4 concentrations and also reproductive performance between treatments. Nutritional status, and consequent BW gain and composition, typically is considered the primary determinant of puberty attainment because it is highly correlated with frequency and amplitude of LH pulses, which stimulate development of ovarian follicles to preovulatory stages (Schillo et al., 1992). Furthermore, blood metabolites and hormones such as glucose, insulin, and IGF -I have been associated with reproductive performance. These substances seem to be the connection between nutritional status and the increase in LH secret ion and activity, by influe ncing hypothalamic-hypophyseal secretory activity (Butler and Smith, 1989; Sc hillo et al., 1992) and also amplifying the effects of LH in ovarian cells (Spicer and Echternkamp, 1995). However, in the present study, neither BW gain nor the glucose/in sulin/IGF-I axis were associated with reproductive performance. Heifers fed CT had greater ( P < 0.05) ADG and blood concentrations of these metabolites/hormone s, but similar reproductive performance

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47 compared to heifers fed ML. When heifer s were divided, disregarding treatments, by puberty attainment and pregnancy status IGF-I was again the only measurement significantly associated with reproductive performance (Table 3-8). Concentrations of IGF-I are correlated positively with insulin co ncentrations (Table 37; Keisler and Lucy, 1996; Webb et al., 2004), that in turn are increased as blood c oncentrations glucose rises, and similarly decreased as blood concentrations of glucose de clines. Therefore, in order to increase blood IGF-I concentrations to enhance BW gain and reproductive performance, glucose synthesis should be maximized. Feeding level is associated positively with blood glucose concentration (Viz carra et al., 1998; La pierre et al., 2000; Hersom et al., 2004). In addition, since propionate accounts for up to 76 % of the glucose synthesized in the liver (Re ynolds et al., 1994), diets contai ning ingredients that produce a greater ratio of propionate in the ru men, such as starches, should be fed. Experiment 2 The same supplement intake behavior fr om Exp. 1 was observed in Exp. 2. Cows were supplemented on Mondays, Wednesdays and Fridays at 0800 h, and cows fed ML required around 48 h to consume the entire am ount of supplement, whereas cows fed CT required 24 to 36 h. Overall forage DMI did not differ betw een treatments (1.16 vs. 1.20 % for CT and ML, respectively; P = 0.52, SEM = 0.08; Figure 3-7). However, a day effect and also a treatment x day interaction were detected ( P < 0.01). Forage DMI was lesser for both groups during the days that supplements we re fed (0.65 and 1.55 % for CT vs. 0.96 and 1.38 % of BW for ML during feeding and non-feeding days, respectively; P < 0.01, SEM = 0.09). This effect likely can be attribut ed to the increased consumption of the supplements during feeding days compared to non-feeding days, since forage DMI is

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48 negatively associated with energy suppl ement intake (Caton and Dhuyvetter, 1997; Kunkle et al., 1999; Bodine and Purvis, 2003). Fu rthermore, forage DMI for cows fed CT was less than for cows fed ML during feed ing days (0.65 vs. 0.96 % of BW, respectively, P < 0.05, SEM = 0.090) but not during non-feeding days ( P = 0.19). This treatment x day interaction can be explained by the greater consumption of supplement during feeding days by CT-fed cows, since these cows c onsumed the entire amount of supplement sooner than cows fed ML (24 to 36 vs. 48 h, re spectively). As cited previously, a greater depression in forage intake usually is obs erved when NSC-based supplements are fed (Sanson et al., 1990; Olson, et al., 1999). However, these results were obtained comparing supplements with similar physical form and intake behavior. In the current study, no differences in overall forage DMI were observed. The differences in supplement physical form and consequent c onsumption behavior led to the significant differences in forage intake pattern between treatments, characterized by the treatment x day interaction. No differences between treatments were detected for BUN concentrations (4.46 vs. 4.19 mg/dL for CT and ML, respectively; P = 0.52, SEM = 0.29) (Table 3-9, Figure 3-8). A time(period) effect ( P < 0.01) was observed because BUN concentrations usually increased after supplements were offered, i llustrating the improvement in the protein status of the cows after s upplements were consumed. A pe riod effect also was observed ( P < 0.01). Cows had greater BUN concentratio ns during the second period, intermediary during the third period, and lesser during the first period (3.45, 5.21 and 4.32 mg/dL, respectively; SEM = 0.23). A treatment x ti me(period) interaction was detected ( P < 0.01). For the second and third periods, cows fed CT had comparable BUN

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49 concentrations during the first 8 h after s upplement feeding, but greater for the following hours compared to ML-fed cows. Although CT-fed cows had greater supplement consumption during feeding days and conseque ntly during the first hours after feeding, citrus pulp undergoes slower ruminal degr adation compared to molasses (NRC, 2001). This effect may have delayed the increas e in BUN associated with the synchrony between ruminal energy and protein supply. A treatment x period interaction also was observed for BUN ( P < 0.01). During the first period, cows fed ML tended to have greater BUN concentrations (3.02 vs. 3.87 mg/dL for CT and ML, respectively; P = 0.06, SEM = 0.33), but cows fed CT had greater B UN concentrations during the third period (4.79 vs. 3.86 mg/dL for CT and ML, respectively; P < 0.05, SEM = 0.33). According to Hammond (1997), BUN concentrations should range between 7 to 8 mg/dL for mature beef cows. Therefore, cows on both treatmen ts would be deficient in protein intake according to their BUN concentrations, since they were often below this optimal range (Table 3-9). However, both supplements were formulated to supply the protein requirements of mature cows, using the NRC model (NRC, 1996) Plasma glucose concentrations did no t differ between treatments (84.85 vs. 85.95 mg/dL for CT and ML, respectively; P = 0.88, SEM = 5.12) (Table 3-9, Figure 3-9). A time(period) effect was observed ( P < 0.01) and can be attribut ed to the fluctuation in glucose synthesis and availabi lity in blood after supplements were offered and consumed. No differences were observed in plasma insulin (1.77 vs. 1.51 ng/mL for CT and ML, respectively; P = 0.33, SEM = 0.19) (Table 3-9, Fi gure 3-10) and plasma IGF-I concentrations (110.11 vs. 100.09 ng/mL for CT and ML, respectively; P = 0.49, SEM = 9.88) (Table 3-9, Figure 3-11). A time(period) e ffect was significant only for insulin ( P <

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50 0.01), and can be associated to the same effect observed for glucose, since the concentration of these substa nces in blood are mutually dependent. For both hormones, a period effect ( P < 0.01) and a treatment x period interaction ( P < 0.05) were detected. The significance in period effects indicates that the energy status of cows from both treatments increased with the advance of the experiment, and can be explained by the adaptation of the cows to the experimental procedures, particularly the frequent blood sampling, since treatments remained unchanged. The differences between treatments for insulin and IGF-I, increased with the a dvance of the experiment, explaining the significance of the trea tment x period interaction. During the second period, CT-fed cows tended to have greater plasma insulin concen trations compared to ML-fed cows (1.97 vs. 1.50 ng/mL, respectively; P = 0.09, SEM = 0.19). According to the NRC (1996), the cows utilized in this experime nt required around 8.5 Mcal/d of NEM. Given that overall forage DMI was similar and treatments were iso-caloric, cows from both treatments consumed approximately 15.0 Mcal/d (175 % of the NEM requirements). Supplements were fed at the same percentage of animal BW during Exp. 1 and Exp. 2. Because energy requirements for growing heifers and mature cows are different, the heifers in Exp. 1 were fed with moderate amounts of ener gy, whereas the cows in Exp. 2 were fed excessive amounts of supplemental energy. This might be an explanation for why the results observed during Exp. 1 were not replicated during Exp. 2. When analyzing initial plasma P4 concen trations, cows from the NCD group were at various stages of the estrous cycle. Howe ver, all cows from the CD group had similar plasma P4 concentrations, mostly originati ng from the exogenous source. Therefore, only data from the CD group were used to compar e if cows fed CT had different plasma P4

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51 concentrations compared to cows fed ML. Plasma P4 concentration did not differ between treatments (4.08 vs. 3.14 ng/mL for CT and ML, respectively; P = 0.12, SEM = 0.34; Figure 3-12). A period effect was observed ( P < 0.01) because plasma P4 concentrations decreased linearly for both groups from the first period to the last period, and this effect can be associated with th e decrease in P4 release from the CIDRs. A time(period) effect also was observed ( P < 0.01). Throughout the entire experiment, plasma P4 concentrations decreased for both groups after the supplements were offered. This decrease can be associated with: a) s upplement intake, which may have increased the rate at which plasma P4 was metabolized in the liver, b) natura l deterioration of the CIDRs, or c) the sum of both factors. A treat ment x period interacti on also was detected ( P < 0.01). Cows fed CT had greater plasma P4 concentration during the first period (5.97 vs. 4.31 ng/mL for CT and ML, respectively; P < 0.05, SEM = 0.31), but not for the second and third periods ( P = 0.20 and P = 0.39, respectively). Concentrations of BUN, insulin and IGF-I were numerically greater fo r CT-fed cows compared to ML-fed cows during the second and third periods, but similar or lesser during the first period. This may imply that during the first period, cows fe d with CT although consumed the entire amount of supplement faster, possibly had lower nutrient absorption by the digestive tract, therefore blood flow to the liver a nd consequent hepatic P4 metabolism were decreased. In conclusion, differences were not observed in plasma P4 concentrations between cows fed CT or ML but for firs t period, even though blood was frequently collected from cows with similar basal plasma P4 concentrations. Further, the differences observed in the first period were contrary to our expectations.

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52 Conclusions The objective of the first experiment was to investigate if heifers fed a molassesbased supplement would experience greater reproductive performance compared to heifers fed with supplements based on dry feed (citrus pulp), as previously observed by our group. To further test our main hypothesi s, we conducted a second experiment with cows, where blood was collected with greater frequency to verify if P4 was being metabolized at a greater exte nt in cows supplemented with citrus pulp. However, the expected supplement intake behavior was not observed in either experiment, and consequently no major differences between treatments were observed in plasma P4 concentrations of heifers and cows. In the first experiment, heifers supplemen ted with CT had greater concentrations of plasma glucose, insulin and IGF-I, which resulted in greater ADG compared to MLsupplemented heifers; however, reproductive performance was not impacted by treatments. In addition, plasma concentrati on of IGF-I was the only plasma measurement significantly correlated with BW gain and positively associated with attainment of puberty and pregnancy. In the second experiment, no major differences between treatments were found for plasma metabolites and hormones. We concluded that the cows were provided excessive supplemental energy, and any poten tial responses to the treatments were suppressed. In addition, although no differences where observed for overall forage DMI, cows supplemented with citrus pulp have a greater daily oscillation in forage intake compared to cows supplemented with molasses.

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53 Table 3-1. Composition of supplements fed to heifers (Exp. 1). Ingredients (%; as-fed) CT 1 ML 2 Blackstrap molasses 0.00 78.30 Citrus Pulp 74.70 0.00 Cottonseed meal 25.30 20.20 Calcium carbonate 0.00 1.50 1 CT = citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays). 2 ML = molasses-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).

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54 Table 3-2. Nutrient composition and intake ra te of supplements fed to heifers (Exp. 1) 1. Nutrient Composition (%; DM) CT ML TDN 70.00 77.00 CP 19.00 20.07 Calcium 1.70 1.80 Phosphorus 0.60 0.50 Supplement Intake (kg/head/d) 2 DM 2.30 2.10 TDN 1.61 1.61 CP 0.43 0.43 Calcium 0.04 0.04 Phosphorus 0.01 0.01 1 Supplements fed on Mondays, Wednesdays, and Fridays (3x/wk). 2 Estimated from the supplement consumption of the pen (5 heifers/pen).

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55 Table 3-3. Composition of supplements fed to cows (Exp. 2). Ingredients (%; as-fed) CT 1 ML 2 Blackstrap Molasses 0.00 75.40 Citrus Pulp 74.75 0.00 Cottonseed Meal 25.25 23.41 Calcium Carbonate 0.00 1.19 1 CT = citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays). 2 ML = molasses-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).

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56 Table 3-4. Nutrient composition and intake rate of supplements fed to cows (Exp. 2) 1. Nutrient Composition (%; DM) CT ML TDN 69.00 76.00 CP 16.60 18.50 Calcium 1.50 1.40 Phosphorus 0.40 0.40 Supplement Intake (kg/head/d) 2 DM 4.53 4.10 TDN 3.13 3.20 CP 0.75 0.75 Calcium 0.06 0.06 Phosphorus 0.02 0.02 1 Supplements fed on Mondays, Wednesdays, and Fridays (3x/wk). 2 Estimated from the supplement consumption of the pen (2 cows/pen).

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57 Table 3-5. Effect of treatments on ADG a nd reproductive performance of heifers in experiment 1. Item CT ML SEM P = ADG, kg/d 1 0.40 0.30 0.02 0.008 Pregnancy rate, % 2 60.0 57.5 8.05 0.830 Puberty rate, % 3,4 80.0 76.6 9.94 0.817 Days to puberty 5 72.4 69.3 5.05 0.650 1 Calculated using initial and final shrunk BW. 2 Pregnant heifers / total heifers. 3 Cycling heifers / total heifers. 4 Puberty = plasma progesterone concentr ation greater than 1.5 ng/mL for two consecutive weeks. 5 Average days to puberty attainment after the beginning of the study.

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581 Blood samples not collected on Monday during the first period. 2 Days at which supplements we re offered are underlined. 3 BUN and Glucose = mg/dL; Insu lin, IGF I, and GH = ng/mL. Table 3-6. Effects of treatments on plasma metabolites and hormones of heifers (E xp. 1) fed citrus pulp(CT) or molasses-based supplement (ML) 1,2.

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59 Table 3-7. Correlations between plasma m easurements and ADG of heifers (Exp. 1) 1,2. Item ADG Glucose Insulin IGF-I Glucose (mg/dL) -0.065 0.62 Insulin (ng/mL) 0.137 0.30 0.140 0.29 IGF-I (ng/mL) 0.414 < 0.01 0.003 0.98 0.329 < 0.05 GH (ng/mL) 0.102 0.44 0.240 0.07 0.031 0.81 0.128 0.33 1 Upper row = correlation coefficients. 2 Lower row = P values.

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60 Table 3-8. Effects of performance and plasma concentration of meta bolites and hormones on reproductive performance of heifers (Exp. 1). Puberty Attainment 1 Pubertal Non-Pubertal SEM P Value Number of animals 45 13 ADG (kg/d) 0.35 0.33 0.06 0.601 Glucose (mg/dL) 78.10 79.05 2.96 0.777 Insulin (ng/mL) 0.83 0.80 0.05 0.643 IGF-I (ng/mL) 120.11 99.10 4.21 < 0.001 Pregnancy Status 2 Pregnant Open SEM P Value Number of animals 33 12 ADG (kg/d) 0.37 0.31 0.07 0.208 Glucose (mg/dL) 79.70 74.97 2.52 0.120 Insulin (ng/mL) 0.83 0.83 0.06 0.987 IGF-I (ng/mL) 123.35 111.38 4.44 0.054 1 Puberty = plasma progesterone concentr ation greater than 1.5 ng/mL for two consecutive weeks. 2 Pubertal heifers only.

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61Table 3-9. Effects of treatments on plasma metabolites and hormones of cows (Exp. 2) fed citrus pulp(CT) or molasses-based supplement (ML) 1. 1 Supplements were offered after blood was sampled at 0 h. 2 BUN and Glucose = mg/dL; In sulin, and IGF I = ng/mL.

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62 Figure 3-1. Outline of the follicular and luteal function sy nchronization protocol. Transr ectal ultrasonography = TUS.

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63 Figure 3-2. Blood urea nitrogen (BUN) concentr ations of heifers supplemented with citr us pulp(CT) or mo lasses-based supplemen t (ML). Days at which supplemen ts were offered are underlined. Heifers fed ML had greater mean BUN concentration (P < 0.05, SEM = 0.24). Period and day(period) effects were si gnificant (P < 0.01). Treatmen t x period and treatment x day(period) interactions were also significant (P < 0.01), and periods with different letters differ (P < 0.05).

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64 Figure 3-3. Plasma glucose concentr ations of heifers supplemented with citrus pul p(CT) or molasses-ba sed supplement (ML). Day s at which supplements were offered ar e underlined. Heifers fed CT had greate r mean glucose concentration (P < 0.05, SEM = 2.46). Period and day(period) effect s were significant (P < 0.01). Treatment x period and treatm ent x day(period) interactions were also significan t (P < 0.01), and periods with diffe rent letters differ (P < 0.05).

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65 Figure 3-4. Plasma insulin concentr ations of heifers supplemented with citrus pulp(C T) or molasses-based supplement (ML). Day s at which supplements were offered are underlined. Heifers fed CT had greater me an insulin concentr ation (P < 0.05, SEM = 0.04). Period and day(period) effects were significant (P < 0.01). Treatment x period interaction was also significant (P < 0.01), and periods with differe nt letters differ (P < 0.01).

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66 Figure 3-5. Plasma insulin-like growth factor I (IGF-I) concentrations of heifers supple mented with citrus pulp(CT) or molass esbased supplement (ML). Days at which s upplements were offered are underlined. He ifers fed CT had gr eater mean IGF-I concentration (P = 0.05, SEM = 4.10). Period and day(period) effects were also si gnificant (P < 0.01), and periods with different letters differ (P < 0.05).

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67 Figure 3-6. Plasma growth hormone (GH) concentrations of heifers supplemented with citrus pulp(C T) or molasses-based supplement (ML). Days at which suppleme nts were offered are underlined. No signi ficant differences were observed (P = 0.62, SEM = 3.20). Period and day(period) effe cts were significant (P < 0.01). Treatm ent x period interaction was also significant (P < 0.05), and periods with di fferent letters differ (P < 0.05).

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68 Figure 3-7. Forage DMI of cows supplement ed with citrus pulp(CT) or molasses-based supplem ent (ML). Days at which supplements were offered are underline d. No significant differences were obs erved (1.16 vs. 1.20 % for CT and ML, respectively; P = 0.52, SEM = 0.08). Day effect and treatment x day inter action were significant (P < 0.01).

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69 Figure 3-8. Blood urea nitrogen (BUN) concentr ations of cows supplemented with citrus pulp(CT) or mola sses-based supplement (ML). Supplements were fed after blood was sampled at 0 h. No significant differences we re observed (P = 0.52, SEM = 0.29). Period and time(period) effects we re significant (P < 0.01) Treatment x period and tr eatment x time(period) interactions were also significan t (P < 0.01), and periods with diffe rent letters differ (P < 0.01).

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70 Figure 3-9. Plasma glucose concentrations of cows supplemented with citrus pulp(CT) or mola sses-based supplement (ML). Supplements were fed after blood was sampled at 0 h. No significant differences were observed (P = 0.88, SEM = 5.12). Time effect was signi ficant (P < 0.01).

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71 Figure 3-10. Plasma insulin conc entrations of cows supplemente d with citrus pulp(CT) or molasses-based supplement (ML). Supplements were fed after blood was sampled at 0 h. No significant differences were observed (P = 0.33, SE M = 0.19). Period and time(period) effects were significant (P < 0.01). Treatment x period interaction was also signif icant (P < 0.05), and periods with different letters differ (P < 0.01).

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72 Figure 3-11. Plasma insulin-like growth fact or I (IGF-I) concentrations of cows supplemented with citrus pulp(CT) or molasses based supplement (ML). Supplements were fed after blood was sampled at 0 h. No significant differences wer e observed (P = 0.49, SEM = 9.88). Pe riod effect was significant (P < 0.0 1). Treatment x period interaction was also significant (P < 0.01) and periods with different letters differ (P < 0.05).

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73 Figure 3-12. Plasma progesterone (P4) concen tration of cows with CIDR and supplemente d with citrus pulp(CT) or molassesbased supplement (ML). No signifi cant differences were observed (P = 0.12, SEM = 0.33). Period and time(pe riod) effects were significant (P < 0.01). Treat ment x period interaction was also si gnificant (P < 0.01), and periods with different letters differ (P < 0.01).

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74 CHAPTER 4 EFFECTS OF SUPPLEMENT TYPE AND FEEDING FREQUENCY ON PERFORMANCE AND PHYSIOLOGI CAL RESPONSES OF YEARLING BRAHMAN-CROSSBRED STEERS Introduction Energy supplementation is required in most of Florida cow-calf operations because the tropical perennial grasses grown in the state usually do not have adequate energy content to meet the requirem ents of the cattle (Moore et al., 1991). The frequency at which supplements are offered depends on the supplement type and also on the management system of the operation. Accord ing to a review compiled by Kunkle et al. (1999), cattle supplemented daily, 3x/wk, or 1x/wk have similar rates of BW gain. However, the majority of these publicatio ns referred to prot ein or grain-based supplements, and none evaluated energy suppl ements based on low-starch by-products, such as molasses or citrus pulp. Citrus pulp and molasses originate fro m orange and sugarcane production, respectively, which are both important and a bundant agricultural ente rprises in Florida. For that reason, these feeds are widely us ed as energy supplements for beef cattle operations across the state. Molasses, despit e its high DM content (approximately 75 %), is classified as a liquid feed, whereas citr us pulp is commonly processed and fed as dry pellets. Differences in physical form betw een citrus pulp and molasses may lead to differences in feed intake behavior (Arthingt on et al. 2004). Molasses and citrus pulp also differ in their carbohydrate pr ofile. Although both are low-starch energy feedstuffs, sucrose is the main carbohydrate of molasses, whereas pectin is the main carbohydrate of

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75 citrus pulp. Pectin and sucrose are fermen ted differently in the rumen, potentially impacting forage intake, diet di gestibility, and en ergy utilization. The objective of this experiment was to i nvestigate the effects of supplement type and feeding frequency on performance, plas ma metabolites and hormones, and voluntary forage intake of yearling steers. Material and Methods This study was conducted at the Univers ity of Florida IFAS, Range Cattle Research and Education Center, Ona. Th e study was conducted during the months of November and December 2004. The animals utilized in this experiment were cared for by acceptable practices as outlined in the Guid e for the Care and Use of Agricultural Animals in Agricultural Resear ch and Teaching (FASS, 1999). Animals Twenty four Brahman x British crossbre d steers (BW SD = 257 26 kg; age SD = 12 1 mo) were utilized in this experime nt. Steers were stratified by initial BW and randomly allocated to 12 pens (2 steers/pen). Pens were th en randomly assigned to one of three treatments: 1) molasses-based supplem ent fed 3x/wk (ML), 2) citrus pulp-based supplement fed 3x/wk (CT), or 3) citrus pul p-based supplement fed daily (CD). Pen was the experimental unit (4 pens/treatment). Diets Limpograss ( Hemarthria altissima ) hay (54 % TDN and 9.1 % CP; DM basis) was coarsely ground and offered at amounts to en sure ad libitum intake throughout the study. Steers also had free access to a complete minera l mix and water. Treatments consisted of three energy supplements (Table 4-1), fe d either 3x/wk (Mondays, Wednesdays and Fridays for ML and CT) or every day (CD) at a daily rate of 2.3 kg of DM per heifer for

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76 CT and CD and 2.1 kg of DM per heifer for ML. Supplement intakes were formulated to be iso-caloric and iso-nitrogenous (Table 4-2) and also balanced for calcium content, given the high concentration of calcium in citrus pulp. Sampling One week before the start and at the e nd of the study, steers were weighed for 2 consecutive days to determine both full and sh runk BW (after 16 h of feed restriction). During the first 3 weeks of the study (d 1 to d 21), blood samples were collected immediately prior and 4, 8, 24, 32 and 48 h after the first supplement feeding of the week (d 1, 8 and 15) for determination of glucose, blood urea nitrogen (BUN), insulin, IGF-I and GH concentrations. Weeks were considered periods (week 1, P 1; week 2, P 2; week 3, P 3) for further st atistical analysis. For the second part of the study (d 22 to d 40), forage DMI was recorded daily. Hay refusal was collected and weighed before ha y and supplement feeding. A sample of the hay was collected twice during this period for nutrient analysis, while samples of the nonconsumed hay were collected daily from each pen to determine DM content. Hay samples were dried for 96 h at 50 C in forced-air ov ens. Random samples of the feedstuffs also were collected during the trial, and all samples were analyz ed for nutrient composition at commercial laboratories (Dairy One Forage Laboratory, Ithaca, NY for hay, cottonseed meal and citrus pulp samples, and SDK Laboratories, Hutchinson, KS for molasses samples). Blood Analysis Blood samples were collected via jugu lar venipuncture into commercial blood collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing

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77 sodium heparin, placed on ice immediately afte r sampling, and centrifuged at 855 g for 30 min for plasma collection. Plasma was frozen at -20C at the same day of collection. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine both plasma glucose (Coulombe and Favreau, 1963; modified and described by Bran + Luebbe Industria l Method #339-01) and BUN (Gochman and Schmitz, 1972; modified and described by Br an + Luebbe Industrial Method #339-19). A double antibody radioimmunoassay (RIA) was used to determine plasma concentrations of insulin (Malven et al., 1987; Badinga et al., 1991), GH (Badinga et al., 1991) and IGFI (Badinga et al., 1991). The extraction proce dure used in the IGF-I assay was modified from Badinga et al. (1991) by using a ethano l:acetone:acetate ratio of 6:3:1. The intraand inter-assay CVs were, respectively, 5.79 and 28.26 % for insulin, 7.80 and 7.86 % for IGF-I, and 13.47 and 18.06 % for GH. Statistical Analysis Data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001). The statistical model used for forage DMI data analysis was: Y = + TRTi + PENj(i) + DAYk + TRTDAYik + Eijkl where Y = response variable = mean TRT = fixed effect of treatment PEN = random effect of pen within treatment DAY = fixed effect of day TRTDAY = effect due to interaction of treatment and day E = residual error

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78 The statistical model used for plasma metabolites and hormones data analysis was: Y = + TRTi + Pj + PENk(i) + STEERl(k) + TIMEm(j) + TRTPij + TRTTIMEim(j) + Eijklm where Y = response variable = mean TRT = fixed effect of treatment P = fixed effect of period PEN = random effect of pen within treatment STEER = random effect of steer within pen TIME = fixed effect of time within period TRTP = effect due to intera ction of treatment and period TRTTIME = effect due to interaction of treatment and time within period E = residual error Results are reported as least square mean s. Means were separated using PDIFF. Significance was set at P 0.05, and tendencies we re determined if P > 0.05 and 0.15. Only significant interactions are reported. Results and Discussion Molasses is a viscous liquid feedstuff, of ten mixed with protein ingredients and fed as slurries. The majority of beef cattle operations offer molasses slurries one to three times per week; therefore we did not incl ude a treatment with the molasses-based supplement fed daily. Supplement intake behavi or was observed for all treatments. Steers

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79 fed ML consumed the whole amount of suppl ement by approximately 48 h after feeding. Steers fed CT consumed their offer by 24 to 36 h, whereas CD-fed steers consumed their offer within 4 h. Mean BW gain was greater ( P < 0.05) for CD vs. ML, and tended to be greater ( P = 0.13) for CD vs. CT (0.30, 0.18 and 0.10 kg/d for CD, CT and ML, respectively; SEM = 0.05). Similar to our previous experience with heifers, feeding citrus pulp-based supplements at the same frequency of mola sses-based supplements numerically increased BW gain. However, differences became significant if supplementation frequency increased. Our findings concur with previous reports indicating inferior BW gain for animals fed molasses compared to animals fe d other energy sources (Pate, 1983; Royes et al., 2001). Feeding citrus pulp-based supplemen ts daily tended to increase BW gains compared to the same supplement fed 3x/wk. To our knowledge, no studies evaluating supplementation frequency with citrus pul p-based supplements have been reported. Overall forage DMI did not differ among treatments (1.50, 1.29 and 1.36 % for ML, CT and CD, respectively; P = 0.16, SEM = 0.07; Figure 4-1), but ML tended to have greater forage DMI than CT ( P = 0.06, SEM = 0.07). From d 31 to the end of the experiment, the collection of forage leftovers and the consequent forage DMI observation were debilitated by inclement weather. Th e differences between ML and CT became intensified during this period a nd therefore, the statistical tendency observed may not be realistic. A significant day effect and also a significant treatment x day interaction were detected ( P < 0.01). Forage DMI was less for CT and ML during the days that these supplements were fed (0.99 and 1.56 for CT vs. 1.41 and 1.59 % BW for ML during feeding days and non-feeding days, respectively; P 0.01, SEM = 0.08). A day effect

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80 was not significant for CD-fed steers ( P = 0.35). These findings concur with our previous experience with mature cows. Supplement c onsumption was greater for CTand ML-fed steers during the days it was offered, and since forage DMI is negativ ely associated with energy supplement intake, forage DMI was reduced (Caton and Dhuyvetter, 1997; Bodine and Purvis, 2003). In addition, forage DMI for steers fed CT was less compared to steers fed ML during feeding da ys only (0.99 vs. 1.41 % BW, respectively, P < 0.01, SEM = 0.08). Greater supplement intake after feeding for CT steers might be the cause for the decreased forage consumption, since these steers consumed the entire amount of supplement sooner than steers fed ML (24 to 36 h vs. 48 h, respectively). Feeding energy supplements daily instead of 3x/wk decreased the oscillation, but not overall forage DMI. This effect may be explained by a reducti on of the substitution effect. During the experiment, supplement consumption was ma inly constant for CD-fed steers, but oscillated significantly for the steers fed CT. Several studies indicated that forage intake was decreased when non-structural carbohydrates -based supplements were fed (Sanson et al., 1990; Olson, et al., 1999). The same res ponse were not realized when fibrous byproduct supplements were fed (Bowman and Sanson, 2000). In the present study, however, this pattern was not observed. Steers fed ML tended ( P = 0.06) to have greater forage intake than CT-fed steers, however, this result should be interpreted with caution. Concentration of BUN tended to be great er for CD vs. ML (3.55 vs. 1.97 mg/dL, respectively; P = 0.06, SEM = 0.53) (Table 4-3, Figure 42), but did not differ for CD vs. CT (2.92 mg/dL for CT; P = 0.42) and CT vs. ML ( P = 0.23). A significant time(period) effect ( P < 0.01) was observed. For all treatments, BUN concentrations increased after feeding. A significant period effect also was detected ( P < 0.01). Mean BUN

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81 concentration of all treatments was greater ( P < 0.01) during the first period compared to the second and third periods (4.02, 2. 25 and 2.17, respectively; SEM = 0.33). Concentrations of BUN are di rectly correlated with the am ount of ammonia produced and absorbed in the rumen (Huntington, 1990). A mmonia is a product of dietary protein degradation by the microbial population in the rumen and is a major precursor for microbial protein synthesis. Concentrati ons of BUN should range between 11 and 15 mg/dL for growing animals to ensure maxi mum rates of gain (Byers and Moxon, 1980). This goal can be achieved by feeding animal s with correct quanti ties of protein and energy (Hammond, 1997; Hunti ngton, 1997), and also synchr onizing the supply of these nutrients in the rumen by feeding energy and pr otein sources with similar rates of ruminal degradability (Spicer et al., 1986; Matras et al., 1991; Tani guchi et al., 1995). In the current study, all treatments would be defici ent in protein content according to the BUN concentrations observed, sin ce they were always below th e optimal range (Table 4-3). However, treatments were formulated to suppl y the requirements of growing steers, using the NRC model (NRC, 1996). Feeding citrus pulp-based supplements daily seemed to improve the protein status of steers when compared to molasses-based supplements fed 3x/wk. This response may be associated with an improved synchrony of protein and energy availability in the rumen. Plasma glucose concentrations were d ecreased for CD vs. CT and ML (66.05, 76.24, and 76.55 mg/dL, respectively; P < 0.05, SEM = 3.02) (Table 4-3, Figure 4-3) but did not differ for CT vs. ML ( P = 0.94). A time(period) effect was detected ( P < 0.01) and can be attributed to the variance in gluc ose synthesis and availa bility in blood after supplements were offered and consumed. A period effect also was detected ( P < 0.01).

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82 Mean plasma glucose concentrati on of all treatments was greater ( P < 0.01) during P 1 and P 2 vs. P 3 (73.85, 74.40 and 70.60 mg/dL, respectively; SEM = 1.80). Plasma insulin concentration was greater for CT vs. CD and ML (0.60, 0.46 and 0.43 ng/mL, respectively; P < 0.05, SEM = 0.04) (Table 4-3, Figure 4-4) but did not differ between CD vs. ML ( P = 0.63). A significant time(per iod) effect was observed ( P < 0.01) and can be associated with fluctuations of plasma glucose concentrations, since the concentration of these substances in blood are mutually dependent. A significant treatment x period interaction was detected ( P < 0.01). From P 1 to P 3, plasma insulin concentration increased for CT ( P < 0.01), was constant for ML, and decreased for CD ( P < 0.01). Plasma IGF-I concentration did not differ among treatments (96.49, 102.71 and 78.65 ng/mL for CD, CT and ML, respectively; P = 0.27, SEM = 10.22) (Table 4-3, Figure 45). A period effect was significant ( P < 0.01). Mean IGF-I concen tration of all treatments increased from P 1 to P 3 (78.00, 97.37 a nd 102.49 ng/mL, respectively; SEM = 5.99). Additionally, steers fed CD ha d the greatest increase in pl asma IGF-I concentrations, reflected in the treatment x period interaction ( P < 0.01). Plasma GH concentrations did not differ among treatments (74.52, 74.85 a nd 76.21 ng/mL for CD, CT and ML, respectively; P = 0.98, SEM = 8.00) (Table 4-3, Fi gure 4-6). A period effect was observed ( P < 0.01) since mean plasma GH concentr ation of all treatments decreased from the first to the third period (84.15, 76.85 and 64.59 ng/mL, respectively, SEM = 5.07). A significant time(period) effect also was detected ( P < 0.01). This was likely due to a pulsatile pattern of GH re lease, ranging from 10 to 15 pul ses/d (Hornick et al., 1998; Bossis et al., 1999). Although blood sampling was relatively frequent in this experiment, it was not frequent enough to identify pulses or basal concentrations of this hormone.

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83 Several studies conducted with beef cattl e reported a relationship between the glucose/insulin/IGF-I/GH axis and animal BW gain. Increased rates of BW gain have been associated with greater concentrations of blood glucose, insulin, and IGF-I, and decreased concentrations of GH (Vizcarra et al., 1998; Bossis et al., 1999; Lapierre et al., 2000). However, in the present study, steers fe d CD had greater BW gain than CTand ML-fed steers, but lesser concentrations of glucose and insulin (only compared to CT), and similar IGF-I and GH concentrations. We theorized that CD-fed steers had a more efficient utilization of energy compared to the other treatments. The lesser plasma glucose concentration may have been the re sult of enhanced cell uptake. Consequently, less insulin was required to activate the recep tors and regulate the transport of glucose into the cells. This effect was mostly noted during the se cond and third period (Figure 44). However, IGF-I concentrations, which usua lly are positively correlated with insulin concentrations (Keisler and Lucy, 1996; Webb et al., 2004), rapidly in creased from the P 1 to P 3 for CD-fed steers (Figure 4-5). This indicates that, althought glucose and insulin concentrations were not as gr eat, energy status was improving at a greater rate for CD-fed steers compared to the other tr eatments, which may have resu lted in the greater BW gain. Conclusions We concluded that feeding a citrus pu lp-based supplement daily improved steer performance compared to the same supplem ent fed 3x/wk or an iso-caloric and isonitrogenous supplement based on liquid molasse s also fed 3x/wk. However, these results were not reflected by the blood hormones and me tabolites usually associated with animal performance, such as glucos e, insulin, IGF-I and GH.

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84 Table 4-1. Composition of supplements fed to steers. Ingredients (%; as-fed) ML 1 CT 2 CD 3 Blackstrap molasses 78.30 0.00 0.00 Citrus pulp 0.00 74.70 74.70 Cottonseed meal 20.20 25.30 25.30 Calcium carbonate 1.50 0.00 0.00 1 ML = citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays). 2 CT = molasses-based supplement fed 3x/ wk (Mondays, Wednesdays and Fridays). 3 CD = citrus pulp-based supplements fed daily.

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85 Table 4-2. Nutrient composition and inta ke rate of supplements fed to steers 1. Nutrient Composition (%; DM) ML CT CD TDN 77.00 70.00 70.00 CP 20.07 19.00 19.00 Calcium 1.80 1.70 1.70 Phosphorus 0.50 0.60 0.60 Supplement Intake (kg/head/d) 2 DM 2.10 2.30 2.30 TDN 1.61 1.61 1.61 CP 0.43 0.43 0.43 Calcium 0.04 0.04 0.04 Phosphorus 0.01 0.01 0.01 2 Estimated from the supplement consum ption of the pen (2 steers/pen).

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86 Table 4-3. Effects of treatment s on plasma metabolites and horm ones of steer fed 3x/wk a citrus pulp(CT) or molasses-based supplements (ML) or fed daily a citruspulp based supplement (CD) 1. 1 Supplements were offered after blood was sampled at 0 h. 2 BUN and Glucose = mg/dL; Insu lin, IGF I, and GH = ng/mL.

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87 Figure 4-1. Forage DMI of steers supplemented 3x/wk with citrus pul p(CT) or molassesbased supplement (ML), or daily with citrus pulpbased supplement (CD). Days at which supplements were offered are underlined. No significant dif ferences were observed (1.50, 1.29 and 1.36 % for ML, CT and CD, respectively; P = 0.16, SEM = 0.07). Day effect and treatment x day interaction were significant (P < 0.01).

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88 Figure 4-2. Blood urea nitrogen (BUN) concentr ations of steers supplement ed 3x/wk with citrus pulp(CT) or molasses-based supplement (ML), or daily with a citrus pulp-based supplement (CD) Supplements were offered after blood was sampled at 0 h. Steers fed CD tended to have greater mean BUN concentration than steers fed ML (P = 0.06, SEM = 0.53). Period and time(period) effects were signi ficant (P < 0.01). Periods with diff erent letters differ (P < 0.01).

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89 Figure 4-3. Plasma glucose concentr ations of steers supplemented 3x/wk with citr us pulp(CT) or mola sses-based supplement (ML) or daily with a citrus pulp-based supple ment (CD). Supplements were offered af ter blood was sampled at 0 h. Steers fed CD had lesser mean glucose concentra tion than steers fed either with ML and CT (P < 0.05, SEM = 3.02). Period and time(period) effects were significan t (P < 0.01), and periods with diff erent letters differ (P < 0.01).

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90 Figure 4-4. Plasma insulin concentr ations of steers supplemented 3x/ wk with citrus pulp(CT) or molasses-based supplement (ML) or daily with a citrus pulp-based supple ment (CD). Supplements were offered af ter blood was sampled at 0 h. Steers fed CT had greater mean insulin concentrat ions than steers fed ML and CD (P < 0.05, SEM = 0.04). A time(per iod) effect and a treatment x period interacti on were significant (P < 0.01).

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91 Figure 4-5. Plasma insulin like growth factor I (IGF-I) concentrations of steers supplemented 3x/wk with citrus pulp(CT) or molasses-based supplement (ML), or daily with a citrus pulp-based supplem ent (CD). Supplements were offered after blood was sampled at 0 h. No si gnificant differences were observed (P = 0.27, SEM = 10.22). Period effect w as significant (P < 0.01). A treatment x period interaction was also significa nt (P < 0.01), and periods with d ifferent letters differ (P < 0.01).

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92 Figure 4-6. Plasma growth hormone (GH) concentrations of steers supplemented 3x/ wk with citrus pulp(CT) or molasses-based supplement (ML), or daily with a citrus pulp-based supplement (CD) Supplements were offered after blood was sampled at 0 h. No significant differences were observed (P = 0.98, SEM = 8.00). Period and time(period) effect s were significant (P < 0.05), and periods with differ ent letters differ (P < 0.05).

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104 Welch, J. G. and A. M. Smith. 1971. Physical stimulation of rumination activity. J. Anim. Sci. 33:1118-1123. Wettemann, R. P. and I. Bossis. 2000. Nutriti onal regulation of ovari an function in beef cattle. J. Anim. Sci. Available:http://www.asas.org/jas/sy mposia/proceedings/0934.pdf Accessed Oct. 25, 2005. Wettemann, R. P., C. A. Lents, N. H. Ciccioli, F. J. White, and I. Rubio. 2003. Nutritionaland suckling-mediated anovulat ion in beef cows. J. Anim. Sci. 81(E. Suppl. 2):E48-E59. Williams, G. L. and D. E. Ray. 1980. Hormonal and reproductive profiles of early postpartum beef heifers af ter prolactin suppression or steriod-induced luteal function. J. Anim. Sci. 50:906-918. Williams, G. L. 1990. Suckling as a regulator of postpartum rebreeding in cattle:A review. J. Anim. Sci. 68:831-852. Wiltbank, J. N. 1970. Research needs in beef cattle reproduction. J. Anim. Sci. 31:755762. Wiltbank, J. N., H. W. Hawk, H. E. Kidder, W. G. Black, L. C. Ulberg, and L. E. Casida. 1956. Effect of progesterone therapy on em bryo survival in cows of lowered fertility. J. Dairy Sci. 39:456-461. Xu, Z. Z., L. J. Burton, and K. L. Macmillan. 1997. Reproductive performance of lactating dairy cows following estrus synchronization regimens with PGF2 and progesterone. Theriogenology 47:687-701.

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105 BIOGRAPHICAL SKETCH Reinaldo Fernandes Cooke was born in So Paulo / SP, Brazil, on May 08, 1979. He is the oldest son of Nelson de Burgos Cooke and Rosangela C. F. Cooke. Reinaldo obtained his B.S. in animal sciences from the So Paulo State University (UNESP) in 2003. In 2004, Reinaldo moved to Gainesville, FL to begin his Master of Science program at the University of Florida An imal Sciences, advised by Dr. John Arthington. His research focused on beef cattle nutri tion. After graduation, Reinaldo will pursue his PhD degree again at University of Florida, further investigating the findings obtained during his MS program.


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Permanent Link: http://ufdc.ufl.edu/UFE0014407/00001

Material Information

Title: Effects of Supplement Type on Performance and Physiological Responses of Brahman-Crossbred Cattle
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014407:00001

Permanent Link: http://ufdc.ufl.edu/UFE0014407/00001

Material Information

Title: Effects of Supplement Type on Performance and Physiological Responses of Brahman-Crossbred Cattle
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014407:00001


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Full Text












EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE AND PHYSIOLOGICAL
RESPONSES OF BRAHMAN-CROSSBRED CATTLE

















By

REINALDO FERNANDES COOKE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Reinaldo F. Cooke
































This thesis is dedicated to my wife Flavia, and also to my entire family.















ACKNOWLEDGMENTS

Firstly, I would like to thank my wife, Flavia Cooke, and my family for all the love,

support and patience during my program.

I also would like to thank Dr. John Arthington for the opportunity, guidance and

also for the excellent mentorship. I would like to thank the members of my committee,

Dr. Cliff Lamb, Dr. William Thatcher and also Dr. Charles Staples, for the support with

my research and class work. Other faculty members not involved in my committee, such

as Dr. Chad Chase, Dr. Matthew Hersom, Dr. Joel Yelich, Dr. Richard Miles, Dr. Gary

Hansen and Dr. Xuejun Qiu, also were fundamental for the accomplishment of my goals.

I would like to thank all the staff that helped me with my research projects, with

special thanks to Mrs. Toni Woods. My former advisor from Brazil, Dr. Jose Luiz

Moraes Vasconcelos, is also very important to my academic and research career, since he

is the one that introduced me to the scientific world. Special thanks are extended to my

fellow graduate students, and also to my Brazilian colleagues Guilherme Marquezini,

Everton Pereira, Ocilon Sa Filho, Davi Brito de Araujo and Dr. Jodo Vendramini for the

indispensable help with my research projects.
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S .............................................. ........................................... vii

LIST OF FIGURES ............ .......... ...... .. ............. viii

A B ST R A C T .......... ..... ...................................................................................... x

1 INTRODUCTION ............... ................. ........... ................. ... .... 1

2 LITERA TURE REVIEW .......................................................... ..............2

Supplementation for Forage-Based Diets ............... .............................................2
E energy Supplem entation ............................................................... ....................... 2
M olasses-B asked Supplem ents......................................................... ................ ..... 6
Supplem enting M olasses to Forage-Based Diets ........................................ ..............8
Citrus Pulp-Based Supplements ........................ ........................... 10
Supplementing Citrus Pulp to Forage-Based Diets .......................................... 13
Energy M etabolism ............................. ........................ ...........16
Energy Metabolism and Beef Cattle Performance ...........................................20
Energy and Nitrogen Synchronization in the Rumen............... ................ ...21
Nutrition and Reproduction in Beef Cattle.....................................................24
Onset of Puberty in Beef Heifers......................... ............. ...............25
Reproductive Performance in Postpartum Beef Cows .......................................26
Feed Intake and Reproductive Performance in Cattle.................................28

3 EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE, REPRODUCTIVE
AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED
F E M A L E S ......................................................... ................ 3 2

In tro du ctio n ...................................... ................................................ 3 2
M material and M methods ........................................................................ ....... ...........33
A n im als................................. ..................................................... ............... 3 3
D ie t s ............................................................................................................... 3 5
S a m p lin g .....................................................................................3 6
B lood A n aly sis ..............................................................37
Statistical A analysis .......................... .......... ............... .... ..... .. 38
R results and D discussion .............................. ........................ .. ...... .... ............4 1









E x p erim ent 1 ....................................................... 4 1
E x p e rim e n t 2 ................................................................................................. 4 7
C o n c lu sio n s........................................................................................................... 5 2

4 EFFECTS OF SUPPLEMENT TYPE AND FEEDING FREQUENCY ON
PERFORMANCE AND PHYSIOLOGICAL RESPONSES OF YEARLING
BRAHMAN-CROS SBRED STEERS.......................................................................74

In tro du ctio n ...................................... ................................................ 7 4
M material and M methods .............................................................................. ........... 75
A n im als................................. ..................................................... ............... 7 5
D ie t s ............................................................................................................... 7 5
S a m p lin g .....................................................................................7 6
B lood A n aly sis ..............................................................76
Statistical A naly sis ........................................ .. .. .... ........... 77
R results and D iscu ssion .............................. ...... .............................. .... ...... ...... 78
C o n c lu sio n s..................................................... ................ 8 3

L IT E R A T U R E C IT E D ............................................................................ ....................93

BIOGRAPH ICAL SKETCH .............................................................. ............... 105
















LIST OF TABLES


Table page

3-1 Composition of supplements fed to heifers......... .. .......... ... .................53

3-2 Nutrient composition and intake rate of supplements fed to heifers .................. 54

3-3 Composition of supplements fed to cows .............. ............................. ....... ....... 55

3-4 Nutrient composition and intake rate of supplements fed to cows .......................56

3-5 Effect of treatments on ADG and reproductive performance of heifers in
ex p erim e n t 1 ....................................................... ................ 5 7

3-6 Effects of treatments on plasma metabolites and hormones of heifers................ 58

3-7 Correlations between plasma measurements and ADG of heifers....................... 59

3-8 Effects of performance and plasma concentration of metabolites and hormones
on reproductive performance of heifers .............. ..... ........... ............... 60

3-9 Effects of treatments on plasma metabolites and hormones of cows ...................61

4-1 Composition of supplements fed to steers...... ............................................ .... .......... 84

4-2 Nutrient composition and intake rate of supplements fed to steers .........................85

4-3 Effects of treatments on plasma metabolites and hormones of steers ......................86















LIST OF FIGURES


Figure p

3-1 Outline of the follicular and luteal function synchronization protocol ....................62

3-2 Blood urea nitrogen (BUN) concentrations of heifers supplemented with citrus
pulp- (CT) or molasses-based supplement (ML) ........................................... 63

3-3 Plasma glucose concentrations of heifers supplemented with citrus pulp- (CT) or
molasses-based supplement (ML)............................. ...............64

3-4 Plasma insulin concentrations of heifers supplemented with citrus pulp- (CT) or
molasses-based supplement (M L)......... ............................... ............... 65

3-5 Plasma insulin-like growth factor I (IGF-I) concentrations of heifers
supplemented with citrus pulp- (CT) or molasses-based supplement (ML)...........66

3-6 Plasma growth hormone concentrations of heifers supplemented with citrus
pulp- (CT) or molasses-based supplement (ML) ........................................... 67

3-7 Forage DMI of cows supplemented with citrus pulp- (CT) or molasses-based
supplem ent (M L) .................. ..................................... ...... ........ .... 68

3-8 Blood urea nitrogen (BUN) concentrations of cows supplemented with citrus
pulp- (CT) or molasses-based supplement (ML) ........................................... 69

3-9 Plasma glucose concentrations of cows supplemented with citrus pulp- (CT) or
molasses-based supplement (ML).... ................. ...............70

3-10 Plasma insulin concentrations of cows supplemented with citrus pulp- (CT) or
molasses-based supplement (M L)......... ............................... ............... 71

3-11 Plasma insulin-like growth factor I (IGF-I) concentrations of cows
supplemented with citrus pulp- (CT) or molasses-based supplement (ML) ............72

3-12 Plasma progesterone (P4) concentrations of cows with CIDR and supplemented
with citrus pulp- (CT) or molasses-based supplement (ML) .................................73

4-1 Forage DMI of steers supplemented 3x/wk with citrus pulp- (CT) or molasses-
based supplement (ML), or daily with citrus pulp-based supplement ...................87









4-2 Blood urea nitrogen (BUN) concentrations of steers supplemented 3x/wk with
citrus pulp- (CT) or molasses-based supplement (ML), or daily with citrus pulp-
based supplem ent .. ....... ...................... ... ....... .......... 88

4-3 Plasma glucose concentrations of steers supplemented 3x/wk with citrus pulp-
(CT) or molasses-based supplement (ML), or daily with citrus pulp-based
su p p le m e n t ...............................................................................................................8 9

4-4 Plasma insulin concentrations of steers supplemented 3x/wk with citrus pulp-
(CT) or molasses-based supplement (ML), or daily with citrus pulp-based
su p p le m e n t ...............................................................................................................9 0

4-5 Plasma insulin-like growth factor I (IGF-I) of steers supplemented 3x/wk with
citrus pulp- (CT) or molasses-based supplement (ML), or daily with citrus pulp-
based supplem ent ......................... .......... .. ...... .... .............. .. 91

4-6 Plasma growth hormone (GH) concentrations of steers supplemented 3x/wk
with citrus pulp- (CT) or molasses-based supplement (ML), or daily with citrus
pulp-based supplem ent........................... .. ..................... ............... 92















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE AND PHYSIOLOGICAL
RESPONSES OF BRAHMAN-CROSSBRED CATTLE

By

Reinaldo Fernandes Cooke

May 2006

Chair: John D. Arthington
Major Department: Animal Sciences

Two studies were conducted to compare the performance and physiological

responses of forage-fed beef cattle supplemented with either molasses-based or citrus

pulp-based supplements. The first study consisted in two separate experiments. In Exp. 1,

we compared BW gain, reproductive efficiency, and plasma concentration of BUN,

glucose, insulin, IGF-I, GH, and progesterone (P4) of 60 Brahman x British heifers

supplemented 3x/wk with either a molasses-based (ML) or citrus pulp-based supplement

(CT). Supplement intakes were formulated to be iso-caloric and iso-nitrogenous.

Reproductive performance was not affected by treatments; however, mean BW gain was

greater (P < 0.05) for heifers fed CT (0.30 and 0.40 kg/d for ML and CT groups; SEM =

0.02). Mean plasma concentrations of glucose, insulin and IGF-I were greater (P < 0.05)

for heifers fed CT, whereas BUN was greater for heifers fed ML. Mean plasma P4

concentration did not differ between treatments. In Exp. 2, we compared forage DMI and

plasma concentration of BUN, glucose, insulin, IGF-I, and P4 of 24 Brahman x British









mature cows supplemented with the same treatments of Exp. 1. Forage DMI did not

differ between treatments; however, a day and treatment x day interaction (P < 0.05)

were detected. No differences were detected in any blood metabolite/hormone. For blood

P4 analysis, the two treatments were further separated into one of two sub-treatments, in

a 2 x 2 factorial arrangement: A) exogenous source of P4 (CD) or B) natural source of P4

(NCD). Only the results from the CD group were reported because cows from the NCD

group were at random stages of the estrous cycle. No differences in plasma P4

concentration were detected between CT and ML-fed cows, but a treatment x period

interaction (P < 0.01) was detected because cows fed CT had greater plasma P4

concentrations during the beginning of the experiment. The second study compared BW

gain, forage DMI, and plasma concentrations of BUN, glucose, insulin, IGF-I, and GH of

24 yearling Brahman x British steers supplemented 3x/wk with either CT or ML, or every

day with a citrus pulp-based supplement (CD). Supplement intakes were again

formulated to be iso-caloric and iso-nitrogenous. Forage DMI did not differ among

treatments; however, a day and treatment x day interaction were detected (P < 0.05).

Mean BW gain was greater (P < 0.05) for CD vs. ML, tended to be greater for CD vs. CT

(P = 0.13), but did not differ for CT vs. ML (0.30, 0.18 and 0.10 kg/d for CD, CT and

ML, respectively; SEM = 0.05). Mean plasma concentration of glucose was lesser (P <

0.05) for CD vs. ML and CT. Mean insulin was greater (P < 0.05) for CT vs. CD and

ML. We concluded that forage-fed cattle offered a citrus pulp-based supplement had

increased BW gain compared to cattle offered a molasses-based supplement.

Additionally, BW gain was further increased if citrus pulp-based supplements were

offered daily.














CHAPTER 1
INTRODUCTION

The beef industry in Florida primarily consists of cow-calf enterprises with forage

as the main component of cattle diets. The reproductive performance of the herd is one of

the most important factors for the overall efficiency of these operations, whereas energy

is the first nutritional concern for cattle reproduction. Forages grown in Florida usually

are lacking in energy density, and consequently, energy supplementation is often

required.

A variety of feedstuffs can be used as ingredients for energy supplements, but price

and availability are highly variable. Citrus pulp and molasses are energy concentrated

byproducts that originate from citrus and sugarcane production, respectively. Both

enterprises are important and extensive agricultural systems in Florida. Despite its

moderate DM content (approximately 75 %), molasses is classified as a liquid feed,

whereas citrus pulp is commonly processed and fed as dry pellets. The different physical

characteristics of these products lead to differences in intake behavior, which have an

important effect in operations where cattle are supplemented with low frequency.

Molasses and citrus pulp also differ in their carbohydrate profile. Sucrose is the

main carbohydrate of molasses, whereas pectin is the main carbohydrate of citrus pulp.

Pectin and sucrose are fermented differently in the rumen, potentially impacting forage

intake, diet digestibility, and energy utilization.

For these reasons, experiments were conducted to compare the effects of

supplement type on performance and physiologic responses of Brahman-crossbred cattle.














CHAPTER 2
LITERATURE REVIEW

Supplementation for Forage-Based Diets

Forage is the cheapest and most available source of nutrition for beef cattle in the

state of Florida. With the purpose of minimizing costs of production, it is the main

dietary component of the cow-calf operations located in the state. However, the forages

grown in Florida usually do not have adequate nutrient density to meet the nutrient

requirements of cattle consuming typical amounts of DM (Moore et. al., 1991),

particularly animals with greater nutrient demands. According to the NRC (1996),

nutritional requirements of beef cattle increase with lactation, pregnancy, and growth

rates. Consequently, growing cattle and lactating beef cows require a high plane of

nutrition to maintain adequate performance. When their diets are based on low-quality

forages, as in the case of Florida, nutrient supplementation is often required.

Energy Supplementation

Energy supplementation is required to maintain optimal animal performance in

areas where energy availability from grazed forages is limited (Caton and Dhuyvetter,

1997). In Florida, even when protein supplements are fed and forage intake is potentially

increased, energy requirements usually are not met because of the lack in forage quality.

Bodine and Purvis (2003) also indicated that although protein is typically considered the

major limiting nutrient for cattle, its supplementation may not result in adequate energy

supply to the animals. Consequently, supplemental energy is required.









Contrary to protein supplements, energy supplements often decrease forage intake

and digestibility (Caton and Dhuyvetter, 1997; Kunkle et al., 1999; Bodine and Purvis,

2003). A data base containing 66 publications concluded that forage intake was decreased

when supplemental TDN intake was greater than 0.7 % of BW (Moore et al., 1999).

These effects are even more pronounced when supplements contain high quantities of

nonstructural carbohydrates (NSC) such as starch and sugars (Sanson et al., 1990; Olson

et al., 1999). The potential mechanisms by which high NSC supplements depress forage

intake and/or digestibility include a decrease in ruminal pH, decrease in synthesis and

activity of cellulolytic enzymes (Martin et. al., 2001), impaired bacterial attachment to

fibrous digesta (Hiltner and Dehority, 1983), and an increase in lag time for fiber

digestion (Mertens and Loften, 1980). Bowman and Sanson (2000) compiled 116

publications and observed that supplements with high quantities of NSC do not have a

negative impact on forage intake when fed less than 0.3 % of BW or when the NSC

fraction of the supplement does not exceed 0.1 % of BW. However, restricting energy

supplementation to these levels may impair animal performance, particularly when

animals are fed low-quality forages and have high nutritional demands.

An alternative to prevent depressed forage intake when energy supplementation is

necessary is the utilization of fibrous by-products feedstuffs as energy sources in the diet.

These feedstuffs usually have reduced NSC with moderate TDN content, are rapidly

digestible in the rumen and also are low-priced when compared to the costs of high NSC

feedstuffs. Some authors have reported no differences in performance when animals were

fed energy supplements with high or low NSC content (Sunvold et al., 1991; Horn et al.,

1995). These data imply that high NSC supplements can be replaced by highly digestible









fibrous supplements without a depression in animal performance. Other authors reported

a small negative effect on forage intake when supplements with low NSC content were

fed (Bowman and Sanson, 1996; Caton and Dhuyvetter, 1997; Royes et al., 2001). A

review by Bowman and Sanson (2000), however, concluded that low NSC energy

supplements, contrary to those with high NSC concentration, increased low-quality

forage intake and digestibility.

According to the NRC (2001), the NSC fraction of a feedstuff is found inside the

plant cells, and is composed of sugars, starches, organic acids and other carbohydrates

such as fructans, serving as the major source of energy for cattle. The NSC fraction is

highly soluble in the ruminal environment. This fraction is degraded rapidly by the

microbial population, and because of this, increases the rate and amount at which VFAs

are formed. Increasing the NSC content or availability in diets typically results in

elevated VFA production/accumulation in the rumen with a decreased acetate:propionate

molar ratio. This subsequently leads to a decrease in ruminal pH that may be detrimental

to fibrolytic bacteria, resulting in impaired forage digestibility (Aldrich et al., 1993; Joy

et al., 1997; Knowlton et al., 1998).

The energy provided by feedstuffs with a low NSC content usually originates

either from NDF or pectin. According to the NRC (2001), NDF is the measurement that

best differentiates structural from nonstructural carbohydrates and constitutes most of the

structural components in plant cells: cellulose, hemicellulose and lignin. On average,

NDF is less digestible than NSC and its concentration in feedstuffs is negatively

associated with energy (NRC, 2001). The chemical profile of NDF (ratio of

cellulose:hemicellulose:lignin) affects its digestibility. Cellulose and hemicellulose are









potentially but incompletely digested and utilized as an energy source by the microbial

population in the rumen. In contrast, lignin is poorly or even not digested at all (Jung and

Allen, 1995). Therefore, feedstuffs with a similar content of NDF will not necessarily

have similar energy concentration. Depending on the lignin concentration within the NDF

fraction, feeds with greater NDF may have more available energy than feeds with lesser

NDF content (NRC, 2001). The concentration of NDF in the diet is positively associated

with ruminal pH since the microbes generally ferment the NDF fraction slower.

Therefore, lesser VFA production/accumulation and a greater acetate:propionate molar

ratio in the rumen may result.

Pectins are found within the plant cell wall, but are not linked to lignin. Pectins

are well digested by the ruminal microbes, but not by mammalian enzymes (Ariza et al.,

2001). Although classified as a dietary fiber, pectins are not represented in the NDF

fraction, and are classified as non-fiber carbohydrates (NFC; Arthington et al., 2002). The

NFC fraction is similar to the NSC. It includes all carbohydrates not included in NDF,

but also includes pectins, which are not classified as part of the NSC fraction (NRC,

2001). Compared to NDF, pectins are rapidly and extensively digested in the rumen, as

reported by Ben-Ghedalia et al. (1989), where the apparent digestibility was 98.7 % for

pectin and 79.4 % for cell wall constituents by sheep fed high-pectin based feed.

Although the digestibility of pectins and NSC components is similar, there are some

major differences between them. When pectin escapes ruminal fermentation, it cannot be

digested in the small intestine, whereas NSC can be digested. Further, when pectins are

fermented in the rumen, they yield greater acetate:propionate ratios compared to NSC

fermentation.









Energy supplementation is used to correct deficiencies in order to maintain or

enhance animal performance. A variety of different feedstuffs can be used as energy

sources in ruminant diets. Supplements with low NSC concentration are a viable

alternative to substitute traditional and costly supplements with high NSC concentration.

When designing and formulating a supplementation program, the source of energy should

be carefully chosen, since the differences in energy fermentation and digestion among

carbohydrate sources may impact animal performance.

Molasses-Based Supplements

According to Curtin (1983), the term molasses referred specifically to the final

effluent obtained in the preparation of sucrose by repeated evaporation, crystallization

and centrifugation of juices from sugarcane or sugar beets. Nowadays, current feed

nomenclature characterizes molasses as any liquid feedstuff containing more than 43 %

sugar (Curtin, 1983). Sugarcane molasses is a by-product of the sugarcane refining

process to obtain sucrose, and must not contain less than 46 % total sugar expressed as

invert. Invert is a 50:50 mixture of glucose and fructose, obtained when sucrose is

hydrolyzed. If molasses moisture content exceeds 27 %, its density is determined by

double dilution and must not be less than 79.50 Brix (Curtin, 1983). The industry uses the

term Brix as a measurement of molasses total solid content. This term originally indicated

the concentration of sucrose in pure sucrose solutions on a weight basis, but molasses

also contains glucose, fructose, raffinose and numerous non-sugar organic materials

(Curtin, 1983). Therefore, the Brix value for molasses often will differ from actual sugar

or total solid content and will represent nothing more than a number denoting specific

gravity, which cannot be related to either sucrose or DM content (Curtin, 1983).









The chemical composition of sugarcane molasses varies widely, similar to that

observed in many other industrial by-products. It is influenced by several factors such as

soil type, temperature, moisture, season of production, plant variety, production practices

at a particular processing plant, and by storage variables (Curtin, 1983). These factors

lead to significant variations in nutrient content, flavor, coloration, viscosity and total

sugar content (Curtin, 1983).

Sugarcane molasses contains large quantities of simple sugars and other

carbohydrates, contributing to its high-energy value. Molasses contains around 88 % of

the TDN of corn (72 vs. 82 % on a DM basis, respectively), and the principal sugar is

sucrose, constituting around 30 to 40 % of molasses on an as-fed basis (Austin, 2003). In

contrast to energy, sugarcane molasses is low in CP content, and its nitrogenous material

mainly consists of non-protein compounds. However, CP content can vary according to

soil type as reported by Chapman et al. (1965), where molasses produced from sugarcane

grown on soils high in organic matter contained 7 to 10 % of CP compared to 3 % for

molasses from sugarcane grown on mineral rich soils. Regarding mineral content, when

compared to other energy sources such as cereal grains, sugarcane molasses is relatively

high in calcium, potassium, magnesium, sodium, chlorine and sulfur, but low in

phosphorus (Curtin, 1983).

According to Kunkle et al. (1995), blackstrap molasses is the by-product of the

sugar industries in south Florida and Louisiana. Most Florida sugarcane is grown on

organic rich soils, resulting in a molasses with greater protein content than molasses

derived from sugarcane grown on soils with low organic matter (Kunkle et al., 1995).

Molasses production is increased during fall and winter, and its price is determined by the









world market. More than 500,000 tons of molasses are produced annually in south

Florida and this makes the state a net exporter of the product (Kunkle et al., 1995).

Blackstrap molasses typically contains 22 % moisture, 60 % TDN and 7 % CP on an as-

fed basis (Kunkle et al., 1995). Molasses slurries are common on many Florida ranches.

Slurries are created by blending molasses with dry ingredients, typically protein sources

such as cottonseed meal or feather meal.

Therefore, molasses-based supplements are classified as energetic supplements,

containing high quantities of NSC derived from its high sugar content.

Supplementing Molasses to Forage-Based Diets

A beneficial use for molasses is its addition to diets based on low quality forages

or roughages, providing a readily available source of energy. However, molasses does not

contain adequate protein to meet the CP requirements of cattle fed low quality forages;

consequently a source of supplemental protein is often required.

Feeding molasses has been studied extensively over the past century. Pate (1983)

compiled the results of several studies where molasses was supplemented to beef cattle

diets based on forage. Supplementation usually resulted in decreased forage intake, but

total DMI was increased. Animal performance was consequently increased, but not

compared to when supplements based on other energy sources, such as cereal grains,

were fed. However, when plant protein was added as a nitrogen source, molasses-based

supplements were similar to corn-based supplements in terms of BW gain by beef cattle.

More recently, a review reported the effects of liquid supplements on the

performance of cattle fed forage-based diets (Moore et al., 1995). Generally, BW gain

was increased but responses were variable, and no consistent relationship between rates

of supplementation and gain were observed. When molasses was the only ingredient of









the supplement, gains were sometimes decreased and at other times increased. However,

when a major protein source was added, BW gains increased considerably.

Supplementation of molasses only increased forage consumption when intake of forage

was less than 1.75 % of BW without supplement; otherwise forage intake was decreased.

A recent study compared sources and rates of energy supplementation for

yearling cattle fed ammoniated hay (Royes et al., 2001). The authors compared

supplements based on sugarcane molasses, corn or soybean hulls, and reported reduced

feed efficiency and consequent less BW gain for animals fed molasses compared to the

other supplements, supporting the findings reported by Pate (1983).

Several authors reported that feeding cattle diets containing high quantities of

sucrose or molasses resulted in a increase in the molar percentage of butyric acid in the

rumen (Owen et al., 1967; Kellogg and Owen, 1969; Hatch and Beeson, 1972). In

addition, Reyes (1974) and Silvestre et al. (1977) also noted a substantial increase in the

molar percentage of butyric acid in the rumen of cattle fed increasing quantities of

molasses. Olbrich and Wayman (1972) compared the molar percentage of butyric acid in

the ruminal fluid of steers fed either molasses or corn, and reported a greater

concentration for the animals fed molasses. Based on these reports, feeding molasses to

cattle increases the molar percentage of butyric acid in the rumen, which usually is at the

expense of propionic acid when molasses substitutes grain or at the expense of acetic acid

when molasses is fed as a supplement in forage-based diets (Pate, 1983). This variation in

VFA profile may be related to differences in ruminal microbial population. Ruminal

protozoa population has been positively correlated with butyrate synthesis in the rumen

(Hristov et al., 2001), whereas defaunation decreased the concentrations of butyrate in the









ruminal environment (Mendoza et al., 1993; Faciola et al., 2004). The inclusion of 35 %

of molasses in beef cattle diets increased the ruminal protozoa population (Foreman and

Herman, 1953). Therefore, an increased protozoa population in the rumen might be one

of the causes to the greater butyrate synthesis observed in cattle fed with molasses-

containing diets.

Most of the available information about the effects of VFA proportion on energy

metabolism involves acetate:propionate ratios, which are negatively correlated to energy

utilization and consequent animal performance. Given that butyrate, like acetate, is not a

glucose precursor, a similar effect can be attributed to butyrate:propionate ratios. This

could explain why animals fed molasses-based supplements typically have reduced

performance compared to animals fed grain-based supplements (Pate, 1983).

In summary, although molasses supplementation usually has a negative impact on

forage DMI, overall performance is often improved by supplementing molasses to cattle

fed energy deficient diets. Further, molasses also can be used as an effective carrier to

deliver other nutrients, such as protein, vitamins and minerals.

Citrus Pulp-Based Supplements

Florida produces more than 80 % of the total United States supply of citrus, and

almost 90 % of this production is processed into juice (Hodges et al., 2001). Citrus pulp

is the residue from citrus processing, and consists of a mixture of peel, rag and seeds.

Annually, the Florida citrus industry produces more than 1 million tons of citrus pulp

(Hodges et al., 2001) and most of this total has been exported to foreign countries

throughout the past years, causing local prices to increase, and resulting in a decrease in

domestic usage. However, recent declines in exports have made citrus pulp an affordable

by-product feed for ruminants in the United States (Arthington and Pate, 2001).









Citrus pulp has been fed to ruminants since the beginning of 20th century, when it

was suggested to have a potential value as a feedstuff Walker (1917) made the initial

observation that cattle readily consumed a mix of fresh citrus pulp and cull fruits, but the

feed often spoiled before animals consumed it all. In the 1930s, dried citrus pulp began to

be produced commercially after Scott (1926) dehydrated grapefruit and fed it to dairy

cattle. Since then, the production and availability of dry citrus pulp has increased steadily

(Arthington et al., 2002). The drying procedure consists of grinding or chopping and then

dehydrating the fresh fruit residue. To aid in the dehydration process, calcium oxide or

calcium hydroxide is mixed in the process (Arthington et al., 2002).

Citrus pulp can be fed wet, as a dry meal, or in dry pellets. The wet product is

usually shipped directly from the juice plant, arranged as piles in the pastures and offered

to cattle. Arthington and Pate (2001) suggested that it is typically uneconomical to feed

wet pulp because of its high wasting rate (up to 30 %) and shipping costs associated to its

low DM content (15 to 20 %). Cattle consume citrus pulp in either the wet or dry form,

but dry citrus pulp is more easily transported and handled (Arthington et al., 2002). Dry

citrus pulp is a bulky feedstuff, having a density of approximately 303 kg/m3. For this

reason and because of economic and transport limitations, pelleting dry citrus pulp is

favorable since it increases density, handling efficiency, and decreases dustiness and

bridging in storage bins (Arthington et al., 2002).

Ammerman et al. (1967) compared performance of steers fed nonpelleted and

pelleted citrus pulp added at different concentrations within the supplement. When fed at

66 % of the total supplement, steers fed pelleted citrus pulp gained more weight (1.38

kg/d) compared to those fed the nonpelleted pulp (1.19 kg/d). The authors attributed the









differences to the greater density of the pelleted form (211 to 374 kg/m3 for nonpelleted

and pelleted form, respectively), increasing its consumption by the steers. A similar study

conducted with lambs also indicated that feeding pelleted citrus pulp increased intake and

animal performance (Loggins et al., 1964). Lambs fed pellets had numerical greater BW

gain (0.12 kg/d) when compared to lambs fed the nonpelleted form (0.11 kg/d) and also

had slightly greater intake (0.06 kg/d). Pinzon and Wing (1976) reported no differences

in the ruminal pH, ruminal ammonia concentration, blood urea nitrogen concentration,

and VFA concentration in the rumen of steers fed pelleted or nonpelleted citrus pulp at

similar rates.

Chemical composition and physical characteristics of citrus pulp mostly depends

on the type of fruit and processing methods used to produce it (Arthington et al., 2002).

The same authors compared the nutritional value of samples collected in two different

studies (Ammerman et al., 1968; Arosemena et al., 1995) and tabular values reported by

NRC (1996). The authors concluded that regardless of some variations among the three

sources attributed to production site, citrus variety and proportion of seeds and peel the

nutritional values were similar.

According to the NRC (1996), citrus pulp usually contains 82 % TDN and 6 to 7

% CP (DM basis). Calcium content is particularly high in citrus pulp because of the

addition of calcium salts to aid in the drying process. The carbohydrate fraction of citrus

pulp is mainly composed of 25 to 44 % pectin, 12 to 40 % sugar and 1 % or less starch on

a DM basis (Hall, 2000). Compared with common cereal grains such as corn, citrus pulp

contains moderate amounts of NSC (sugars and starch) and high concentration of pectins.

For that reason, citrus pulp contains a different carbohydrate profile compared to









traditional energy feeds. Citrus pulp-based supplements are classified as energy

supplements, and despite the modest content of NSC, digestibility is great because of the

large concentration of pectins.

Supplementing Citrus Pulp to Forage-Based Diets

Several studies have been conducted to investigate citrus pulp and subsequent

pectin fermentation in the ruminal environment. Welch and Smith (1971) compared

rumination patterns when sheep and cattle were fed pelletized citrus pulp, beet pulp or

alfalfa meal in addition to mixed hay. Cell wall content was 27, 50, 48, and 68 % of DM

for citrus pulp, beet pulp, alfalfa meal, and mixed hay, respectively. Citrus pulp decreased

rumination time for sheep and cattle when compared to mixed hay fed alone (0.34 vs.

0.16 min/g of DM consumed for mixed hay and citrus pulp, respectively). However,

when data were analyzed per unit of cell wall content, no difference in rumination time

was found (0.50 vs. 0.56 min/g of cell wall content consumed for mixed hay and citrus

pulp, respectively). Alternatively, beet pulp and alfalfa meal reduced rumination time

when data were analyzed as total DM or cell wall content consumed. The authors

attributed these effects to the lack of physical form required to stimulate rumination in

beet pulp and alfalfa meal, which were greater in cell wall content compared to citrus

pulp. The beef pulp and alfalfa meal were finely ground, and consequently did not

contribute substantially to the rumination process. The authors concluded that addition of

citrus pulp to forage-based diets did not influence rumination process and time, despite its

low cell wall content compared to forages. Fiber digestibility increased when citrus pulp

was added to sheep diets in a dose-dependent pattern (Sudweeks, 1977). Strobel and

Russell (1986) added different sources of carbohydrate to cultures of ruminal bacteria and

measured the fermentation process and products. The authors reported that carbohydrate









fermentation was decreased when pH became more acidic, particularly when pectin was

the major carbohydrate source. Pectin utilization decreased by 53 % when culture pH

dropped from 6.7 to 6.0, indicating that organisms that degrade pectins are pH sensitive.

At neutral culture pH (around 6.7), the authors reported that addition of starch and

sucrose increased lactic acid production, whereas little lactic acid was produced when

pectin was added. When culture pH was lowered to 5.5, lactate production quadrupled for

the starch, doubled for sucrose and did not change for pectin. Acetic acid production was

always doubled for pectin compared to all other carbohydrates, even when pH dropped

below 6.0. Therefore citrus pulp may be less likely to cause lactic acidosis and

consequential impaired fiber digestibility when compared to these other feedstuffs high in

NSC. Several other researches also have reported that feeding citrus pulp increased the

concentrations of acetic acid in the rumen and also increased the acetic to propionic acid

ratio when compared to traditional energy sources such as cereal grains (Loggins et al.,

1964; Hentges et al., 1966; Drude et al., 1971). Although greater ruminal propionate

production usually was associated with high concentrate diets and negatively associated

with high forage diets, when forage (corn silage) was replaced with increasing amounts

of citrus pulp (0 %, 33 % 67 %, and 100 %) in steers diets, only a moderate decrease in

the ruminal pH (7.12, 6.97, 6.81, and 6.76, respectively) was reported (Schaibly and

Wing, 1974). Furthermore, acetate synthesis was the greatest and propionate was the

poorest when diets contained 100 % citrus pulp, even if compared to 100 % corn silage

diets. The authors concluded that citrus pulp, though not considered a forage in the

strictest sense, does contain some forage-like properties which promote less acidic

ruminal pH values. All these data imply that citrus pulp, rich in pectin, can have less









detrimental effects on ruminal pH and fermentation products compared to other energy

sources, such as high NSC containing feedstuffs.

Research was conducted in the 1950s and 1960s to investigate the impact of

supplementing citrus pulp on the performance of beef cattle fed forage-based diets.

Chapman et al. (1953) reported no significant differences in gain or feed efficiency when

steers grazing St. Augustine pasture were supplemented with 2.3 kg/d of citrus pulp or

snapped corn, or had free-choice access to sugarcane molasses. But in a similar study,

steers on pasture fed citrus pulp tended to have greater gains (0.78 kg/d) compared to

steers fed ground snapped corn (0.72 kg/d), sugarcane molasses (0.65 kg/d), or a mixture

of corn, citrus pulp and cottonseed meal (0.75 kg/d) (Chapman et al., 1961).

Research resulting from several studies indicated positive results from feeding

citrus pulp as an energy source to feedlot animals. Kirk and Davis (1954) fed steers for

120 d with either ground snapped corn or citrus pulp and reported similar gains (1.08 and

0.99 kg/d, respectively), but animals fed citrus pulp had greater feed efficiency (5.40 and

4.70 kg of required TDN / kg of BW gain, respectively). Peacock and Kirk (1959)

conducted three 140 d trials in which steers were fed either corn or citrus pulp at 70 % of

the total diet, and did not detect differences in gain or feed efficiency. Ammerman et al.

(1963) replaced ground corn with citrus pulp at 22, 44 and 66 % of the total diet of

feedlot steers and did not find differences in gain across treatments, but gains were

numerically greater for the 22 and 44 % inclusion rate (1.35 and 1.48 kg of ADG,

respectively). Kirk and Roger (1970) published a review that contained 23 separate trials

conducted at the University of Florida Range Cattle Research and Education Center in

Ona, Florida, which used citrus products in beef finishing diets. Citrus pulp was the main









energy source in 20 of these studies. The authors concluded that the amount of citrus pulp

fed was positively associated with feedlot performance.

Research demonstrates that citrus pulp is a good energy source for ruminants,

particularly for animals fed forage-based diets, since it does not seem to interfere with

fiber digestion and rumen health. With a large citrus industry in Florida, citrus pulp may

provide an economical advantage for producers in the southeastern portion of the U.S.

Energy Metabolism

According to an extensive review by Bergman (1990), the VFAs, also known as

short-chain fatty acids, are mainly produced in the rumen by microbial fermentation of

carbohydrates, and contribute approximately 70 % to the caloric requirements of

ruminants. The major VFAs are acetate, propionate and butyrate, produced in a ratio

varying from approximately 75:15:10 to 40:40:20 (acetate:propionate:butyrate). After

synthesis, VFAs are absorbed across the ruminal epithelium, from which they are either

carried by ruminal veins to the portal vein and hence through the liver or are retained and

metabolized for energy by the ruminal epithelial cells (Bergman, 1990). Acetate and

butyrate are oxidized to produce energy whereas propionate is reserved for

gluconeogenesis. Approximately 90 % of the butyrate formed in the rumen is retained by

epithelial tissue and converted to ketone bodies or carbon dioxide whereas the remainder

is removed by the liver (Bergman, 1990). Little acetate approximately 30 % is

retained in the rumen compared to butyrate (90 %). Most of the acetate is oxidized

throughout body tissues to generate ATP. Acetate also is the major source of acetyl CoA

for lipogenesis (Bergman, 1990). Propionate is 50 % retained in the rumen and the

remainder is removed from portal blood by the liver, where it serves as a major substrate

for gluconeogenesis (Bergman, 1990). This process is essential to glucose status of the









ruminant animal because almost all dietary glucose is fermented in the rumen and does

not reach the small intestine for absorption. In summary, propionate is directed to glucose

synthesis whereas acetate and butyrate are oxidized for energy generation, but butyrate is

highly retained in the rumen and poorly contributes as an energy source for other tissues.

Although acetate is the largest single energy source absorbed through the portal

system, accounting for more than half of the total energy absorbed as VFA (Bergman et

al., 1990; Baird et al., 1975), it is inefficiently utilized as an energy source by tissues

when compared to glucose, which is effectively synthesized from propionate in the liver.

Glucose is crucial for maintenance and productive functions in ruminants such as

growing and lactating cattle (Reynolds, 2005), and propionate accounts for up to 76 % of

the glucose synthesized in the liver (Reynolds et al., 1994). Ruminants eating forage-

based diets depend on liver synthesis of glucose to meet their metabolic requirements

(Huntington, 1997), and increased liver glucose synthesis is positively associated with

energy balance and animal performance. Research by Aiello et al. (1989) also reported

detrimental effects of butyrate on VFA metabolism by restraining hepatic propionate

utilization, thus inhibiting the conversion of propionate to glucose. In addition, the

inhibition is specific and not due to a general competition among VFAs for metabolism.

High energy diets and supplements selected to enhance animal performance are typically

associated with increased VFA production, particularly propionate, leading to increased

glucose synthesis. Even when these are based on ingredients that are not propiogenic,

such as citrus pulp or molasses, overall formation of propionate is increased in the rumen

as a consequence of increased total VFA production.









Insulin is a hormone synthetized by beta-cells in the Islets of Langerhans in the

pancreas, and primarily regulates glucose uptake by cells (Austgen et al., 2003). This

hormone also has an important effect on lipogenesis and cellular uptake of amino acids

and some electrolytes (Austgen et al., 2003). Although there always is a low rate of

insulin secretion in the pancreas, the amount secreted into the blood increases as blood

concentrations of glucose rises, and similarly decreases as blood concentrations of

glucose falls. Therefore, insulin is important for body homeostasis because it maintains

blood glucose under constant concentrations and also has an anabolic role because it

increases cellular uptake of nutrients. The mechanism by which insulin promotes glucose

uptake by cells consists in the activation of insulin receptors embedded in the plasma

membrane, which regulates the number and operation of protein molecules also in the

plasma membrane that transport glucose into the cell (Austgen et al., 2003). Once inside

the cells, glucose enters glycolysis and the respiratory cycle to generate ATP molecules.

Insulin-like growth factor I is a protein hormone that resembles insulin on its

structure, and is mainly synthetized by the liver in response to GH (Gluckman et al.,

1987). However, several studies in cattle demonstrated that IGF-I concentrations in blood

were positively correlated to feed intake (Bossis et al., 1999; Armstrong et al., 2001;

Rausch et al., 2002) and blood insulin concentration (Keisler and Lucy, 1996; Webb et

al., 2004). The IGF-I molecule binds to specific receptors present on many cell types in

several tissues and has an anabolic function promoting cell growth and multiplication

(Quin, 1992).

Growth hormone, also known as somatotropin, is a protein hormone synthesized

and secreted by cells called somatotrophs in the anterior pituitary (Austgen et al., 2003).









It assists in the control of several physiologic processes, particularly stimulating anabolic

processes such as cell growth and multiplication. Growth hormone can stimulate tissue

growth by two different mechanisms; 1) direct effects by binding with receptors on target

cells, or 2) indirect effects by stimulating IGF-I secretion by the liver. The majority of the

growth-promoting effects of GH are actually due to IGF-I acting on target cells,

especially in muscle and bone (Austgen et al., 2003). Growth hormone also affects

nutrient metabolism since it stimulates protein anabolism in tissues, enhances the

utilization of fat by stimulating triglyceride oxidation in adipocytes, and also is one of a

series of hormones that serves to maintain blood glucose within a normal range (Austgen

et al., 2003). Synthesis and secretion of GH are regulated primarily by two well-

characterized hypothalamic peptides: stimulated by GHRH or inhibited by somatostatin.

In addition, GH synthesis and secretion also are regulated by IGF-I concentrations in

blood. Increased concentrations of IGF-I in blood decrease the secretion of GH by

directly suppressing its synthesis in the somatotrophs and by stimulating the release of

somatostatin from the hypothalamus (Austgen et al., 2003). Since insulin concentration

and feed intake are positively correlated with IGF-I, they consequently are negatively

associated with GH secretion and concentrations in blood. The integration of all the

factors that affect GH synthesis and secretion lead to a pulsatile pattern of release of this

hormone (Austgen et al., 2003). Studies with beef cattle demonstrated that frequency of

GH pulses is not affected, but pulse height, amplitude and overall GH concentration in

blood are inversely correlated with energy intake (Nolan et al., 1990; Hornick et al.,

1998; Bossis et al., 1999).









Energy Metabolism and Beef Cattle Performance

Feed intake, blood metabolites associated with nutritional status such as the

glucose/insulin/IGF-I/GH axis, and consequent animal performance are usually

associated in cattle. Research by Vizcarra et al. (1998) analyzed the influence of BW gain

on concentrations of blood glucose and insulin in primiparous beef cows. The authors fed

cows in order to attain gains of 0.45 or 0.90 kg/d and reported that animals under a higher

plane of nutrition had greater concentrations of glucose and insulin in blood. In a similar

study with beef steers, blood glucose, insulin, and IGF-I typically increased with rate of

gain (Hersom et al., 2004). Animals fed to 60, 100 or 160 % of maintenance energy and

protein requirements also had increased blood concentrations of glucose, insulin and IGF-

I when feeding level increased (Lapierre et al., 2000). Ellenberger et al. (1989) compared

serum concentration of glucose, IGF-I and GH in steers fed either ad libitum gaining 1.40

kg/d, or restricted amounts gaining 0.37 kg/d. Mean serum concentrations of GH were

elevated (45.6 vs. 23.4 ng/mL) and serum concentrations of IGF-I were decreased (108

vs. 167 ng/mL) for feed-restricted steers. Bossis et al. (1999) compared blood

concentrations of glucose, insulin, IGF-I and also blood concentration, pulse frequency

and pulse amplitude of GH from heifers fed either to maintain body condition score or

restricted diets to induce negative energy balance. Decreased blood glucose (58.7 vs. 71.5

mg/dL), insulin (0.95 vs. 1.85 ng/mL), and IGF-I (15.35 vs. 94.1 ng/mL) concentrations

but increased GH concentration (38.7 vs. 12.1 ng/mL) and pulse amplitude (36.3 vs. 17.6

ng/mL) for feed-restricted heifers were noted. Pulse frequency of GH did not change

between groups.

It can be concluded that animal performance is associated with energy intake and

metabolism. There is a direct relationship among diet and blood concentrations of









glucose, insulin, IGF-I and GH, and these metabolites also have direct effects on each

other. Additionally, blood concentration of IGF-I is typically associated with BW gain

and therefore, should be maximized.

Energy and Nitrogen Synchronization in the Rumen

Ruminants have a high rate of ammonia absorption from the rumen, which like

peptides and amino acids, is a product of dietary protein degradation by the microbial

population in the rumen (Huntington, 1990). Ammonia is a major precursor for microbial

protein synthesis, and its excess is absorbed by ruminal epithelium and transported

mostly to the liver. In liver, ammonia is converted into urea, which can be re-utilized as a

nitrogen source in the rumen via saliva or circulatory system (blood urea nitrogen; BUN),

or be excreted in urine by the kidneys. Hepatic ammonia metabolism and renal excretion

of urea require energy. Therefore, elevated amounts of ammonia produced in and

absorbed by the rumen increase the animal's requirement for energy (Huntington, 1990).

Microbial protein synthesis is optimal when both energy and nitrogen sources are

available in the rumen (Hammond, 1997). Diets rich in protein content but lacking either

in energy availability or synchrony between ruminal energy and nitrogen supplies may

limit the utilization of available nitrogen by ruminal microorganisms (Huntington, 1997),

and also increase BUN concentrations (Santos, 2004). Alternatively, diets with

inadequate protein concentrations limit ruminal microbial growth and compromise fiber

digestion. Therefore, to increase efficiency of nitrogen utilization in the rumen and

consequent microbial protein synthesis, energy and nitrogen should be available in the

rumen in a coordinated pattern. Ruminal degradability and digestibility of dietary

carbohydrates and protein differ among feedstuffs (NRC, 2001) and these differences

may influence microbial fermentation and protein synthesis in the rumen. Diets or









supplements containing highly degradable energy sources, such as high NSC feedstuffs,

should also contain protein sources with high rumen degradability in order to grant a

synchronized availability of energy and nitrogen to the microbial population in the

rumen. Conversely, when fibrous ingredients such as citrus pulp are the main energy

source, protein sources with lower degradation rates should be added.

Several studies examined the effects of synchronizing energy and nitrogen

supplies in the rumen. When steers were infused with starch and casein in their rumen or

abomasum, they had reduced ammonia absorption and increased nitrogen retention when

both nutrients were delivered in the rumen (Taniguchi et al., 1995). In research with

lambs, Matras et al. (1991) fed three different sources of energy varying in ruminal

degradability (barley > steam-flaked corn > dry rolled sorghum) combined with three

different sources of protein also varying in ruminal degradation rates (urea > 50:25:25 of

urea, blood meal and corn gluten meal > 50:50 blood meal and corn gluten meal) in a 3 x

3 Latin square design. Nitrogen utilization was most efficient when rapidly degraded

sources (barley and urea) and slowly degraded sources (sorghum and blood + corn gluten

meal) of energy and protein were fed together. The authors concluded that it was

advantageous to feed animals sources of protein and energy containing similar ruminal

degradation rates. Spicer et al. (1986) fed feed ingredients differing in ruminal

degradation rates to beef steers (barley > corn > sorghum) and reported that microbial

protein synthesis and efficiency of nitrogen utilization was positively associated with

protein and carbohydrate degradability of the feedstuff. Studies with dairy cattle indicated

that synchronizing ruminal fermentability of carbohydrate and protein sources increased

nitrogen utilization and consequent outflow of bacterial protein from the rumen. Herrera-









Saldana et al. (1990) fed lactating dairy cows barley or milo as energy sources (90.5 vs.

70.5 % of ruminal carbohydrate degradability, respectively) and cottonseed meal or

brewers dried grains as protein sources (56.6 vs. 38 % of ruminal protein degradability,

respectively). The authors reported increased microbial protein synthesis when animals

were fed synchronized diets, particularly diets with highly degradable ingredients, which

resulted in increased milk production. In a similar study, Aldrich et al. (1993) fed

lactating dairy cows four diets formulated for high and low ruminal degradability of

carbohydrate and protein. The authors reported that passage of bacterial N to the small

intestine was the greatest (262 g/d) when highly ruminally-degradable nonstructural

carbohydrate was combined with high rumen-available protein, and was the least when

high rumen-available nonstructural carbohydrate was combined with low rumen-

available protein (214 g/d).

Monitoring BUN is a technique that can be used for measuring protein and energy

status in cattle and is highly correlated with the amount of ammonia produced and

absorbed in the rumen (Hammond, 1997). Therefore, in healthy ruminants, BUN

concentrations are indicative of the protein to energy ratio in the diet, and furthermore,

synchrony between dietary energy and protein degradability in the rumen. Little work has

been reported in beef cattle with regard to the influence of ruminal energy and nitrogen

synchronization with BUN concentrations. Most of the work analyzed the effects of

dietary protein concentration or degradability. Hammond (1983) fed steers isocaloric

diets having three concentrations of CP and reported that BUN concentrations increased

from 2.6 to 11.1 mg/dL as dietary CP increased from 6 to 18 % (DM basis). Burris et al.

(1975) fed steers isocaloric and isonitrogenous corn-cottonseed hull based diets









supplemented with either urea or soybean meal. Urea increased BUN values (6.7 vs. 4.8

mg/dL). Similar to the studies examining ruminal synchrony of energy and nitrogen,

Chase et al. (1993) fed bulls diets formulated to provide 75 or 150 % of maintenance

energy requirements but equal CP intake. The concentrations of BUN averaged 5.6

mg/dL in bulls fed the highest energy diet and 19.7 mg/dL at the lowest energy intake,

demonstrating that increasing dietary energy intake, while holding protein intake

constant, decreased BUN concentrations. In other studies with beef cattle, BUN

concentrations between 11 and 15 mg/dL were associated with maximum rates of gain

for growing animals (Byers and Moxon, 1980) and concentrations of BUN of 7 to 8

mg/dL were associated with maximum performance of finishing steers (Preston et al.,

1978) or mature cows (Hammond, 1997). The variance in BUN concentration reported in

these studies may be related to the different protein requirements for each animal

category. Optimal concentrations of BUN can be associated with maximized microbial

protein synthesis, leading to increased performances. Concentrations that are less than

these optimal values may indicate a deficiency in dietary protein relative to the energy

intake, while excessive values of BUN may indicate unnecessary dietary protein and/or

lack of either energy availability or synchrony between ruminal energy and nitrogen

supplies, leading to increased ammonia formation and absorption in the rumen.

Nutrition and Reproduction in Beef Cattle

The primary nutrient consideration for optimum reproductive performance in beef

cattle, particularly females, is energy (Mass, 1987). Inadequate energy intake is

correlated to decreased reproductive performance, delayed onset of puberty, extended

postpartum anestrous and decreased conception and pregnancy rates in cattle (Santos,

2004). Therefore, one important goal for beef cattle operations, particularly cow-calf









enterprises in Florida, is the development of nutrition programs that emphasize energy

supplementation to maintain or enhance the reproductive efficiency of the herd.

Onset of Puberty in Beef Heifers

Age at puberty is a major determinant of lifetime reproductive performance of

beef cows, and is defined as the age at which heifers have their first ovulation and

consequential luteal phase (Santos, 2004). Sexual maturity is defined as the age at which

animals reach their maximum reproductive potential and are definitely able to conceive

and gestate. In cattle, sexual maturity is associated with onset of puberty, and heifers

typically attain sexual maturity around three or four normal estrous cycles (around 60 to

90 d) after the onset of puberty (Santos, 2004). One of the most important goals for cow-

calf operations is to develop heifers so that they are able to conceive at 14 to 16 mo of

age and calve at approximately 2 yr of age (Schillo et al., 1992). Heifers should also

experience two or three estrous cycles before the onset of the breeding season in order to

be sexually mature. For that reason, replacement heifers should attain puberty around 12

mo of age to achieve optimal reproductive performance during their first breeding season

(Schillo et al., 1992). Heifers that conceive early in their first breeding season and

therefore calve early in their first calving season wean more and heavier calves during

their productive lives (Lesmeister et al., 1973).

Age at puberty is directly associated with BW, body composition, and breed type.

Furthermore, heifers with greater energy intake and consequent greater BW gain attain

puberty earlier (Schillo et al., 1992). The onset of puberty seems to be associated with

increased frequency and amplitude of LH pulses that stimulates development of ovarian

follicles to the preovulatory stages. These LH pulses are clearly associated with

nutritional status (Schillo, 1992). As explained previously, energy intake increases VFA









production in the rumen, particularly propionate, which increases glucose synthesis and

consequent insulin and IGF-I secretion. These hormones and metabolites appear to

increase gonadotropin secretion (LH and FSH) by influencing hypothalamic-hypophyseal

secretary activity (Butler and Smith, 1989; Schillo et al., 1992), and also appear to

amplify the effects of these hormones in ovarian cells (Spicer and Echternkamp, 1995).

The onset of puberty is a gradual process. During the pre-pubertal period, ovarian

follicles secrete basal quantities of estrogens which inhibit LH secretion. This negative

feedback declines as puberty approaches due to increased pulsatile secretion of LH (Day

et al., 1984). An increased nutritional plane may decrease the sensitivity to the negative

feedback from estradiol and enhance concentrations of LH released (Foster, 1988).

In summary, the exact mechanism by which a higher plane of nutrition anticipates

puberty is not well known, but the increase in the availability of metabolites and

hormones (glucose, insulin, and IGF-I) that may enhance gonadotropin secretion/activity

and decrease the sensitivity of the hypothalamic-hypophyseal tissues to the negative

feedback from estrogens may be potential explanations.

Reproductive Performance in Postpartum Beef Cows

Postpartum interval, characterized as the period from parturition to resumption of

estrus, largely determines the likelihood of cows becoming pregnant during the breeding

season (Wiltbank, 1970). Calves derived from cows that faced long postpartum intervals

tend to be younger and lighter at weaning (Ferrell, 1991). Therefore, decreased

postpartum interval is a major goal to be attained in cow-calf enterprises in order to

increase the efficiency of the operation.

The mechanisms associated with the acquisition and maintenance of reproductive

capability in postpartum cows results from functional integration of the hypothalamic-









hypophyseal-ovarian axis (Hess et al., 2005). During late gestation, elevated production

of steroids by placenta and maternal ovaries, particularly estradiol and progesterone (P4),

exerts intense negative feedback on the hypothalamus, decreasing the release of GnRH

(Short et al., 1990; Lucy, 2003) and therefore depleting hypophyseal reserves of LH

(Wettemann et al., 2003). Despite the relatively fast restoration of LH supply in the

hypophysis after calving, postpartum anestrous is sustained because of the sensitivity to

negative feedback effects from estradiol (Short et al., 1990). Postpartum anestrous can be

even prolonged due to the negative effects of calf-nursing (Williams, 1990) and severe

negative energy balance on the amplitude and frequency of LH secretion (Schillo, 1992).

During the early postpartum period, when the requisites for energy increase primarily due

to lactation, energy intake often does not satisfy energy requirements. Consequently,

decreased blood concentration of glucose, insulin, IGF-I, P4 and also GnRH and LH

pulse frequency are observed (Santos, 2004). The mechanism by which energy affects

GnRH and gonadotropins secretion is yet unknown, but blood concentrations of glucose,

insulin and IGF-I seem to mediate this process. Glucose is the primary metabolic fuel for

the central nervous system, and low glucose concentrations appear to reduce

hypothalamic secretion of GnRH (Wettemann et al., 2003). Due to its impact in glucose

metabolism, insulin may be an important link between nutritional and reproductive

processes. Insulin combined with glucose may stimulate GnRH release from the

hypothalamus (Arias et al., 1992). Insulin mediates follicular dynamics (Webb et al.,

2004), stimulates cell proliferation and steroid synthesis in the ovary (Wettemann and

Bossis, 2000) and also facilitates hepatic production of IGF-I (Keisler and Lucy, 1996;

Webb et al., 2004). Blood concentrations of IGF-I were increased for beef cows that









resumed estrous cycles early postpartum, but not for those that remained in anestrous

(Roberts et al., 1997). Other studies also reported the presence of IGF-I receptors in the

ovaries and dominant follicles of beef cows (Armstrong and Benoit, 1996; Bao and

Garverick, 1998). Therefore, IGF-I may be a positive signal to the hypothalamo-

hypophyseal-ovarian axis to resume normal functionality after parturition.

Feed Intake and Reproductive Performance in Cattle

The endocrine events that lead to resumption of luteal activity in postpartum cows

and the attainment of puberty in heifers are similar (Looper et al., 2003). Elevations in

blood P4 concentrations were detected during the period that immediately preceded

puberty in heifers (Gonzalez-Padilla et al., 1975), and this P4 is originated from ovaries

(Berardinelli et al., 1979), specifically from compact luteal tissues embedded within the

ovary. The same pattern was observed in postpartum beef cows, since first ovulation after

parturition was usually followed by a temporary increase (4 to 5 d of duration) in P4

(Williams and Ray, 1980; Perry et al., 1991). The increase in concentrations of P4

appears to be a necessary factor for the resumption of normal estrous cycles. Looper et al.

(2003) indicated that 81 % of cows that had a rise in P4 before first estrus had normal

length estrous cycles (19 to 23 d) whereas only 36 % of cows that did not have increased

P4 had normal estrous cycles. The rise in P4 before the onset of puberty in heifers and

first estrus in cows may have a role in the endocrinal changes leading to the

establishment of gonadotropins and gonadal hormone secretions, preparing the

reproductive system to attain normal and fertile estrous cycles (Kinder et al., 1987).

Circulating concentration of P4 is also critical for the onset and maintenance of

pregnancy, especially during the early phases (Spencer and Bazer, 2002). Progesterone

prepares the uterine environment for gestation, and also inhibits the release of hormones









that may disrupt pregnancy. In cattle, blood concentration of P4 before or after breeding

has been associated with conception rates. Conception rates were influenced by plasma

concentrations ofP4 before artificial insemination in dairy cows (Folman et al., 1990).

Cows were estrus synchronized by two injections of PG, and cows with P4 concentration

below 5 ng/mL 2 d before second PG administration had conception rates of 36 %,

whereas cows with concentration of P4 above 5 ng/mL had conception rates of 75 %. Xu

et al. (1997) also synchronized estrous in lactating dairy cows with two treatments of PG

13 d apart, but half of the animals were supplemented with exogenous P4 for 5 d before

second injection. The authors reported improved estrous response (89.6 vs. 82.9 %) and

conception rate from first AI (65.1 vs. 59.7 %) for animals that received exogenous P4.

Robinson et al. (1989) supplemented dairy cows with exogenous P4 between d 5 to 12 or

d 10 to 17 after insemination, and detected increased pregnancy rates compared to

untreated cows (60 to 30 %). These findings are also supported by several other studies

(Wiltbank et al., 1956; Folman et al., 1973; Fonseca et al., 1983), validating the

importance of P4 for the onset and maintenance of pregnancy, and subsequent conception

rates.

Several studies with dairy cattle reported that high feed intake may decrease

circulating P4 concentrations, and therefore impair reproductive efficiency. Vasconcelos

et al. (2003) suggested that feeding pattern may alter blood P4 concentration in pregnant

lactating Holstein cows. In this study, feed was withheld 12 h before the experiment

started and then cows were provided with either 100 % of a total mixed ration (TMR) at

once, half of TMR every 12 h, a quarter of TMR every 6 h, or leaving animals unfed for

an additional 12 h (control). After first feeding, blood was sampled hourly for 24 h. The









authors reported that supplying either 100 % or 50 % of TMR decreased circulating P4

concentration by 1 h after feeding and concentrations remained depressed until 8-9 h after

feeding, whereas feeding 25 % of TMR did not reduce concentrations of P4. In a similar

study, Sangsritavong et al. (2002) aspirated follicles and regressed the corpus luteum of

non-lactating Holstein cows in order to synchronize the follicular wave, and then treated

animals with no feed, feed intake at 0.5 of maintenance requirements (M) or at 1.5 M.

The authors determined that blood P4 concentrations were inversely correlated with

feeding regimen at 4 h after feeding (4.14, 3.33 and 2.70 ng/mL of P4 for unfed, 0.5 M

and 1.5 M, respectively). The same authors compared circulating P4 concentrations in

lactating cows either unfed or fed 0.5 M, 1.5 M, or 2.2 M. Again, feed intake decreased

P4 concentrations between 1 h and 4 h after feeding, although no differences were found

between cows fed 1.5 or 2.2 M. Research by Rabiee et al. (2001) compared P4

concentration of non-lactating grazing dairy cows with full or restricted access (2 h/d) to

pasture. Animals were drenched 2x/d with chromic oxide capsules to allow daily feed

intake estimation. Circulating P4 was supplied by exogenous sources and initial blood

concentrations of P4 were similar among animals, since endogenous production was

eliminated prior to the study via GnRH-agonist implants. The average daily DMI of

pasture was greater for cows in the full access group (15.9 vs. 6.3 kg of DM) and their

plasma P4 concentrations were less (1.08 vs. 1.71ng/mL) compared to the restricted

group. In research with beef cows, Pescara et al. (2005) synchronized the ovulation of

364 cows. After AI, cows were fed daily 2 kg or 6 kg of corn (as-fed basis). Blood

concentrations of P4 were greater for animals fed 2 kg of corn (1.7 vs. 1.3 ng/mL) on d 7

after AI, but conception rates did not differ between the treatments. Arthington et al.









(2004) supplemented beef heifers (3x/wk) with either a molasses/cottonseed meal slurry

or range cubes. Heifers supplemented with range cubes readily consumed the entire

amount of feed, whereas molasses-fed heifers had a longer period for total supplement

consumption (48 h). Animals had similar rates of BW gain, but pregnancy rates were

different (76.3 vs. 49.2 % for molasses and range cube-fed heifers, respectively). The

authors attributed this effect to the different supplement intake pattern, associating faster

feed intake with acute decreases in circulating P4 and consequently impaired pregnancy

rates, although blood concentrations of P4 were not determined.

The mechanism by which feed intake decreases blood P4 concentration in cattle is

well defined by Sangsritavong et al. (2002). After meal consumption, liver blood flow

increases in order to transport digested nutrients from the gut through the liver and on to

the rest of the body (Huntington, 1990). Liver is the main site of peripheral steroid

inactivation and catabolism by enzymatic processes, converting biologically active

hormones into inactive forms, which are then eliminated primarily in the urine (Steimer,

2003). Blood flow to the liver is substantially increased when large amounts of feed are

ingested, therefore increasing the rate by which P4 is inactivated and catabolized, causing

acute and chronic reductions in circulating concentrations of P4.

In summary, high planes of nutrition may be detrimental to reproductive

efficiency if not managed correctly. During periods where P4 is particularly fundamental,

either for priming the hypothalamus-hypophysis axis for the onset/re-attainment of

estrous cycles or for the onset/maintenance of pregnancy, feed intake should be carefully

controlled in order to avoid sharp decreases in circulating P4.














CHAPTER 3
EFFECTS OF SUPPLEMENT TYPE ON PERFORMANCE, REPRODUCTIVE AND
PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED FEMALES

Introduction

The primary nutritional consideration for optimum reproductive performance of

beef females is energy (Mass, 1987). With greater energy intake and consequent

improved energy status, heifers attain puberty earlier (Schillo et al., 1992) whereas cows

resume postpartum estrous cycles earlier (Roberts et al., 1997). Alternatively, inadequate

energy intake is correlated highly with delayed onset of puberty, extended postpartum

intervals and decreased conception and pregnancy rates (Santos, 2004). In Florida, where

the beef cattle industry is primarily constituted of cow-calf operations with diets based on

forages lacking in energy availability (Moore et al., 1991), nutritional programs with

emphasis on energy supply are therefore required.

Citrus pulp and molasses are energy by-products originated from orange and

sugarcane industries, respectively, two of the most important agricultural operations in

Florida. Sucrose is the main carbohydrate of molasses, whereas pectin is the main

carbohydrate of citrus pulp. Pectin and sucrose are fermented differently in the rumen,

potentially impacting forage intake, diet digestibility, and energy utilization.

Molasses is classified as a liquid feed, whereas citrus pulp is processed and fed

commonly as dry pellets. Previous research by our group (Arthington et al., 2004)

reported that differences in physical form of the supplement led to variances in intake

behavior and reproductive performance. Beef heifers supplemented with dry feed









consumed the entire amount of supplement faster than heifers supplemented with an

equivalent amount of CP and energy from a liquid supplement. Although no differences

in BW gain were detected, heifers fed the liquid supplement had greater overall

pregnancy rates. Supported by studies that indicated that infrequent and/or acute feed

intake is associated negatively with circulating progesterone (P4) concentration

(Sangsritavong et al., 2002; Vasconcelos, et al., 2003), we hypothesized that heifers

supplemented with dry feed had decreased circulating P4 and consequent impaired

pregnancy rates.

The objective of the present experiments was to investigate the effects of

supplement type on performance and reproductive efficiency of Brahman-crossbred

cattle, including plasma metabolites and hormones.

Material and Methods

Two experiments were conducted at the University of Florida IFAS, Range

Cattle Research and Education Center, Ona. The first experiment was conducted from

September to December 2004 and was divided into a sampling phase (September and

October) and a breeding phase (November and December). The second experiment was

conducted during the months of October and November 2004. The animals utilized in

these experiments were cared for by acceptable practices as outlined in the Guide for the

Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS,

1999).

Animals

Experiment 1. Sixty Brahman x British crossbred heifers (BW + SD = 246 23 kg;

age SD = 10 1 mo) were utilized in this experiment. For the sampling phase (d 0 to d

45), heifers were stratified by initial BW and age, and randomly allocated to 12 pens (5









heifers/pen). Pens were assigned randomly to one of two treatments: 1) molasses-based

supplement (ML) or 2) citrus pulp-based supplement (CT). Pen was considered the

experimental unit (6 pens/treatment) and each pen was a 1.3 ha pasture ofbahiagrass

(Paspalum notatum). For the breeding phase (d 46 to d 107), heifers were re-allocated by

treatment into two bahiagrass pastures and exposed to bulls for a 60-d breeding season.

Experiment 2. Initially, 40 non-lactating and non-pregnant multiparous Brahman x

British crossbred cows had follicular and luteal function synchronized (Figure 3-1) by

treatment with GnRH (100 ug; Cystorelin; Merial Ltd., Duluth, GA) and insertion of an

intra-vaginal P4 releasing device containing 1.38 g ofP4 (Eazi-Breed CIDR; Pfizer

Animal Health, New York, NY) on d -21, followed by treatment with of PGF2a (25 mg;

Lutalyse; Pfizer Animal Health, New York, NY) and CIDR removal on d -14, and a

second treatment with GnRH (100 ug) 48 h after the PGF2a injection (d -12). Transrectal

ultrasonography was performed immediately and 48 h after the second GnRH injection (d

-10). Cows that were detected at the same stage of the follicular wave were then chosen

to be enrolled in the experiment (Figure 3-1). Thirty-four cows were selected and

stratified by age, parity, BW, and BCS (1 = emaciated, 9 = obese; Wagner et al., 1988)

and randomly assigned to one of the two treatments: 1) exogenous source of P4 (CD) or

2) natural source of P4 (NCD). In order to obtain 12 cows at the beginning of the estrous

cycle in each group at the start the experiment (d 0), all cows were again treated with

PGF2a (25 mg) on d -5 (Figure 3-1). At this time, two CIDRs were inserted in the vagina

of the cows from group CD, and remained throughout the entire experiment. Two days

later (d 3), cows from NCD were treated with GnRH (100 ug) (Figure 3-1).









At the beginning of the experiment (d 0), both groups CD and NCD containing

twelve cows each (BW + SD = 502 55 kg; age SD = 5 2 yr) were stratified by BW

and randomly allocated to 12 feedlot pens (2 cows/pen, 6 pens/group). Pens were then

randomly assigned to one of the two treatments in a 2 x 2 factorial arrangement of

treatments: A) molasses-based supplement (ML) or B) citrus pulp-based supplement

(CT). Pen was considered the experimental unit.

Diets

Experiment 1. Pasture quality was estimated to be 56 % TDN and 7.4 % CP (DM

basis) from samples analyzed by a commercial laboratory (Dairy One Forage Laboratory,

Ithaca, NY). A complete mineral mix and water were offered ad libitum throughout the

experiment. Stargrass (Cynodon nlemfuensis) hay was offered when pasture availability

was limited. Hay quality was estimated to be 52 % TDN and 6.5 % CP (DM basis) from

samples also analyzed by commercial laboratory (Dairy One Forage Laboratory).

Treatments consisted of two energy supplements (Table 3-1), fed 3x/wk (Mondays,

Wednesday and Fridays) at a rate of 2.1 and 2.3 kg of DM per heifer daily for ML and

CT, respectively. Supplement intakes were formulated to be iso-caloric and iso-

nitrogenous (Table 3-2) and also balanced for calcium content, given the high

concentration of calcium in citrus pulp.

Experiment 2. Limpograss (Hemarthria altissima) hay was offered ad libitum

throughout the entire experiment, and hay quality was estimated to be 54 % TDN and 9.1

% CP (DM basis) from samples analyzed by commercial laboratory (Dairy One Forage

Laboratory). Cows had free access to a complete mineral mix and water. Treatments

consisted of two energetic supplements (Table 3-3), fed 3x/wk (Mondays, Wednesdays

and Fridays) at a rate of 4.1 and 4.5 kg of DM per cow daily for ML and CT,









respectively. Supplement intakes were formulated to be iso-caloric and iso-nitrogenous

(Table 3-4) and also balanced for calcium content, given the high concentration of

calcium in citrus pulp.

Sampling

Experiment 1. One week before the start (d -7 and d -6) and at the end of the

experiment (d 107 and d 108), heifers were weighed for 2 consecutive days to determine

both full and shrunk (after 16 h of feed restriction) BW. Heifers also were weighed every

other week to monitor average daily gain (ADG) rates, but overall ADG was calculated

using initial and final shrunk BW values. Blood samples were collected weekly (on

Wednesday) throughout the entire experiment to determine onset of puberty using blood

P4 concentrations. Heifers were considered pubertal if P4 value was greater than 1.5

ng/mL for 2 consecutive weeks.

During the sampling phase, in addition to the weekly collections, blood samples

were obtained once a day during four consecutive days, every other week, starting at 4 h

after supplements were offered for determination of glucose, blood urea nitrogen (BUN),

insulin, IGF-I, GH, and P4 concentrations. These samples were collected from d 0 to d 3,

d 14 to d 17, d 28 to d 31 and d 42 to d 45, which were classified as periods (P 1, P 2, P 3

and P 4, respectively) for further statistical analysis. Periods always started on Mondays

and ended on Thursdays.

During the breeding season, heifers were exposed to mature Angus bulls from d

46 to d 107. Each group was exposed to 2 bulls at the same time (1:15 bull to heifer

ratio), and bulls were rotated weekly between groups to account for potential effect of

bull. Heifer pregnancy status was verified by presence of fetus using transrectal









ultrasonography (5.0 MHz transducer, Aloka 500V, Wallingford, CT) 70 d after the end

of the experiment (d 177).

Random samples of feedstuff, pasture, and hay were collected during the trial and

analyzed for nutrient composition at commercial laboratories (Dairy One Forage

Laboratory for cottonseed meal and citrus pulp samples, and SDK Laboratories,

Hutchinson, KS for molasses samples).

Experiment 2. During the first 3 weeks of the experiment (d 1 to d 21), blood

samples were collected immediately prior and 4, 8, 24, 32 and 48 h after the first

supplement feeding of the week (d 1, 8 and 15) for determination of glucose, BUN,

insulin, IGF-I and P4 concentrations. Weeks were classified as periods (week 1 = P 1;

week 2 = P 2; week 3 = P 3) for further statistical analysis.

During the second part of the experiment (d 22 to d 37), daily forage DMI was

recorded. Hay was offered in ad libitum amounts, and refusal was collected and weighed

daily. A sample of the offered hay was collected twice during this period for nutrient

analysis, while samples of refusal were collected daily from each pen to determine DM

content. Hay samples were dried for 96 h at 500 C in forced-air ovens. Random samples

of the feedstuffs also were collected during the trial, and all the samples obtained for

nutrient composition were analyzed at commercial laboratories (Dairy One Forage

Laboratory for cottonseed meal and citrus pulp samples, and SDK Laboratories for

molasses samples).

Blood Analysis

Blood samples were collected via jugular venipuncture during Exp. 1 and from

coccygeal vein or artery during Exp. 2 into commercial blood collection tubes

(Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin,









placed on ice immediately, and centrifuged at 855 g for 30 min for plasma collection.

Plasma was frozen at -200C on the same day of collection.

A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was

used to determine both plasma glucose (Coulombe and Favreau, 1963; modified and

described by Bran + Luebbe Industrial Method #339-01) and BUN (Gochman and

Schmitz, 1972; modified and described by Bran + Luebbe Industrial Method #339-19). A

double antibody radioimmunoassay (RIA) was used to determine plasma concentrations

of insulin (Malven et al., 1987; Badinga et al., 1991), IGF-I (Badinga et al., 1991) and

GH (Badinga et al., 1991). The extraction procedure used in the IGF-I assay was

modified from Badinga et al. (1991) by using a ethanol:acetone:acetate ratio of 6:3:1.

Concentrations of plasma P4 were determined using Coat-A-Count Kit (DPC

Diagnostic Products Inc., Los Angeles, CA) solid phase 125I RIA. The intra- and inter-

assay CV for Exp. 1 were, respectively, 9.66 and 10.05 % for insulin, 8.13 and 5.87 % for

IGF-I, 5.95 and 18.71 % for GH, and 4.48 and 8.15 % for P4. The intra- and inter-assay

CV for Exp. 2 were, respectively, 12.89 and 15.92 % for insulin, 9.90 and 11.00 % for

IGF-I, and 2.41 and 11.19 % for P4.

Statistical Analysis

Experiment 1. Data were analyzed using the PROC MIXED procedure of SAS

(SAS, 2001).

The statistical model used was:

Y = u + TRTi + Pj + PENk(i) + HEIFERl(k) + DAYm() + TRTPij + TRTDAYim,) + Eijklm

where

Y = response variable

S= mean









TRT = fixed effect of treatment

P = fixed effect of period

PEN = random effect of pen within treatment

HEIFER = random effect of heifer within pen

DAY = fixed effect of day within period

TRTP = effect due to interaction of treatment and period

TRTDAY = effect due to interaction of treatment and day within period

E = residual error



Results are reported as least square means. Means were separated using LSD.

Significance was set at P < 0.05, and tendencies were determined ifP > 0.05 and < 0.10.

Only significant interactions are reported.

Experiment 2. Data were analyzed using the PROC MIXED procedure of SAS

(SAS, 2001).

The statistical model used for forage DMI data analysis was:

Y = k + TRTi + PENj(i) + DAYk + TRTDAYik + Ejkl

where

Y = response variable

t = mean

TRT = fixed effect of treatment

PEN = random effect of pen within treatment

DAY = fixed effect of day

TRTDAY = effect due to interaction of treatment and day









E = residual error



The statistical model used for plasma hormones and metabolites data analysis

was:

Y = t + TRTi + Pj + PENk(i) + COW1(k) + TIMEm() + TRTPij + TRTTIMEim,) + Eijklm

where

Y = response variable

t = mean

TRT = fixed effect of treatment

P = fixed effect of period

PEN = random effect of pen within treatment

COW = random effect of cow within pen

TIME = fixed effect of time within period

TRTP = effect due to interaction of treatment and period

TRTTIME = effect due to interaction of treatment and time within period

E = residual error



Results are reported as least square means. Means were separated using LSD.

Significance was set at P < 0.05, and tendencies were classified at P > 0.05 and < 0.10.

Only significant interactions are reported. All plasma metabolites and hormones (but P4)

data were analyzed comparing CT vs. ML without distinction between CD and NCD.

Progesterone data compared CT vs. ML within CD and NCD groups.









Results and Discussion

Experiment 1

Heifers were provided supplement treatments on Mondays, Wednesdays and

Friday at 0700 h throughout the entire experiment. Heifers fed ML required

approximately a 48 h period to completely consume the supplement, whereas heifers fed

CT consumed the whole amount of supplement 24 to 36 h after supplements were

offered. This intake behavior was unexpected, since previous research by our group

(Arthington et al., 2004) reported that heifers in similar conditions of management,

consumed entire amounts of dry supplement within 2 to 3 h after feeding, whereas liquid

supplement also was entirely consumed in approximately 48 h. Therefore, we concluded

that intake behavior did not differ between treatments as expected, and the results

obtained for heifer performance and reproductive efficiency in this experiment should be

primarily attributed to the nutritional differences between CT and ML.

For all plasma metabolites and hormone data, day(period) and period effects were

significant (P < 0.01). The significance in day(period) effect can be explained by the

daily variability in animal behavior and consequent supplement consumption,

considerably affected by the environment and management activities, although

supplement feeding and blood samplings always started at the same time throughout the

entire experiment. Regarding the significance in period effect, it can be attributed to

animal maturation and also to the adaptation of the animals to the experimental

procedures such as feeding and handling management, since it was observed that both

energy and protein status of the animals improved with the advance of the experiment

without any modification in the treatments.









Heifers fed CT had greater ADG compared to heifers fed ML (0.40 vs. 0.30 kg/d,

respectively; P < 0.05, SEM = 0.02; Table 3-5), concurring with previous data reporting

that animals fed molasses-based supplements usually have inferior BW gain compared to

animals fed supplements based on other energy sources, such as corn or soybean hulls

(Pate, 1983; Royes et al., 2001). However, the superiority in performance from CT-fed

heifers was not reflected in reproductive efficiency. There were no treatment effects on

pregnancy rate, puberty rate and number of days required to attain puberty (Table 3-5).

Heifers fed ML had greater overall BUN concentrations (P < 0.05) compared to

heifers fed CT (5.17 vs. 4.17 mg/dL, respectively; SEM = 0.24) (Table 3-6; Figure 3-2).

However, this effect was mainly observed during the first two periods of the experiment.

During periods 3 and 4, the significance was less apparent (P = 0.07 and P = 0.60,

respectively). Therefore, a treatment x period interaction was detected (P < 0.01). A

treatment x day(period) interaction also was detected (P < 0.01) and can be mainly

attributed to the differences in supplement intake behavior and supplement ruminal

degradability, leading to differences in daily BUN concentrations between the treatments.

Monitoring BUN is a technique used for measuring protein status in cattle and is directly

correlated with the amount of ammonia produced and absorbed in the rumen

(Huntington, 1990). Ammonia is a product of dietary protein degradation by the

microbial population in the rumen and is a major precursor for microbial protein

synthesis. Concentrations of BUN should range between 11 and 15 mg/dL for growing

animals to ensure maximum rates of gain (Byers and Moxon, 1980). This goal can be

achieved by feeding animals with correct quantities of protein and energy (Hammond,

1997; Huntington, 1997), and also synchronizing the supply of these nutrients in the









rumen by feeding energy and protein sources with similar rates of ruminal degradability

(Spicer et al., 1986; Matras et al., 1991; Taniguchi et al., 1995). In the current study, both

treatments would be deficient in protein according to the BUN concentrations observed,

since they were consistently below the optimum (Table 3-6). However, supplements were

formulated to supply the protein requirements of developing heifers gaining similar

weight as observed in this experiment, using the NRC model (NRC, 1996). Therefore,

BUN might not be the most accurate indicator of the protein status in our case, since the

BW gain of heifers were in an expected range and the negative effects of protein

deficiency were thus not observed. The differences between treatments may be attributed

to the faster rate of ruminal degradation of sucrose compared to pectin (NRC, 2001),

optimizing synchrony of protein and energy supply in the rumen of heifers fed ML during

the initial periods.

Plasma glucose (83.30 vs. 74.68 mg/dL for CT and ML, respectively; SEM =

2.46), insulin (0.89 vs. 0.75 ng/mL for CT and ML, respectively; SEM = 0.04) and IGF-I

(121.53 vs. 108.91 ng/mL for CT and ML, respectively; SEM = 4.10) concentrations

were greater for heifers fed CT (P < 0.05; Table 3-6; Figure 3-3, Figure 3-4 and Figure 3-

5, respectively). The differences between the treatments increased with the advance of the

experiment for glucose and insulin concentrations; therefore, treatment x period

interactions were detected (P < 0.01). A treatment x day(period) interaction was only

observed for glucose (P < 0.01), and similar to BUN, may also be attributed to the

differences in supplement intake behavior. Several researchers have reported that feeding

diets containing high molasses or sucrose inclusion rates resulted in an increase in butyric

acid concentration in the rumen of cattle (Owen et al., 1967; Kellogg and Owen, 1969;









Hatch and Beeson, 1972), whereas diets rich in citrus pulp, consequently pectin,

produced a greater proportion of acetic acid (Loggins et al., 1964; Hentges et al., 1966;

Drude et al., 1971). Both butyric acid and acetic acid are VFAs that oxidized for energy

production (Bergman, 1990). The epithelial tissue of the rumen retains approximately 90

% of the butyrate produced and converts it to ketone bodies or carbon dioxide, whereas

only 30 % of acetate is metabolized by ruminal epithelium, and the remaining is oxidized

throughout most of the body tissues to generate ATP (Bergman, 1990). In addition,

butyrate also has a detrimental effect on VFA metabolism by restraining hepatic

propionate utilization, thus inhibiting gluconeogenesis and restraining glucose synthesis

and availability (Aiello et al., 1989). Ruminal pH and forage intake and/or digestibility

were not measured in the present experiment. However, several authors reported that

feeding supplements based on non-structural carbohydrates (NSC), such as sucrose,

depressed forage intake (Sanson et al., 1990; Olson, et al., 1999) and digestibility by

decreasing ruminal pH and consequent microbial activity (Mertens and Loften, 1980;

Hiltner and Dehority, 1983; Martin et. al., 2001). Alternatively, when supplements based

on fibrous by-products such as citrus pulp were fed, forage intake and digestibility was

less negatively affected (Bowman and Sanson, 1996; Caton and Dhuyvetter, 1997; Royes

et al., 2001) or even increased (Bowman and Sanson, 2000).

The positive relationship between glucose, insulin, and IGF-I concentrations with

ADG, observed in this experiment, is also supported by others (Vizcarra et al., 1998;

Lapierre et al., 2000; Hersom et al., 2004). Some authors also included analysis for

plasma GH concentration and observed a significant inverse relationship between GH

concentrations and animal performance/energy status (Ellenberger et al., 1989; Bossis et









al., 1999). In these studies, blood was sampled in serial collections (every 20 and 10 min,

respectively) to accurately measure GH concentrations, which is released in a pulsatile

pattern (10 to 15 pulses/d; Hornick et al., 1998; Bossis et al., 1999). This might be an

explanation to why no differences were detected in GH concentrations between CT- and

ML-fed heifers (41.45 vs. 39.27 ng/mL, respectively; P = 0.62, SEM = 3.20) (Table 3-6;

Figure 3-6), given that blood was sampled once a day and we were not able to identify

pulses or basal concentrations of this hormone. Therefore, GH analysis in this experiment

was not accurate enough to be associated directly to the glucose/insulin/IGF-I axis. The

correlation between all plasma metabolites and hormones with ADG also was determined

with Pearson correlation coefficients (Table 3-7). The only significant correlation was

observed for IGF-I (P < 0.01).

In order to verify if plasma P4 concentrations would be influenced by treatments

and consequent intake behavior, only samples from pubertal heifers with plasma P4

greater than 2 ng/mL during the sampling period were used. In view of the fact that these

heifers were at random stages of the estrous cycle and consequently concentrations of P4

varied significantly across heifers, we analyzed the difference of P4 concentration on a

percentage basis, comparing the values from days that supplements were offered to

values from days that supplements were not offered (set as 100 %). Both treatments had a

decrease in P4 concentration during feeding days (P < 0.01; SEM = 2.9); however, no

differences were observed between treatments (89.5 and 90.3 % for CT and ML,

respectively; P = 0.66, SEM = 4.1). Elevated or infrequent feed intake may decrease

circulating concentrations of P4, and therefore impair reproductive efficiency given that

P4 is essential for the onset of puberty (Gonzalez-Padilla et al., 1975) and for the









recognition and maintenance of early pregnancy (Spencer and Bazer, 2002). Vasconcelos

et al. (2003) fed pregnant lactating Holstein cows with either 100 % of the daily diet at

once, half of the diet every 12 h, a quarter of diet every 6 h or left animals unfed. The

authors reported that the first two treatments decreased circulating concentrations of P4

by 1 h after feeding and concentrations remained depressed until 8 to 9 h after feeding.

An inverse relationship between feed intake and blood P4 concentrations also was

reported by others (Rabiee et al., 2001; Sangsritavong et al., 2002; Pescara et al., 2005).

In the current experiment, the differences observed in feed intake behavior (24 to 36 h

and 48 h for total supplement consumption for CT and ML, respectively) were not as

substantial as expected, and might explain why no differences were found in blood P4

concentrations and also reproductive performance between treatments.

Nutritional status, and consequent BW gain and composition, typically is

considered the primary determinant of puberty attainment because it is highly correlated

with frequency and amplitude of LH pulses, which stimulate development of ovarian

follicles to preovulatory stages (Schillo et al., 1992). Furthermore, blood metabolites and

hormones such as glucose, insulin, and IGF-I have been associated with reproductive

performance. These substances seem to be the connection between nutritional status and

the increase in LH secretion and activity, by influencing hypothalamic-hypophyseal

secretary activity (Butler and Smith, 1989; Schillo et al., 1992) and also amplifying the

effects of LH in ovarian cells (Spicer and Echtemkamp, 1995). However, in the present

study, neither BW gain nor the glucose/insulin/IGF-I axis were associated with

reproductive performance. Heifers fed CT had greater (P < 0.05) ADG and blood

concentrations of these metabolites/hormones, but similar reproductive performance









compared to heifers fed ML. When heifers were divided, disregarding treatments, by

puberty attainment and pregnancy status, IGF-I was again the only measurement

significantly associated with reproductive performance (Table 3-8). Concentrations of

IGF-I are correlated positively with insulin concentrations (Table 3-7; Keisler and Lucy,

1996; Webb et al., 2004), that in turn are increased as blood concentrations glucose rises,

and similarly decreased as blood concentrations of glucose declines. Therefore, in order

to increase blood IGF-I concentrations to enhance BW gain and reproductive

performance, glucose synthesis should be maximized. Feeding level is associated

positively with blood glucose concentration (Vizcarra et al., 1998; Lapierre et al., 2000;

Hersom et al., 2004). In addition, since propionate accounts for up to 76 % of the glucose

synthesized in the liver (Reynolds et al., 1994), diets containing ingredients that produce

a greater ratio of propionate in the rumen, such as starches, should be fed.

Experiment 2

The same supplement intake behavior from Exp. 1 was observed in Exp. 2. Cows

were supplemented on Mondays, Wednesdays and Fridays at 0800 h, and cows fed ML

required around 48 h to consume the entire amount of supplement, whereas cows fed CT

required 24 to 36 h.

Overall forage DMI did not differ between treatments (1.16 vs. 1.20 % for CT and

ML, respectively; P = 0.52, SEM = 0.08; Figure 3-7). However, a day effect and also a

treatment x day interaction were detected (P < 0.01). Forage DMI was lesser for both

groups during the days that supplements were fed (0.65 and 1.55 % for CT vs. 0.96 and

1.38 % of BW for ML during feeding and non-feeding days, respectively; P < 0.01, SEM

= 0.09). This effect likely can be attributed to the increased consumption of the

supplements during feeding days compared to non-feeding days, since forage DMI is









negatively associated with energy supplement intake (Caton and Dhuyvetter, 1997;

Kunkle et al., 1999; Bodine and Purvis, 2003). Furthermore, forage DMI for cows fed CT

was less than for cows fed ML during feeding days (0.65 vs. 0.96 % of BW, respectively,

P < 0.05, SEM = 0.090) but not during non-feeding days (P = 0.19). This treatment x day

interaction can be explained by the greater consumption of supplement during feeding

days by CT-fed cows, since these cows consumed the entire amount of supplement

sooner than cows fed ML (24 to 36 vs. 48 h, respectively). As cited previously, a greater

depression in forage intake usually is observed when NSC-based supplements are fed

(Sanson et al., 1990; Olson, et al., 1999). However, these results were obtained

comparing supplements with similar physical form and intake behavior. In the current

study, no differences in overall forage DMI were observed. The differences in

supplement physical form and consequent consumption behavior led to the significant

differences in forage intake pattern between treatments, characterized by the treatment x

day interaction.

No differences between treatments were detected for BUN concentrations (4.46

vs. 4.19 mg/dL for CT and ML, respectively; P = 0.52, SEM = 0.29) (Table 3-9, Figure

3-8). A time(period) effect (P < 0.01) was observed because BUN concentrations usually

increased after supplements were offered, illustrating the improvement in the protein

status of the cows after supplements were consumed. A period effect also was observed

(P < 0.01). Cows had greater BUN concentrations during the second period, intermediary

during the third period, and lesser during the first period (3.45, 5.21 and 4.32 mg/dL,

respectively; SEM = 0.23). A treatment x time(period) interaction was detected (P <

0.01). For the second and third periods, cows fed CT had comparable BUN









concentrations during the first 8 h after supplement feeding, but greater for the following

hours compared to ML-fed cows. Although CT-fed cows had greater supplement

consumption during feeding days and consequently during the first hours after feeding,

citrus pulp undergoes slower ruminal degradation compared to molasses (NRC, 2001).

This effect may have delayed the increase in BUN associated with the synchrony

between ruminal energy and protein supply. A treatment x period interaction also was

observed for BUN (P < 0.01). During the first period, cows fed ML tended to have

greater BUN concentrations (3.02 vs. 3.87 mg/dL for CT and ML, respectively; P = 0.06,

SEM = 0.33), but cows fed CT had greater BUN concentrations during the third period

(4.79 vs. 3.86 mg/dL for CT and ML, respectively; P < 0.05, SEM = 0.33). According to

Hammond (1997), BUN concentrations should range between 7 to 8 mg/dL for mature

beef cows. Therefore, cows on both treatments would be deficient in protein intake

according to their BUN concentrations, since they were often below this optimal range

(Table 3-9). However, both supplements were formulated to supply the protein

requirements of mature cows, using the NRC model (NRC, 1996)

Plasma glucose concentrations did not differ between treatments (84.85 vs. 85.95

mg/dL for CT and ML, respectively; P = 0.88, SEM = 5.12) (Table 3-9, Figure 3-9). A

time(period) effect was observed (P < 0.01) and can be attributed to the fluctuation in

glucose synthesis and availability in blood after supplements were offered and consumed.

No differences were observed in plasma insulin (1.77 vs. 1.51 ng/mL for CT and ML,

respectively; P = 0.33, SEM = 0.19) (Table 3-9, Figure 3-10) and plasma IGF-I

concentrations (110.11 vs. 100.09 ng/mL for CT and ML, respectively; P = 0.49, SEM =

9.88) (Table 3-9, Figure 3-11). A time(period) effect was significant only for insulin (P <









0.01), and can be associated to the same effect observed for glucose, since the

concentration of these substances in blood are mutually dependent. For both hormones, a

period effect (P < 0.01) and a treatment x period interaction (P < 0.05) were detected.

The significance in period effects indicates that the energy status of cows from both

treatments increased with the advance of the experiment, and can be explained by the

adaptation of the cows to the experimental procedures, particularly the frequent blood

sampling, since treatments remained unchanged. The differences between treatments for

insulin and IGF-I, increased with the advance of the experiment, explaining the

significance of the treatment x period interaction. During the second period, CT-fed cows

tended to have greater plasma insulin concentrations compared to ML-fed cows (1.97 vs.

1.50 ng/mL, respectively; P = 0.09, SEM = 0.19). According to the NRC (1996), the

cows utilized in this experiment required around 8.5 Mcal/d of NEM. Given that overall

forage DMI was similar and treatments were iso-caloric, cows from both treatments

consumed approximately 15.0 Mcal/d (175 % of the NEM requirements). Supplements

were fed at the same percentage of animal BW during Exp. 1 and Exp. 2. Because energy

requirements for growing heifers and mature cows are different, the heifers in Exp. 1

were fed with moderate amounts of energy, whereas the cows in Exp. 2 were fed

excessive amounts of supplemental energy. This might be an explanation for why the

results observed during Exp. 1 were not replicated during Exp. 2.

When analyzing initial plasma P4 concentrations, cows from the NCD group were

at various stages of the estrous cycle. However, all cows from the CD group had similar

plasma P4 concentrations, mostly originating from the exogenous source. Therefore, only

data from the CD group were used to compare if cows fed CT had different plasma P4









concentrations compared to cows fed ML. Plasma P4 concentration did not differ

between treatments (4.08 vs. 3.14 ng/mL for CT and ML, respectively; P = 0.12, SEM =

0.34; Figure 3-12). A period effect was observed (P < 0.01) because plasma P4

concentrations decreased linearly for both groups from the first period to the last period,

and this effect can be associated with the decrease in P4 release from the CIDRs. A

time(period) effect also was observed (P < 0.01). Throughout the entire experiment,

plasma P4 concentrations decreased for both groups after the supplements were offered.

This decrease can be associated with: a) supplement intake, which may have increased

the rate at which plasma P4 was metabolized in the liver, b) natural deterioration of the

CIDRs, or c) the sum of both factors. A treatment x period interaction also was detected

(P < 0.01). Cows fed CT had greater plasma P4 concentration during the first period

(5.97 vs. 4.31 ng/mL for CT and ML, respectively; P < 0.05, SEM = 0.31), but not for the

second and third periods (P = 0.20 and P = 0.39, respectively). Concentrations of BUN,

insulin and IGF-I were numerically greater for CT-fed cows compared to ML-fed cows

during the second and third periods, but similar or lesser during the first period. This may

imply that during the first period, cows fed with CT although consumed the entire

amount of supplement faster, possibly had lower nutrient absorption by the digestive

tract, therefore blood flow to the liver and consequent hepatic P4 metabolism were

decreased. In conclusion, differences were not observed in plasma P4 concentrations

between cows fed CT or ML but for first period, even though blood was frequently

collected from cows with similar basal plasma P4 concentrations. Further, the differences

observed in the first period were contrary to our expectations.









Conclusions

The objective of the first experiment was to investigate if heifers fed a molasses-

based supplement would experience greater reproductive performance compared to

heifers fed with supplements based on dry feed (citrus pulp), as previously observed by

our group. To further test our main hypothesis, we conducted a second experiment with

cows, where blood was collected with greater frequency to verify if P4 was being

metabolized at a greater extent in cows supplemented with citrus pulp. However, the

expected supplement intake behavior was not observed in either experiment, and

consequently no major differences between treatments were observed in plasma P4

concentrations of heifers and cows.

In the first experiment, heifers supplemented with CT had greater concentrations

of plasma glucose, insulin and IGF-I, which resulted in greater ADG compared to ML-

supplemented heifers; however, reproductive performance was not impacted by

treatments. In addition, plasma concentration of IGF-I was the only plasma measurement

significantly correlated with BW gain and positively associated with attainment of

puberty and pregnancy.

In the second experiment, no major differences between treatments were found

for plasma metabolites and hormones. We concluded that the cows were provided

excessive supplemental energy, and any potential responses to the treatments were

suppressed. In addition, although no differences where observed for overall forage DMI,

cows supplemented with citrus pulp have a greater daily oscillation in forage intake

compared to cows supplemented with molasses.






53


Table 3-1. Composition of supplements fed to heifers (Exp. 1).
Ingredients (%; as-fed) CT 1 ML 2
Blackstrap molasses 0.00 78.30
Citrus Pulp 74.70 0.00
Cottonseed meal 25.30 20.20
Calcium carbonate 0.00 1.50
SCT = citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).
2 ML = molasses-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).









Table 3-2. Nutrient composition and intake rate of supplements fed to heifers (Exp. 1) .
Nutrient Composition (%; DM) CT ML
TDN 70.00 77.00
CP 19.00 20.07
Calcium 1.70 1.80
Phosphorus 0.60 0.50

Supplement Intake (kg/head/d) 2
DM 2.30 2.10
TDN 1.61 1.61
CP 0.43 0.43
Calcium 0.04 0.04
Phosphorus 0.01 0.01
1 Supplements fed on Mondays, Wednesdays, and Fridays (3x/wk).
2 Estimated from the supplement consumption of the pen (5 heifers/pen).






55


Table 3-3. Composition of supplements fed to cows (Exp. 2).
Ingredients (%; as-fed) CT 1 ML 2
Blackstrap Molasses 0.00 75.40
Citrus Pulp 74.75 0.00
Cottonseed Meal 25.25 23.41
Calcium Carbonate 0.00 1.19
SCT = citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).
2 ML = molasses-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).









Table 3-4. Nutrient composition and intake rate of supplements fed to cows (Exp. 2) 1
Nutrient Composition (%; DM) CT ML
TDN 69.00 76.00
CP 16.60 18.50
Calcium 1.50 1.40
Phosphorus 0.40 0.40

Supplement Intake (kg/head/d) 2
DM 4.53 4.10
TDN 3.13 3.20
CP 0.75 0.75
Calcium 0.06 0.06
Phosphorus 0.02 0.02
1 Supplements fed on Mondays, Wednesdays, and Fridays (3x/wk).
2 Estimated from the supplement consumption of the pen (2 cows/pen).






57


Table 3-5. Effect of treatments on ADG and reproductive performance of heifers in
experiment 1.
Item CT ML SEM P=
ADG, kg/d1 0.40 0.30 0.02 0.008
Pregnancy rate, % 2 60.0 57.5 8.05 0.830
Puberty rate, % 3,4 80.0 76.6 9.94 0.817
Days to puberty 5 72.4 69.3 5.05 0.650
1 Calculated using initial and final shrunk BW.
2 Pregnant heifers / total heifers.
3 Cycling heifers / total heifers.
4 Puberty = plasma progesterone concentration greater than 1.5 ng/mL for two
consecutive weeks.
5 Average days to puberty attainment after the beginning of the study.
















Table 3-6. Effects of treatments on plasma metabolites and hormones of heifers (Exp. 1) fed citrus pulp- (CT) or molasses-based
supplement (IML) 1,2


Period 1
(d) Tu Wed Th


Period 2
Mo Tu Wed Th


Period 3
Mo Tu Wed Th


Period 4
Mo Tu Wed Th


BUN


5.7 10.4
6.4 7.9


Glucose
CT
ML


Insulin 3


91.2 81. 1 83.6
80 6 76 9 81.3




0.83 0.86 0 92
0.72 0.89 0 87


80.3 85.4 83.6 87.7
72 9 77.6 77.1 78.1




0.86 0 96 077 086
0.87 074 074 077


77.4 84.4 88.8 81.S
68.6 73 5 70 5 71 2




0 99 0.97 0.96 0.84
075 0.70 0.70 0.71


76.6 88.9 73.6 82.8
69.4 74.7 71.9 70.6




0 90 0.97 080 0 86
0 68 0.69 064 064


IGF- I


121 8 114 5 120.0
1105 102 5 106.0




50 7 42 6 35.7
53.6 43.6 38.8


118.3 117.6 109.7 117.3
110.3 107.1 102.6 104.9




39 9 43.5 45.8 46.3
32.2 47.4 39.3 42.7


124.2 1279 1132 1188
104.3 114 5 104 5 106 2




34.1 42 7 47 6 42 6
29.5 32.8 41.3 34.8


125.5 140.3 125.5 1307
109.5 123.9 113.6 115 3




34.0 35.7 42.8 35.4
32.8 41.7 35.5 36.4


1 Blood samples not collected on Monday during the first period.

2 Days at which supplements were offered are underlined.
3 BUN and Glucose = mg/dL; Insulin, IGF I, and GH = ng/mL.


SE P=


0 015


0.043


0.049






59


Table 3-7. Correlations between plasma measurements and ADG of heifers (Exp. 1) 1,2.
Item ADG Glucose Insulin IGF-I
Glucose (mg/dL) -0.065
0.62
Insulin (ng/mL) 0.137 -0.140
0.30 0.29
IGF-I (ng/mL) 0.414 0.003 0.329
< 0.01 0.98 < 0.05
GH (ng/mL) 0.102 0.240 0.031 0.128
0.44 0.07 0.81 0.33
1Upper row = correlation coefficients.
2 Lower row = P-values.









Table 3-8. Effects of performance and plasma concentration of metabolites and hormones
on reproductive performance of heifers (Exp. 1).
Puberty Attainment1
Pubertal Non-Pubertal SEM P Value
Number of animals 45 13 -
ADG (kg/d) 0.35 0.33 0.06 0.601
Glucose (mg/dL) 78.10 79.05 2.96 0.777
Insulin (ng/mL) 0.83 0.80 0.05 0.643
IGF-I (ng/mL) 120.11 99.10 4.21 < 0.001

Pregnancy Status 2
Pregnant Open SEM P Value
Number of animals 33 12 -
ADG (kg/d) 0.37 0.31 0.07 0.208
Glucose (mg/dL) 79.70 74.97 2.52 0.120
Insulin (ng/mL) 0.83 0.83 0.06 0.987
IGF-I (ng/mL) 123.35 111.38 4.44 0.054

Puberty = plasma progesterone concentration greater than 1.5 ng/mL for two
consecutive weeks.
2 Pubertal heifers only.
















Table 3-9. Effects of treatments on plasma metabolites and hormones of cows (Exp. 2) fed citrus pulp- (CT) or molasses-based
supplement (ML)1.


Period 1
(h) 0 4 8 24 32 48


Period 2
0 4 8 24


Period 3
32 48 0 4 8 24


32 48 SE P


BUN2


0 520


0.8 1.2 1.8 3.7 4.3 6.1
1.7 2.3 2.6 4.8 5.5 6.1


890 81.5 985 844 80.9 793
938 885 962 86 1 83.5 79 9




0.92 1.19 1 08 1.25 1 37 179
1.04 1.23 1.24 1.11 1.14 1.13


88 1 87.5 884 86.3 78.1 77.2
89 9 80.4 907 88.8 78.6 76.4




1.69 2 35 2 22 2 26 1 80 148
1.34 1.61 1.80 1.41 1.57 1.27


79.0 883 89 6
86.3 850 908




2 34 2.53 2 69
2.14 1.97 2.28


95 1 90 7 89 4 97 8 90.6 95 4
94.9 86.4 89.9 90.7 79.8 90.8


1147 112 1 1084 117 3 110 1 108 1 119.2 115.7 123 9 132.8 125 5 124 9 9.98
97.8 101.3 101.6 104.0 99.5 99.1 103.2 107.4 106.4 112.6 106.9 114.1 9.98


1 Supplements were offered after blood was sampled at 0 h.
2 BUN and Glucose = mg/dL; Insulin, and IGF I = ng/mL.


Glucose 2
CT
ML


Insulin 2
CT
ML


IGF-I


0 339


0483












PGF2a GnRH


GnRH
GnRH PGF2a + TUS TUS


C-- CIDR -


40 cows


Figure 3-1. Outline of the follicular and luteal function synchronization protocol. Transrectal ultrasonography = TUS.


Sampling
Period


34 cows
34 cows


24 cows
24 cows











Blood Urea Nitrogen


N~


z2


MIo Tu We Th
Period 1 A


Mo Tu We Th
Period 2 B


Mo Tu We Th
Period 3 c


Mo Tu We Th (d)
Period 4 c


Blood urea nitrogen (BUN) concentrations of heifers supplemented with citrus pulp- (CT) or molasses-based supplement
(ML). Days at which supplements were offered are underlined. Heifers fed ML had greater mean BUN concentration
(P < 0.05, SEM = 0.24). Period and day(period) effects were significant (P < 0.01). Treatment x period and treatment x
day(period) interactions were also significant (P < 0.01), and periods with different letters differ (P < 0.05).


-Molasses
-Citrus Pulp









1 1 I1 1 1 11 1 1 11 1 1
?0. '. .'. y


Figure 3-2.










Glucose


100 -
0- Molasses
95
Citrus Pulp
90
S85
E 80
Q 75
o 70-
u 65-

60 --
55


Mo Tu We Th Mo Tu We Th Mo Tu We Th Mo Tu We Th (d)
Period 1 A Period 2 B Period 3 c Period 4 c

Figure 3-3. Plasma glucose concentrations of heifers supplemented with citrus pulp- (CT) or molasses-based supplement (ML). Days
at which supplements were offered are underlined. Heifers fed CT had greater mean glucose concentration (P < 0.05,
SEM = 2.46). Period and day(period) effects were significant (P < 0.01). Treatment x period and treatment x day(period)
interactions were also significant (P < 0.01), and periods with different letters differ (P < 0.05).










Insulin


1.10
-Molasses
1.00 Citrus Pulp

0.90 -7 ----

S0.80

Z 0.70

0.60

0.50


Mo Tu We Th
Period 1 A


I I I I I I I I I I I I I


Mo Tu We Th
Period 2 A


Mo Tu We Th
Period 3 B


Mo Tu We Th (d)
Period 4 c


Figure 3-4. Plasma insulin concentrations of heifers supplemented with citrus pulp- (CT) or molasses-based supplement (ML). Days
at which supplements were offered are underlined. Heifers fed CT had greater mean insulin concentration (P < 0.05, SEM
= 0.04). Period and day(period) effects were significant (P < 0.01). Treatment x period interaction was also significant (P
< 0.01), and periods with different letters differ (P < 0.01).


0.40


'6










Insulin-like growth factor I


Mo Tu We Th
Period 1 AB


Mo Tu We Th
Period 2 A


Mo Tu We Th
Period 3 c


Mo Tu We Th
Period 4 c


Figure 3-5. Plasma insulin-like growth factor I (IGF-I) concentrations of heifers supplemented with citrus pulp- (CT) or molasses-
based supplement (ML). Days at which supplements were offered are underlined. Heifers fed CT had greater mean IGF-I
concentration (P = 0.05, SEM = 4.10). Period and day(period) effects were also significant (P < 0.01), and periods with
different letters differ (P < 0.05).


150

140

130

120

110

100

90


Molasses
Citrus Pulp

-------^--VA'


(d)










Growth Hormone


z

0
cJD
s.
a
o


Mo Tu We Th
Period 1 A


Mo Tu We Th
Period 2 A


Mo Tu We Th
Period 3 B


Mo Tu We Th (d)
Period 4 B


Figure 3-6. Plasma growth hormone (GH) concentrations of heifers supplemented with citrus pulp- (CT) or molasses-based
supplement (ML). Days at which supplements were offered are underlined. No significant differences were observed (P
0.62, SEM = 3.20). Period and day(period) effects were significant (P < 0.01). Treatment x period interaction was also
significant (P < 0.05), and periods with different letters differ (P < 0.05).


Molasses
-Citrus Pulp






1 1 1 1 11 1 1 1 11 1 1 1 1











Forage DMI


2.00%
1.80%
1.60%
1.40%
1.20%
1.00%
0.80%
0.60%
0.40%
0.20%
0.00%


22 23


24 25 26 27 28 29 30 31 32 33 34 35 36 37


Days of Study


Figure 3-7. Forage DMI of cows supplemented with citrus pulp- (CT) or molasses-based supplement (ML). Days at which
supplements were offered are underlined. No significant differences were observed (1.16 vs. 1.20 % for CT and ML,
respectively; P = 0.52, SEM = 0.08). Day effect and treatment x day interaction were significant (P < 0.01).


.- -Molasses T
- Citrus Pulp T


T *




S, t
-P










Blood Urea Nitrogen


12
10 --Mo1i sse %
S CititnL, Pulp
~ $8



0 4

2 .


0 4 8 24 32 48 0 4 8 24 32 48 0 4 8 24 32 48 (h)
Period 1 A Period 2 B Period 3 c


Figure 3-8. Blood urea nitrogen (BUN) concentrations of cows supplemented with citrus pulp- (CT) or molasses-based supplement
(ML). Supplements were fed after blood was sampled at 0 h. No significant differences were observed (P = 0.52, SEM =
0.29). Period and time(period) effects were significant (P < 0.01). Treatment x period and treatment x time(period)
interactions were also significant (P < 0.01), and periods with different letters differ (P < 0.01).











Glucose


110-
Molasses
105 Citrus Pulp
100







75
70 ,
0 4 8 24 32 48 0 4 8 24 32 48 0 4 8 24 32 48 (h)
Period 1 Period 2 Period 3


Figure 3-9. Plasma glucose concentrations of cows supplemented with citrus pulp- (CT) or molasses-based supplement (ML).
Supplements were fed after blood was sampled at 0 h. No significant differences were observed (P = 0.88, SEM = 5.12).
Time effect was significant (P < 0.01).











Insulin


Molasses
Citrus Pulp




TT0..



* I I I I


0 4 8 24 32 48

Period 1 A


0 4 8 24 32 48


0 4 8


Period 2 B


24 32 48 (h)


Period 3 c


Figure 3-10. Plasma insulin concentrations of cows supplemented with citrus pulp- (CT) or molasses-based supplement (ML).
Supplements were fed after blood was sampled at 0 h. No significant differences were observed (P = 0.33, SEM = 0.19).
Period and time(period) effects were significant (P < 0.01). Treatment x period interaction was also significant (P <
0.05), and periods with different letters differ (P < 0.01).


3.40

2.90

2.40

1.90

1.40

0.90

0.40











Insulin-like growth factor I


24 32 48


0 4 8


24 32 48


0 4 8


Period 2 B


24 32 48 (h)


Period 3 c


Figure 3-11.


Plasma insulin-like growth factor I (IGF-I) concentrations of cows supplemented with citrus pulp- (CT) or molasses-
based supplement (ML). Supplements were fed after blood was sampled at 0 h. No significant differences were
observed (P = 0.49, SEM = 9.88). Period effect was significant (P < 0.01). Treatment x period interaction was also
significant (P < 0.01), and periods with different letters differ (P < 0.05).


140

130

120

110

100


N-,

0


-Molasses
Citrus Pulp











I I I I I I I I I I I I I I I I I I I I I I I I


0 4 8


Period 1 A











Progesterone


8

7

6

5
gL4


2
1

0


24 32 48

Period 1 A


0 4 8


24 32


0 4 8


Period 2 B


24 32


Period 3 c


Figure 3-12.


Plasma progesterone (P4) concentration of cows with CIDR and supplemented with citrus pulp- (CT) or molasses-
based supplement (ML). No significant differences were observed (P = 0.12, SEM = 0.33). Period and time(period)
effects were significant (P < 0.01). Treatment x period interaction was also significant (P < 0.01), and periods with
different letters differ (P < 0.01).


S- Molasses
Citrus Pulp





* -


0 4 8


48 (h)














CHAPTER 4
EFFECTS OF SUPPLEMENT TYPE AND FEEDING FREQUENCY ON
PERFORMANCE AND PHYSIOLOGICAL RESPONSES OF YEARLING
BRAHMAN-CROSSBRED STEERS

Introduction

Energy supplementation is required in most of Florida cow-calf operations because

the tropical perennial grasses grown in the state usually do not have adequate energy

content to meet the requirements of the cattle (Moore et al., 1991). The frequency at

which supplements are offered depends on the supplement type and also on the

management system of the operation. According to a review compiled by Kunkle et al.

(1999), cattle supplemented daily, 3x/wk, or lx/wk have similar rates of BW gain.

However, the majority of these publications referred to protein or grain-based

supplements, and none evaluated energy supplements based on low-starch by-products,

such as molasses or citrus pulp.

Citrus pulp and molasses originate from orange and sugarcane production,

respectively, which are both important and abundant agricultural enterprises in Florida.

For that reason, these feeds are widely used as energy supplements for beef cattle

operations across the state. Molasses, despite its high DM content (approximately 75 %),

is classified as a liquid feed, whereas citrus pulp is commonly processed and fed as dry

pellets. Differences in physical form between citrus pulp and molasses may lead to

differences in feed intake behavior (Arthington et al. 2004). Molasses and citrus pulp also

differ in their carbohydrate profile. Although both are low-starch energy feedstuffs,

sucrose is the main carbohydrate of molasses, whereas pectin is the main carbohydrate of









citrus pulp. Pectin and sucrose are fermented differently in the rumen, potentially

impacting forage intake, diet digestibility, and energy utilization.

The objective of this experiment was to investigate the effects of supplement type

and feeding frequency on performance, plasma metabolites and hormones, and voluntary

forage intake of yearling steers.

Material and Methods

This study was conducted at the University of Florida IFAS, Range Cattle

Research and Education Center, Ona. The study was conducted during the months of

November and December 2004. The animals utilized in this experiment were cared for by

acceptable practices as outlined in the Guide for the Care and Use of Agricultural

Animals in Agricultural Research and Teaching (FASS, 1999).

Animals

Twenty four Brahman x British crossbred steers (BW + SD = 257 26 kg; age +

SD = 12 1 mo) were utilized in this experiment. Steers were stratified by initial BW and

randomly allocated to 12 pens (2 steers/pen). Pens were then randomly assigned to one of

three treatments: 1) molasses-based supplement fed 3x/wk (ML), 2) citrus pulp-based

supplement fed 3x/wk (CT), or 3) citrus pulp-based supplement fed daily (CD). Pen was

the experimental unit (4 pens/treatment).

Diets

Limpograss (Hemarthria altissima) hay (54 % TDN and 9.1 % CP; DM basis) was

coarsely ground and offered at amounts to ensure ad libitum intake throughout the study.

Steers also had free access to a complete mineral mix and water. Treatments consisted of

three energy supplements (Table 4-1), fed either 3x/wk (Mondays, Wednesdays and

Friday for ML and CT) or every day (CD) at a daily rate of 2.3 kg of DM per heifer for









CT and CD and 2.1 kg of DM per heifer for ML. Supplement intakes were formulated to

be iso-caloric and iso-nitrogenous (Table 4-2) and also balanced for calcium content,

given the high concentration of calcium in citrus pulp.

Sampling

One week before the start and at the end of the study, steers were weighed for 2

consecutive days to determine both full and shrunk BW (after 16 h of feed restriction).

During the first 3 weeks of the study (d 1 to d 21), blood samples were collected

immediately prior and 4, 8, 24, 32 and 48 h after the first supplement feeding of the week

(d 1, 8 and 15) for determination of glucose, blood urea nitrogen (BUN), insulin, IGF-I

and GH concentrations. Weeks were considered periods (week 1, P 1; week 2, P 2; week

3, P 3) for further statistical analysis.

For the second part of the study (d 22 to d 40), forage DMI was recorded daily. Hay

refusal was collected and weighed before hay and supplement feeding. A sample of the

hay was collected twice during this period for nutrient analysis, while samples of the non-

consumed hay were collected daily from each pen to determine DM content. Hay samples

were dried for 96 h at 500 C in forced-air ovens. Random samples of the feedstuffs also

were collected during the trial, and all samples were analyzed for nutrient composition at

commercial laboratories (Dairy One Forage Laboratory, Ithaca, NY for hay, cottonseed

meal and citrus pulp samples, and SDK Laboratories, Hutchinson, KS for molasses

samples).

Blood Analysis

Blood samples were collected via jugular venipuncture into commercial blood

collection tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing









sodium heparin, placed on ice immediately after sampling, and centrifuged at 855 g for

30 min for plasma collection. Plasma was frozen at -200C at the same day of collection.

A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was

used to determine both plasma glucose (Coulombe and Favreau, 1963; modified and

described by Bran + Luebbe Industrial Method #339-01) and BUN (Gochman and

Schmitz, 1972; modified and described by Bran + Luebbe Industrial Method #339-19). A

double antibody radioimmunoassay (RIA) was used to determine plasma concentrations

of insulin (Malven et al., 1987; Badinga et al., 1991), GH (Badinga et al., 1991) and IGF-

I (Badinga et al., 1991). The extraction procedure used in the IGF-I assay was modified

from Badinga et al. (1991) by using a ethanol:acetone:acetate ratio of 6:3:1. The intra-

and inter-assay CVs were, respectively, 5.79 and 28.26 % for insulin, 7.80 and 7.86 % for

IGF-I, and 13.47 and 18.06 % for GH.

Statistical Analysis

Data were analyzed using the PROC MIXED procedure of SAS (SAS, 2001).

The statistical model used for forage DMI data analysis was:

Y = t + TRTi + PENj(i) + DAYk + TRTDAYik + Eijj

where

Y = response variable

t = mean

TRT = fixed effect of treatment

PEN = random effect of pen within treatment

DAY = fixed effect of day

TRTDAY = effect due to interaction of treatment and day

E = residual error












The statistical model used for plasma metabolites and hormones data analysis

was:

Y = L + TRTi + Pj + PENk(i) + STEERI(k) + TIMEm() + TRTPij + TRTTIMEim,) + Eijklm

where

Y = response variable

t = mean

TRT = fixed effect of treatment

P = fixed effect of period

PEN = random effect of pen within treatment

STEER = random effect of steer within pen

TIME = fixed effect of time within period

TRTP = effect due to interaction of treatment and period

TRTTIME = effect due to interaction of treatment and time within period

E = residual error



Results are reported as least square means. Means were separated using PDIFF.

Significance was set at P < 0.05, and tendencies were determined ifP > 0.05 and < 0.15.

Only significant interactions are reported.

Results and Discussion

Molasses is a viscous liquid feedstuff, often mixed with protein ingredients and

fed as slurries. The majority of beef cattle operations offer molasses slurries one to three

times per week; therefore we did not include a treatment with the molasses-based

supplement fed daily. Supplement intake behavior was observed for all treatments. Steers









fed ML consumed the whole amount of supplement by approximately 48 h after feeding.

Steers fed CT consumed their offer by 24 to 36 h, whereas CD-fed steers consumed their

offer within 4 h.

Mean BW gain was greater (P < 0.05) for CD vs. ML, and tended to be greater (P

= 0.13) for CD vs. CT (0.30, 0.18 and 0.10 kg/d for CD, CT and ML, respectively; SEM

= 0.05). Similar to our previous experience with heifers, feeding citrus pulp-based

supplements at the same frequency of molasses-based supplements numerically increased

BW gain. However, differences became significant if supplementation frequency

increased. Our findings concur with previous reports indicating inferior BW gain for

animals fed molasses compared to animals fed other energy sources (Pate, 1983; Royes et

al., 2001). Feeding citrus pulp-based supplements daily tended to increase BW gains

compared to the same supplement fed 3x/wk. To our knowledge, no studies evaluating

supplementation frequency with citrus pulp-based supplements have been reported.

Overall forage DMI did not differ among treatments (1.50, 1.29 and 1.36 % for

ML, CT and CD, respectively; P = 0.16, SEM = 0.07; Figure 4-1), but ML tended to have

greater forage DMI than CT (P = 0.06, SEM = 0.07). From d 31 to the end of the

experiment, the collection of forage leftovers and the consequent forage DMI observation

were debilitated by inclement weather. The differences between ML and CT became

intensified during this period and therefore, the statistical tendency observed may not be

realistic. A significant day effect and also a significant treatment x day interaction were

detected (P < 0.01). Forage DMI was less for CT and ML during the days that these

supplements were fed (0.99 and 1.56 for CT vs. 1.41 and 1.59 % BW for ML during

feeding days and non-feeding days, respectively; P < 0.01, SEM = 0.08). A day effect









was not significant for CD-fed steers (P = 0.35). These findings concur with our previous

experience with mature cows. Supplement consumption was greater for CT- and ML-fed

steers during the days it was offered, and since forage DMI is negatively associated with

energy supplement intake, forage DMI was reduced (Caton and Dhuyvetter, 1997;

Bodine and Purvis, 2003). In addition, forage DMI for steers fed CT was less compared

to steers fed ML during feeding days only (0.99 vs. 1.41 % BW, respectively, P < 0.01,

SEM = 0.08). Greater supplement intake after feeding for CT steers might be the cause

for the decreased forage consumption, since these steers consumed the entire amount of

supplement sooner than steers fed ML (24 to 36 h vs. 48 h, respectively). Feeding energy

supplements daily instead of 3x/wk decreased the oscillation, but not overall forage DMI.

This effect may be explained by a reduction of the substitution effect. During the

experiment, supplement consumption was mainly constant for CD-fed steers, but

oscillated significantly for the steers fed CT. Several studies indicated that forage intake

was decreased when non-structural carbohydrates-based supplements were fed (Sanson et

al., 1990; Olson, et al., 1999). The same response were not realized when fibrous by-

product supplements were fed (Bowman and Sanson, 2000). In the present study,

however, this pattern was not observed. Steers fed ML tended (P = 0.06) to have greater

forage intake than CT-fed steers, however, this result should be interpreted with caution.

Concentration of BUN tended to be greater for CD vs. ML (3.55 vs. 1.97 mg/dL,

respectively; P = 0.06, SEM = 0.53) (Table 4-3, Figure 4-2), but did not differ for CD vs.

CT (2.92 mg/dL for CT; P = 0.42) and CT vs. ML (P = 0.23). A significant time(period)

effect (P < 0.01) was observed. For all treatments, BUN concentrations increased after

feeding. A significant period effect also was detected (P < 0.01). Mean BUN









concentration of all treatments was greater (P < 0.01) during the first period compared to

the second and third periods (4.02, 2.25 and 2.17, respectively; SEM = 0.33).

Concentrations of BUN are directly correlated with the amount of ammonia produced and

absorbed in the rumen (Huntington, 1990). Ammonia is a product of dietary protein

degradation by the microbial population in the rumen and is a major precursor for

microbial protein synthesis. Concentrations of BUN should range between 11 and 15

mg/dL for growing animals to ensure maximum rates of gain (Byers and Moxon, 1980).

This goal can be achieved by feeding animals with correct quantities of protein and

energy (Hammond, 1997; Huntington, 1997), and also synchronizing the supply of these

nutrients in the rumen by feeding energy and protein sources with similar rates of ruminal

degradability (Spicer et al., 1986; Matras et al., 1991; Taniguchi et al., 1995). In the

current study, all treatments would be deficient in protein content according to the BUN

concentrations observed, since they were always below the optimal range (Table 4-3).

However, treatments were formulated to supply the requirements of growing steers, using

the NRC model (NRC, 1996). Feeding citrus pulp-based supplements daily seemed to

improve the protein status of steers when compared to molasses-based supplements fed

3x/wk. This response may be associated with an improved synchrony of protein and

energy availability in the rumen.

Plasma glucose concentrations were decreased for CD vs. CT and ML (66.05,

76.24, and 76.55 mg/dL, respectively; P < 0.05, SEM = 3.02) (Table 4-3, Figure 4-3) but

did not differ for CT vs. ML (P = 0.94). A time(period) effect was detected (P < 0.01)

and can be attributed to the variance in glucose synthesis and availability in blood after

supplements were offered and consumed. A period effect also was detected (P < 0.01).









Mean plasma glucose concentration of all treatments was greater (P < 0.01) during P 1

and P 2 vs. P 3 (73.85, 74.40 and 70.60 mg/dL, respectively; SEM = 1.80). Plasma

insulin concentration was greater for CT vs. CD and ML (0.60, 0.46 and 0.43 ng/mL,

respectively; P < 0.05, SEM = 0.04) (Table 4-3, Figure 4-4) but did not differ between

CD vs. ML (P = 0.63). A significant time(period) effect was observed (P < 0.01) and can

be associated with fluctuations of plasma glucose concentrations, since the concentration

of these substances in blood are mutually dependent. A significant treatment x period

interaction was detected (P < 0.01). From P 1 to P 3, plasma insulin concentration

increased for CT (P < 0.01), was constant for ML, and decreased for CD (P < 0.01).

Plasma IGF-I concentration did not differ among treatments (96.49, 102.71 and 78.65

ng/mL for CD, CT and ML, respectively; P = 0.27, SEM = 10.22) (Table 4-3, Figure 4-

5). A period effect was significant (P < 0.01). Mean IGF-I concentration of all treatments

increased from P 1 to P 3 (78.00, 97.37 and 102.49 ng/mL, respectively; SEM = 5.99).

Additionally, steers fed CD had the greatest increase in plasma IGF-I concentrations,

reflected in the treatment x period interaction (P < 0.01). Plasma GH concentrations did

not differ among treatments (74.52, 74.85 and 76.21 ng/mL for CD, CT and ML,

respectively; P = 0.98, SEM = 8.00) (Table 4-3, Figure 4-6). A period effect was

observed (P < 0.01) since mean plasma GH concentration of all treatments decreased

from the first to the third period (84.15, 76.85 and 64.59 ng/mL, respectively, SEM =

5.07). A significant time(period) effect also was detected (P < 0.01). This was likely due

to a pulsatile pattern of GH release, ranging from 10 to 15 pulses/d (Hornick et al., 1998;

Bossis et al., 1999). Although blood sampling was relatively frequent in this experiment,

it was not frequent enough to identify pulses or basal concentrations of this hormone.









Several studies conducted with beef cattle reported a relationship between the

glucose/insulin/IGF-I/GH axis and animal BW gain. Increased rates of BW gain have

been associated with greater concentrations of blood glucose, insulin, and IGF-I, and

decreased concentrations of GH (Vizcarra et al., 1998; Bossis et al., 1999; Lapierre et al.,

2000). However, in the present study, steers fed CD had greater BW gain than CT- and

ML-fed steers, but lesser concentrations of glucose and insulin (only compared to CT),

and similar IGF-I and GH concentrations. We theorized that CD-fed steers had a more

efficient utilization of energy compared to the other treatments. The lesser plasma

glucose concentration may have been the result of enhanced cell uptake. Consequently,

less insulin was required to activate the receptors and regulate the transport of glucose

into the cells. This effect was mostly noted during the second and third period (Figure 4-

4). However, IGF-I concentrations, which usually are positively correlated with insulin

concentrations (Keisler and Lucy, 1996; Webb et al., 2004), rapidly increased from the P

1 to P 3 for CD-fed steers (Figure 4-5). This indicates that, although glucose and insulin

concentrations were not as great, energy status was improving at a greater rate for CD-fed

steers compared to the other treatments, which may have resulted in the greater BW gain.

Conclusions

We concluded that feeding a citrus pulp-based supplement daily improved steer

performance compared to the same supplement fed 3x/wk or an iso-caloric and iso-

nitrogenous supplement based on liquid molasses also fed 3x/wk. However, these results

were not reflected by the blood hormones and metabolites usually associated with animal

performance, such as glucose, insulin, IGF-I and GH.






84


Table 4-1. Composition of supplements fed to steers.
Ingredients (%; as-fed) ML 1 CT 2 CD 3
Blackstrap molasses 78.30 0.00 0.00
Citrus pulp 0.00 74.70 74.70
Cottonseed meal 20.20 25.30 25.30
Calcium carbonate 1.50 0.00 0.00


1ML
2CT:
3 CD


- citrus pulp-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).
molasses-based supplement fed 3x/wk (Mondays, Wednesdays and Fridays).
citrus pulp-based supplements fed daily.






85


Table 4-2. Nutrient composition and intake rate of supplements fed to steers .
Nutrient Composition (%; DM) ML CT CD
TDN 77.00 70.00 70.00
CP 20.07 19.00 19.00
Calcium 1.80 1.70 1.70
Phosphorus 0.50 0.60 0.60

Supplement Intake (kg/head/d) 2
DM 2.10 2.30 2.30
TDN 1.61 1.61 1.61
CP 0.43 0.43 0.43
Calcium 0.04 0.04 0.04
Phosphorus 0.01 0.01 0.01
2 Estimated from the supplement consumption of the pen (2 steers/pen).
















Table 4-3. Effects of treatments on plasma metabolites and hormones of steer fed 3x/wk a citrus pulp- (CT) or molasses-based
supplements (ML), or fed daily a citrus-pulp based supplement (CD) .


Period 1
(h) 0 4 8 24 32 48


Period 2
0 4 8 24


Period 3
32 48 0 4 8 24


32 48 SE P=


BUN2


0 161


3.6 3.4 4 9 4 1 46 56
1.2 2.6 3.7 2.9 3.7 3.9
3.6 3.9 27 56 49 69



72.7 73.7 76.0 75 2 80 1 81 7
75.9 75.6 72.6 79 5 81 0 82 1
59.1 65.6 70.7 66.9 69.4 70.7



0.41 0.61 0.66 0.50 0.61 0.49
040 047 044 0.38 0.44 0.32
047 055 068 0.46 0.44 0.41



86.6 87.2 84.0 90 2 86 93.2
76.4 76.0 73.1 66.4 63.6 65.2
85.5 82.6 70.5 742 678 74 2



80.9 81.4 75.6 97 6 91 1 744
74.8 1100 70.3 1009 94 4 80 5
75.1 97.3 61.8 72.9 74.8 100.2


2.4 3 6
1.1 2.1
3.7 2 2


0 063


76 8 78.2
72 7 72.2
68.0 63.2



0.74 0.74
0.45 041
0.45 040


1070 106.9 106 6 1117
82.6 81.1 1.S 8 6.7
1055 104.6 95 7 100 8



61.8 55 2 84 7 73.3
63.6 78 8 71 9 92.1
71.0 54.4 70.0 90.5


70 9 74 0 779 82 5
717 74 6 76 5 76 9
60.8 63.5 62.6 68.1



0.70 0.71 0.72 0.64
051 0.57 042 0.44
043 0.45 034 0.44


107.2 101 5
80.5 81.2
104.8 105 9



100.5 70.8
89 4 82.3
83.1 89.4


1105 115.1 1071 1125 123.0
84.0 85.4 76.8 S6.9 87.0
1083 110.3 1064 1103 111.2



64.5 63 7 66 7 68 1 62 7
49.2 48 8 64 8 70 0 776
77.0 63.4 81.4 57.3 59.1


1115
80.4
1177


0.987


SSupplements were offered after blood was sampled at 0 h.
2 BUN and Glucose = mg/dL; Insulin, IGF I, and GH = ng/mL.


Glucose2
CT
ML
CD

Insulin 2
CT
ML
CD

IGF- 12


0 034











Forage DMI


--ML -CT -CD *-P






S* *


*

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Days of Study


Forage DMI of steers supplemented 3x/wk with citrus pulp- (CT) or molasses-based supplement (ML), or daily with
citrus pulp-based supplement (CD). Days at which supplements were offered are underlined. No significant differences
were observed (1.50, 1.29 and 1.36 % for ML, CT and CD, respectively; P = 0.16, SEM = 0.07). Day effect and
treatment x day interaction were significant (P < 0.01).


2.30%
2.10%
1.90%
1.70%
1.50%
1.30%
1.10%
0.90%
0.70%
0.50%
0.30%


Figure 4-1.











Blood Urea Nitrogen


0 4 8 24 32 48

Period 1 A


0 4 8 24 32 48


0 4 8


Period 2 B


Period 3 B


Figure 4-2. Blood urea nitrogen (BUN) concentrations of steers supplemented 3x/wk with citrus pulp- (CT) or molasses-based
supplement (ML), or daily with a citrus pulp-based supplement (CD). Supplements were offered after blood was sampled
at 0 h. Steers fed CD tended to have greater mean BUN concentration than steers fed ML (P = 0.06, SEM = 0.53). Period
and time(period) effects were significant (P < 0.01). Periods with different letters differ (P < 0.01).


-ML -CT -CD





T TL

1 1 1 1 1 1 1 1 11 11 1 1 111


24 32 48 (h)











Glucose


I --ML --CT --CD


0 4 8


24 32 48


0 4 8


24 32


48 (h)


Period 1A


Period 2A


Period 3 B


Figure 4-3. Plasma glucose concentrations of steers supplemented 3x/wk with citrus pulp- (CT) or molasses-based supplement (ML),
or daily with a citrus pulp-based supplement (CD). Supplements were offered after blood was sampled at 0 h. Steers fed
CD had lesser mean glucose concentration than steers fed either with ML and CT (P < 0.05, SEM = 3.02). Period and
time(period) effects were significant (P < 0.01), and periods with different letters differ (P < 0.01).


0 4 8


24 32