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1 EFFECTS OF DRIED DISTILLERS GRAIN AS A SUPPLEMENT TO ROUND BALE SILAGE BASED SUB TROPICAL FORAGE DIETS By ERIN NICOLE ALAVA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLM ENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Erin Nicole Alava
3 To my loving husband for his support, encouragement, and collaboration
4 ACKNOWLEDGMENTS I would like to express my sincer est gratitude to my co advisors Drs. Matt Hersom and Joel Yelich, both offering necessary guidance and support from their own disciplines to allow me to successfully complete my PhD. I would also like to offer my genuine appreciation to the members of my committee, Drs. Gbola Adesogan, Cliff Lamb and Joao Vendramini for their dedication to my program and pushing me to be the best graduate student I can be. A special thank you to all the staff at the Santa Fe Research Unit for all their time, effort, and sw eat to help me complete my research. I also would like to acknowledge the tremendous amount of help I receive d from my lab mates Aline DeLucia Cody Welchons, Megan Thomas, and Regina Esterman. Without them my work would still be unfinished. Also, I woul d like to thank all of the undergraduates who assisted me with the not so fun and very dirty work. Thanks to all of the rest of my fellow graduate students who provided friendship and uplifting support during the four years of completing my degree. Finall y, I would like to thank my parents, Michael and Lynn McKinniss as well as my brothers Matthew and Stephen, for continuing to support me as I attain my goals, of what they refer to as being a professional student. Lastly, to my husband, Eduardo for knowin g the best balance of when to push me, challenge me, and comfort me.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTERS 1 INTRODUCTION ................................ ................................ ................................ .... 13 2 REVIEW OF LITERATURE ................................ ................................ .................... 16 Heifer Development ................................ ................................ ................................ 16 Puberty ................................ ................................ ................................ ................... 17 Factors Affecting Onset of Puberty ................................ ................................ ......... 19 Nutrition ................................ ................................ ................................ ............ 19 Body weight and composition ................................ ................................ ........... 20 Rate and time of growth ................................ ................................ ................... 21 Breed ................................ ................................ ................................ ................ 22 Forages ................................ ................................ ................................ ................... 24 Florida Forages ................................ ................................ ................................ 24 Conserved Forages ................................ ................................ .......................... 25 Supplementation ................................ ................................ ................................ ..... 27 Energy Supplementation ................................ ................................ .................. 27 Carbohydrate Supplementation ................................ ................................ ........ 30 Fat Supplementation ................................ ................................ ........................ 31 Protein Supplementation ................................ ................................ .................. 33 Rumen Degradable Protein ................................ ................................ .............. 34 Rumen Undegradable Protein ................................ ................................ .......... 37 Energy and Protein Supplement Interactions ................................ ................... 39 Dried Distillers Grains ................................ ................................ ....................... 41 Soybean Meal ................................ ................................ ................................ .. 44 Dietary effects on metabolic based hormones and substrates ................................ 45 Rumen Dynamics ................................ ................................ ................................ ... 51 3 EFFECT OF ADDING RUMEN DEGRADABLE PROTEIN TO A DRIED DISTILLERS GRAIN SUPPLEMENT ON GROWTH, BODY COMPOSITION, BLOOD METABOLITES, AND REPRODUCTIVE PERFORMANCE IN YEARLING ANGUS AND BRANGUS HEIFERS. ................................ ................... 56 Introduction ................................ ................................ ................................ ............. 56 Materials and Methods ................................ ................................ ............................ 57
6 Animals ................................ ................................ ................................ ............. 57 Treatments ................................ ................................ ................................ ....... 58 Feed Sampling ................................ ................................ ................................ 59 Sample Collection and Analysis ................................ ................................ ....... 60 Breeding ................................ ................................ ................................ ........... 61 Statistical Analysis ................................ ................................ ............................ 64 Results and Discussion ................................ ................................ ........................... 65 Growth Performance and Body Composition ................................ ................... 65 Blood Metabolites ................................ ................................ ............................. 69 Reproductive Performance ................................ ................................ ............... 72 Implications ................................ ................................ ................................ ............. 76 4 EFFECT OF AMOUNT OF INCLUSION OF DRIED DISTILLERS GRAIN SUPPLEMENT ON ADAPTATION, INTAKE, DIGESTIBILITY AND RUME N PARAMETERS IN STEERS CONSUMING BERMUDAGRASS ROUND BALE SILAGE. ................................ ................................ ................................ .................. 90 Introduction ................................ ................................ ................................ ............. 90 Materials and Methods ................................ ................................ ............................ 91 Animals ................................ ................................ ................................ ............. 91 Adaptation Experiment ................................ ................................ ..................... 92 Treatments ................................ ................................ ................................ 92 Sample collection and analysis ................................ ................................ .. 92 Statistical analysis ................................ ................................ ...................... 94 Digestibility Experiment ................................ ................................ .................... 95 Treatments ................................ ................................ ................................ 95 Sample collection and analysis ................................ ................................ .. 95 Statistical analysis ................................ ................................ ...................... 96 Results and Discussion ................................ ................................ ........................... 97 Adaptation Experiment ................................ ................................ ..................... 97 Digestibility Ex periment ................................ ................................ .................. 102 Implications ................................ ................................ ................................ ........... 108 5 CONCLUSIONS ................................ ................................ ................................ ... 119 APPENDIX A NUTRI TIONAL COMPOSITION OF MINERAL VITAMIN MIX FOR ALL EXPERIMENTS ................................ ................................ ................................ .... 125 B OVERALL REPRODUCTIVE RESPONSES FROM 77 DAY BREEDING SEASON ................................ ................................ ................................ ............... 126 C TEMPERATURES DURING THE DIGESTIBILITY EXPERIMENT ....................... 127 D GLUCOSE ASSAY PROTOCOL ................................ ................................ .......... 128 E NON ESTERIFIED FATTY ACIDS (NEFA) ASSAY PROTOCOL ........................ 130
7 F BLOOD UREA NITROGEN (BUN) ASSAY PROTOCOL ................................ ...... 133 G LIST OF REFERENCES ................................ ................................ ....................... 135 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 161
8 LIST OF TABLES Table page 3 1 Nutritional composition of dried distillers grain (DDG), soybean meal (SBM ), bermudagrass round bale silage (RBS), and pasture offered to yearling Angus and Brangus heifers throughout the experiment. ................................ ..... 77 3 2 Amount of rumen degradable protein (RDP) and rumen undegrad able protein (RUP) provided in supplement diets throughout the supplementation period. .... 78 3 3 Growth characteristics on d 0 and 140 of the experiment for heifers consuming round bale silage su pplemented with dried distillers grain (DDG) or DDG plus soybean meal at two amounts ................................ ...................... 79 3 4 Body ultrasound measurements on d 0 and 140 of the experiment for heifers consumin g round bale silage supplemented with dried distillers grain (DDG) or DDG plus soybean meal at two amounts ................................ ...................... 80 3 5 Body ultrasound measurements on d 0 and 140 of the experimen t for Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) or DDG p lus soy bean meal at two amounts .................... 81 3 6 Estrous response, fir st service conception rate, first service AI pregnancy rate, and final AI pregnancy rate from phase 1 of the breeding season for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amounts. ................................ ................................ .............. 88 3 7 Estrous response conception rate, timed AI pregnancy rate, and synchronized pregnancy rate from phase 2 of the breeding season for yearling Angus an d Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal at two amounts. ................................ ................................ ......................... 89 4 1 Chemical composition of dried distillers gra in (DDG), and bermudagrass round bale silage (RBS) fed to steers during the Adaptation and Digestibility Experiments. ................................ ................................ ................................ ..... 109 4 2 Effect of dried distillers grain (DDG) level on round bale silage (RBS) DMI and total DMI in steers during the Adaptation Experiment (LS means). 1 .......... 110 4 3 Effect of dried distillers grain (DDG) level on ruminal pH collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the Adaptation Experiment. ................................ .............. 111
9 4 4 Effect of dried distillers grain (DDG) level on rumen NH3 N concentrations (mg/dL) collected fo r 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the Adaptation Experiment. ..... 112 4 5 Effect of dried distillers grain (DDG) level on NEFA concentra tions (mEq/ml) collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the Adaptation Experiment. ..... 113 4 6 Effect of dried distillers grain (D DG) level on plasma urea nitrogen (PUN) concentrations (mg/dL) collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the Adaptation Experiment. ................................ ................................ ................................ ...... 114 4 7 Effect of dried distillers grain (DDG) on intake, fecal output, and digestibility in steers consuming bermudagrass round bale silage (RBS) during the Digestibility Experiment. ................................ ................................ ................... 116 4 8 Effects of dried distillers grain (DDG) fed at 4 amounts during the Digestibility Experiment on ruminal pH collected over 15 h in steers consuming bermudagrass round bale silage. ................................ ................................ ..... 117 4 9 Effects of dried distillers grain (DDG) fed at 4 amounts during the Digestibility Experiment on ruminal NH3 N collected over 15 h in steers consuming bermudagrass round bale silage. ................................ ................................ ..... 118 A 1 N utritional composition of mineral vitamin mix for all experiments. .................. 125 B 1 Overall estrous response, conception rate, synchronized pregnancy rate, AI pregnancy rate, and final pregnancy rate f rom phase 1 and 2 of the breeding season for yearling Angus and Brangus heifers consuming round bale silage (RBS) and supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amounts. ................................ .............................. 126
10 LIST OF FIGURES Figure page 3 1 NEFA concentrations by treatment breed and day of experiment for yearling Angus and Brangus heifers consuming round bale silage and supplemented with dried distillers grain (DDG) or DDG plus soybean meal (SBM) a t two amount ................................ ................................ ................................ .............. 82 3 2 Plasma urea nitrogen (PUN) concentrations by breed and day of experiment for yearling Angus and Brangus heife rs consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soyb ean meal (SBM) at two amounts ................................ ................................ .............. 83 3 3 Glucose concentrations by treatment and day of experim ent for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soyb ean meal (SBM) at two amounts. ................................ ................................ ................................ ............. 84 3 4 Survival curve for proportion of non pubertal heifers by treatment across day of experiment for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soyb ean meal (SBM) at two amounts. ................................ ................................ 85 3 5 Survival curve for proportion of non pregnant heifers by breed across day of experiment for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soyb ean meal (SBM) at two amounts. ................................ ................................ 86 3 6 Survival curve for proportion of non pregnant heifers by treatment across day of breeding season for yearling Angus and Brangu s heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soyb ean meal (SBM) at two amounts. ................................ ................................ 87 4 1 Effect of dried distillers grain (DDG) level on mean glucose concentrations in steers consuming bermudagrass round bale silage (RBS) dur ing the Adaptation Experiment. ................................ ................................ .................... 115
11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF DRIED DISTILLERS GRAIN AS A SUPPLEMENT TO ROUND BALE SILAGE BASED SUB TROPICAL FORAGE DIETS By Erin Nicol e Alava August 2012 Chair: Matt Hersom Cochair : Joel Yelich Major: Animal Science s Three experiments were conducted to evaluate the effect of supplementing dried distillers grain (DDG) to cattle fed bermudagrass round bale silage (RBS) on performance, reproduction, intake, digestibility, and rumen pa rameters. In Experiment 1 Angus (n = 30) and Brangus (n = 30) heifers were supplemented with: 1) DDG at 0.75% BW; 2) DDG at 0.75% BW + soybean meal (SBM) a t 7.5% of DDG; or 3) DDG at 0.75% BW + SBM at 15% of DDG. No treatment ( P > 0.05) effects were obse rved for BW, BCS, ADG, or hip height. Treatments were similar ( P > 0.05) for REA, REA/cwt, IMF, RIBFT, and RMPFT Mean NEFA PUN, and glucose concentrations were similar ( P > 0.05) between treatments There were no treatment effects ( P > 0.05) on pubertal status and reproductive responses associated with breeding. In Experiment 2 and 3 Angus (n = 4) and Brangus (n = 4) steers were utilized and received RBS only on d 2 and 1, d 0, 1.13 kg of DDG d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and d 12 4.52 kg until d 15 As DDG intake increased, dry matter intake (DMI) of RBS was not affected ( P > 0.05), total DMI increased ( P ruminal pH decreased ( P Steers consuming 3.39 and 4.52 kg DDG, had
12 increased ( P NH 3 N. Level of DDG affected PUN ( P ( P 0.09) to effect NEFA and glucose concentrations Experiment 3, a Latin square desig n, all steers received RBS and were assigned to: 1 ) RBS only; 2) DDG supplement at 0.33% BW; 3) DDG supplement at 0.66% BW; or, 4) DDG supplement at 1% BW. There was a quadratic ( P ) response to the amount of DDG supplemented on RBS DMI, total DMI i ntake, and total tract apparent DM digestibility and a linear ( P < 0.01) response of fecal output. Mean pH had an inverse linear relationship ( P = 0.02) with amount of DDG and a cubic response ( P < 0.01) for NH 3 N concentrations. In conclusion, addition of SBM had no effect on heifer growth or reproductive performance, and increasing levels of DDG increased total DMI, digestibility, fecal output, and altered rumen parameters.
13 CHAPTER 1 INTRODUCTION Southeastern United States beef cattle production is c lose to 100% dependent upon grass based systems (Burns, 2006). The majority of permanent pastures consist of warm season tropical or sub tropical perennial grasses. Characteristically, these forages have increased annual DM yield, but lower feeding value as compared to temperate forages (Skerman and Riveros, 1990). These grasses are also sensitive to changes in temperature, rainfall, and in some instances day length. In North Florida, during periods of cool weather and shortened day length between Novemb er and April, most grasses are dormant or have hindered productivity. In addition, decreased rainfall typical from April to early June also create s forage quantity shortages (Chambliss et al., 1998; Sollenberger and Chambliss, 1991). In these instances a lternatives to grazing must be sought. Typica l alternative forages resources include hay, silage, or round bale silage (RBS). Round bale silage is a viable option when rain events prohibit proper amount of drying time for traditional haymaking, allowing for capture of optimum nutritive value of the forage. Whether through grazing or utilizing conserved forage, warm season grasses often cannot meet the nutritional requirements of cattle due to ty to consume enough to meet nutritional demands (Moore et al., 1991). Therefore, supplementation strategies must be considered. The primary cost of heifer development is feed cost (Hersom et al., 2010), thus minimizing feed input costs while still ensurin g that heifers meet target weight gains and attain puberty by the start of the breeding season is critical. Development of ethanol plants across the country has provided a unique opportunity to utilize dried distillers
14 grains (DDG) as an alternative feed. Dried distillers grains consist of 27 to 36% CP (24 to 40% RDP), 29 to 39% NDF, 78 to 88% TDN, and 9 to 16% fat (Dairy One, 2012 ) Due to the high protein and energy composition of DDG, it may be an economical supplement for growing heifers. Dried distil lers grain diets commonly provide excess rumen undegradable protein (RUP). There are inconsistent reports about the effect of excess RUP may have on heifer performance published in the literature Lalman et al. (1993) observed increased BW at puberty, an d fewer heifers in estrus during the first 21 d of the breeding season when fed 421 g of RUP/d primarily from blood meal compared to heifers fed 231 g RUP/d. However, Martin et al. (2007) observed no differences in BW, ADG, BCS, age at puberty, or BW at p uberty, but observed increased AI conception and pregnancy rates in heifers fed a DDG diet providing an average 267 g RUP/d compared to a control diet consisting mostly of corn gluten feed which provided an average of 90 g RUP/d. Additionally, DDG diets w hen fed in forage based systems often have a negative RDP balance. Meeting RDP requirements is important as it is metabolized by rumen microbes and converted to ammonia or amino acids, which can be further utilized by rumen microbes to produce microbial p rotein (Butler, 1998). Supplementation of RDP has been associated with improvement in cattle performance through increase of forage digestibility, DMI, and improvement of metabolizable energy (Owens et al., 1991). However, there is limited research evalu ating the relationship of DDG in the diet with breed, stage of production, reproductive function, metabolic parameters, and rumen parameters in cattle consuming sub tropical forages and supplemented with DDG.
15 In summary, beef cattle production in the Sou theast relies on forage based systems, however the forage available may not provide the quantity, quality, or both to support the nutritional needs of growing cattle; therefore, low cost supplementation strategies must be evaluated and developed One possi ble option is utilizing DDG as both an energy and protein supplement. Considerations for meeting RDP demands in forage based diets when feeding DDG as a supplement must also be made. Therefore, the objectives of the research presented in this dissertation were to evaluate possible breed effects and the potential differences in growth, body composition, blood metabolites, and reproductive performance of Angus and Brangus heifers fed RBS supplemented with DDG or DDG in addition to 2 amount s of SBM. A secon d objective was to evaluate the blood metabolites, forage intake, and rumen parameters in steers fed an adaptation diet of bermudagrass RBS supplemented with DDG. And a third objective was to evaluate the effect of amount of DDG supplementation on forage intake, digestibility, and rumen parameters in steers consuming bermudagrass RBS.
16 CHAPTER 2 REVIEW OF LITERATUR E Heifer Development The reproductive system is the last major organ system to mature in mammals (Ramaley, 1979) Therefore, beef producers mu st be aware of factors that influence development of the reproductive system in prepubertal yearling heifers, in order to creat e a management strategy that can economically optimize attainment of puberty. The age at which heifers attain puberty and becom e pregnant can affect the 1973) and is therefore identified as a crucial production trait. Heifers that have their first calf at 2 yr of age produce more calves during t heir lifetime compared to heifers that have their first calf at 3 yr of age or greater (Pope, 1967; Donaldson, 1968, Chapman et al., 1978). In addition, cows that calve late in the calving season tend to calve late or not at all in subsequent years (Burri s and Priode, 1958); consequently, heifers that calve early in their first calving season have a greater lifetime calf production compared to those that calve late (Lemeister et al., 1973). Many production practices in the United States impose restricted b reeding seasons, which demand heifers calve a t 2 y r of age Attainment of puberty before the breeding season is therefore crucial. Byerly et al. (1987) reported that heifers allowed 3 post pubertal estruses before being bred had greater pregnancy rates (7 8%) compared to those bred at their pubertal estrus (58%). This improved pregnancy rate is likely due to maturation of the uterine environment and synchronous communication between the uterus and ovaries (Del Vechhio et al., 1991; Staigmiller et al., 1993 ). Additionally, in pre pubertal heifers, transient elevations of progesterone have been observed prior to the first normal luteal phase (Gonzalez
17 1969) and essentia l for the first estrous cycle which is normal in length. The Lemeister et al. (1973) study also demonstrated that calves born late in the calving season usually weigh less and are worth less economically, which tends to decrease the total productivity of the dam compared to dams that calve early in the calving season. Puberty Puberty can be defined as the first behavioral estrus accompanied by the development of a corpus luteum (CL) that is maintained for a period characteristic of a particular species ( Kinder et al., 1987; Moran et al., 1989). The process of attainment of puberty actually starts before birth and continues throughout pre pubertal development and is the eventual maturation of the endocrine and reproductive systems of the young heifer. Af ter the attainment of puberty, the endocrine and reproductive systems eventually function similar ly to those of an adult. as a classical hypothesis that explains the endocrine me chanisms associated with the onset of puberty in heifers. The theory suggests that the pre pubertal increase in LH secretion is the result of a decline in the negative feedback of estradiol on hypothalamic centers that control LH secretion. Day et al. (1 987) proposed that the reason for the changes in LH secretion could be due to a decline in estradiol receptors in the hypothalamus and pituitary, which results in a decline in the efficacy of estradiol to exert negative feedback effects on LH secretion. P ulses of LH have been observed in heifers as young as one month of age (Schams et al., 1981), but in pre pubertal heifers the LH pulses are infrequent and irregular, once every 4 to 24 h (Day et al., 1987). The decline in estradiol receptors permits an in crease in pulsatile secretion of LH. The peri pubertal
18 increase in mean LH may result from either a decreased ability of estradiol to inhibit GnRH secretion, a decrease in negative feedback of estradiol at the pituitary or both thereby increasing pituita ry responsiveness to GnRH. Circulating estradiol concentrations will eventually reach concentrations sufficient to cause the pre ovulatory surge of LH. Day et al. (1984) observed an increase in LH pulse frequency and an increase in mean LH concentration du ring the 126 d preceding puberty. As a result, approximately 50 d before the onset of puberty LH pulses increase in frequency to approximately 1 pulse every hour in the days prior to puberty (Day et al., 1987). Induction of the pre ovulatory LH surge by estradiol is an essential component for puberty to occur (Kinder et al., 1987). During the peri pubertal period concentrations of metabolic hormones are significantly altered as well. Increases in concentrations of GH, IGF 1, insulin, and leptin have be en observed (Jones et al., 1991; Yelich et al., 1995; Monget and Martin, 1997; Garcia et al., 2002), and have been linked to LH secretion (Schillo et al., 1992; Lin et al., 2000; Garcia et al., 2002). These hormonal changes are probably responsible for th e pubertal growth spurt and possibly for the maturation of the gonadal axis (Monget and Martin, 1997). Modifications in hormone concentrations are sensitive to nutrition, and can impact pubertal status by mediating LH secretion (Steiner et al., 1983). Cir culating concentrations of nonesterified fatty acids (NEFAs) and GH increase (Gill and Hart, 1981; Peters, 1986; Canfield and Butler, 1991) and insulin and IGF 1 decrease (Bassett et al, 1971; Breir et al., 1986; Jones et al., 1991; Rutter et al., 1989) du ring periods of suppressed nutrient intake. Schillo et al. (1992) observed that NEFAs and GH are
19 possibly inhibitory and IGF 1 and insulin are stimulatory to LH release. A nutritionally induced delay in puberty was associated with low serum IGF 1 and hig h serum GH in heifers (Granger et al., 1989). Day et al. (1986) observed heifers on a low energy diet (gain ing 0 .2 kg/d) failed to exhibit an increase in LH pulse frequency compared to heifers fed a growing diet (gain ing 0.9 kg/d), which exhibited increa sed LH pulse frequencies and attained puberty. In contrast, during periods of increased nutrient intake (160% maintenance diet) circulating concentrations of insulin decline (Richards et al., 1989) where as glucose and IGF 1 increase (Granger et al., 1989; Yelich et al., 1996), which subsequently led to increased LH concentrations. Factors Affecting Onset of Puberty There are numerous factors that affect the onset of puberty in beef heifers including age, BW, breed, level of nutrition, rate of post weaning g ain, environment, and interactions of any of the aforementioned factors. Nutrition The age at which heifers begin regular estrous cycles is correlated with BW gains from birth to puberty (Plasse et al., 1968; Arije and Wiltbank, 1971). Nutrition is a key factor that can dictate the onset of puberty in beef heifers (Sorensen et al., 1959; Bellows et al., 1965; Wiltbank et al., 1969). In brief, as yearling heifer nutrition is increased, the age at which heifers attain puberty is decreased. As early as 191 5, Eckles (1915) theorized that heifers fed high energy diets attain puberty at earlier ages and have heavier BW than heifers fed low energy diets. Subsequent research has validated that e nergy intake is positively related to growth rate and inversely rel ated to age of puberty (Wiltbank et al., 1969; Arije and Wiltbank, 1971). Recent research by Gasser et al. (2006), demonstrated that precocious puberty could be induced in early
20 weaned heifers fed a high concentrate diet by increasing LH pulse frequency, which supports previous work by Day et al. (1984, 1986) on the importance of LH pulse frequency on onset of puberty. Restriction of dietary energy intake shifts secretion of LH that precedes puberty (Day et al., 1986, Yelich et al., 1996), which results i n delayed ovarian follicular development (Bergfeld et al., 1994) and ultimately delayed puberty. Body weight and composition Body condition can be used as in indirect indicator of nutritional status as it estimates the amount of fat that an animal contain s (Wagner et al., 1988). Body condition score or changes in body condition are a more reliable guide to evaluate the nutritional status of a cow when compared to live weight due to factors such as gut fill and pregnancy (Herd and Sprott, 1986). Body cond ition score, when taken regularly along with other major factors such as lactation status and forage quality, can be an important tool to a producer when determining the nutritional management of the cow herd. Body condition score is often used as a measu rement to estimate reproductive potential (Randel, 1990). However, the effect of composition of body tissues is not as well understood as a predictor of puberty in beef cattle. B ody fat has been observed as a good indicator of puberty in humans (Frische et al., 1970) as well as in rats (Kennedy and Mitra, 1963). Inconsistency in body fat measurements at attainment of puberty in heifers has been noted (Hall et al, 1995; Yelich et al., 1995). le explains that puberty can be expec ted to occur at a genetically predetermined size among individual animals, and only when heifers reach their predetermined target BW can high pregnancy rates be obtained (Patterson et al., 1992). Furthermore, the target weight principal dictates that heife rs need to be approximately 60 to 65% of their mature BW to attain puberty depending on frame size
21 and breed (Steward et al., 1980; Ferrell, 1982; Sacco et al., 1987; Funston and Deutscher, 2004). In general, there appears to be a positive relationship of mature body size and the age at the onset of puberty where heifers with large mature sizes tend to be older and heavier at the onset of puberty (Martin et al., 1992). Therefore, a itional plan by the start of the breeding season. While this is a good management strategy it should be noted that biologically it is not likely that BW alone dete rmines puberty, but a group of physiologic conditions (BW, gain, management) in tandem are responsible including heifer age (Greer et al., 1983). Rate and time of growth Both rate and timing of post weaning growth has been shown to affect age and weight at puberty. Heifers developed on a slow gain diet with lower energy density reached puberty at significantly older ages and had decreased pregnancy rates compared to heifers developed on high gain diets (Wiltbank et al., 1969; Ferrell, 1982). Yelich et al. (1995) observed that increased rate of gain (1.36 kg/d) resulted in increased BW and BCS and a decreased age at puberty in Angus Hereford heifers compared to heifers fed at lower rate of gains (0.68 kg/d). Pre pubertal heifers maintained on a low gain ( 0.2 kg/d) diet failed to exhibit an increase in LH pulse frequency compared to contemporaries fed a growth diet (0.9 kg/d), which resulted in increased LH pulse frequency and decreased age at puberty (Day et al., 1986). Martin et al. (1992) reported posit ive genetic correlations showing that heifers growing more rapidly from birth to yearling reach puberty at younger ages.
22 There appears to be latitude in the timing of gain in yearling beef heifers relative to when gain occurs during the 90 d directly prec eding the breeding season. This strategy may be sufficient for attainment of puberty and reproductive efficiency as well as a more economic solution to decreasing the cost of supplementing heifers. Clanton et al. (1983) conducted an experiment to determin e if timing and rate of gain effected performance and reproduction of heifers. Forty five days after weaning until breeding heifers were fed 1 of 3 diets: 1) no gain for the first half and 0.91 kg/d the second half, 2) 0.91 kg/d for entire feeding period, or 3) 0.91 kg/d for first half and no gain in the second. They observed no differences in any of the growth measures, age at puberty, conception rate, or subsequent calf production between any of the diets. Monari, (2010) fed heifers to gain either cons tantly 0.84 kg/d for 140 d or 0.28 kg/d for the first 70 d followed by 1.4 kg/d for the final 70 d of the feeding period. No differences were found in percentage of heifers attaining puberty or pregnancy rates between heifers in the two dietary treatments Similarly, Lynch et al. (1997) observed less total feed input costs were spent and no negative effects on reproductive performance when developing heifers to gain 0.11 kg/d for 112 d followed by 0.91 kg/d for 47 d compared to heifers on a constant gain o f 0.45 kg/d. In summary, feeding at a high rate of gain during the weaning to breeding period can be delayed until the latter part of the period without altering heifer growth and reproductive performance. Breed The literature is conclusive that breed can impact attainment of puberty. For example, Charolais and Chianina tend to be older and heavier at puberty compared to Hereford or Angus heifers. However, genetic selection toward different production goals can offset this relationship. For example, bree ds selected for milk production
23 (Holstein, Simmental, Brown Swiss, or Gelbvieh) reach puberty at younger ages compared to those with minimal selection pressure for milk like Charolais or Chianina (Gregory et al., 1991; Martin et al., 1992). Bos indicus b reeds reach puberty at older ages and heavier weights compared to Bos taurus cattle (Warnick et al., 1956; Wiltbank et al., 1966; Plasse et al., 1968; Laster et al., 1978; Gregory et al., 1979; Nelsen et al., 1982; Dow et al., 1982). Gregory et al. (1979) reported that Angus Hereford heifers bred to Brahman sires reached puberty at a mean age of 398 5.8 d, an average of 66 d later than Angus Hereford heifers sired by Angus, Hereford, Sahiwal, Pinzgauer, or Tarentaise sires. The Brahman cross heifers averaged 40 kg more BW at the onset of puberty compared to the Angus Hereford heifers. In addition, Angus Hereford heifers mated to Sahiwal sires, another Bos indicus breed, were older at puberty compared to Angus, Hereford, Pinzgauer and Tarentaise s ired heifers. Nelsen et al. (1982) reported Brahman heifers were 428 d old and weighed 287 kg at the onset of puberty compared to Angus heifers that were 343 d old and weighed 227 kg. Dow et al. (1982) observed that only 17% of Brahman Hereford heifers reached puberty by 15 mo of age compared to 92% of Hereford Red Poll heifers. Heterosis derived from crossbreeding Bos indicus with Bos taurus cattle decreases the age at puberty compared to the age at puberty in the straightbred Bos indicus heifers. Plasse et al. (1968) observed Brahman British heifers achieved puberty at younger ages compared with Brahman heifers. Reynold s et al. (1963) observed the onset of puberty in Angus, Brahman, Brangus, and Angus Brahman heifers over 4 y. Angus heifers a ttained puberty at the earliest age (433 d), followed by Angus Brahman (460 d), Brangus (531 d), and Brahman (816 d).
24 Inclusion of Bos taurus Bos indicus crossbreeding programs in the Southeast can be utilized as a management strategy to maintain prod uction traits from Bos taurus animals (ex. marbling, early age at puberty, etc.) and environmental adaptation traits of Bos indicus animals (ex. parasite and disease resistance, heat tolerance, etc.) while capturing the benefits of heterosis as well. For ages Florida Forages Beef cattle production in the Southeastern United States relies primarily on grass based systems (Burns, 2006). In Florida, most pastures are perennial grasses of tropical or sub tropical origin. Covering approximately 1 million ha, b ahiagrass ( Paspalum notatum ) is the predominant grass species used for grazing in Florida (Chambliss and Sollenberger, 1991). Bahiagrass actively grows in north Florida from April to November. Other popular tropical forages utilized throughout Florida in clude: bermudagrass ( Cyndon dactylon ), stargrass ( Cynodon nlemfuensis ), and limpograss ( Hemarthria altissmia ; Arthington and Brown, 2005). Characteristically, tropical forages have greater annual DM yield, but lower feeding value compared to temperate for ages (Skerman and Riveros, 1990). These grasses often cannot meet the nutrient requirements of growing cattle due to either inadequate nutrient composition of the demands (Mo ore et al., 1991). Warm season grasses are more slowly degraded in the rumen than cool season grasses (Akin, 1989). Nutritive value of tropical grasses is typically highest in the spring but declines rapidly when growth rates increase during the summer m onths (Sollenberger et al., 1988; Williams et al., 1991; Williams, 1994; Williams and Hammond, 1999). Furthermore, Moore et al. (1991) reported the
25 aforementioned grasses may contain 48 to 51% of TDN (DM basis) and 5 to 7% of CP (DM basis) on a yearly bas is. I n contrast values necessary to met requirements for growing heifers ( NRC 2000) are higher, 55% TDN and 8% CP. These grasses are also sensitive to changes in temperature, rainfall, and in some instances day length. In North Florida, warm season gr asses are dormant or have hindered productivity during periods of cool weather and shortened day length between November and April In addition, decreased rainfall typical from April to early June will also create s shortages in forage quantity (Chambliss et al., 1998; Sollenberger and Chambliss, 1991). In these instances alternatives to grazing must be sought either in the form o f conserved forages, supplements or both Conserved Forages While Florida does have an extended growing season for warm season grasses due to the warm climate, forages may have limited quantity or quality during the cool season. Therefore, cattle producers should conserve forages during the late fall, winter, and early spring to have feasible supply of forage during those months Typically this is in the form of hay, silage or round bale silage (RBS). Total production of all hay in Florida ranges from 600,000 to 800,000 tons per year (Chambliss et al., 1998). There are 3 elements that can influence the nutritional value of c onserved forage (in order of magnitude): maturity, grass species (to a lesser extent cultivar), and fertilization rate (Brown and Kalmbacher, 1998). Arthington and Brown (2005) observed an average decrease of 37.8% in CP concentration in bahiagrass, limpo grass, bermudagrass, and stargrass when maturity increased from 4 to 10 wk maturity Furthermore, decrease s in IVOMD for stargrass and bermudagrass w ere observed between the 2 maturities. Mislevy et al. (1989) observed a decline from 18 to 8% in CP
26 and a 68 to 58% drop in TDN when comparing 2 versus 7 wk maturity in stargrass. Moore et al. (1981) evaluated multiple cultivars of bahiagrass, bermudagrass, and stargrass and observed a decrease in the values of TDN as maturity increased in age from 2 to 8 wk maturity as well as numerical differences between the TDN of bahiagrass compared to stargrass and bermudagrass. At 4 wk maturity the bermudagrass (57.3%) and stargrass (59.4%) had greater TDN compared to bahiagrass (55.8%), however as the time progressed TDN values of the 8 wk maturity were greater in bahiagrass (53.1%) than bermudagrass (43.8%) and stargrass (51.0%). The effect of maturity on optimum nutritive value is species specific. Typically, to supplement low winter production of pasture forage, fo rage is harvested during peak periods of production and conserved for later use. However, in the Southeastern United States the peak pasture production occurs from the middle of June through the end of August, which also coincides with the lowest probabil ity of encountering a 3 d dry period with conditions suitable for making hay (Bates et al., 1989). Frequent rain events can delay harvest and/or cause field losses of forages (Hersom and Kunkle, 2003). Moore et al. (1979) observed a rapid decline in fora ge quality in most tropical forages due to rain delayed harvest. Therefore, an alternative storage method to traditional haymaking is needed to make substantial differences in the end product by timely harvesting the grass. One such method is through sto rage as RBS. Round bale silage can be made with both grasses and legumes and can be baled with greate r moisture con centration allowed to ferment in the absence of oxygen to preserve the forage. This process wa s developed in Europe, which utilizes the same harvesting equipment as traditional hay,
27 and Kunkel, 2003). Hersom et al. (2011) conducted a demonstration comparing two forage management systems of hay harvest only (harvested as growing and weather conditions allowed) or a complimentary RBS harvest system (harvested on a 4 wk cutting schedule) The authors observed that the inclusion of the option to make RBS allowed mo re cutttings (5 vs. 3), increased number of bales harvested (479 vs. 259), and increased the mean bale CP (12.9 vs. 10.1%) and TDN content of the bales (57.1 vs. 53.8%). Additionally, when comparing the final cost economics, RBS production was cheaper on an as fed basis and the DM forage cost in dollars per ton and the cost of DM, TDN, and CP in dollars per pound were less expensive compared to the traditionally baled hay. While the technique of making RBS does not improve the forage quality, this process allows capturing of the high nutritive value by harvesting at the appropriate time. This provides an opportunity in Florida to utilize RBS as a medium to high quality cost competitive forage for heifer development. Supplementation Supplementation strate gies for forage based diets is important for correcting nutrient deficiencies, improving forage utilization and animal performance, increasing economic return, and managing animal behavior (Kunkle et al., 1999). Energy Supplementation Nutrient requirement s of cattle for energy vary based on body type, breed, genotype, sex, age, season, temperature, physiological state, and previous nutrition (NRC, 2000) Warm season forages typically do not contain enough energy to meet nutrient requirements of developing heifers; therefore energy is usually the limiting factor to achieving adequate ADG and target BW (Rice, 1991) and must be
28 supplemented. A balance must be struck when determining how much energy to supplement. Providing inadequate energy may restrict the reproductive development and future performance of heifers. For example, increased age at puberty, reduced conception rates, and inadequate udder development has been observed when energy intake was limited (Sorenson et al., 1959; Wiltbank et al., 1966; Short and Bellows, 1971). In addition, Gombe and Hansel (1973) fed heifers isonitrogenous diets with different levels of TDN and observed increased plasma LH concentrations but decreased progesterone concentrations and smaller CL in heifers fed low TDN. Heifers maintained on a low energy diet exhibited a decreased LH pulse frequency compared to heifers fed an adequate energy that exhibited increased LH pulse and attained puberty (Day et al., 1986). Harrison and Randel (1986) observed heifers fed a diet c ontaining 75% of the NRC requirements for maintenance had decreased BW and BCS, in addition to alterations in ovarian response, and lower progesterone content of the CL compared to heifers receiving a diet containing 180% of NRC requirements for maintenanc e Providing excessive energy can also have deleterious effects such as a weak display of estrus, reduced conception rates, high rates of embryonic mortality, and disruption of mammary gland development (Sorenson et al., 1959; Wiltbank et al., 1966; Short and Bellows, 1971). In addition, high feeding levels reduced mammary growth during the pre pubertal period (Sejrsen et al., 1982; Mntysaari et al., 1995; Sejrsen et al., 1998). Capuco et al. (1995) observed the negative effect of high feeding level on m ammary growth was more severe in a high energy diets (corn based) compared to an alfalfa based diet.
29 Energy supplementation, when balanced with other nutrients typically will increase performance of cattle fed forages. However, a substitution of suppleme ntal energy may occur due to decreases in forage intake and utilization. In a review of 66 publications in nonlactating cattle consuming forgage, it was concluded that energy based supplements decreased volunta ry forage intake when supplemental TDN intake was greater than 0.7% BW, the TDN:CP was less than 7, or when voluntary forage intake was greater than 1.75% of BW (Moore et al., 1999). Horn and McCollum (1987) summarized that in grazing ruminants, concentrat es can be fed up to 0.5% of BW without causin g large decreases in forage intake. Likewise, supplementing grain based supplements above 0.25% of BW resulted in negative effects on forage utilization (Bowman and Sanson, 1996). Bodine and Purvis, (2003) fed steers consuming low quality forage one of 4 diets: 1) 7.5 g of dry rolled corn DM/(kg of BW d) and added an adequate amount of soybean meal to balance total diet RDP requirements (CRSBM), 2) 7.5 g of dry rolled corn DM/(kg of BW d ), and added soybean hulls to achieve an equal amount of supplemental TDN, g/(kg of BW d ) as CRSBM (CORN), 3) soybean meal to supply an equal amount of supplemental RDP g/(kg of BW d ) to CRSBM (SBM), or 4) a cottonseed hull based control supplement (CONT) to evaluate the effects of balancing total diet RDP with dietary TDN. They observed the greatest ADG in steers consuming a diet balanced for RDP and TDN as well as greater total diet digestibility and digestible OM intake compared to steers consuming diets that were equal in TDN or RDP to the balanced diet. They concluded f eeding large amounts of supplement that balances both energy and RDP will allow animals to achieve greater rates of gain while grazing low quality forages. In agreement, Bodine et al. (2001) concluded providing an adequate
30 RDP:TDN balance can decrease the negative associative effects observed when high starch supplements are fed to animals consuming low quality forage. In summary, total dieta ry RDP requirements need to be considered when evaluating supplementation programs when animals are consuming low or medium quality forages, and a balance between RDP and energy must be achieved for animals to maximize performance. Carbohydrate Supplementation The main source of energy supplementation for ruminants is carbohydrates, which are divided into structural c arbohydrates (SC) and non structural carbohydrates (NSC). Structural carbohydrates are made primarily of cellulose and hemicellulose. Examples of sources of SC include soybean hulls, wheat middlings, beet pulp, and alfalfa hay. The structural components a re associated with slow digestion and are accounted for as neutral detergent fiber (NDF) and acid detergent fiber (ADF) in fiber analysis. Examples of sources NSC include corn, barley and sorghum grains as well as molasses based liquids or blocks that are high in starches and sugars (Bowman and Sanson, 1996). The NSC are found in the leaves and seeds of plants are readily degraded in the rumen. The type of carbohydrate supplement fed has a major impact on the rate and extent of forage digestion (Bowman and Sanson, 1996), as well as quality of the base forage (Garcs Ypez et al., 1997; Mount et al., 2009). Sources of highly degraded fiber generally do not reduce forage intake and in fact when fed with low quality forages, forage intake and utilization ma y increase. Garcs Ypez et al. (1997) evaluated 3 different energy concentrates (corn SBM, wheat middlings, and soybean hulls) offered at 2 levels (high and low) to growing steers and sheep consuming chopped bermudagrass hay. They observed supplements co ntaining highly digestible fiber
31 (soybean hulls) produced less negative associative effects than high starch supplements (corn SBM) when fed with bermudagrass at the high level (0.8 to 1% of BW), but no differences were observed when fed at the low level ( 0.4 to 0.5% of BW) Mount et al. (2009) evaluated a fibrous (beet pulp) and non fibrous (corn) supplement in heifers grazing high quality small grain pasture and observed no differences in ADG for either supplement. Additionally, NSC based supplements m ay depress intake and digestibility ( Tamminga, 1993 ; Caton and Dhuyvetter, 1997). Chase and Hibberd (1987) observed a linear decrease in digestibility of hemicellulose and cellulose as corn increased in the diet. The level of inclusion in the diet can al so have an impact on animal response. For example, limited quantities of NSC may stimulate fiber digestion mediated by increases in microbial activity and attachment to digesta (Demeyer, 1981; Hiltner and Dehority, 1983), however, as mentioned previously, large amounts of NSC usually will depress forage digestion and intake. Fat Supplementation Fat supplementation has a place in ruminant diets to increase the energy density of the diet. Energy values reported in the NRC ( 2000 ) are at least 2 times greate r for lipid feedstuffs compared to cereal grains Energy requirements can often be met with supplementation of a high fat feedstuff such as whole oilseeds (soybeans, cottonseed, and canola seed), tallow, and vegetable fat. As a guideline, no more than 20 % of dietary ME should be introduced into the diet from fat due to deleterious effects (Palmquist, 1994). These effects include reduction of DM digestibility, decreased DMI, and depression of overall performance when included at levels greater than 5% of t he total rations (Brooks et al., 1954; Erwin
32 et al., 1956; Davison and Woods, 1960; Esplin et al., 1963). If fat is added to the diet at a rate of 5% or greater of total DMI, fiber digestion can be decreased (Kowalczyk et al., 1977; Doreau and Chilliard, 1 997; Williams and Stanko, 2000). This effect is due to inhibition of the celluloytic bacteria (Kowalczyk et al., 1977) and protozoa (Doreau and Chilliard, 1997). Hess et al. (2007) after conducting a review of over 10 yr of research results, concluded th at an optimal inclusion rate of supplemental fat is less than 3% DM to maximize intake of forage based diets, should be limited to 2% or less of DMI to prevent substitution effects of forage with supplemental fat, and should not exceed 4% of DMI if goal is to increase dietary DE with fat supplementation. Composition of fatty acids is the primary property that influences whether dietary fats affect ruminal fermentation. Most of the naturally occurring plant oils contain a large profile of unsaturated fatty acids. Whole soybeans and whole cottonseed have a fatty acid profile of 85% and 71% of unsaturated fatty acids and 15% and 29% of saturated fatty acids, respectively. Unsaturated fats contribute greater inhibitory effects on ruminal fiber digestion compa red to saturated fats (Harfoot and Hazlewood, 1988; Eastridge and Firkins, 1991; Pantoja et al., 1994) and can depress DMI (Pantoja et al., 1994). As saturation increases, fats become more inert in the rumen, and their absorption in the small intestine is also decreased (Eastridge and Firkins, 1991, Pantoja et al., 1994) Additionally, an increase of unsaturated fatty acids in the rumen decreases the acetate to propionate ratio (Jenkins, 1997; Jenkins et al., 2000; Jenkins and Adams, 2002), which can also be accompanied by reduced OM digestion (Doreau and Chilliard, 1997).
33 Feeding increased levels of dietary fat has been associated with positive effects on reproduction, including larger dominant follicles (Lammoglia, 1996; Mattos et al., 2000), increased t otal number of follicles (Lucy et al. 1991a,b; Wehrman et al ., 1991; Thomas and Williams, 1996) and enhanced steroidogenic capacity of the CL (Williams, 1989). Whitney et al. (2000) observed heifers fed a 3% soybean oil diet conceived approximately 11 d sooner compared to heifers fed diets with 0 or 6% soybean oil. Hess et al. (2002) summarized nine experiments o n dietary fat supplementation and noted that fat supplementation increased overall pregnancy rates from 63.8% in non fat supplemented heifers to 73.6% in heifers supplemented with fat. Protein Supplementation Generally speaking, when low quality forages are not limited in quantity, protein is the most beneficial nutrient, responses to which are usually observed when the CP content of the forage i s less than 6 to 8% (Campling, 1970; Kartchner, 1981). Young growing animals as well as animals in a high production state are most likely to respond with increased intake and gain when provided with protein supplementation and will typically respond when consuming higher quality basal diets (DelCurto et al., 1999). Protein supplementation has been shown to stimulate forage intake, digestion, and animal performance (Guthrie and Wagner, 1988; Del Curto et al., 1990; Kster et al., 1996; Bandyk et al., 2001 ) as well as reproductive efficiency in cows (Clanton, 1982; Wallace, 1987) and even increased weaning weights in calves (Clanton, 1982; Lee et al., 1985). Patterson et al. (1992), concluded that diets high in protein support faster growth resulting in ea rlier onset of puberty, and greater pregnancy rates compared to restricted protein diets.
34 Dietary protein is composed of two types, including rumen degradable protein and rumen undegradable protein (RUP). Rumen degradable protein is broken down by rumen m icrobes and converted to ammonia or amino acids, which can be further utilized by rumen microbes to produce microbial protein (Butler, 1998). Rumen undegradable protein is not metabolized by rumen microbes and is absorbed by the small intestine. In order to function properly, ruminants must consume both RDP and RUP to satisfactorily meet requirements set forth by the demands of each life stage. Rumen Degradable Protein Rumen degradable protein is typically the portion of protein needed for improving low qu ality forage intake and its digestion by cattle (Heldt, 1998; Olson, 1998). Supplementation of low quality forages with an RDP source can improve forage utilization and OM digestion (Bandyk et al., 2001) and improve metabolizable energy use (Owens et al., 1991), which results in improved performance of cattle. Providing RDP to ruminants fed low quality forage increases forage intake and flow of nutrients to the small intestine (Hannah et al., 1991; Lintzenich et al., 1995). Kster et al. (1996) infused s upplemental RDP (sodium caseinate) at levels of 0, 180, 360, 540, and 720 g/d. The authors observed a quadratic increase in OM intake, with increasing RDP supplementation, with the peak at 540 g/d. True ruminal OM and NDF digestion increased up to 180 g/ d RDP, with a cubic response at the higher levels. An increase in total ruminal VFA and ammonia concentrations was also observed as RDP increased. The authors concluded that responses to RDP were maximized when the digestible OM contains 11% RDP. Mathis et al. (2000) reported some variability in response to supplementation of RDP (sodium caseinate) in low and medium quality forage diets. When bermudagrass (8.2% CP) was supplemented with 0, 0.041, 0.082, or 0.124% of
35 BW, RDP had no effect on forage OM i ntake or total OM intake. Total OM digestion tended to respond cubically, with the 0.082% having the great est digestibility value. However, when they used the same RDP supplement levels in bromegrass (5.9% CP) and forage sorghum (4.3% CP), total OM intak e and total OM digestion increased with increasing amounts of supplemental RDP. The differing responses in intake and digestion are likely due to differences in forage characteristics. The main characteristic that may have contributed to variability in r esponses is differences in CP content and potential for some of the RUP to contribute to ruminally available nitrogen via urea recycling. Van Soest (1994) indicated N recycling as the cause for minimal improvement in intake and digestion in cattle in respo nse to CP supplementation when the basal forage has > 7 % CP Diets containing excess amounts of RDP can also be undesirable due to excess N, which is excreted resulting in a net N loss (Poos et al., 1979). When protein intake exceeds requirements, or whe n the protein is highly degradable, large amounts of ammonia are produced, which will be detoxified into urea in the liver. This process requires energy could potentially alter the energy balance in animals (Oldham, 1984) and could subsequently have a n egative impact on animal performance. Excess RDP in the diet can also have a negative impact on fertility. Elrod and Butler (1993) fed Holstein heifers to either meet RDP requirements (73% of CP; normal) or to exceed RDP requirements (82.3% of CP; high) to determine effects of RDP on fertility measures. Heifers that received the high RDP had decreased uterine pH values on d 7 of the estrous cycle compared to the normal RDP heifers. In addition, first service conception rates were decreased in the high RD P heifers (61%) compared to the
36 normal RDP heifers (82%). Concentrations of plasma urea nitrogen (PUN) were elevated in the high RDP heifers compared to the normal RDP heifers (14.8 vs. 10.2 mg/dL respectively). Alterations in PUN will be discussed at gr eater lengths further in the review. They speculated that fertilization rate was similar between treatments, but due to prolonged luteal length in 7 of the heifers fed high protein diets it is likely that embryonic death occurred sometime after d 20 in th ese heifers. The authors concluded that the excess RDP could have deleterious effects on fertility mediated through changes in the uterine environment, but assumptions about the role these alterations play in tissue urea, glutamine, NH 4 concentrations emb ryo survival, or sperm integrity could not be determined from this study. This concurs with the findings of Jordan and Swanson (1979) and Jordan et al. (1983) who reported that excess dietary protein altered the ionic composition of uterine fluid during t he luteal phase resulting in decreased fertility, although, no uterine changes were observed during the follicular phase of the estrous cycle. Canfield et al. (1990) established that in lactating cows, conception rate to first service was decreased in cow s on a high protein diet that exceeded RDP requirements compared to cows fed a diet that met requirements. rates it does appear to reduce plasma progesterone concentrations which is possibly linked to effects of negative energy balance created by increased RDP values. Blanchard et al. (1990) fed lactating cows a diet containing either 64 or 73% RDP (16% CP). As RDP increased from 64 to 73%, there were decreases in fertilized ova recovered (54.8 vs. 79.2%, respectively) as well as percentage of transferable ova (44.2 vs. 66.9%, respectively). They concluded that excess RDP could contribute to fertilization failure or
37 early degeneration of embryos. However, it should be noted, a similar study utilizing non lactating cows failed to produce differences in embryo quality or number of embryos produced (Garcia Bojalil et al. 1994). The authors suggested that excess RDP in addition to a negative energy balance (e.g. during lactation) m ay exacerbate factors a ffecting nutrition, metabolism, or energy balance resulting in lower reproductive performance and may explain differences in embryo number and quality observed therein. In low quality forage diets, considerations for meeting RDP req uirements must be managed to maximize animal performance, but RDP should not be supplemented in excess. Rumen Undegradable Protein Rumen undegradable protein in the ruminant diet s is typically from by products such as fishmeal, meat and bone meal, feather meal, blood meal, corn gluten meal (CGM), dried distillers grains (DDG), and brewers dried grains (Santos et al., 1998 ). Increasing the quantity of RUP (288 g RUP) consumed by heifers fed a low quality roughage diet improved their ability to utilize dieta ry energy for BW gain, shown by requiring less TDN to gain 0.5 kg/d than did heifers fed 38 g RUP (Lalman et al., 1993). Furthermore, MacDonald et al. (2007) observed differences in ADG between heifers fed equal amounts of RUP, but from different sources. The ADG from heifers consuming DDG, was greater compared to heifers consuming CGM. Response to RUP supplementation in cattle consuming actively growing forages is due to the protein in the forage being highly degraded in the rumen, causing a metabolizab le protein (MP) deficiency (Klopfenstein, 1996: Creighton et al., 2003). Feeding RUP in excess of requirements also appears to alter metabolic and reproductive hormones as well as ovarian and pituitary function, which ultimately can
38 impact reproductive suc cess in cattle. Kane et al. (2004) supplemented post pubertal heifers with RUP (115, 216, or 321 g/d) from either feather meal or fishmeal sources and observed decreased basal serum FSH, reduced FSH area under the curve and increased IGFBP 2 and 4 in th e follicle during d 12 and 14 of the estrous cycle in heifers supplemented at 321 g/d compared to 115 g/d. Therefore, uptake of high levels of RUP may likely impair gonadotropin secretion and follicle development. Additionally, excess RUP also appears to alter steroid metabolism due to reduced cholesterol concentrations in cattle supplemented with RUP (Wiley et al., 1991; Lalman et al., 1993). This is important since cholesterol is a precursor of estrogen and progesterone, which are major reproductive ste roids. Lalman et al. (1993) hypothesize d that low stimulation of the LH release required for follicle development and ovulation required for the heifer to attain puberty, which parallels with the observation that RUP supplemented heifers reached puberty at a heavier BW and later age. In contrast, Martin et al. (2007) fed heifers 267 g/d of RUP and observed no differences in attainment of puberty between the control diet (9 0 g/d RUP) and the treated group. In the postpartum cow, it also appears that additional RUP may have positive effects on reproduction. Additional protein suppl emented as RUP not in excess of requirements has been shown to decrease the duration of postpa rtum anestrus and BW loss (Wiley et al., 1991; Appeddu et al., 1996; Anderson et al., 2001) and improve conception rates (McCormick et al., 1999). Triplett et al. (1995) fed an isonitrogenous diet with three levels of RUP (low = 38.1, medium = 56.3, and h igh = 75.6% of CP) to Brahman cows and observed decreased first service conception rates in the low RUP
39 group (29.2%) compared to the medium (57.6%) and high (54.6%) RUP groups. Overall pregnancy rates tended to be greater in the high (56.4%) and medium ( 61.5%) RUP groups as well. In summary, addition of RUP not in excess of requirements in grazing cattle can improve animal performance and reproduction, however feeding RUP in excess can be detrimental to metabolic and reproductive hormones, which can nega tively impact reproduction. Energy and Protein Supplement Interactions Microbial protein synthesis is the most important process in maximizing the amino acid supply to a ruminant. High microbial protein production can decrease the need for supplementing R UP (Blummel et al., 1999) by supplying between 70 to 100% of amino acids to the ruminant (AFRC, 1992). Therefore delivery of a balanced or synchronized supply of protein and energy to maximize microbial protein synthesis is crucial. ynchroniza tion is utilized when the provision of both RDP (non protein N and true protein) and energy (ruminally fermentable carbohydrates) to the rumen so microorganisms can utilize both simultaneously (Seo et al., 2010). DelCurto et al. (1990) observed that incr easing supplemental energy in the diet without adequate protein availability was associated with depressed intake and digestibility. Synchrony of protein and energy can enhance the efficiency of microbes in capturing N and utilizing ATP for microbial grow th (Herrera Saldana et al., 1990; Sinclair et al., 1993), which should translate into increased microbial protein production, enhanced rumen fermentation efficiency, and ultimately enhanced nutrient utilization and animal performance. Synchronization can also mean reduction of urinary N excretion (Sinclair et al., 1993) and reduction of fermentative losses through CO 2 and CH 4 (Blummel et al., 1999). Chumpawaddee et al. (2006) formulated diets according to a synchrony index
40 (SI; Sinclair et al., 1993), wher e values of 1.0 represented perfect synchrony and values < 1.0 indicate a degree of asynchrony. The experimental treatments were organized in 4 levels of SI (0.39, 0.50, 0.62, and 0.74). As SI increased, researchers observed a linear increase in DM, OM, and ADF digestibility, which were a result of increased microbial populations and fermentation. In addition, cattle fed a diet with a higher SI index had decreased blood urea nitrogen (BUN) concentrations, which they indicated were a more efficient utiliza tion of N in the rumen. Seo et al. (2010) observed no differences in apparent total tract digestibility of DM, CP, NDF, or ADF among different SI diets, however this may be due to SI that were higher and not as divergent (0.77, 0.81, and 0.83) as in the s tudy by Chumpadwaddee et al. (2006). Although, total VFA production was increased, urinary N was decreased, and total N excretion was decreased in cattle fed the 2 higher SI diets compared to the low SI (Seo et al., 2010). While physiologic measurements of metabolites, digestibility, and rumen parameters may be altered by degree of synchrony, published results regarding those effects on animal performance are quite variable. One approach to synchronizing nutrients is through timing of feed delivery. Ri chardson et al. (2003) provided either a synchronous, intermediate, or asynchronous supply of OM and N to the rumen by shifting feed ingredients between morning and evening feedings in lambs. The synchronous diet received equal amounts of raw ingredients at both feedings, the intermediate diet received the more slowly degradable protein source in the morning feeding and the more rapidly degradable protein source in the evening feeding, and the asynchronous diet received all the protein sources in the morni ng feeding. Average daily gain and efficiency of gain w ere not affected by treatment, however, lambs fed the
41 asynchronous diet tended to have less fat in their carcasses, which could be interpreted as underutilization of energy. Another approach to synch rony is to utilize the SI as previously described (Chumpawaddee et al. 2006). Witt et al. (1999) reported greater live weight gain, growth efficiency, and feed conversion efficiency in synchronous dietary treatments. Stateler et al. (1995) achieved synchr ony through supplementing protein in different forms. By feeding a molasses slurry containing urea and a mixture of blood and feather meal, steers increased ADG by 0.05 kg/d in yr 1 and 0.08 kg/d in yr 2 compared to steers offered a molasses soybean meal t reatment. Substitution as defined by Bowman and Sanson (1996) is the change in forage DMI (kg) per kg supplement DM fed. Positive associative effect takes place when the supplement increased total intake or digestibility of the forage. A negative associa tive effect occurs when the supplement decreases total intake or digestibility of the forage so that the intake of digestible nutrients is less than would be expected from the forage and supplement separately (Bowman and Sanson, 1996). Dried Distillers Gr ains Distillers grains are a product of the dry milling industry for ethanol production (Stock et al., 2000). As demand for ethanol has increased along with rising costs of corn, by products of corn and other grains have been considered as an alternative nutrient source for supplementation. Nutritionally, DDG has 118 to 130% the energy value of corn when supplemented in a forage based diet (Loy et al., 2008). Distillers grains are low in starch, but are high in digestible fiber and they also contain 11 to 12% fat (Lodge et al., 1997). Crude protein concentration of DDG is close to 30% and approximately 50% or more of the CP is RUP (Benton, et al., 2006), with values ranging from 47 to 69% (Schingoethe, 2006). In summary, DDG could be an effective
42 supplement particularly in high forage production systems because it provides high energy, protein, and phosphorus concentrations, all necessary nutritional factors when considering supplementation. Martnez Prez et al. (2010) demonstrated that DDG could be fed up to 0.4% of BW in steers grazing medium to high quality forage without negatively impacting forage intake. The authors theorized that total tract digestibility was increased as supplementation of DDG increased and this is what enhanced animal performance. Leupp et al. (2009) observed a linear increase in total tract OM digestibility as DDG supplementation was increased to 1.2% BW in steers consuming smooth brome hay. Supplementing DDG at a rate up to 0.6% to cattle grazing small grain pastures, which are h igh in CP and digestibility, can increase fat intake and fat and NDF digestibility with no adverse effects on forage or total intake, digestibility, and ruminal fermentation (Islas and Soto Navarro, 2011). Gadberry et al. (2010) reported a significant incr ease in ADG (0.9 kg/d DDG = 1.00 kg/d gain and 1.8 kg/d DDG = 1.05 kg/d gain) for steer calves grazing medium to high quality bermudagrass pasture supplemented with DDG compared to unsupplemented steers (0.74 kg/d). However, there was no added benefit in ADG between feeding rates of 0.90 kg/d or 1.8 kg/d of DDG. In an economic evaluation, the 0.9 kg/d supplemented steers added $19.28 per calf in return. The same authors also conducted an experiment with low quality bermudagrass hay supplemented with dif fering rates of DDG (0, 0.3 0.6 and 1.2% of BW), and ob s erve d a cubic relationship between rate of supplementation and ADG, with the 1.2% supplemented steers reaching 0.82 kg/d, which was a 0.77 kg/d increase over the non supplemented group. Added return w hich is defined as
43 return of 0.0% BW DDG treatment was greatest in the 0.3% supplemented group at an additional $20.54 per calf over the non supplemented calves. Others have also observed increases in ADG when supplementing DDG on low and high quality forages in both steer and heifer calves (Morris et al., 2005; Winterholler et al., 2009). Feeding DDG has been shown to improve reproductive and growth performance in heifers. Martin et al. (2007) performed a study to determine if excess RU P (267 g/d) from DDG fed during development would affect heifer growth or reproduction. There were no differences in final BCS or ADG between the DDG fed heifers, or in the control heifers, which were fed corn gluten feed and whole corn germ. H owever, h e ifers fed DDG had greater AI conception rate (75%) compared to control heifers (52.9%) as well as increased AI pregnancy rate (57% vs. 40.1%; respectively). Harris et al. (2008) observed no differences in reproductive performance between DDG fed heifers o r heifer s fed a whole soybean diet; however the DDG heifers had increase d ADG throughout the feeding period compared to control heifer s MacDonald et al. (2007) supplemented heifers grazing smooth bromegrass with no supplement, DDG, CGM, or corn oil to de termine what effect the contributions of fat and RUP contained in DDG had on heifer performance. Supplementation of DDG increased ADG compared to non supplemented heifers, while daily forage intake was decreased. These authors e stimate d that DDG will rep lace grazed forage at a rate approximately 50% of the amount supplemented for cattle receiving up to 7.5 g of DDG per kg of BW. Additionally, p roviding RUP equal in concentration to DDG (CGM diet) resulted in gains only 39% as great as those observed in t he DDG heifers, suggesting one third of the response to DDG may be due to meeting a MP deficiency. Providing equal amounts of
44 fat to DDG (corn oil diet) provided no additional gain leading the investigators to conclude that the associative effect from pro tein and energy from RUP and fat found in DDG may be responsible for the additional gain. Addition of DDG in forage diets, may cause decreases in forage intake, however it will not negatively impact and may improve animal performance. Soybean Meal Soybean meal (SBM) is the most widely utilized oilseed meal in the United States and is used in feed rations as a protein source in the ruminant diet and provides approximately 51.8% CP, 34% RUP, and 1.67% fat (NRC, 1996). Titgemeyer et al. (1989) evaluated dif ferent protein sources (SBM, CGM, blood meal, and fish meal) and their effect on ruminal dynamics and nitrogen degradation. They observed SBM had the lowest proportion of N escaping ruminal degradation, had the highest increase in ruminal NH 3 N, and appro ximately 13% of the SBM N was absorbed from the small intestine. Addition of SBM to diet s has been associated with positive effects on intake, digestibility, and rumen NH 3 concentrations. Guthrie and Wagner (1988) conducted an experiment to evaluate the influence of 2 levels of protein (low and high) utilizing a SBM based diet as well as an energy supplement composed of corn grain. They observed the high level protein supplement increased forage intake, digestibilities of ADF, DM, OM, CP, and cellulose, as well as increased ruminal NH 3 concentrations compared to the low level of inclusion. In a second experiment, they included SBM at (0, 121, 241, 362, and 603 g DM/d) and observed a quadratic increase in forage intake, CP and OM digestibility, and rumin al NH 3 concentrations. When they compared observed vs. expected DM digestibility (DMD), observed values were greater at all levels of feeding
45 SBM, indicating a positive associative effect of protein from SBM on digestibility of the forage. Mathis et al. (2000) conducted an experiment to examine effects of supplemental RDP in the form of SBM on intake and digestibility of steers fed a low quality forage. Soybean meal was supplemented at five rates (0, 0.08, 0.16, 0.33, or 0.50% of BW/d). A cubic increase in forage OM intake was observed as level of SBM increased. Digestibility of OM also increased quadratically as SBM was increased. There was a tendency for an inverse relationship between ruminal pH and level of SBM and a n increase in rumen NH 3 concentr ations as level of SBM increased in the diet. Using s oybean meal as a protein supplement in the diet can increase i ntake, digestibility, and rumen parameters. Dietary effects on metabolic based hormones and substrates Shifts in metabolic hormones and subst rates in response to supplementation and plane of nutrition have been well documented. Steiner et al. (1983) were the first to suggest that circulating concentrations of metabolic substances such as insulin, NEFAs, and certain amino acids could act as sig nals reflecting metabolic status. Ellenberger et al. (1989) conducted a comprehensive study that represents changes in metabolism under different nutrient intake scenarios. This study was designed to compare 3 growth phases (normal, restricted, and compe nsatory) and evaluate shifts in hormones and metabolic substrates during these phases. Normal fed steers were offered ad libitum access to diet (70% cracked corn and 30% alfalfa pellets) from 240 kg to 510 kg of BW, whereas others were fed a restricted di et at 50% of ad libitum consumption from BW of 240 kg to 307 kg, and steers in the compensatory treatment group were on the restricted diet then r ealimented to ad libitum feeding until 510 kg. Plasma glucose concentrations were decreased in the restricted steers, with a linear and quadratic
46 increase in glucose concentrations once steers were realimented. No differences were observed in NEFA concentrations between ad libitum and restricted steers, however once realimentation occurred, NEFA concentrations d ecreased, and were maintained at decreased concentrations compared to normal fed steers. Serum concentrations of insulin like growth factor I (IGF I) changed in approximately direct proportion to dietary intake, with the restricted steers having 45% lower IGF I concentrations and a linear increase in concentrations as steers were realimented. There were no differences in PUN concentrations for steers fed ad libitum compared to limit fed steers. However, there was a sharp decline, then a cubic increase wh en limit fed steers were realimented to an ad libitum diet. The liver detoxifies NH 4 and forms urea, a metabolite of dietary protein. The concentrations of plasma urea nitrogen (PUN) can be reflective of the quantity and degradability of the protein consu med, a measure of the negative energy balance in a fasted animal, or a combination of the two aforementioned factors. Kohn et al. (2005) observed that measuring PUN concentrations could be a useful indicator of protein status in a group of animals. Plasm a urea nitrogen has also been used as an indicator of adequate DIP relative to TDN in the diet. Hammond et al. (1993) summarized that PUN concentrations of cattle fed subtropical forages should be between 8 to 12 mg/dL. Concentrations of PUN < 8 mg/dL co uld be indicative of a protein deficiency while concentrations > 12 mg/dL an energy deficiency. Plasma urea nitrogen has often been used as an indicator of circulating protein concentrations and quite possibly fertility of the animal. For example heifer conception rates that were at least 30% lower compared to heifers with PUN
47 concentration s < 16 mg/dL. Also, it has been suggested that when serum urea nitrogen (SUN) concentrations are > 20 mg/dL, fertili ty could be impaired (Ferguson et al., 1991). Kaim et al. (1983) reported that cows with PUN concentrations of 16.8 mg/dL fed a 20% CP diet had lower pregnancy rates compared to cows with PUN concentrations of 9.0 mg/dL fed a 15% CP diet. However, other reports have shown elevated PUN (> 24 mg/dL) in cows fed high protein diets that did not cause a decline in reproductive responses (Carroll et al., 1988; Howard et al., 1987). Variability in the literature can potentially due to source of protein in the d iet and stage of animal production and energy demands from the animal. Non esterified fatty acids are the major component of triglycerides. They can be used as an energy source by many tissues including skeletal muscle and hepatocytes. Hydrolysis of sto red triglycerides in adipose tissue by the enzyme lipase can liberate NEFAs and glycerol. Hormones such as glucagon, which is released when blood glucose concentrations are low, can stimulate the hormone sensitive lipase causing a release of NEFA. Theref ore, circulating NEFA concentrations can be use d as a marker of fat tissue mobilization. Additionally, when an animal is in a negative energy balance there are typically a correlated increase in concentrations of NEFAs (Breie r et al., 1986; Peters, 1986; Richards et al., 1989) due to increased release of fatty acids from adipose tissue (Bines and Hart, 1982) as well as mobilization of fat (Blum et al., 1985; Ellenberger et al., 1989). Several studies have documented level of feeding is inversely related t o circulating NEFA concentrations (Gill and Hart, 1981; Peters, 1986; Yelich et al., 1996). Yambayamba et al. (1996) fed heifers an ad libitum diet or a restricted diet formulated for zero gain. The restricted heifers were realimented (for an additional 100
48 d) to an ad libitum diet after 95 d of restriction Concentrations of NEFAs were greater (538 mEq/mL) in restricted heifers compared to the ad libitum fed heifers (257 mEq/mL), however once the restricted heifers recovered after realimentation, there were no difference s in NEFA concentrations between the 2 groups. So evaluation of NEFA can be a tool utilized to determine energy status and degree of fat mobilization in cattle. Like the other metabolic hormones and (or) substrates, glucose is commonly m onitored as an indicator of nutritional status. Glucose is a simple sugar and a source of energy, which serves as a metabolic intermediate. Circulating glucose concentrations are positively associated with nutrient intake and BW gain (Vizcarra et al., 19 98; Hersom et al., 2004; Cooke et al., 2007; Richards et al., 1989; Yelich et al., 1996) and Adams ( 1987 ) concluded that glucose is essential for maintenance of body and reproductive function. Low blood glucose concentrations have been associated with red uced fertility in both beef and dairy cattle (Oxenreider and Wagner, 1971) and glucose concentrations probably control the synthesis and secretion of reproductive hormones like the steroids and gonadotropins (Lynn et al., 1965). Hess et al. (2005) reporte d that secretion of GnRH was interrupted by low glucose concentrations but recovered when glucose was adequate in the blood. However, they concluded that the effects might not be due directly to increased glucose availability via gluconeogenesis, but due t o overall improvements to energy status and other metabolic factors. So evaluation of glucose concentrations can be utilized to measure influence of nutrient intake and nutrient status of an animal. Insulin concentrations are directly proportional to leve l of feed intake and BW gain (Bassett et al. 1971; Blauweikel and Kincaid, 1986; Bossis et al., 2000; Lapierre et
49 al., 2000; Cooke et al., 2007). Enhanced reproductive function throughout the hypophyseal hypothalamic ovarian axis has been associated with insulin (Butler, 2000; Gong, 2002). Insulin may serve as a nutritional signal influencing LH release (Schillo, 1992), however conflicting results have been presented in the literature and therefore it is hard to draw conclusions regarding direct links bet ween insulin and LH. Cooke et al. (2007) observed no relationship between insulin on attainment of puberty or pregnancy rates in heifers. Likewise, Harrison and Randel (1986) reported no influence of insulin infusion on LH patterns or on serum progestero ne concentrations of heifers during the estrous cycle. In contrast, postpartum anestrous beef cows with low insulin concentrations failed to ovulate the first dominant follicle in response to restricted calf access in comparison to cows with moderate plas ma insulin values (Sinclair, 2002). Bovine follicular cells in vitro can be stimulated to produce estradiol when cultured with insulin and FSH (Spicer et al., 1993). Insulin can increase the diameter of large follicles and increase estradiol concentratio ns in follicular fluid in super ovulated cows (Simpson et al. 1994). During low energy balance LH pulses as well as reduced ovarian responsiveness to LH stimulation has been observed and plasma insulin is also reduced (Beam and Butler, 1999; Butler, 2000 ). Insulin like growth factor I is produced predominantly in the liver and is associated with GH and other metabolic hormones (Anderson et al., 1988). Elsasser et al. (1989) noted positive associations among plasma IGF 1 concentrations, protein and energ y intake and protein accretion. Restriction of feed intake is shown to reduce concentrations of IGF I (Breier et al., 1986) L ikewise intake and BW gain have a positive association with IGF I (Bossis et al., 2000; Armstrong et al., 2001). Wettemann
50 and Bossis, (2000) concluded that IGF I is a major metabolic signal regulating reproduction in cattle. They propose d that IGF I may be one of many signals that influence pulsatile secretion of GnRH and LH, controlling follicular growth in cattle. The major e ffect of nutrition on ovarian function is via alteration of LH secretion; however, nutrition may have modulating effects on the ovary through metabolic signals such as IGF I. Cooke et al. (2007) observed positive correlations between increased IGF I conce ntrations and ADG, attainment of puberty, and pregnancy rates in heifers. Additionally, other reports have observed an increase in circulating IGF 1 concentration in heifers as puberty approached (Jones et al., 1991; Yelich et al., 1996; Daz Torga et al. 2001). Beam and Butler, (1997, 1998) observed in postpartum d ai ry cows higher levels of IGF I in cows in which the dominant follicle ovulated compared to levels in cows who did not ovulate. In addition, they also noted a significant correlation between plasma estradiol concentrations and IGF 1 levels. In the postpartum cow during negative energy balance, the ability for estradiol production seems to be dependent on availability of insulin and IGF 1 (Beam and Butler, 1999) In summary, when an animal is in a n egative energy balance there is typically a correlated increase in concentrations of NEFAs due to the mobilization of triglycerides to meet energy requirements. The concentr ations of PUN can be reflective of the quantity and degradability of the pr otein consumed, a measure of the negative energy balance in a fasted animal, or a combination of the two aforementioned factors. Concentrations of PUN are typically increased during feed restriction; however, if feed restriction is not severe to lead to pr otein mobilization PUN concentrations do not increase Circulating glucose concentrations are positively associated with nutrient
51 intake and BW gain Additionally differences reported in the literature in metabolite concentrations among animals and stud ies are primarily because animals can respond differently to dietary differences and design of experiments. Rumen Dynamics Bergman et al. (1990) described volatile fatty acids ( VFA ) also known as short chain fatty acids, as products of microbial fermentat ion of carbohydrates and endogenous substrates in the gastrointestinal tract, predominantly in the forestomach but also in intestines. Volatile fatty acids are a major source of energy for ruminants and VFA synthesized in the rumen account for approximate ly 70% of the caloric requirements of ruminants. The primary forms of VFA that occur in the rumen or large intestine are acetate, propionate, and butyrate. They are produced in varying ratios from 75:15:10 to 40:40:20 and can be manipulated by diet. Yost et al. (1977) observed a direct response of feed intake to propionate production, where increased intake of a high grain diet increased propionate production. McCollum and Galyean, (1985) observed a shift toward higher propionate production when prairie hay was supplemented with cottonseed meal, a protein source. The different VFAs also have different metabolic fates. Butyrate is converted to ketone bodies or CO 2 by the epithelial cells or removed by the liver. The ketones are oxidized in cardiac and sk eletal muscle, and are used for fatty acid synthesis in adipose and mammary gland tissue (Fahey and Berger, 1988). Propionate is an important precursor for glucose in gluconeogenesis and propionate accounts for up to 76% of the glucose synthesized in the l iver (Reynolds et al., 1994). Whereas, a small amount of acetate is absorbed through the rumen wall and converted to ketone bodies, most is carried by portal circulation to the liver. Approximately 80% of acetate
52 that reaches the liver escapes into periph eral circulation and is oxidized via the tricarboxylic acid cycle or used for fatty acid synthesis (Fahey and Berger, 1988). Bagely, (1993) concluded that high energy, high quality supplements shift rumen fermentation pattern toward propionate production. Shifting the production of VFA toward propionate production and away from acetate and butyrate can have positive outcomes on animal performance and reproductive function of the animal. McCartor et al. (1979) observed a decrease in age and BW at puberty in heifers with higher propionate concentrations. Infusing propionate into the abomasum of pre pubertal heifers enhanced blood glucose and release of LH following a GnRH challenge (Rutter et al., 1983). Likewise, when ionophores are fed to pre pubertal heifers to increase ruminal propionate concentrations, ovarian response to gonadotropins is increased (Bushmich et al., 1980). An unfavorable rumen environment can have consequences on intake and digestibility. Optimization of pH and ammonia concentrations is vital for cellul ol ysis. Diet can influence rumen pH, which can alter microbial fermentation, and ultimately the end products (VFAs, CH 4 CO 2 NH 3 N) (Hungate, 1966). Rapid fermentation of starch often exceeds the ability of ruminants to maintain a st able ruminal pH (rskov and Fraser, 1975), and as pH decreases the function of cellulolytic bacteria is impaired which can decrease fiber digestion. When rumen pH was reduced below about 6.2, cellulolysis was reduced which resulted in up to a 40% reduction in digestibility of hay (Mould et al., 1982). Feed intake is depressed when the rumen pH falls bel ow 5.5 (Fulton et al., 1979). Therefore, maintenance of an optimum rumen environment is important for intake and digestibility.
53 Rumen microbes utilize free ammonia (NH 3 ) for protein synthesis growth and fermentation of feeds. A range of values for rumen NH 3 that optimizes microbial growth has been reported ranging from 1.4 mg/dL (Schaeffer et al., 1980) in vitro to 19.4 mg/dL (Mehrez and rskov, 1977). How ever, Erdman et al. (1986) concluded that rumen function of the digestibility of the diet. A number of factors affect rumen ammonia N in ruminants including: forage intake, ti me after feeding, location of ruminal sampling, type of diet, concentration of protein in the diet, protein solubility, rumen volume, and pH (Adams and Kartcher, 1984; Wohlt et al., 1976; Elliot and Topps, 1964; Haaland et al., 1982; El Shazly, 1958; Harro p, 1974; Lana et al., 1998;). Lana et al. (1998) reported when steers were fed increasing amounts of concentrate and decreasing amounts of forage that VFA concentration increased while and ruminal pH, acetate:propionate, and dissociated ammonia decreased. Furthermore, the acetate:propionate and ammonia concentrations were highly correlated with ruminal pH. The regulating role pH played on ammonia production was replicated by in vitro studies in which ruminal bacteria from cattle fed forage diets were incu bated in oxygen free nitrogen or Trypticase ; ammonia product ion decreased from 28 to 2 nmol mg protein 1 min 1 as pH dropped from 6.5 to 5.7. The fat content of the diet can also alter digestibility Accumulation of unsaturated fatty acids inhibits ruminal fibrolytic activity resulting in decreased diet digestibility (Palmquist and Jenkins, 1980 ). Devendra and Lewis (1974) summarized 4 theories to explain this effect: 1) physical coating of the fiber with fat preventing microbial attack; 2) a modification of the rumen microbial population from possible toxic
54 effects of fat on certain microorgani sms; 3) inhibition of microbial activity from surface active effects of fatty acids on cell membranes; 4) reduced cation availability from formation of insoluble complexes with long chain fatty acids. The last effect could be acting directly on availabilit y of cations for microbial function or indirectly by affecting rumen pH. Others have also reported decreased fiber digestion when fats were supplemented (Kowalczyk et al., 1977; Doreau and Chilliard, 1997; Williams et al., 2000). Increased rate of digest ion and passage are believed to be responsible for increased voluntary intake of low quality forages when supplemented with protein (Ellis, 1978). Gut fill is considered the most limiting factor for intake of forage diets (Campling, 1970; Freer, 1981). C learances of digesta from the rumen and particle reduction are the primary processes that affect forage intake by ruminants (Campling et al., 1961; Ulyatt et al., 1986; Van Soest, 1982). Before forage particles can leave the rumen they must be reduced to a critical size (Troelsen and Campell, 1968; Poppi et al., 1980). Digestion by the ruminal microorganisms and rate and extent of particle size reduction are of major importance in regards to clearance rate (Mosely, 1982). Many authors have observed rate o f particle size reduction to be the main factor, which limits digesta clearance from the rumen (Balch and Campling, 1962; Welch, 1982; Ulyatt et al., 1986). Reducing length of forage or processing, like pelleting, can increase intake and passage rate in l ow quality forages (Campling and Freer, 1966; Martz and Belyea, 1986). However, in the instance of high quality forage, clearance may be limited by rate of small particle removal (Shaver et al., 1988). Forage maturity can impact rate of passage and forag e intake. Bowman et al. (1991) observed faster NDF and ADF
55 disappearance and increased passage rate of small and large particles of early maturity orchardgrass compared to late maturity orchardgrass. Feeding late maturity orchardgrass also depressed inta ke and the authors concluded that this might be a result of a decrease in the rate of large particle size reduction and the flow of small particles from the rumen.
56 CHAPTER 3 EFFECT OF ADDING RUMEN DEGRADABLE PROTEIN TO A DRIED DISTILLERS GRAIN SUPPLEMEN T ON GROWTH, BODY COMPOSITION, BLOOD METABOLITES, AND REPRODUCTIVE PERFORMANCE IN YEARLING ANGUS AND BRANGUS HEIFERS. Introduction The majority of replacement beef heifers in the Southeastern United States are developed on forage based diets. Most of the forages are warm season perennial grasses that are grazed or fed as conserved forage in the form of hay or round bale silage (RBS). Utilization of RBS has gained popularity due to the frequency of rain events during the peak growing season, which can delay harvest, cause field losses of forages ( Hersom and Kunkle 2003) and a rapid decline in forage quality (Moore et al., 1979). While preserving forage as RBS does not improve quality, harvesting the forage on an regrowth interval allows for capturing of th e optimum nutritive value of the forage Due to the low to mid quality nutritive value of warm season forages, CP and TDN feed resource. Therefore, growing heifers m ust be supplemented with protein and (or) energy. The primary cost of heifer development is feed cost (Hersom et al., 2010), therefore minimizing feed input costs while still ensuring that heifers meet target weight gains and attain puberty by the start of the breeding season is critical. Development of ethanol plants across the country has provided a unique opportunity to utilize dried distillers grains (DDG) as an alternative feed. Dried distillers grains consist of 27 to 36% CP (24 to 40% RDP), 29 to 39 % NDF, 78 to 88% TDN, and 9 to 16% fat (Dairy One, 2012 ) Due to the relatively low price and high protein and energy composition of DDG, it may be an economical supplement for growing heifers.
57 A negative RDP balance often occurs in the total diet when DD G is supplemented in forage based diets. Meeting RDP requirements is critical for the production of ammonia or amino acids, which are utilized by rumen microbes to produce microbial protein (Butler, 1998). Supplementation of additional RDP to forage based diets has been associated with improve d cattle performance through increase s of forage digestibility, DMI and improvement of metabolizable energy (Owens et al., 1991). Therefore, we hypothesized that adding soybean meal (SBM), a source of RDP, to a DDG su pplement program w ould enhance the growth and reproductive performance of Angus and Brangus heifers fed bermudagrass RBS. The objectives of this research were to evaluate the effect of supplementing DDG alone or DDG in addition to 2 levels of SBM on growt h, body composition, blood metabolites, and reproductive performance in Angus and Brangus heifers fed RBS. Materials and Methods The experiment was conducted at University of Florida Santa Fe Beef Research Unit, north of Alachua, FL from October 2009 until June 2010. The experiment was divided into a supplement and sampling period (d 0 to 140), and a breeding period (d 140 to 217). The experiment was conducted in accordance with acceptable practices as outlined by Guide for the Care and Use of Agricultura l Animals in Agricultural Research and Teaching (FASS, 1999) and University of Florida IFAS Non regulatory Animal Research System protocol number 011 09ANS. Animals Angus (n = 30) and Brangus (n = 30) heifers with initial BW of 229 4 kg and 250 4 kg, respectively were utilized. After the initiation of the experiment, a Brangus heifer broke her leg and was removed from the trial. On d 0, mean age of the heifers
58 was 259 21 d. A full BW was taken 7 d prior to start of experiment, and heifers were blo cked by BW and breed and stratified by sire and allocated to one of twelve 1.2 ha p en s with five heifers per pen. The p asture s were composed of a mixture of dormant bahiagrass ( Paspalum notatum ) and bermudagrass ( Cynodon dactylon ). The pastures received no fertilization previous to or during the experiment. The mean forage mass per pen in October was estimated at 1,683 DM kg/ha, in November 1,922 DM kg/ha, in December 1,545 DM kg/ha, and in April 763 DM kg/ha. Pasture samples were not collected January through March due to insufficient forage growth. Heifers remained in the same pen from d 0 to 178 of the experiment. Treatments Heifers were supplemented 3 d/wk, based on mean pen BW, and supplement amount was adjusted on a 28 d basis. Pens were randomly assigned to 1 of 3 treatments: 1) supplementation with dried distillers grain (DDG) at 0.75% of BW; 2) supplementation with DDG at 0.75% of BW plus soybean meal at 7.5% of the DDG amount (D DG+7.5); 3) and supplementation with DDG at 0.75% of BW plus SBM at 15% of the D DG supplement amount (DDG+15). The diets were formulated for nominal gain requirements for the heifers to reach a target BW based on the NRC (2000). Soybean meal amount of 7.5% was chosen to eliminate a rumen degradable protein (RDP) deficien cy and 15% to provide a linear dose amount. Heifer s also received ad libitum access to Tifton 85 bermudagrass ( Cynodon dactylon ) round bale silage (RBS ), water, and custom made mineral vitamin mix (appendix A). Heifers were offered DDG at 1.81 kg heifer 1 d 1 from d 14 to 1 and were maintained on treatment diets from d 0 to 172 at which time on d 172, 174, and 176 the supplement offered was offered in
59 decreas ing intervals (0.6, 0.45, and 0.3% BW respectively ) to prepare heifers for breeding pastures. Feed Sampling Samples of RBS were collected from each bale throughout the experiment as it was fed, frozen, and pooled monthly for analysis Consumption of RBS was estimated for each pen using individual bale weights and a monthly weigh back weight. Samp les of DDG and SBM were collected monthly. Pasture samples were also obtained in October, November, December, and April from each pen to estimate forage quantity and quality by hand clipping three 0.25 m 2 areas and compositing the samples. Samples were d ried at 60 C in forced air oven for approximately 72 h. Dried samples were ground to pass through a 1 mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA). Samples were analyzed for residual DM and OM (AOAC, 2007). Total nitrogen was determined by the combustion method using a macro N analyzer (Elementar Vario Max CN instrument, Elementar Americas, Mount Laurel, NJ, USA) and used to calculate CP (N x 6.25). In vitro dry matter digestibility (IVDMD) of samples was determined using an ANKOM DAISY II incubator (ANKOM Technology Corp., Fairport, NY) using the ANKOM Technology Method for In Vitro Digestibility Rumen fluid inoculum for this procedure was obtained from a ruminally fistulated, non lactating Holstein cow consuming a diet o f ad libitum bermudagrass hay and 450 g soybean meal daily. Total digestible nutrient was calculated for RBS and pasture using a formula for warm season grasses (Fike et al., 2003). Total digestible nutrient values for DDG and SBM were determined by a co mmercial laboratory (Dairy
60 One Forage Laboratory, Ithaca, NY). The n utritional composition for DDG, SBM, RBS, and pasture are presented in Table 3 1. Sample Collection and Analysis Heifers were weighed every 2 wk from d 0 to 70, weekly from d 70 to 140. B ody weights are reported in 28 d intervals for d 0 to 140. Average daily gain was calculated for each 28 d period during the supplement ation period (d 0 to 140. Hip height (HH) and BCS (1 = severely emaciated; 5 = moderate; 9= very obese; Wagner et al., 1988) were measure d every 28 d until d 140. A BCS was also collected at final pregnancy diagnosis. Body condition score was recorded by two observers and averaged at each collection. Ultrasound measurements (Pie Medical 200 ultrasound machine utilizing th e ASP 18 probe Pie Medical Benelux BV, Maastricht, The Netherlands) of LM area (REA) at the 13 th rib, 13 th rib fat thickness (RIBFT), rump fat thickness (RMPFT), and intramuscular fat of the REA (IMF) were taken on d 0 and 140. The REA and BW measurement s were used to calculate REA/cwt, which adjusted REA per 100 kg of BW. Blood samples were collected via jugular venipuncture into 6.0 mL polypropylene syringes containing 10.8 mg potassium EDTA as an anticoagulant (BD Vacutainer, BD Diagnostics, Franklin L akes, NJ) on d 14 and 7, every 2 wk from d 0 to 70, and weekly from d 70 to 140 during the sampling period of the experiment. Blood samples were placed on ice immediately after collection and transported to the lab for further processing. Blood samples were centrifuged at 3,000 g for 15 min at 5 C to obtain plasma, which was placed in sample vials and frozen at 20 C for subsequent analysis. Concentrations of progesterone in plasma samples were analyzed by RIA using Coat A Count kit (Diagnostic Produ cts Corp., Los Angeles, CA; Seals et al., 1998).
61 Assay sensitivity for a 100 l sample was 0.1 ng/ml. The intra and inter assay CV were 6 and 11%, respectively. Pubertal status was determined d 14, and 7 for analysis of heifers pubertal at d 0 of the experiment. Any heifers pubertal at d 0 were not included in further analysis of puberty. Pubertal status was also determined using 2 week samples from d 0 to d 168. Angus heifers were considered pubertal when progesterone concentrations were 1.0 ng/ mL of progesterone Brangus heifers were considered pubertal when progesterone concentrations were 1.5 ng/mL of progesterone (Cooke and Arthington, 2009). Date of puberty was defined as the first progesterone concentration 1.0 ng/mL for Angus and 1 .5 ng/mL for Brangus. Plasma NEFA concentrations were determined using a Wako HR Series NEFA HR kit (Wako Diagnostics, Richmond, VA). Plasma urea nitrogen concentrations were determined using a BioAssay Systems QuantiChrom Urea Assay Kit series DIUR 500 k it (BioAssays Systems, Hayward, CA). Plasma glucose concentrations were determined using a Cayman Chemical Co. Glucose Analysis kit (Cayman Chemical Co., Ann Arbor, MI). The intra and inter assay CV were 6.6% and 13.3% for NEFA, 7.3% and 20.5% for PUN, an d 3.2% and 17.7% for glucose, respectively. Breeding Breeding occurred in two phases; in phase 1, heifers were synchronized without a progesterone source (d 140 to 178) and in phase 2, heifers were synchronized with a progesterone source (d 168 to 178). T he first synchronization phase was introduced to avoid any confounding effects of progesterone priming on the induction of puberty (Short et al., 1976; Anderson et al., 1996; Imwalle et al., 1998). Whereas, the second synchronization phase was utilized to satisfy the reproductive management demands of
62 the research unit since only 32% of heifers were inseminated during phase 1 of the synchronization program. During phase 1, estrous detection patches (Rockway, Inc., Spring Valley, WI) were applied to heifers on d 140 of the experiment. Estrus was visually detected twice daily for 7 d and heifers were AI 8 to 12 h after an observed estrus. On d 147 all heifers received radiotelemetric estrous detection devices (HeatWatch, Cow Chips, Denver, CO; Dransfield et al., 1998) and 2 doses of PG were administered at 0700 and 1900 h (25 mg i.m.; Lutalyse Sterile Solution, Pfizer Animal Health, New York, NY) to heifers not previously AI HeatWatch was checked twice daily for 31 d for heifers in estrus, and heifers obser ved in estrus were AI 8 to 12 h after observed estrus. Estrus was defined as > three 1 second mounts in an 8 hour period (Dransfield et al. 1998) If heifers returned to estrus at any point during the AI breeding season, they were allowed a second AI 8 to 12 h after an observed estrus. All heifers were AI by a single AI technician throughout the study. The AI sires were pre assigned to heifers prior to the start of the breeding season. During phase 2, which began on d 168, heifers not previously detected in estrus and AI were administered 100 g (i.m) of GnRH (Cystorelin, Merial, Inc., Duluth, GA) and a progesterone device was inserted (EAZI BREED CIDR; Pzifer Animal Health, New York, NY). Seven days later, heifers received 25 mg PG (i.m.) and CIDR were removed. Estrus was detected utilizing the HeatWatch system for 72 h after CIDR removal and heifers were AI 8 to 12 h after the onset of estrus. Heifers not exhibiting estrus by 72 h were administered 100 g (i.m.) GnRH and AI at 72 to 76 h after PG. All heifers were AI by a single AI technician. Seven days later, heifers were divided into
63 respective breed groups and exposed to like breed bulls for an additional 30 d. The total length of th e breeding season lasted 77 d Pregnancy was diagnosed on d 175 207, and 249 by transrectal ultrasonography, using a real time, B mode ultrasound (Aloka 500v, Corometrics Medical Systems, Wallingford, CT) equipped with 5.0 MHz transducer. Day of conception during the breeding season was equivalent to AI date for hei fers confirmed pregnant to AI. To determine day of conception for heifers conceiving to natural service, a mean gestation length was calculated for all heifers and was used to back calculate conception date from calving date. Response variables to the syn chronization phases will be presented separately for phase 1 and phase 2. Phase 1 response variables included: estrous response (number of heifers that exhibited estrus once during the 38 d breeding phase divided by total number of heifers), first servi ce conception rate (number of heifers that became pregnant to the AI divided by total that exhibited estrus once), first service pregnancy rate (number of heifers that became pregnant to the AI in phase 1 divided by total number of heifers), final AI pregn ancy rate (number of heifers pregnant to first or second AI divided by total number of heifers AI during the 38 d breeding phase). Phase 2 response variables included: estrous response (number of heifers that exhibited estrus during the 3 d after PG divid ed by total synchronized in phase 2), conception rate (number of heifers that became pregnant to the AI divided by total displaying estrus during phase 2), two timed AI pregnancy rate (number of heifers which failed to display estrus and timed AI divided b y total number timed AI), synchronized pregnancy rate (number of heifers that became pregnant to AI divided by total synchronized in phase
64 2). A final pregnancy rate (total number of heifers pregnant during a 77 d breeding season divided by total number of heifers) for phase 1 and phase 2 was also calculated. Statistical Analysis This experiment was conducted as a randomized block design with pen as the experimental unit. Heifer growth performance, body composition, and blood metabolite data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The model stateme nt used for BW, ADG, BCS, HH IMF, REA, REA/cwt, RIBFT, and RMPFT included the effect of treatment, breed, and the interaction of treatment and breed. All variables were anal yzed using pen(breed treatment) as the random statement.. The model statement used for glucose, NEFA, and PUN contained the effect of treatment, breed, time, and all appropriate interactions. Data were analyzed as repeated measures using pen(breed tr eatment) as the random statement and heifer(pen breed trt) as the subject. For the blood metabolites t he appropriate covariance structure of the data was chosen for each analysis from the structures of autoregressive one (AR(1)), heterogeneous (ARH(1) ), and Heterogeneous TOEP (TOEPH), as these were the most appropriate for the data set. Criterion (AIC) and Bayesian Information Criterion (BIC) were utilized to select for the best fit model Contrast statements were included in the statistical model to compare the effect of DDG vs. DDG+7 and DDG+15 as well as DDG+7 vs. DDG+15. Reproductive data were analyzed using GLIMMIX procedure of SAS. The model statement used for all reproductive response variables contained the effect of t reatment, breed, and interaction between treatment and breed. To determine the effect of pubertal status on response variables, it was also included in the model where appropriate. Data were analyzed using pen(breed treatment) as the random variable.
65 C ontrast statements were included in the statistical model to compare the effect of DDG vs. DDG+7 and DDG+15 as well as DDG+7 vs. DDG+15. The rate of attainment of puberty during the experiment and rate of conception during the breeding season were analyze Kaplan Meyer survival curves (PROC LIFETEST; SAS). puberty by d 168 and those who were not pregnant in the 77 d breeding season were censored. del included effects of treatment, breed, tre atment breed, and age at d 0 of the experiment was included as a covariate. When interactions were P > 0.10 they were dropped from the model. The adjusted hazard ratios (AHR) and the 95% CI were calculated. For all analysis, mean comparisons were made using the PDIFF statement associated with generation of least square means. Results are reported as least square means, significance was set as P de clare d if P > 0.05 and Re sults and Discussion Growth Performance and Body Composition At the initiation of the experiment, BW were similar ( P > 0.05; Table 3 3) across treatments and breeds during the 140 d supplementation period and intervening 28 d periods ( data not shown). Tre atment breed effects were similar ( P > 0.05) as were contrasts of DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 ( P > 0.05). Mean weight gain of heifers was 119 kg during the 140 d supplement period. Additionally, estimated RBS consumption was simila r ( 7457 kg/pen; P > 0.05) during the supplementation period among treatments. This provides additional evidence as to why no differences between treatments were observed. Martin et al. (2007) supplemented
66 heifers with 0.59% BW DDG (supplement offering hi gh rumen un degradable protein (RUP)) or 0.78% BW corn gluten meal (CGM) based diet (s upplement offering high rumen degradable protein (RDP)) and observed no difference in BW between the treatments after a 196 d supplementation. Heifers offered DDG in the Martin et al. (2007) study had a negative RDP balance as estimated by the NRC (2000) model level 1 compared to heifers offered CGM based diets which had a positive balance of 176 1 1 Similarly, by our estimates from the Beef Cattle NRC (2000), heifers in our study supplemented with DDG alone averaged 1 1 RDP balance with a range of 1 1 1 1 Rumen undegradable protein may indirectly supply RDP via the urea cycle (Archibeque et al., 2008). It is likely the high amount of RUP in the diet of the present study was compensating for the low RDP via N recycling through the urea cycle which could explain the similar BW observed when additional SBM (a source of RDP) was added to the diets. Body condition scores for d 0 and 140 are presented in Table 3 3. On d 0 and 140 BCS were similar across treatments and breeds ( P > 0.05) as well as for the intervening 28 d periods (data not shown) other than o n d 56, Brangus heifers tende d ( P = 0.09) to have greate r BCS compared to Angus heifers (5.5 vs. 5.3 respectively ; data not shown ). T reatment breed effects were similar ( P > 0.05) for all measured time points as well as for contrasts of DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 on BCS ( P > 0.05) Si milar to BW, Martin et al. (2007) observed no differences in final BCS between DDG and CGM. This further confirms no additional benefits off adding SBM as a source of RDP to the diet in the growing heifer.
67 There was no effect ( P > 0.05) of treatment, bree d, and treatment breed on ADG during an y of the 28 d sampling periods or throughout the supplement period (data not shown). Contrasts of DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 were also similar ( P > 0.05). Across treatments, heifers gained 0. 77 kg/d from d 0 to 140 of the experiment. The ADG observed were within anticipated gains predicted by the Beef Cattle NRC (2000) at the start of the experiment by providing supplementation at 0.75% BW. The ADG are reflective of other reports in the lite rature when heifers were supplemented with DDG. Martin et al. (2009) observed ADG of 0.64 and 0.72 kg/d when DDG were fed at 0.59% of BW. Additionally, Loy et al. (2008) fed DDG at 0.81% of BW with predicted gains of 0.80 kg/d and achieved 0.89 kg/d when offered 3 times per wk. So these results in addition to what we observed would suggest that DDG alone is an effective supplement in growing yearling heifers developed on forage based diets. Hip height was similar ( P > 0.05; Table 3 3) across treatments f or each 28 d sampling (data not shown) up to d 140 and there was no treatment breed effect ( P > 0.05) on HH. Brangus heifers had a greater HH at each sampling time point from d 0 to d 140 ( P 0.05 ; data not shown ). This is consistent with observations that growing Brahman influenced cattle have greater hip height compared to Angus cattle (Gregory et al., 1979; Monari, 2010). Effects of treatment on ultrasound measurements taken on d 0 and d 140 of the experiment are presented in Table 3 4. The REA/cw t was calculated to adjust REA per 100 kg of BW to normalize any differences associated with heifer BW. There were no treatment, treatment breed effects or contrasts of DDG vs. DDG+7.5 and DDG+15
68 and DDG+7.5 vs. DDG+15 effects ( P > 0.05) on REA, REA/cw t, IMF, RIBFT, and RMPFT on d 0 or 140 of the experiment. The similarity between treatments for body composition measures is supported by similar BW and BCS measures observed between treatments, indicating there was no difference in mobilization or deposi tion of fat or muscle between treatments. This implies additional RDP provided through SBM did not elicit an additive effect for heifer performance as measured by BW, BCS, or measure of body composition, which resulted in a similar pattern of growth among treatments. The effects of breed on ultrasound measurements taken on d 0 and d 140 are presented in Table 3 5. There was no breed effect ( P > 0.05; Table 3 5) on REA, REA/cwt, RIBFT, or RMPFT on d 0. However, Angus heifers tended ( P = 0.08; Table 3 5) t o have a greater percent IMF on d 0 compared to Brangus heifers and the breed effect on IMF was still present on d 140 of the experiment as Angus heifers had a greater ( P 0.05) percent IMF compared to Brangus heifers. These results are consistent with observations made in similar breeds of heifers from the same location in previous years (Monari, 2010). Additionally, numerous authors have reported lower marbling scores in Brahman or crossbred Brahman cattle compared to cattle of Bos taurus br eeding (Adams et al., 1982; Koch et al., 1982; Huffman et al., 1990; DeRouen et al., 2000). Relative to ultrasound measurements associated with fat thickness, there was no breed ef fect ( P > 0.05) on RIBFT but Brangus heifers tended to have a greater ( P = 0.08) RMPFT thickness compared to Angus heifers on d 140 of the experiment. Rump fat is positively correlated with BC S (Domecq et al., 1995; Houghton and Turlington, 1992; Monari, 2 010), which indicates that BCS can be a reflect ion of the
69 amount of subcutaneous fat. This is correlation can be observed in our study as Brangus tended to have greater BCS on d 56 and had numerically higher BCS on d 140. On d 140, there was a tendency ( P = 0.08) for Brangus heifers to have a larger REA compared to Angus heifers. When REA was adjusted for BW, Brangus heifers had a larger ( P 0.05) REA/cwt compared to Angus heifers. The REA/cwt measurement is a better indicator of lean:bone in the animal compared to REA, as REA is more a reflection of BW. Blood Metabolites Plasma NEFA concentrations are presented in Figure 3 1. Mean NEFA concentrations (313 mEq/ml) were not different ( P > 0.05) among treatments from d 0 to 140. There was also no effect ( P > 0.05) of treatment breed or contrasts of DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 on NEFA concentrations. There was an eff ect ( P 0.05) of breed, time, time treatment, time breed, and time treatment breed on NEFA concentrations. Concentrations of NEFA were greatest on d 0, and were greater ( P 0.05) than all times points except d 56 and d 140. Increased NEFA concentratio ns are typically indicative of a negative energy balance (Breie r et al., 1986; Peters, 1986; Richards et al., 1989) and have been observed in feed restricted heifers (Yambayamba et al., 1996). Heifers in this study were on a lower plane of nutrition befor e the start of this trial, and despite a 14 d adaptation period, the greater NEFA concentrations on d 0 suggest that may have still been in a negative energy state. Yambayamba et al. (1996) reported the metabolic shift that occurs after realimentation take s up to 10 d. While we did begin adaptation to DDG supplementation over a 14 d peri od, the feeding amount (1.81 kg heifer 1 d 1 ) was lower than any amount given during the experimental supplementation period, and could still possibly
70 not be enough suppleme ntation to lower NEFA concentrations and in part could explain why the response among treatments is similar on d 0. It should be noted that NEFA concentrations on d 0 and throughout the supplement period were well below values reported in studies in which mobilization of fat or a negative energy balance were observed (Yelich et al. 1995; Yambayamba et al., 1996). Monari (2010) reported a peak NEFA concentration of 526 mEq/ml after 70 d of RBS feeding in heifers on a diet formulated for low gain (0.28 kg/d ). In contrast, heifers fed RBS and supplemented with DDG to achieve a consistent gain of 0.84 kg/d had peak NEFA concentrations of 347 mEq/ml (Monari, 2010). Concentrations of NEFAs were similar among treatments, this is further evidence to support the similarities among treatments for BCS, BW or ADG observed throughout d 140, indicating all three diets were able to meet growth requirements of these heifers. Brangus heifers (342 mEq/ml) had greater ( P 0.05) mean NEFA concentrations compared to Angus heifers (284 mEq/ml) similar to a report by Monari (2010) in Angus and Brangus heifers fed RBS and supplemented with a comparable amount of DDG. A ddtionally, Obeidat et al. (2002 ) reported that Brahman co ws during three different physiological states (late gestation, early and late lactation) had greater NEFA concentrations compared to A ngus cows. Obeidat et al. (2002 ) proposed a different mechanism exists in adipose tissue metabolism between Brahman and Angus cattle. Plasma urea nitrogen (PUN) concentrations are presented in Figure 3 2. Mean PUN concentrations (30.80 mg/dL) did not differ ( P > 0.05) between treatments from d 0 to d 140. There were no breed, treatment breed, or time treatment time treatment breed effects ( P > 0.05) or contrasts of DDG vs. DDG+7.5 and DDG+15 and
71 DDG+7.5 vs. DDG+15 ( P > 0.05). However, there were time and time breed effects ( P 0.05). Concentrations of PUN of all three treatments were greater compared to thos e observed in similar studies in heifers fed high RUP in the form of blood meal (Lalman et al., 1993), and high RUP in the form of DDG (Monari, 2010). In the present study, protein was overfed for all treatments, which likely contributed to the greater in PUN concentrations. Dhuyvetter et al. (1993) reported feeding high levels of RUP, which is provided in high amounts from DDG, is shown to increase PUN concentrations due to deamination of excess MP. By d 140, the DDG heifers were receiving 2.53 kg heifer 1 d 1 of DDG which provided 0.65 kg CP heifer 1 d 1 the DDG+7.5 heifers were receiving 2.43 kg heifer 1 d 1 of DDG and 0.17 kg heifer 1 d 1 of SBM which provided 0.70 kg CP heifer 1 d 1 and the DDG+15 heifers were receiving 2.33 kg heifer 1 v d 1 o f DDG and 0.35 kg heifer 1 d 1 of SBM which provided 0.77 kg CP heifer 1 d 1. Suggested ranges of 11 to 15 mg/dL (Byers and Moxon, 1980) and 8 to 12 mg/dL (Hammond et al., 1994) of PUN for maximizing animal performance have been described, the higher PUN concentrations observed in our study probably indicate an energy imbalance. Based on ultrasound measurements and animal performance measurements, the PUN values do not reflect a situation where the heifers are catabolizing body stores. Additionally, PUN concentrations were higher from d 80 to d 140 compared to the first 70 d of the supplement period. All three diets provided high levels of protein, and increasing PUN values could be indicative of an accumulation of protein in the blood due to increased d ietary levels of protein as diets were adjusted to increasing BW over time. Similar responses were observed in heifers fed DDG SBM diets where PUN values increased after d 84 of feeding (Austin, 2009).
72 Plasma glucose concentrations are presented in Figur e 3 3. Mean plasma glucose concentrations (95.08 mg/dL) were not different ( P > 0.05) across treatments from d 0 to d 140 of the experiment. There were time and time treatment effects ( P 0.05) observed. There were no ( P > 0.05) tre atment breed, time breed, time treatment breed effects or contrasts of DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 observed The glucose concentrations observed here are greater than what is o bserved in the literature for animals consuming DDG and warm season forages (Monari, 2010; Wahrmund et al., 2011), however, concentrations are similar to those observed in steers grazing winter wheat pasture (Hersom et al., 2004). Also, Brangus heifers (98 .11 mg/dL) tended ( P = 0.06) to have increased glucose concentrations compared to Angus heifers (92.05 mg/dL). These results are consistent with observations that cattle with Bos indicus breeding have greater glucose concentrations compared to Angus cattl e (Alvarez et al., 2000; Williams et al. 2002; Obeidat et al., 2002). Reproductive Performance Four heifers were pubertal before the beginning of the experimental period and they were excluded from the puberty analysis. The percentage of heifers that we re pubertal (29.1%; n = 55) at the start of the breeding season (d 140) was not different ( P > 0.05) between treatments. Additionally, treatment and treatment breed had no effect ( P > 0.05) on rate of heifers attaining puberty during 168 d that pubertal status was monitored (Figure 3 4). The fact that such a low number of heifers were pubertal at the start of the breeding season is intriguing. All heifers were greater than 65% of their mature BW (based on a 545 kg cow) on d 140 suggesting that BW was a dequate to attain puberty. Also, BCS average d 5.5 or greater in these heifers; suggesting body
73 composition would not hinder attainment of puberty. Martin et al. (2007) developed composite Bos taurus heifers on prairie hay and supplemented with DDG fed at 0.59% BW to gain 0.68 kg/d and observed 77.2% of heifers had obtained puberty by the start of the breeding season. These heifers were on average 358 days old and 329 kg, younger and lighter than those in this trial (399 days old and 357 kg at d 140 ) despi te being fed DDG at similar rates. There tended ( P = 0.06) to be a greater percentage of Brangus heifers (41.0%; n = 26) that attained puberty at the start of the breeding season compared to Angus heifers (17.4%; n = 29; data not shown). Additionally bre ed tended ( P = 0.07 ) to effect rate of attaining puberty during the 168 d pubertal status was monitored (Figure 3 5). Brangus heifers began to attain puberty sooner throughout the experiment, and more total were pubertal. Purebred and crossbreed cattle of Bos indicus breeding typically reach puberty at older ages than those of Bos taurus breeding (Plasse et al., 1968; Nelson et al., 1982; Dow et al., 1982), which contradict s observations in the present study. There was a tendency for mean glucose concen trations across the supplementation period to be greater in Brangus heifers compared with the Angus heifers. Glucose has been positively associated with rate of steriodogenesis and gonadotropin synthesis and secretion (Lynn et al., 1965; Sen et al., 1979) Hileman et al. (1991) demonstrated in lambs that availability of metabolic fuels such as glucose may influence reproductive activity via effects on pulsatile LH secretion. Perhaps of LH secretory patterns are decreased in the Angus heifers, which impact ed their timely ability to attain puberty.
74 There were no ( P > 0.05; Table 3 6) treatment, breed, treatment breed effects on phase 1 estrous response, first service conception rate, first service AI pregnancy rate, or final AI pregnancy rate. Contrasts between DDG vs. DDG+7 and DDG+15 and DDG+7 vs. DDG+15 were not different ( P > 0.05). We recognize that interpretation of this data is limited due to the small number of heifers, which responded to the first phase synchronization. However, the small numb er of heifers that responded is predominantly due to most heifers not attaining puberty by the beginning of the breeding season. There was a significant effect of pubertal status ( P (data not shown). Of the heifers that were p ubertal, 52.1% (n = 16) displayed estrus compared to 20.5% (n = 39) of the non pubertal heifers. Pubertal status also affected ( P pregnant to either the first or second AI com pared to 10.7% of the non pubertal heifers Phase 2 breeding season results are presented in table 3 7. There were no ( P > 0.05) treatment, breed, or treatment breed effects. Contrasts between DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 were not different ( P > 0.05). Pubertal status at d 168 was calculated only from the number of heifers that received synchronization in phase 2 (30.8%; n = 12/39), and was not affected by breed or treatment ( P > 0.05). Pubertal status did not affect ( P > 0.05) es trous response, conception rate, or synchronized pregnancy rate in phase 2. This is likely due to the addition of a CIDR in the second synchronization protocol. Progestogens, like those found in CIDR, have been shown to induce estrous cycles by providing a priming effect to the reproductive system (Short et al., 1976; Anderson et al., 1996; Imwalle et al.,
75 synchronization. This could be a reflection of the fertility of the ovu lation induced from the synchronization protocol. The heifers, which were induced to cycl e by the CIDR, would have ovulated a follicle from their first pubertal cycle. Studies have illustrated that conception rate of heifers bred at their first spontaneou s estrus were 21% lower than those bred on their third estrus (Byerly et al. 1987; Perry et al., 1991). There were no ( P > 0.05) differences observed among DDG (95%), DDG+7.5 (79%), or DDG+15 (80%) for final pregnancy rate, which included pregnancy rates f rom the entire 77 d breeding season. There were also no ( P > 0.05) breed, treatment, or treatment breed effects on final pregnancy rate (Appendix B). Contrasts between DDG vs. DDG+7 and DDG+15 were also not significant. Treatment, breed, and treatment breed did not a ffect ( P > 0.05) the rate of conception during the 77 d breeding season (Figure 3 7). In summary, addition of SBM (a source of RDP) at either 7 or 15% of the total supplement had no beneficial effects on animal performance or reproduction While the heifers supplemented with DDG alone had an average 1 1 RDP balance with a range of 1 1 1 this does not appear to have had a negative impact on animal performance. Perhaps, the over supplementa tion of total CP in the diet compensated for this small deficiency in RDP. Rumen undegradable protein may indirectly supply RDP via the urea cycle as well (Archibeque et al., 2008). The addition of SBM did provide a positive RDP balance as intended but did not appear to enhance characteristics of growth, body composition, or reproduction. Perhaps some of the positive effects of meeting or exceeding RDP in the diet, such as forage utilization and animal performance observed in other studies (Owens, et al ., 1991; Bandyk et al., 2001) are due to feeding lower quality forages, whereas the RBS in this study was of
76 medium quality. Additionally, the TDN:CP for the RBS was 4.28. Moore et al. (1999) stated that forage TDN:CP was < 7 and animals were supplemente d voluntary forage intake was decreased. This could be additional reason why animal performance did not differ across treatments. Brangus and Angus heifers when fed similarly had no difference in BW, ADG, or BCS, but grew to different heights and had onl y slight differences in body composition by the end of the supplementation period. Treatments had no effect on percentage of heifers that attained puberty at the start of the breeding season, however overall reproductive performance was less than anticipa ted even though the diet contained adequate TDN and CP to meet the nutrient requirements of growing heifers. H eifers managed under this nutritional system and in this environment had trouble attaining puberty by the start of the breeding season despite ma intaining a target ADG and meeting target BW by the start of breeding, which held further consequences that persisted throughout the synchronized breeding season. Although, breeding season pregnancy rates were similar between breeds and treatments. Implic ations Addition of SBM to compensate for a RDP deficiency in diets growing heifers consuming medium quality forage supplemented with DDG provide d no additional benefit to growth performance. Therefore, DDG can function as single source ingredient in suppl ementation diets for yearling heifers when consuming bermudagrass RBS
77 Table 3 1. Nutritional composition of dried distillers grain (DDG), soybean meal (SBM), bermudagrass round bale silage (RBS), and pasture offered to yearling Angus and Brangus heifers throughout the experiment. Item DDG SBM RBS Pasture 1 DM % 91.5 0 .00 91.0 0.01 44.2 0.12 49.1 0.21 DM basis CP, % 25.6 0.02 48.8 0.03 12.1 0.03 11.7 0.19 RDP, % CP 48.0 0.00 66.0 0.00 69.0 0.02 IVDMD, % 77.7 0.04 94.2 0 .01 44.7 0.07 42.8 0.19 TDN, % 81.3 0.01 78.0 0.00 60.3 0.02 51.4 0.18 Sulfur, % 0.4 0.03 0.4 0.01 0.2 0.23 1 Dormant mixture of bahiagrass and bermudagrass forage.
78 Table 3 2. Amount of rumen degradable protein (RDP) and rumen und egradable protein (RUP) provided in supplement diets throughout the supplementation period. Item RDP, kg heifer 1 d 1 RUP, kg heifer 1 d 1 DDG 1 0.12 0.17 0.35 0.48 DDG+7.5 2 0.15 0.22 0.35 0.49 DDG+15 3 0.20 0.27 0.37 0.50 1 DDG: Supplement at 0.75% BW w ith dried distillers grain (DDG) 2 DDG+7.5: Total supplement at 0.75% BW with DDG plus 7.5% of supplement soybean meal (SBM) 3 DDG+15: Total supplement at 0.75% BW with DDG plus 15% of supplement SBM
79 Table 3 3. Growth characteristics on d 0 and 140 of t he experiment for heifers consuming round bale silage supplemented with dried distillers grain (DDG) or DDG plus soybean meal at two amount s (LS means SE). 1 Day of Experiment Item 0 140 BW, kg DDG 3 DDG+7.5 4 DDG+15 5 232 13 233 13 249 13 346 17 358 17 366 17 BCS 2 DDG DDG+7.5 DDG+15 5.1 0.1 5.0 0.1 5.2 0.1 5.5 0.1 5.6 0.1 5.8 0.1 Hip height, cm DDG DDG+7.5 DDG+15 111 1.8 11 3 1.8 11 3 1.8 12 1 1.4 122 1.5 12 1 1.4 1 Treatment ( P > 0.05); Contrast DDG v s. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 ( P > 0.05) 2 BCS: 1 = severely emaciated; 5 = moderate, 9 = obese 3 DDG: Supplement at 0.75% BW with dried distillers grain 4 DDG+7.5: Total supplement at 0.75% BW with DDG plus 7.5% of supplement soybean meal (SBM) 5 DDG+15: Total supplement at 0.75% BW with DDG plus 15% of supplement SBM
8 0 Table 3 4. Body ultrasound measurements on d 0 and 140 of the experiment for heifers consuming round bale silage supplemented with dried distillers grain (DDG) or DDG pl us soybean meal at two amount s (LS means SE). 1 Day of Experiment 0 140 IMF 2 % DDG 7 DDG+7.5 8 DDG+15 9 3.85 0.24 4.10 0.25 3.64 0.24 3.91 0.21 4.01 0.22 3.85 0.21 RIBFT 3 cm DDG DDG+7.5 DDG+15 0.39 0.02 0.35 0 .02 0.39 0.02 0.53 0.03 0.47 0.03 0.53 0.03 RMPFT 4 cm DDG DDG+7.5 DDG+15 0.35 0.02 0.31 0.02 0.36 0.02 0.60 0.03 0.55 0.03 0.57 0.03 REA 5 cm 2 DDG DDG+7.5 DDG+15 42.03 2.77 42.22 2.78 45.00 2.77 57.16 3.50 57.29 3. 51 61.48 3.50 REA/cwt 6 cm 2 DDG DDG+7.5 DDG+15 17.96 0.64 18.06 0.65 17.91 0.64 16.72 0.52 16.28 0.52 17.09 0.52 1 Treatment ( P > 0.05) for all variables, Contrast DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 ( P > 0.05) 2 IMF: intr amuscular fat in LM 3 RIBFT: 13 th rib subcutaneous fat thickness 4 RMPFT: rump subcutaneous fat thickness 5 REA: LM area 6 REA/cwt: REA adjusted for 100 kg of BW 7 DDG: Supplement at 0.75% BW with dried distillers grain 8 DDG+7.5: Total supplement at 0.75 % BW with DDG plus 7.5% of supplement soybean meal (SBM) 9 DDG+15: Total supplement at 0.75% BW with DDG plus 15% of supplement SBM
81 Table 3 5. Body ultrasound measurements on d 0 and 140 of the experiment for Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) or DDG plus soybean meal at two amount s (LS means SE). Day of Experiment Item 0 140 IMF 1 % Angus Brangus 4.66 0.20 x 3.07 0.20 y 4.79 0.17 a 3.06 0.18 b RIBFT 2 cm Angus Brangus 0. 36 0.02 0.39 0.02 0.51 0.02 0.51 0.02 RMPFT 3 cm Angus Brangus 0.33 0.02 0.35 0.02 0.54 0.02 x 0.61 0.03 y REA 4 cm 2 Angus Brangus 40.78 2.26 45.38 2.27 54.34 2.86 x 62.94 2.86 y REA/cwt 5 cm 2 Angus Brangus 17.83 0.53 18.13 0.53 15.83 0.42 a 17.57 0.43 b a,b Breed means within same item in a column with different superscript differ ( P 0.05) x,y Breed means within same item in a column with different superscript differ ( P = 0.08) 1 IMF: intramuscular fat in LM 2 RIBFT: 13 th rib subcutaneous fat thickness 3 RMPFT: rump subcutaneous fat thickness 4 REA: LM area 5 REA/cwt: REA adjusted for 100 kg of BW
82 Figure 3 1. NEFA concentrations by treatment breed and day of experiment for yearling Angus and Brangus heifers consuming round bale silage and supplemented with dried distillers grain (DDG) or DDG plus soybean meal (SBM) at two amount s. DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supp lement at 0.75% BW plus 15% SBM). Treatment (P > 0.05), time breed (P = 0.09), time, breed, time treatment, time breed treatment (P 0.05). 0 100 200 300 400 500 600 0 14 28 42 56 70 84 98 112 126 140 AN DDG BN DDG AN DDG+7.5 BN DDG+7.5 AN DDG+15 BN DDG+15
83 Figure 3 2. Plasma urea nitrogen (PUN) concentrations by breed and day of experiment for yearling Angu s and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amount s. DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). Treatment, breed, treatment breed, time treatment time treatment breed (P > 0.05) t ime, time breed and breed (P 0.05).
84 Figure 3 3. Glucose concentrations by treatment and day of experiment for yearling Angus and Brangus heife rs consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amount s. DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM).Treatment, treatment breed, time breed, time breed treatment (P > 0.05) t ime, time treatment (P 0.05) 60 70 80 90 100 110 120 130 0 14 28 42 56 70 84 98 112 126 140 Glucose, mg/dL Day of Experiment DDG DDG+7.5 DDG+15
85 Figure 3 4. Survival curve for proportion of non pubertal heifers by treatment across day of experiment for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean m eal (SBM) at two amounts. DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75 % BW plus 15% SBM). The effect of treatment on rate of attainment of puberty (P > 0.05; adjusted hazard ratio DDG+7.5 = 2.0, 95% CI = 0.70 to 5.87; adjusted hazard ratio DDG+15 = 1.85, 95% CI = 0.63 to 5.4). 0 10 20 30 40 50 60 70 80 90 100 0 25 50 75 100 125 150 Proportion of non pubertal heifers Day of Experiment DDG DDG+7.5 DDG+15
86 Figure 3 5. Survival curve for proportion of non pregnant heifers by breed across day of experiment for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amounts. DDG (supplement at 0.75% BW), DDG +7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). The effect of breed on rate of attainme nt of puberty ( P = 0.07 ; adjusted hazard ratio = 2.02, 95% CI = 0.95 to 4.31 ). 0 10 20 30 40 50 60 70 80 90 100 0 25 50 75 100 125 150 Proportion of non pubertal heifers Day of Experiment Angus Brangus
87 Figure 3 6 Survival curve for proportion of non pregnant heifers by treatment across day of breeding season for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amounts. DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). DDG (supplement at 0.75% BW), DDG+7.5 (supplement at 0.75% BW plus 7.5% SBM), DDG+15 (supplement at 0.75% BW plus 15% SBM). The effect of treatment on rate of pregnancy during the breeding season ( P > 0.05 ; adjusted h azard ratio DDG+7.5 = 0.86 95% CI = 0.42 to 1.77 ; adj usted hazard ratio DDG+15 = 0.89, 95% CI = 0.44 to 1.78 ). 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 Proportion of non pregnant heifers Day of the Breeding Season DDG DDG+7.5 DDG+15
88 Table 3 6. Estrous response, first service conception rate, first service AI pregnancy rate, and final AI pregnancy rate from phase 1 of the breeding season for yearling Angus and Brangus heifers consuming round bale silage supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amount s. 1 Item Estrous response, % 2 First service conception rate, % 3 First service AI pregnancy rate, % 4 Final AI pregnancy rate, % 5 DDG 6 20 (4/20) 7 5 (3/4) 15 (3/20) 15 (3/20) DDG+7.5 7 39 (7/19) 57 (4/7) 21 (4/19) 27 (5/19) DDG+15 8,9 40 (8/20) 63 (5/8) 25 (5/20) 35 (7/20) Angus 20 (6/30) 50 (3/6) 10 (3/30) 13(4/30) Brangus 46 (13/29) 69 (9/13) 31 (9/29) 38 (11/29) P value Treatment Breed Trea tment Breed 0.70 0.27 0.70 0.96 0.71 0.96 0.86 0.21 0.86 0.66 0.20 0.83 1 Heifers estrous detected for 7 d followed by 2 doses PG (25 mg i.m.) 12 h apart followed by 31 d of estrous detection. Allowed two AI if returned to estrus during 38 d breedi ng period. 2 Percentage of heifers displaying one estrus out of the total treated. 3 Percentage of heifers pregnant to AI of the total that exhibited estrus once. 4 Percentage of heifers pregnant to first service AI. 5 Percentage of heifers pregnant to f irst and second AI during 38 d breeding period. 6 DDG: Supplemented at 0.75% BW 7 DDG+7.5: Total supplement at 0.75% BW plus 7.5% of supplement SBM 8 DDG+15: Total supplement at 0.75% BW plus15% of supplement SBM 9 Contrast DDG vs. DDG+7.5 and DDG+15 and D DG+7.5 vs. DDG+15 ( P > 0.05)
89 Table 3 7. Estrous response conception rate, timed AI pregnancy rate, and synchronized pregnancy rate from phase 2 of the breeding season for yearling Angus and Brangus heifers consuming round bale silage supplemented with dri ed distillers grain (DDG) alone or DDG plus soybean meal at two amount s. 1 Item Estrous response, % 2 Conception rate, % 3 Timed AI pregnancy rate, % 4 Synchronized pregnancy rate, % 5 DDG 6 75 (13/16) 43 (6/13) 0 (0/3) 38 (6/16) DDG+7.5 7 69 (8/12) 52 (4/8) 25 (1/4) 46 (5/12) DDG+15 8,9 58 (7/12) 55 (4/7) 0 (0/5) 32 (4/12) Angus 76 (19/24) 54 (10/19) 20 (1/5) 46 (11/24) Brangus 58 (9/16) 46 (4/9) 0 (0/7) 32 (4/16) P value Treatment Breed Treatment Breed 0.65 0.27 0.29 0.93 0.78 0.53 0.77 0.52 0.52 0.88 0.52 0.61 1 Heifers synchronized using Select Synch/CIDR + timed AI: 100 g (i.m) of GnRH and a CIDR were administered on d 168, seven days later, heifers received 25 mg PG (i.m.) and CIDR were removed. Estrus was detected utilizing the HeatWatch sy stem for 72 h after CIDR removal and heifers were AI 8 to 12 h after the onset of estrus. Heifers not exhibiting estrus by 72 h were administered 100 g (i.m.) GnRH and timed AI at 72 to 76 h after PG. 2 Percentage of heifers displaying estrus out of the t otal treated. 3 Percentage of heifers pregnant to AI of the total that exhibited estrus. 4 Percentage of heifers pregnant to the timed AI 5 Percentage of heifers pregnant to synchronization protocol. 6 DDG: Supplement at 0.75% BW with dried distillers gra in 7 DDG+7.5: Total supplement at 0.75% BW with DDG plus 7.5% of supplement soybean meal (SBM) 8 DDG+15: Total supplement at 0.75% BW with DDG plus 15% of supplement SBM 9 Contrast DDG vs. DDG+7.5 and DDG+15 and DDG+7.5 vs. DDG+15 ( P > 0.05)
90 CHAPTER 4 E FFECT OF AMOUNT OF INCLUSION OF DRIED DISTILLERS GRAIN SUPPLEMENT ON ADAPTATION, INTAKE, DIGESTIBILITY AND RUMEN PARAMETERS IN STEERS CONSUMING BERMUDAGRASS ROUND BALE SILAGE. Introduction Beef cattle production in the Southeastern United States is almost 100% dependent upon grass based systems (Burns, 2006), and the majority of these grasses are perennial grasses of tropical or sub tropical origin. Characteristically, tropical forages have increased annual DM yield, but lower feeding value compared to tem perate forages (Skerman and Riveros, 1990). In addition, between November and April these grasses are dormant or have decreased productivity. In these instances alternatives to grazing must be sought whether that be in the form of conserved forages, such as round bale silage (RBS), supplements, or both. Increased demand for ethanol production has led to an increase in availability of dried distillers grains (DDG). Dried distillers grains consist of 27 to 36% CP (24 to 40% RDP), 29 to 39% NDF, 78 to 88% TDN and 9 to 16% fat (Dairy One, 2012 ), and therefore can provide both energy and protein which may be deficient in forage based diets. Leupp et al. (2009) reported that feeding DDG up to 1.2% BW to beef cattle consuming moderate quality brome hay (10.6% C P) had no adverse effects on forage digestion or ruminal fermentation a lthough, hay intake decreased linearly with increasing amount s of DDG while total diet intake increased linearly. However, little is known about the relationship of amount of DDG in t he diet and metabolic and rumen parameters of cattle consuming sub tropical forages. Therefore we hypothesized that DDG supplementation of steers consuming bermudagrass RBS would not adversely
91 affect total diet digestion and rumen function, but would decr ease forage intake and increase total intake of the diet. The objectives of these experiments were to evaluate the response of blood metabolites, forage intake, and rumen parameters to an adaptation diet of bermudagrass RBS supplemented with DDG and to ev aluate the effect of amount of DDG supplementation on forage intake, digestibility, and rumen parameters in steers consuming bermudagrass RBS. Materials and Methods Two experiments were conducted at University of Florida Department of Animal Sciences, Gai nesville, FL, from October until December 2009. The research was divided into two sequential experiments: adaptation (d 3 to 15) and digestibility (d 0 to 84). The experiments were conducted in accordance with acceptable practices as outlined in the Guid e for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999) and University of Florida IFAS Non regulatory Animal Research System protocol number 011 09ANS. Animals Ruminally cannulated Angus (n = 4) and Brangus (n = 4) steers (age 2 to 4 yr, 500 kg 100 kg) and Tifton 85 bermudagrass ( Cynodon dactylon ) round bale silage (RBS) were utilized for both experiments. Individual bales were ground to a 7.5 to 15 cm chop length using a bale grinder and stored for < 7 d in a co vered barn until feeding. Visibly spoiled RBS was disposed of. For both experiments steers had ad libitum access to water and a custom mineral vitamin premix was mixed into the dried distiller grain (DDG) and fed daily at a rate of 57 g steer 1 d 1 (appe ndix A).
92 Adaptation Experiment Treatments Twenty eight days prior to beginning of the adaptation experiment, steers were stalled individually and randomly assigned to a pen (9.1 2.4 m) for acclimation to the pens and fed a ground bermudagrass hay diet. Hay was ground in a similar fashion to the RBS as previously described. Nine days prior to initiation of the adaptation experiment steers began adaption to RBS diets and RBS was offered 110% of the approximately 75% of the RBS fed at 0730 and 25% of the RBS fed at 1900 h. The adaptation experiment diets were designed to simulate a 14 d step up adaption to a concentrate diet. Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg o f DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15 Any feed refusal was collected and recorded daily. Sample collection and analysis Full BW were obtained on d 3. Sample and feed collection coincided with diet changes wi th d 2 and 1 representing RBS only diets. Samples of DDG and RBS were collected, weighed, and dried at 60C for 72 h and stored for further analysis. Rumen fluid was collected at 0, 3, 6, 12, 24, and 36 h and blood samples were collected at 0, 6, 12, 24 and 36 h after new diets were first offered at the AM feeding. Rumen samples were collected from the ventral sac of the rumen and removed through the rumen fistula. Approximately 100 ml of rumen fluid was obtained, directly filtered through 2 layers of cheesecloth, and pH was measured immediately using a pH meter (Denver Instrument, model UP 5, Denver Instrument Company, Denver, CO). Rumen fluid was stored in duplicate 50 mL polypropylene conical centrifuge tubes and placed on ice until
93 they could be f rozen ( 20C) and stored for subsequent analysis. The concentration of NH 3 N in ruminal fluid was determined using the Alpkem RFA 300 Rapid Flow Analyzer and an adaptation of the Noel and Hambleton (1976) procedure that involved colorimetric quantificatio n of N. Blood samples were collected via jugular venipuncture into 6.0 mL polypropylene syringes containing 10.8 mg potassium EDTA as an anticoagulant (BD Vacutainer, BD Diagnostics, Franklin Lakes, NJ). Samples were placed on ice immediately after colle ction and transported to the lab for further processing. Blood samples were centrifuged at 3,000 g for 15 min at 5 C to obtain plasma, which was placed in sample vials and frozen at 20 C for subsequent analysis. Plasma urea nitrogen concentrations wer e determined using BioAssay Systems QuantiChrom Urea Assay Kit series DIUR 500 kit (BioAssays Systems, Hayward, CA). Plasma glucose concentrations were determined using Cayman Chemical Co. Glucose Analysis kit (Cayman Chemical Co., Ann Arbor, MI). Plasma NEFA concentrations were determined using Wako HR Series NEFA HR kit (Wako Diagnostics, Richmond, VA). The intra and inter assay CV were 8.6% and 6.4% for NEFA, 6.8% and 19.5% for PUN, and 3.9% and 10.1% for glucose, respectively. Dried samples of RBS a nd DDG were ground to pass through a 1 mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA). Samples were analyzed for residual DM and OM (AOAC, 2007). Total nitrogen was determined by the combustion method using a macro N analyzer (Elementar Vario Max CN instrument, Elementar Americas, Mount Laurel, NJ, USA) and used to calculate CP (N x 6.25). In vitro dry matter digestibility (IVDMD) of RBS, DDG, and orts was determined using an
94 ANKOM DAISY II incubator (ANKOM Technology Corp., F airport, NY) using the ANKOM Technology Method for In Vitro Digestibility Rumen fluid inoculum for this procedure was obtained from a ruminally fistulated, non lactating Holstein cow consuming a diet of ad libitum bermudagrass hay and 450 g soybean meal daily. Total digestible nutrient concentration was calculated for RBS using a formula for warm season grasses described by Fike et al. ( 2003) whereas TDN values for DDG were analyzed by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). Nutritional composition for DDG and RBS are presented in Table 4 1. Statistical analysis Dry matter intake was analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The model statement contained the effect of level of DDG. For all variabl es breed was initially included in the model, but was removed and BW was included as a covariate to control for confounding effects of breed and BW. Steer was utilized as the random statement. Rumen pH and NH 3 N and blood metabolite concentration were an alyzed with the MIXED procedure of SAS. The model statement contained the effect of level and hour(level) and BW was also utilized as a covariate. The interaction of level and hour was not included in the analysis because comparisons of hours across leve ls were not of interest. Data were analyzed as repeated measures using steer as the random statement and steer(level) as the subject. The appropriate covariance structure of the data was chosen for each analysis from the structures of autoregressive one (AR(1)), heterogeneous (ARH(1)), and Heterogeneous TOEP (TOEPH) as these were the most appropriate for the data set. Information Criterion (BIC) were utilized to select for the best fit model
95 For all analysis, mean comparisons were made using the PDIFF statement associated with generation of least square means. Results are reported as least square means, significance was set as P clared if P > 0.05 and Digestibility Experiment Treatments An 8 x 4 latin square design with 4 periods (n = 8 for each treatment) was utilized. Steers were housed in the same facilities and assigned to pens similarly to the adaptation experiment. On d 0 of the experiment, steers were assigned to 1 of 4 dietary treatments: 1) Tifton 85 bermudagrass round bale silage (RBS); 2) RBS + DDG supplement offered at 0.33% BW (RBS+0.33); 3) RBS + DDG supplement offered at 0.66% BW (RBS+0.66); 4) RBS + DDG supplement offered at 1% BW (RBS+1). Steers received the supplement at 0730 h and RBS was offered at intake divided into two daily feedings with approximately 75% of the RBS fed at 0730 and 25% of th e RBS fed at 1900 h. Sample collection and analysis Each period was 21 d in length and consisted of an 11 d diet adaptation, a 2 d fecal bag adaptation period, a 5 d data collection period for total intake, total fecal output, rumen fluid, and feed samplin g, and a 3 d in situ degradation period. Full BW were obtained on the first day of each period and at the conclusion of the entire trial. Rumen s amples were taken on d 17 of each period and collected from the ventral sac and removed through the rumen fist ula hourly for 12 h beginning 2 h before feeding of supplement (0530 h). Collecting, processing, and storage of rumen samples were conducted as described for the adapta tion experiment. Steers were fitted with
96 custom canvas feces collection bags for the 5 d total fecal collection. Fecal collection bags were emptied twice daily (0700 and 1900 h), weighed for calculation of total daily fecal output, and a 10% subsample was obtained. Feces subsamples were dried at 60C for 72 h in a forced air oven, and sto red for subsequent analysis. Feces samples were pooled by day for analysis. Samples of DDG and RBS were obtained daily during the 5 d sample collection period and daily refusals were also collected. All samples were weighed and dried at 60C for 72 h the n stored for further analysis. Dried samples of RBS, DDG, orts, and feces were ground to pass through a 1 mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA, USA). Samples were analyzed for residual DM and OM, CP, IVDMD of RBS, DDG, an d orts, and TDN of RBS and DDG using previously described protocols for the adaptation experiment Nutritional composition of RBS and DDG are presented in Table 4 1. Total intake was measured during the 5 d sampling period and utilized with total fecal o utput to calculate total tract apparent DM digestibility (TTADMD). Apparent DM digestibility was calculated using the mean daily DMI and the mean daily fecal DM output (FO) using the equation: DM digestibility = (DMI FO)/DMI. Statistical analysis Dry matter intake, FO, and TTADMD were analyzed using the MIXED procedure of SAS. The model statement contained the effect of treatment, period, treatment period For all variables breed was initially included in the model, but was removed and BW was inclu ded as a covariate to control for confounding effects of breed and BW. Steer was utilized as the random statement. Polynomial orthogonal contrasts were used to test for linear, quadratic, and cubic effects of increasing supplemental DDG amount.
97 Rumen pH and NH 3 N were analyzed with the MIXED procedure of SAS. The model statement contained the effect of treatment, period, hour, treatment period, treatment hour, and BW was also utilized as a covariate. Data were analyzed as repeated measures using ste er as the random variable and steer(treatment period) as the subject. The appropriate covariance structure of the data was chosen for each analysis from the structures of autoregressive one (AR(1)), heterogeneous (ARH(1)), and Heterogeneous TOEP (TOEPH) as these were the most appropriate for the data set. were utilized to select for the best fit model For all analysis, mean comparisons were made using the PDIFF statemen t associated with generation of least square means. Results are reported as least square means, significance was set as P de clared if P > 0.05 and Results and Discussion Adaptation Experiment Effect of level of DDG offered on intake is presented in Table 4 2. Dry matter intake of RBS was not affected ( P > 0.05) by level of DDG supplementa tion. However, there was a response ( P As DDG level increased, total DMI increased. The increase in total DMI in this study is a reflection of increasing levels of DDG, as RBS DMI did not change. In contrast, steer s offered a medium quality hay and supplemented up to 1.2% BW of DDG, had a linear decrease in hay intake (Leupp et al., 2009). Likewise, Loy et al. (2007) observed a decrease in grass hay DMI supplemented with DDG compared to non supplemented steers. We acknowledge the Leupp et al. (2009) and the Loy et al. (2007) were not designed as
98 adaptation trials, but contain results, which are the closest possible comparisons in the literature. In the current study it is possible that the effect DDG had on RBS DM I required more time to emerge, as feeding amounts were changed on 4 d intervals and intake data was only collected during that period of time. Therefore, steady state intake may not have been achieved. There was an effect ( P ed and hour within level of DDG offered on rumen pH (Table 4 3). Mean ruminal pH decreased as DDG offered increased. Mean pH when no DDG was supplemented was higher (6.44) than mean pH for any other level of DDG offered, with steers supplemented at 4.52 kg having the lowest mean pH (6.02). By h 6 after supplementation ruminal pH concentrations from any diets with DDG reached a minimum, however, steers consuming RBS alone had the lowest pH at h 12 When steers were consuming RBS alone, the pH was the mos t stable across the 36 h and was never < 6.3 7 Hour 24 collection occurred just prior to supplementation on the 2nd day of steers receiving a particular level of DDG and is reflective of h 0 pH where the pH rebounded to similar concentrations. Loy et al. (2007) reported that heifers supplemented with DDG had decreased mean pH compared to unsupplement ed heifers. Additionally, Calla way et al. (2010) reported a decreased ruminal pH in cattle consuming a 50% DDG ration compared with cattle consuming no DDG i n the ration. However, in the current experiment it does not appear that decreases in pH were profound enough to negatively impact forage intake during this time frame. T here was an effect ( P DDG offered on rumen NH 3 N ( Table 4 4 ) No differences ( P > 0.05) were observed for
99 mean rumen NH 3 N across the 36 h sampling for steers consuming 0, 1.13, or 2.26 kg of DDG (20.7, 20.8, 21.4 mg/dL, respectively) but concentrations were decreased ( P 0.05) compared to steers consuming 3.39 and 4.52 kg (29.6 and 34.8 mg/dL, respectively) Other than a latent peak from steers consuming 3.39 kg at h 6, all other levels of rumen NH 3 N peaked at h 3 po st supplementation, and NH 3 N concentrations decreased following the initial peak. In animals fed diets with plant proteins at high levels, rumen NH 3 N peaks are usually observed 3 to 5 h post feeding (Owens and Zinn, 1988). R umen NH 3 N concentrations ra ng ing from 5 to 29 mg/dL has been reported as optimum for maximum microbial growth (Satter and Slyter, 1974; Miller 1973) and 23.5 mg/dL for maximum rate of fermentation (Mehrez et al.,1977). Values observed in the present study are well below toxic levels enough to not have a negative impact on rate of digestion or feed intake (Owens and Zinn, 1988). Level of DDG offered ( P = 0.08) tended to a ffect NEFA concentrations and there was an effect ( P G offered on NEFA concentrations (Table 4 5). Steers consuming 0 and 1.13 kg of DDG had greater NEFA mean concentrations ( 300.0 and 308.5 mEq/ml respectively ; P offered 2.26 kg (242 .2 mEq/ml), but w ere not different ( P > 0.05) compared to steers consuming RBS alone 3.39, or 4.52 kg (300 .0, 263.1, 261.1 mEq/ml respectively ). For steers consuming 0, 2.26, 3.39, and 4 .52 kg of DDG a similar pattern for NEFA concentrations occurred where h 0 was highest and by 12 h post feeding t he lowest concentrations were observed, then concentrations increased again by h 24 Likewise, in general, NEFA concentrations were similar for h 12 and h 36 as they represent the
100 sa me time after supplementation was offered. When Blum et al. (2000) fed l actating cows diets containing free fatty acids, crystalline triglycerides or starch NEFA concentrations declined after the morning feeding. Additionally, Nikkhah et al. (2008) observed a decrease in plasma NEFA concentrations after feeding either a low concentrate or high concentrate total mixed ration, then a n increase in plasma NEFA concentrations at 16 to 18 h after feed delivery in primiparous cows The increased NEFA concentrations are probably explained by a decreased feed intake which result s in decreased nutrient availability during this period (Nikkhah et al. 2011) further resulting in decreased insulin secretion and increased lipolysis and blood NEFA concentrations ( Brockman, 1978; Sutton et al., 1988). Steers supplemented with 1.13 kg respo nded in a manner where at h 12, NEFA concentration s were at a peak and then declined to concentration s similar to h 0 between 24 and 36 h post feeding. It is plausable this response could possibly be due to introduction of a high fat supplement for the fi rst time in these steers and digestion and metabolization of the fat in the rumen is not complete allowing greater post ruminal absorption of intact lipids. Palmquist and Mattos (1978) have shown that majority of NEFA present in blood lipoproteins are of dietary origin. Increased NEFA results from incomplete uptake of NEFA by peripheral tissues after hydrolysis of triglycerides in chylomicrons or very low density lipoproteins by lipoprotein lipase (Chilliard, 1993; Grummer et al., 1987). Mean PUN concentr ations were increased ( P ; Table 4 6 ) in steers consuming 3.39 kg (45.1 mg/dL) compared with steers consuming 2.26 kg and 4.52 kg (37.4 and 33.7 mg/dL, respectively). However PUN concentration of steers consuming 3.39 kg was not different ( P > 0.05) compared to steers consuming 0 or 1.13 kg (38.99
101 and 40.64 mg/dL, respectively). Generally, PUN concentrations increased 6 to 12 h post feeding in steers consuming all diets except for steers consuming 1.13 kg, where an inverse response was observed. Pl asma urea nitrogen concentration s ha ve been shown to increase shortly after feeding in dairy (Gustafsson and Palmquist 1993; Nikkhah et al., 2011) and beef cows (Coggins and Field 1976), and is related to increases in rumen ammonia concentrations due to r umen fermentation (Gustafsson and Palmquist, 1993; Blum et al., 2000; Plaizier et al., 2005 ). The effect of level of DDG tended ( P = 0.08) to effect glucose concentrations however; hour within level of DDG offered was not different ( P > 0.05 ; Figure 4 1 ) Mean glucose concentrations in steers offered RBS only (65.9 mg/dL) were increased ( P mg/dL, respectively), however there was no difference ( P > 0.05) in glucose concentrations between steers receiving any other amount of DDG. The lower glucose concentration s in steers which were supplemented DDG is contrary to reports in the literature which describes positive associations of DDG intake with glucose concentrations and nutrient intake (Richards et al., 1989; Yelich et al., 1996). The means only represent glucose concentrations for the 36 h after supplement was offered perhaps the decline may be attributed to an increase in plasma insulin due to an increase in propionate availability and the insulin increase d glucose utilization by the periphery (Sutton et al., 19 88; Blum et al., 2000; Nikkhah et al., 2008). In summary, over the short time period DDG were introduced at increasing levels to steers consuming RBS it appears that total intake, rumen pH, and rumen NH 3 N are relatively comparable with observations in an imals fed supplement over longer periods
102 of time. Additionally, the period of time in which RBS DMI was measured, did not result in any difference between DDG level. Bl ood metabolite data was dependent upon level of DDG intake and time after supplementat ion. Digestibility Experiment There was a linear ( P < 0.01) and quadratic ( P = 0.01) response of amount of DDG supplemented on round bale silage RBS DMI (Table 4 7 ). The greatest numerical RBS DMI occurr ed in steers fed the 0.33% DDG and the least at the 1% DDG inclusion. Effects of period and treatment period ( P Period 1 had decreased ( P ; 6.90 kg ) RBS DMI compared to any other period, which were similar ( P > 0.05) to each other (period 2, 8.48; period 3, 8.19; and period 4, 7.91 kg) Despite the 11 d diet adaptation phase before sampling started, numerous environmental factors could explain the differences observed between periods. The temperature during period 1 was the greatest, with maximum temperatures reaching 35C (Florida Automated Weather Network, 2012; Appendix C) In addition, steers were exposed to fecal collection bags for the first time during period 1, with a 2 d adaptation just prior to collection of intake and fecal data, which could also have contributed to decreased RBS intake. The treatment period effect wa s a reflection of steers on DDG+1 during period 1 having depressed RBS intake ( P steers on DDG+1 from the other 3 periods. Despite a period for adaptation perhaps the introduction of 1% of BW DDG for the 1 st time in addition to above e nvironmental factors could have caused steers to have depressed RBS intake. When DDG was fed at 1% of BW, RBS DMI was less ( P amounts of 0, 0.33, or 0.66%. Likewise, there was a linear ( P < 0.01) and quadratic ( P = 0.02) response of amount of DDG supplemented on total DMI (Table 4 7 ). Steers consuming only RBS had the least
103 total DMI and the greatest numerical total DMI occurred at the 1% DDG inclusion. Morris et al. (2006) supplemented DDG (0 to 1% BW) to steers grazing summer S andhills range and observed forage intake declined by 0.53 kg for every 1 kg of DDG offered. In addition, Leupp et al. (2009) reported a linear decrease in hay OM intake and a linear increase in total OM intake in steers consuming bromegrass hay supplemen ted with increasing amount s of DDG (0 to 1.2% BW). Garcs Ypez et al. (1997) evaluated 3 different energy concentrates (corn SBM, wheat middlings, and soybean hulls) offered at 2 levels (high and low) to growing steers and sheep consuming chopped bermuda grass hay. When supplementation was < .5% of body weight, these supplements did not reduce forage intake. When supplementation was 0.8 to 1% of body weight, forage intake was decreased. In part, the observed reductions in RBS DMI for DDG+.66 and DDG+1 cou ld be explained by concurrent drop in ruminal pH measurements (Figure 4 2) as the pH observed for these two DDG amounts dropped below 6.2 for the longest periods of time Ruminal pH depression has been reviewed as an explanation for reduction of forage i ntake with energy supplementation (Horn and McCollum, 1987; Caton and Dhuyvetter, 1997) and pH in the ranges of 5.7 to 6.2 populations of cellulytic bacteria diminish accounting for reductions in forage fiber digestibility (Russell and Dombrowski, 1980). Effects of period and treatment period ( P parallel the period and treatment period effects observed for RBS DMI. Fecal DM output increased linearly ( P < 0.01) as amount of DDG supplement increased (Table 4 7 ) but there w ere no period and treatment period ( P observed. Martnez Prez et al. (2010) observed a quadratic response for fecal OM
104 output in steers supplemented differing amount s of DDG (0, 0.2, 0.4, or 0.6% of BW) grazing native range pastures. H ighest fecal OM output occurred in steers supplemented at 0.4% BW DDG. In contrast, no differences in fecal OM outpu t were observed by Islas and Sotto Navarro (2011 ) in steers supplemented up to 0.6% BW of DDG grazing small grain pastures. The difference s in fecal OM output observed in the current study are likely a refl ection of total DMI. Both Islas and Sotto Navarro (2011) and Martnez Prez et al. (2010) observed no differences in total intake among amount s of DDG offered whereas we observed a quadr atic increase of total DMI as amount of DDG increased. In both Martnez Prez et al. (2010) and Islas and Sotto Navarro (2011) higher quality basal forage diets were fed, compared to our study, which could account for the differences in total DMI observed Total tract apparent DM digestibility also increased in a linear ( P < 0.01) and quadratic ( P manner as amount of DDG supplement increased (Table 4 7 ). In addition, steers consuming DDG+1 had a tendency ( P = 0.09) to have increased TTADMD compared to steers consuming DDG+.33 There were no period and treatment period ( P > 0.05) effects observed. The increase in TTADMD is likely a reflection of the DDG being more digestible than the RBS, which is of moderate quality. Leupp et al. (2009) observed linear increases in OM total tract digestibility when DDG supplementation increased from 0 t o 1.2% of BW in steers fed smooth bromegrass hay. Reed et al. (2007) observed an increase in total tract OM digestion in steers consuming grass hay supplemented with diets containing increasing levels of RUP in the form of bloodmeal compared to the steer s fed no R U P supplement, and also reported greater digestibility in steers consuming medium and high R U P levels compared to those
105 supplemented with a low level Bandyk et al. (2001) reported that supplementation of cracked corn or whole shelled corn to a low quality tall grass prarie hay (3.4% CP) basal diet, increased OM digestibility in steers. Similarly, Sanson et al. (1989) observed an increase in diet DM digestibility as they increased corn supplement ation in the diet. Mean pH values responded in a l inear ( P = 0.02) and quadratic manner ( P = 0.0 03 ) A s DDG amount increased mean pH values decreased. Steers fed RBS had greater pH ( P (6.28). The effect of treatment across the 15 h sam pling period is presente d in Table 4 8. There was an effect ( P period, hour, and treatment hour but n o treatment period effects ( P > 0.05). Steers consuming the RBS+1 diet had the greatest variation in pH across the sampling per iod. Two hours prior to diets being fed steers fed RBS+1 had a greater pH ( P RBS+.66, this difference continued to the sampling just prior to feeding (0 h). Immediately after diets were fed, steers consuming RBS+1 ha d decreasing pH, which reached a minimum at 3 h post feeding (6. 74 at 0 h to 5.7 5 at 3 h), which was less ( P 0.05) compared to steers consuming RBS or RBS+.33 The pH of steers consuming RBS+1 began to recover after h 3, but never increased to a similar pH as was observed at 0 h. Steers consuming the RBS diet had the smallest change in pH across the sampling times, reaching a peak 2 h post feeding (6. 59 ) and decreasing ( P the sampling at 12 h (6.2 3 ). Steers consuming RBS+.33 and RBS+.66 were intermediate to either RBS or RBS+1. Loy et al. (2007) reported decreased mean pH in heifers offered grass hay (8.2% CP) and supplemented daily with DDG at 0.4% of BW or 0.8% of BW on alternating d compared with un supplemented heifers. Moreover,
106 Calla wa y et al. (2010) reported a decreased ruminal pH in cattle consuming a grass hay diet supplemented with 50% DDG ration compared with cattle consuming a 0% DDG ration. Conversely, Leupp et al. (2009) reported mean ruminal pH of 6.5 with no effect of DDG sup plementation up to 1.2% BW in steers consuming bromegrass hay (10.6% CP) Likewise, Islas and Soto Navarro (2011) also observed no differences in ruminal pH in heifers fed up to 0.6% DDG grazing small grain pastures (17.7% CP) Differences observed acros s the literature are likely indicative of interactions between the basal forage diet and the amount of supplementation. Despite the decrease in pH, digestibility in our study was not impaired. Russell et al. (1979) reported reduction in cellulolytic bact erial numbers when pH fell between 5.7 to 6.2, which has been implicated in reducing digestibility (Horn and McCollum, 1987). Perhaps the pH in the study was not sustained at low enough concentration s for a sufficient amount of time to cause a shift in mi crobial populations, negating any influence on digestibility. Rumen NH 3 N concentrations tended to respond in a cubic manner ( P = 0.0 7 ) as DDG supplementation increased in the diet. Steers consuming RBS had decreased ( P 2 5.45 mg/dL ) mean rumen NH 3 N compared to steers consuming RBS+.66 (33. 01 mg/dL) and RBS+1 (33. 20 mg/dL) The effect of treatment across the 15 h sampling period is presented in Table 4 9 There tended to be an effect of treatment ( P = 0.09), t he re was an effect ( P period, treatment hour, and treatment period The interaction o f treatment and period is a reflection of the response from steers consuming RBS+1. The rumen concentrations of NH 3 N in these steers were greatest ( P ; 49.38 mg/dL ) during period 1 compared to all other periods (period 2, 33.17; period 3, 22.80; period 4, 27.47) and no differences ( P > 0.05 ) were observed
107 for steers consuming any other diet across periods. This is likely attributed to the reduct ion in RBS DMI and the possible environmental conditions previously discussed. For the 2 h prior to feeding steers co nsuming RBS+1 had increased ( P NH 3 N compared with RBS RBS+.33 or RBS+.66 From h 1 through 5 treatment had no effect ( P > 0.05 ) on rumen NH 3 N concentrations. Steers consuming RBS had the lowest numerical peak concentration of rumen NH 3 N with a concentration of 40.43 mg/dL and steers consuming DDG+.66 had the highest numerical peak concentration of 52.03 mg/dL at h 3. Pe ak concentrations were observed for all treatments between h 3 and 4 post feeding, with steers consuming RBS+.66 the highest at 53.5 mg/dL. After peaking, rumen NH 3 N concentrations dropped rapidly returning to pre feeding concentrations by 7 h post feedi ng (data not shown). Leupp et al. (2009) also reported a similar pattern for rumen NH 3 N as in this study, with steers consuming 0.9% BW DDG having greater rumen NH 3 N concentrations immediately before feeding observing peak concentrations between 2 and 4 h post feeding for all treatments and a rapid return to pre feeding cocentrations by 12 h post supplementation Additionally, these authors observed greatest numerical rumen NH 3 N concentrations in steers fed 0.9% BW DDG and the lowest when fed 0% DDG, similar to the findings in this study. Others have also observed increases in rumen NH 3 N concentration with DDG supplementation (Loy et al., 2007; Islas and Soto Navarro, 2011). In animals fed diets with plant proteins at high levels, rumen NH 3 N peaks are usually observed 3 to 5 h post feeding (Owens and Zinn, 1988). A range from 5 to 29 mg/dL has been reported for rumen NH 3 N concentrations as optimum for maximum microbial growth (Satter and Slyter, 1974; Miller 1973) and 23.5 mg/dL for maximum rate of fermentation (Mehrez et
108 al., 1977). Mean ruminal NH 3 N concentrations observed in this study fit into the ranges likely optimum for microbial growth and fermentation. In summary, supplementing DDG up to 1% of BW in the diet depressed RBS intake through a substitution of DDG for RBS. The increase in DDG offered did depress ruminal pH for a discreet amount of time, however supplementing DDG up to 1% of BW increased total DMI, TTADMD, and rumen NH 3 N. Implications Feeding DDG up to 1% of BW had no adver se effects on total intake and digestion. While decreases in ruminal pH were observed, they did not appear to impact total intake and digestibility of the diet. The decrease in RBS consumption due to DDG supplementation could lend itself to be used in a management scenario where forage is limited, or when it is more cost effective to increase DDG supplementation.
109 Table 4 1. Chemical composition of dried distillers grain (DDG), and bermudagrass round bale silage (RBS) fed to steers during the A daptatio n and D igestibility E xperiments. Adaptation Digestibility Item DDG RBS DDG RBS DM % 91. 7 0 .0 48.5 0.1 92.7 0.3 46.6 0.1 DM basis CP, % 25.0 0.2 10.0 0.4 24.2 0.1 9.2 0. 1 IVDMD, % 77.7 0. 7 44.7 1.4 77.7 0. 7 44.7 1.4 TDN, % 8 1 .0 0 .0 61.0 0.1 80. 7 0.3 59.7 0 .0 Sulfur, % 0.4 0 .0 0.2 0 .0 0.4 0 .0 0.2 0 .0 Ammonia, % 0.5 0.1 1.1 0.2 Lactic acid, % 2.0 0. 7 1.6 0.3 Acetic acid, % 0. 6 0.4 1.4 0. 0 VFA score 6.4 1.4 4. 5 0. 4
110 Tabl e 4 2. Effect of dried distillers grain (DDG) level on round bale silage (RBS) DMI and total DMI in steers during the A daptation E xperiment (LS means). 1 Level of DDG Intake, kg/d RBS DMI, kg/d Total DMI, kg/d 0 8.85 8.85 1.13 9.42 10.55 2.2 6 9.59 11.86 3. 39 9.94 13.33 4.5 2 9.26 13.26 SE 2 1.31 1.31 P value 0.42 <0.01 1 The adaptation diets simulated a 14 d step up adaption phase of a concentrate diet. Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg o f DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15 2 Pooled across means
111 Table 4 3. Effect of dried distillers grain (DDG) level on ruminal pH collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) d uring the A daptation E xperiment. Hours since supplement offered Supplement level, kg 1,2 0 3 6 12 24 36 Mean pH 3 0 6.48 a 6.49 a 6.37 b 6.32 c 6.50 a,b 6.51 a 6.44 v 1.13 6.45 a 6.30 b 6.29 b 6.32 b 6.45 a,b 6.33 a,b 6.36 w 2.26 6.36 a 6.15 b 5.95 c 6.27 a,d 6.27 a,d 6. 08 b 6.18 x 3.39 6.44 a 5.98 b,c 5.90 b 5.99 c 6.53 a 6.07 c 6.15 y 4.52 6.34 a 5.81 b 5.68 c 6.00 d 6.26 a 6.02 e 6.02 z 1 Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15. 2 SE pooled across hour within level means = 0.08 3 SE = 0.05 a, b, c, d, e Means within a row with different superscripts differ ( P v, w, x, y, z Means within a column with different superscripts differ ( P
112 Table 4 4. Effect of dried distillers grain (DDG) level on rumen NH3 N concentrations (mg/dL) collected for 36 h post feeding supplement in steers consuming berm udagrass round bale silage (RBS) during the A daptation E xperiment. Hours since supplement offered Supplement level, kg 1,2 0 3 6 12 24 36 Mean NH 3 N 3 0 11.1 a 39.4 b 25.1 c 14.9 a,f 17.3 d,f 16.5 d,e,f 20.7 x 1.13 14.8 a 37.0 b 26.9 c 15.3 a 15.9 a 15.0 a 20.8 x 2.26 14.8 a 40.9 b 29.1 c 14.9 a 13.8 a 14.6 a 21.4 x 3.39 16.7 a,e,f 45.9 b 52.5 c 23.3 d 21.4 e 17.9 f 29.6 y 4.52 25.9 a 48.7 b 44.6 b 26.2 a 32.9 c 30.3 d 34.8 z 1 Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15. 2 SE pooled across hour within level means = 2.3 3 SE = 1.0 a, b, c, d, e, f Means within a row with different superscripts differ ( P x, y, z Means within a column with different superscripts diff er ( P
113 Table 4 5. Effect of dried distillers grain (DDG) level on NEFA concentrations (mEq/ml) collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the A daptation E xperiment. Hours since suppl ement offered Suppplement level, kg 1,2 0 6 12 24 36 Mean NEFA 3 0 418.1 a 329.2 b 219.9 c 263.6 c,d 269.1 c,e 300.0 y 1.13 293.4 a 274.3 a 385.1 b 320.2 a 269.6 a,c 308.5 y 2.26 369.5 a,b,c 194.8 a,b,d 159.6 e 257.6 b 229.4 a,c,d,e 242.2 z 3.39 343.5 a 211.6 b 221.3 b,c 2 92.3 a,d 246.1 b,e 263.1 y,z 4.52 296.2 a 221.7 b 254.7 a,b 279.1 a 253.8 a,b 261.1 y,z 1 Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15 2 SE pooled acr oss hour within level means = 34.3 3 SE = 28.0 a, b, c, d, e Means within a row with different superscripts differ ( P y, z Means within a column with different superscripts differ ( P
114 Table 4 6. Effect of dried distillers grain (DDG) level on plasma urea nitrogen (PUN) concentrations (mg/dL) collected for 36 h post feeding supplement in steers consuming bermudagrass round bale silage (RBS) during the A daptation E xperiment. Hours since supplement offered Supplement level, kg 1,2 0 6 12 24 36 Mean PUN 3 0 33.6 a,b 45.5 a 41.6 a,b 31.8 b 42.6 a,b 39.0 w 1.13 51.3 a 38.3 b 37.0 b 46.2 a,b 30.5 b 40.6 w,y 2.26 31.7 42.4 43.5 31.4 37.8 37.4 w 3.39 51.3 a 49.2 a 50.8 a 42.4 a,b 31.8 b 45.1 x,y 4.52 35.6 37.3 28.2 34.4 32.9 33.7 z 1 Day 2 and 1 steers receive d RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15 2 SE pooled across hour within level means = 4.7 3 SE = 2.2 a, b Means within a row with different superscripts differ ( P 05) w, x, y, z Means within a column with different superscripts differ ( P
115 Figure 4 1. Effect of dried distillers grain (DDG) level on mean glucose concentrations in steers consuming bermudagrass round bale silage (RBS) during the A daptation E xperiment. Day 2 and 1 steers received RBS only, o n d 0, steers received 1.13 kg of DDG on d 4 2.26 kg of DDG, d 8 3.39 kg of DDG, and on d 12 4.52 kg until d 15 Level ( P = 0.08) and hour ( P > 0.05). a,b letters differ ( P .05)
116 Table 4 7 Effec t of dried distillers grain (DDG) on intake, fecal output, and digestibility in steers consuming bermudagrass round bale silage (RBS) during the D igestibility E xperiment. DDG 1 % of BW Contrast 3 Item 0 0.33 0.66 1 SE 2 P value Linear Quadratic Cubic RB S DMI, kg/d 8.28 8.73 7.87 6.59 0.26 < 0.01 < 0.01 0.01 0.51 Total DMI, kg/d 8.29 10.59 11.47 12.09 0.27 < 0.01 < 0.01 0.02 0.38 Fecal DM, kg/d 4.13 4.69 4.80 5.02 0.59 < 0.01 < 0.01 0.21 0.36 TTADMD 4 % 52.97 57.58 60.24 60.68 1.10 < 0.01 < 0.01 0.05 0 .83 1 DDG supplementation: 0 = no supplement, 0.33 = DDG offered at 0.33% of BW, 0.66 = DDG offered at 0.66% of BW, 1 = DDG offered at 1% BW. 2 n = 8 3 Probabilities for the linear, quadratic, or cubic effects of amount of DDG. 4 Total tract apparent DM d igestibility
117 Table 4 8. Effects of dried distillers grain (DDG) fed at 4 amounts during the D igestibility E xperiment on ruminal pH collected over 15 h in steers consuming bermudagrass round bale silage. DDG 1 % BW Hour 2,3 0 0.33 0.66 1 2 6.42 a 6.48 a 6 .50 a 6.74 b 1 6.50 a 6.50 a 6.55 a 6.82 b 0 6.53 a 6.55 a 6.58 a 6.91 b 1 6.57 a 6.30 b 6.20 b 6.15 b 2 6.59 a 6.26 b 6.00 c 5.91 c,d 3 6.56 a 6.29 b 5.91 c 5.75 c,d 4 6.48 a 6.22 b 5.96 c 5.83 c,d 5 6.43 a 6.17 b 6.01 b 6.00 b 6 6.39 a 6.13 b 6.03 b 6.05 b 7 6.32 a 6.13 b 5.99 c 6 .21 a,b 8 6.36 a 6.17 b 6.06 c 6.26 a,b 9 6.32 a,b 6.18 a,b 6.14 a 6.36 b 10 6.28 a,b 6.13 a 6.11 a 6.41 b 11 6.26 a,b 6.15 a 6.10 a 6.41 b 12 6.23 a 6.15 a 6.13 a 6.46 b 1 DDG supplementation: 0 = no supplement, 0.33 = DDG offered at 0.33% of BW, 0.66 = DDG offered at 0 .66% of BW, 1 = DDG offered at 1% BW. 2 Hours since supplementation 3 Pooled SE = 0.08 a, b, c, d Means within a row with different superscripts differ ( P
118 Table 4 9. Effects of dried distillers grain (DDG) fed at 4 amounts during the D igestibility E xperiment on ruminal NH3 N collected over 15 h in steers consuming bermudagrass round bale silage. DDG 1 % BW Hour 2,3 0 0.33 0.66 1 2 19.37 a 19.61 a 23.03 a 34.76 b 1 19.68 a 20.11 a 25.32 a 38.64 b 0 23.93 a 22.54 a 27.74 a,b 38.14 b 1 33.24 36.32 47.32 4 5.14 2 38.87 42.61 50.10 47.45 3 40.43 46.04 52.03 47.57 4 39.80 47.82 49.82 42.70 5 36.68 41.68 45.46 38.76 6 28.37 a 33.61 a 37.24 b 31.64 a 7 22.68 a 26.04 a 30.46 b 25.33 a 8 17.87 a 20.32 a 24.74 b 23.01 a 9 16.62 17.61 20.53 20.89 10 15.49 a 16.61 a,b 19. 67 a,b 22.58 b 11 14.87 a 20.68 a,b 20.67 a,b 21.76 b 12 13.80 a 18.18 a,b 20.96 b 19.70 b 1 DDG supplementation: 0 = no supplement, 0.33 = DDG offered at 0.33% of BW, 0.66 = DDG offered at 0.66% of BW, 1 = DDG offered at 1% BW. 2 Hours since supplementation 3 Po oled SE = 2.27 a, b Means within a row with different superscripts differ ( P
119 CHAPTER 5 CONCLUSIONS In C hapter 3, an experiment was conducted to evaluate the effect of feeding heifers bermudagrass round bale silage (R BS ) and supplemented with dried distillers grain ( DDG ) alone or DDG in addition to 2 amount s of soybean me al (SBM) on growth, body composition, blood metabolites, and reproductive performance in Angus and Brangus heifers. Diets offered were: 1) supplementation with DDG at 0.75% of BW; 2) supplementation with DDG at 0.75% of BW plus soybean meal at 7.5% of DDG amount (DDG+7.5); 3) and supplementation with DDG at 0.75% of BW plus SBM at 15% of DDG supplement amount (DDG+15) and all treatments received ad libitum access to RBS. We hypothesized that adding soybean meal (SBM), a source of rumen degradable protein ( RDP ) to a DDG supplement program could enhance heifer growth and reproductive performance in Angus and Brangus heifers fed bermudagrass RBS. Many forage based diets result in a negative RDP balance, and therefore choosing a supplement to overcome this is an important consideration in a feeding management program. Results observed from the experiment in C hapter 3 did not support this hypothesis. Similar ( P > 0.05 ) responses were observed between treatments for BW, BCS, ADG, hip height (HH), LM area (REA) at the 13 th rib, 13 th rib fat thickness (RIBFT), rump fat thickness (RMPFT), int ramuscular fat of the REA (IMF), REA adjusted per 100 kg of BW ( REA/cwt ), NEFA concentrations, plasma urea nitrogen (PUN) concentrations, and glucose concentrations. Addition ally, treatments were similar ( P > 0.05 ) for attainment of puberty and responses to synchronization and breeding.
120 Heifers receiving the DDG treatment, like many cattle consuming forage based diets, had a negative rumen degradable protein (RDP) balance base d on estimations from the beef cattle NRC (2000), to correct this we added soybean meal to the supplement. Reports in the literature suggest that supplying the appropriate amount of RDP is critical for microbial protein synthesis, and additional RDP impro ved animal rumen undegradable protein (RUP); heifers were receiving an indirect supply of RDP through N recycling via the urea cycle. This would explain why the negative RDP balance did not negatively impact the growth performance of heifers in the DDG treatment. Additionally, the small amount of SBM added to the supplement provided no positive associative effects for the DDG+7.5 and DDG+15 heifers. All three treatments start of breeding season. Therefore, it is concluded that DDG as a supplement to yearling heifers as a single source ingredient when supplemented at 0.75% of BW can support heifer growth when consuming a medium quality forage, such as bermudagrass RBS. The interpretation of the reproductive data is challenging due to limited number of experimental units In addition, only 29.1% of the heifers were pubertal at the start of t he breeding season, which contributed to our need to provide a 2 nd synchronization phase with a progesterone source to meet the beef unit management needs. The fact that only 29.1% were pubertal is still disconcerting from a management perspective. Heife rs were on a high plane of nutrition, attained calculated ADG, were of the appropriate age (13 to 15 mo), and reached a target weight of at least 65% of the
121 mature BW for Brangus and Angus at the start of the breeding season. In previous heifer developmen t studies at this location, a similar percentage of pubertal heifers at the start of the breeding season have been observed. Additionally, there tended ( P = 0.0 6 ) to be a greater percentage of Brangus heifers pubertal at the start of the breeding season c ompared compared to Angus heifers. Despite the convention that Bos taurus animals typically reach puberty at lighter and earlier ages the Brangus heifers responded better. This is likely due to the better adaptation of the Brangus heifers to the Florida environment, when provided similar nutrition to the Angus heifers. However, it is unexpected to observe so few Angus heifers attaining puberty. Perhaps the Angus genetics used in this herd have been selected for too much growth and eventual mature size re sulting in older ages at the onset of puberty or perhaps there are some unknown environmental interactions that were not measured in this experiment that are contributing to these numbers. Nevertheless by the end of the breeding season, acceptable final pregnancy rates were observed for a 77 d breeding season and no differences between treatments on proportion of heifers conceiving throughout the breeding season. Breed differences were observed for HH, IMF, RMPFT, REA, REA/cwt, NEFA concentrations and glu cose concentrations. These differences in growth and metabolites between breeds are consistent with the literature. Overall implications for C hapter 3, are addition of SBM, to compensate for a RDP deficiency in supplementation diets of DDG to growing heif ers consuming medium quality forage provide d no additional benefit to growth performance under the conditions of this study Therefore, DDG can function as single source ingredient when
122 supplemented at 0.75% of BW in supplementation diets for yearling hei fers fed a medium quality RBS of a bermudagrass origin. This results in a relatively simple management scenario for beef producers when developing yearling replacement heifers to where multiple ingredients do not need to be fed. The objectives of the exp eriments in C hapter 4 were two fold. First, to evaluate blood metabolites, forage intake, and rumen parameters of steers fed an adaptation diet of bermudagrass RBS supplemented with DDG. And secondly, to evaluate the effect of amount of DDG supplementati on on forage intake, digestibility, and rumen parameters in steers consuming bermudagrass RBS. We hypothesized that DDG supplementation of steers consuming bermudagrass RBS would not adversely affect total diet digestion and rumen function, but would decre ase forage intake and increase total intake of diet. In the A daptation E xperiment no effect ( P > 0.05) of DDG level was observed on RBS DMI and there was an increase ( P D igestibility E xperiment, a linear ( P < 0.01) and quadratic ( P = 0.01) response of amount of DDG supplemented on RBS DMI was observed. The D igestibility E xperiment was consistent with our h ypothesis. It is possible in the adaptation experiment that the effect DDG had on RBS DMI required more time to emerge, as feeding amounts were changed on 4 d intervals and intake data was only collected during that period of time. Therefore, steady stat e intake may not have been achieved. For both the A daptation and D igestibility experiments mean pH decreased ( P 0.05) as DDG offered increased. In both experiments, steers fed only RBS maintained the most consistent pH throughout the sampling period, and the steers consuming the
123 highest amounts of DDG had the most dynamic pH profile, with the lowest drops in pH. In the A daptation E RBS DMI, perhaps due to the short amount of time the steers were exposed to lower pH concentrations. However, the lower pH values may in part be an explanation to depres sion in RBS DMI in the D igestibility Experiment as they were exposed to the DDG amount over a longer period of time. Additionally, the depression in pH in the digestibility trial was an anticipated response based on our hypothesis. Rumen NH 3 N in the A da ptation Experiment increased ( P and tended ( P = 0.07) to increase in the D igestibility E xperiment with greater amount s of DDG supplementation. Additionally, rumen NH 3 N concentrations peaked around 3 to 4 h post feeding, which is a consistent resp onse with that observed in the literature. The increased concentrations of NH 3 N are not surprising as DDG is high in protein, which can contribute to the N pool in the rumen. In addition, the rumen NH 3 N concentrations observed in these experiments fit into the ranges likely optimum for microbial growth and fermentation. Which further supports our hypothesis that we would not see any adverse effects on rumen function. In the D igestibility E xperiment, t otal tract apparent DM digestibility ( TTADMD ) increa sed as amount of DDG supplement ation increased in a linear ( P < 0.01) and quadratic ( P likely a reflection of the DDG being more digestible than the RBS, which is of moderate quality. Additionally, despite the de creased pH when feeding DDG there was no negative effect on digestibility. These findings are consistent with the literature and support our hypothesis that DDG would not depress digestibility when steers consume bermudagrass RBS.
124 In the A daptation E xper iment, level of DDG tended ( P = 0.08 ) to effect NEFA and glucose concentrations and a ffected ( P concentrations The metabolites were affected ( P The response within a level was often difficult to describe, as the patterns were not always consistent with observations in the literature. This may be due to the short period of time that transpired from when a new level of DDG was introduced to time of sample collection. Our data collection was designed to capture the immediate response once DDG was introduced, and DDG levels subsequently increased in the diet. In most studies, the animals are already adapted to a particular diet, and often samples are not as intensively collected. More work looking at the effect of addition of DDG and how the rumen environment and metabolic system are effected immedi ately after introduction and how the animal adapts to these changes is warranted. Feeding DDG up to 1% of BW had no adverse effects on intake and digestion. While decreases in ruminal pH were observed, it does not appear to impact total intake and digesti bility of the diet. The decrease in RBS consumption due to feeding DDG could lend itself to be used in a management scenario where forage is limited, or when it is more cost effective to increase DDG supplementation. Overall, when supplemented to cattle c onsuming warm season forages such as bermudagrass RBS, DDG can be a supplement option that provides energy and protein to support growing cattle and have no negative impact on total intake and digestibility of the diet.
125 APPENDIX A NUTRITIONAL COMPOSITION OF MINERAL VITAMIN MIX FOR ALL EXPERIMENTS Table A 1. Nutritional composition of mineral vitamin mix for all experiments. Item Amount Calcium (Ca) Max 14.00% Calcium (Ca) Min 12.00% Phosphorus (P) Min 6.00% Salt (NaCl) Max 21.00% Salt (NaCl) Min 19. 00% Potassium (K) Min 0.8% Magnesium (Mg) Min 1.00% Sulfur (S) Min 0.40% Iron (Fe) Min 0.40% Copper (Cu) Min 2,000 ppm Cobalt (Co) Min 200 ppm Manganese (Mn) Min 2,200 ppm Iodine (I) Min 175 cpm Selenium (Se) Min 48 ppm Zinc (Zn) Min 9,500 ppm F luorine (F) Max 800 ppm Vitamin A Min 100,000 IU/lb Vitamin D3 Min 20,000 IU/lb
126 APPENDIX B OVERALL REPRODUCTIVE RESPONSES FROM 77 DAY BREEDING SEASON Table B 1. Overall estrous response, conception rate, synchronized pregnancy rate, AI pregnancy rate, and final pregnancy rate from phase 1 and 2 of the breeding season for yearling Angus and Brangus heifers consuming round bale silage (RBS) and supplemented with dried distillers grain (DDG) alone or DDG plus soybean meal (SBM) at two amount s. Item Overal l estrous response, % 1 Overall conception rate, % 2 Overall synchronized pregnancy rate, % 3 Overall AI pregnancy rate, % 4 Final pregnancy rate, % 5 DDG 6 85 (17/20) 53 (9/17) 45 (9/20) 50 (10/20) 95 (19/20) DDG+7.5 7 79 (15/19) 53 (8/15) 47 (9/19) 53 (10/1 9) 79 (15/19) DDG+15 8 75 (15/20) 60 (9/15) 45 (9/20) 55 (11/20) 80 (16/20) Angus 83 (25/30) 52 (13/25) 47 (14/30) 50 (15/30) 83 (25/30) Brangus 76 (22/29) 59 (13/22) 45 (13/29) 55 (16/29) 86 (25/29) P value Treatment Breed Treatment Breed 0.79 0 .57 0.30 0.90 0.71 0.78 0.99 0.94 0.82 0.97 0.71 0.78 0.38 0.80 0.86 1 Percentage of heifers displ aying estrus combined for phase 1 and 2 of the breeding season. 2 Percentage of heifers pregnant to AI of the total that exhibited estrus for phase 1 an d 2 of the breeding season. 3 Percentage of heifers pregna nt to synchronization for phase 1 and 2 of the breeding season. 4 Percentage of heifers pregnant to any AI including: phase 1 and 2 of the breeding season. 5 Percentage of heifers pregnant during the entire 77 d breeding season. 6 DDG: Supplement at 0.75% BW with dried distillers grain 7 DDG+7.5: Total supplement at 0.75% BW with DDG plus 7.5% of SBM 8 DDG+15: Total supplement at 0.75% BW with DDG plus 15% of supplement SBM
127 APPENDIX C TEMPERATURE S DURING THE DIGESTIBILITY EXPERIMENT Item 1 Period 1 Period 2 Period 3 Period 4 Average temperature, C 25 22 16 13 Minimum temperature, C 10 3 0.4 3 Maximum temperature, C 35 34 33 28 1 Data from Florida Automated Weather Network Alachua, Flor ida
128 APPENDIX D GLUCOSE ASSAY PROTOCOL Materials: Microcentrifuge tubes Rack for microcentrifuge tubes 12 x 75 mm test tubes 18 x 150 mm test tubes Ultra Pure water Control sample (pool of plasma samples) Rack for test tubes Incubator Plate reader 10 l Pi pette 1000 l Pipette 200 l Pipette Pipette tips Cayman Glucose Kit Glucose Assay Standard Glucose Assay Buffer Glucose Enzyme Mixture Two 96 well plates Assay: Glucose Assay Kit Catalog No. 10009582, Cayman Chemical Company Each plate allows for 39 unknow n plus 1 control all in duplicate. Before start of the assay: Glucose assay buffer should be thawed and equilibrated to 4 C. All reagents and ultra pure water should be equilibrated to 4 C before the start of the assay. Turn on incubator set to 37 C. Sta ndard preparation: Take eight 12 x 75 mm test tubes and label them A H. Place tubes in a rack. Add the amount of glucose standard and assay buffer to each tube as described in the table below. Make sure to mix well!
129 Tube Glucose Standard ( l) Ass ay Buffer ( l) Glucose Concentration (mg/dl) A 0 200 0 B 2.5 197.5 12.5 C 5 195 25 D 10 190 50 E 20 180 100 F 30 170 150 G 40 160 200 H 50 150 250 Performing the assay part I: Label first 8 microcentrifuge tubes from A H where the standards are g oing to be placed. The following microcentrifuge tubes are labeled according to samples ID. Label the last microcentrifuge tube of each well plate as control. Add 5 l of plasma sample, control sample and standard to labeled microcentrifuge tube. Enzyme m ixture preparation: One vial of the enzyme is sufficient to evaluate 48 wells. Add 500 l of 4 C UltraPure water to the enzyme vial. Transfer the reconstituted solution to an 18 x 150 mm test tube. Add 12 ml of assay buffer to the reconstituted solution an d mix well. NOTE: A portion of the 12 ml should be used to rinse any residual solution from the vial. This reconstituted solution is now ready to be used in the assay. NOTE: The reconstituted solution is stable for at least one hour when stored at 4 C. Per forming the assay part II: Add 500 l of the enzyme mixture forcefully down the side of each microcentrifuge tube (standard and samples). Tap tubes a couple of times to mix thoroughly. Place tubes in a 37 C incubator for 10 minutes. After 10 minutes, remov e tubes from incubator. Load 150 l (in duplicate) from each tube to the 96 well plate. Read the absorbance at 500 520 nm using a plate reader.
130 APPENDIX E NON ESTERIFIED FATTY ACIDS (NEFA) ASSAY PROTOCOL Materials: Color reagent A Solvent A Color reagen t B Solvent B Serum samples Control sample (Pool sample) Kit standards Saline Deionized water 10 l pipette 1000 l pipette Pipette tips 96 well plates with lids flat bottom plates Colored paper Plate reader Saline Solution: Prepare 0.9% saline solution b efore the start of the assay. Procedure: Mix 0.9g of NaCl with 100 ml of deionized water. Assay: Each plate allows for 42 unknown samples plus 1 control all in duplicate. NEFA HR kit Code No. 999 34691, 991 34891, 993 35191, Wako Diagnostics. Assay proce dure: Turn on heat control for incubator to 37 C. Sort samples and prepare assay sheet. Randomly select samples and place in rack, recording the order on your assay sheet. Obtain plates. Wells 1A and 2A are blanks. Wells 3A 12A are standards. Wells H11 and H12 are pools. Remaining wells are duplicate samples. Repeat blanks, standards and pool for each plate. Prepare reagents: Add one bottle of Solvent A to one vial of Color Reagent A. Add one bottle of Solvent B to one vial of Color Reagent B. Mix gently by inverting the vials until contents are completely dissolved. Prepare the standard curve dilutions in test tubes and transfer 5 l into the following wells.
131 Standard (mEq/ml) Ultra pure water ( l) Standard Solution ( l) (blank) 0 500 0 (Std 2) 200 4 00 100 (Std 3) 400 300 200 (Std 4) 600 200 300 (Std 5) 800 100 400 (Std 6) 1000 0 500 Pipette 5 l of Control (pool) in wells H11 and H12. Pipette 5 l of serum sample in duplicate as per the order on your assay sheet. Pipette 200 l Color Reagent A Sol ution to all wells. for the plate reader. Press turn on button for temperature on the plate reader. Once the temperature has reached 37 C place plate in the plate reader and incu bate for 5 minutes. After 5 minutes, press read on the computer. Measure and record absorbance of each well at 560 nm (sub: 660nm) for Abs 1 (only with reagent A). Remove well plate from the plate reader and add 100 l of Color Reagent B Solution to all wel ls. Place well plate in the plate reader and incubate for 5 minutes again. After 5 minutes, press read on the computer. Measure and record absorbance of each well at 560nm (sub:660nm) for Abs 2 (reagent A and reagent B). Calculations: The results obtained from the plate reader are not the final results. Calculation of final absorbance: The absorbance 1 from the first measurement should be multiplied by a factor (F) in order to correct for changes in volume, as follows: F = (Sample volume + Solution A vol ume) / (Sample volume + Solution A volume + Solution B volume) F = (4 + 200) / ( 4 + 200 + 100) = 0.67 Therefore: Final sample absorbance = Absorbance 2 (Absorbance 1 0.67)
132 Plot absorbance vs. concentration of standards to generate a linear regress ion curve and equation. The equation will be used to calculate the final results Use the excel template to calculate final results.
133 APPENDIX F BLOOD UREA NITROGEN (BUN) ASSAY PROTOCOL Materials: Reagent A Reagent B Urea standard Ultra pure water 1000 l pipette Pipette tips 96 well plates round bottom plates Serum or plasma samples Control sample Plate reader Assay: Allows for 42 unknown samples plus 1 control all in duplicate. BioAssay Systems Quantichrom Urea Ass Kit series DIUR 500, BioAssays Systems. Standard preparation: Standard (mg/dl) Saline Solution ( l) Urea standard ( l) (Std 1) 0 100 0 (Std 2) 12.5 75 25 (Std 3) 25 50 50 (Std 4) 37.5 25 75 (Std 5) 50 0 100 Assay procedure: Reagent preparation: Equilibrate reagents to room temperature. Prepare enough working reagent by combining equal volumes of Reagent A and Reagent B. Use working reagent within 20 minutes after mixing. Add 5 l water (blank) to wells A1 and A2. Add 5 l standard (50mg/dL) to wells B1, B2 through E1, E2 Add 5 l of contr ol sample to wells F1 and F2. Add 5 l samples in duplicate into following wells. Add 200 l working reagent and tap tightly to mix. Incubate for 20 minutes at room temperature. Turn on plate reader. Turn on computer and open the program for the plate reader Select BUN protocol.
134 Read optical density at 520nm. The results are obtained are not the final results since it is based on urea concentrations. The following conversion is necessary. Conversions: BUN (mg/dL) = [Urea] / 2.14
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161 BIOGRAPHICAL SKETCH Erin Alava was born in Athens, Ohio, in 1984. She is the only daughter of Michael and Lynn McKinniss and sister to Matthew and Stephen McKinniss. After graduation from Northmont High School in 2002, she pursued a Bachelor of Science degree at The Ohio State University in a nimal s ciences. She graduated in August of 2006. In the fall of 2006 she began her Master of Science degree under Dr. Joel Yelich. She completed her work Summer 2008 with her th term progestogen based estrous synchronization protocols in yearling heifers and suckled postpartum cows of Bos indicus x Bos taurus She was awarded a graduate alumni fellowship through the University of F lorida, which provided her the opportunity to begin her PhD in the fall of 2008. Dr. Matthew Hersom and Dr. Joel Yelich co advised her in the fields of beef cattle nutrition and reproduction for PhD research. As a graduate student, Erin was very active i n the department as a teaching assistant to various undergraduate and graduate level courses as well as serving multiple offices and committees for the graduate student association. In September 2009, she married a fellow University of Florida graduate stu dent, Eduardo Alava and they are currently expecting their first child in August 2012. After graduation Erin plans to apply for a fellowship through Prometeo Ecuador to continue her work in the field of beef cattle production and research.