2015 Florida Beef Research Report

Transcription

1 2015 Florida Beef Research Report Department of Animal Sciences

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3 Table of Contents Genetics Genomic-Polygenic and Polygenic Evaluation of Multibreed Angus-Brahman Cattle for Direct and Maternal Growth Traits Under Subtropical Conditions...1 Genomic Variability and Predictions for Postweaning Ultrasound Traits Using Actual and Imputed Illumina50k SNP Markers in Angus-Brahman Multibreed Cattle... 7 Reproduction Effects of Administration of Prostaglandin F2α at Initiation of the 7-d CO-Synch+CIDR Ovulation Synchronization Protocol for Suckled Beef Cows and Replacement Beef Heifers Effects of Recombinant Bovine Somatotropin Administration at Breeding on the Cow, Conceptus, and Subsequent Offspring Performance of Beef Cattle Utilization of Fixed-time Artificial Insemination (TAI) to Reduce Breeding Season Length and its Effects on Subsequent Calf Value: A Case Study Resynchronization as a Strategy to Increase the Percentage of Replacement Beef Heifers Conceiving to Artificial Insemination (AI) After an Initial Fixed-time AI (TAI) Forages Clover-annual Ryegrass Mixtures to Extend the Grazing Season in North Florida Seeding Rates of Ball Clover in Mixtures with Annual Ryegrass in North Florida Red Clover Varieties for North-Central Florida White Clover Varieties for North-Central Florida Alfalfa Production in North-Central Florida Microbiology Prevalence of Shiga-Toxin Producing Escherichia Coli in Two Cohorts of Beef Cattle is Associated with Diversity of Microflora and Animal Age Prevalence of Cefotaxime Resistant Enterobacteriaceae in Beef Cattle in Florida i

4 Nutrition Effect of Prenatal Trace Mineral Source on Neonatal and Growing Calf Liver and Serum Mineral Status Effects of Trace Mineral Source on Cow Performance and Mineral Status During a Production Cycle Effects of Prepartum Mineral Supplement Source and Cow Breed on Cow Colostrum Composition and Neonatal Calf Serum Immunoglobulin Concentration Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Ruminal Fermentation of Beef Steers Consuming Bermudagrass Hay Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Performance and Total Tract Digestibility of Beef Heifers Consuming Bermudagrass Hay Effect of Frequency of Supplementation with Megalac-R on Non-esterified Fatty Acids and Blood Urea Nitrogen Concentration in Lactating Beef Cows Effects of Prepartum Supplementation of a Rumen Fermentation Enhancer on Subsequent Beef Cow Performance Effects of Pre- and Post-Breeding Supplementation of a Ruminally Protected Lipid on Subsequent Beef Cow Performance Effect of Rate of Inclusion of Fermenten in a Backgrounding Diet on Performance and Carcass Characteristics in Growing Angus Crossbred Steers Evaluation of Brassica Carinata as a Protein Supplement for Growing Beef Heifers Effects of Chitosan on Ruminal Fermentation and In Situ Nutrient Degradability Effects of Chitosan on Enteric Methane Production and Nutrient Digestibility of Beef Heifers Effect of Residual Feed Intake Classification on Maintenance Energy Requirements and Efficiency of Energy Use in Growing Heifers The use of trade names in this publication is solely for the purpose of providing specific information. UF/IFAS does not guarantee or warranty the products named, and references to them in this publication does not signify our approval to the exclusion of other products of suitable composition. ii

5 Genomic-Polygenic and Polygenic Evaluation of Multibreed Angus-Brahman Cattle for Direct and Maternal Growth Traits Under Subtropical Conditions M. A. Elzo 1, M. G. Thomas 2, D. D. Johnson 1, C. A. Martinez 1, G. C. Lamb 3, D. O. Rae 4, J. G. Wasdin 1, and J. D. Driver 1 Synopsis High rank correlations existed between estimated breeding value for birth weight direct, weaning weight direct, postweaning gain direct, birth weight maternal, and weaning weight maternal from a genomicpolygenic model that used all phenotypic, pedigree, and genotypic information and a polygenic model. Variance components and genetic parameters from these two models were similar for all traits. Incomplete pedigree or lack of it resulted in lower rank correlations and poor estimates of genetic parameters particularly for maternal traits. Thus, to maximize the benefits of genotyping, commercial producers would need to keep complete pedigree records as well as individual animal phenotypes. Summary The objectives of this research were to compare variance components, genetic parameters, and estimated breeding value (EBV) rankings for birth weight (BW) direct and maternal, weaning weight (WW) direct and maternal, and postweaning gain from 205 d to 365 d (PWG) direct using three genomic-polygenic and one polygenic model representing four plausible beef cattle genetic evaluation scenarios for growth traits under subtropical conditions in the US southern region. The dataset included 5,264 animals from a multibreed Angus-Brahman population born from 1987 to Genomic-polygenic models 1 (GP1; pedigree relationships for all animals; genomic relationships for genotyped animals), 2 (GP2; pedigree relationships for non-genotyped animals; genomic relationships for genotyped animals), and 3 (GP3; no pedigree relationships; genomic relationships for genotyped animals) used actual and imputed genotypes from 46,768 SNP markers. Restricted maximum likelihood variance components and genetic parameters from GP1 were the most similar to those from the polygenic model, followed by those from GP2, and the least similar (especially for maternal traits) were those from GP3. The highest rank correlations were those between animal EBV from the polygenic model and GP1, followed by those between animal EBV from GP1 and GP2 and between the polygenic model and GP2. Model GP3 performed poorly for maternal traits due to lack of calf-dam relationships. These results indicated that the polygenic model and GP1 should be preferred, although high genotyping costs still make the polygenic model preferable for commercial beef cattle operations. Introduction Utilization of genotype information for genetic evaluation of cattle has become widespread in beef and dairy cattle. Currently, genomic evaluations are routinely conducted in dairy cattle in the US and other countries. Conversely, the US beef industry has only recently begun to implement national genomic evaluations that combine phenotypic, pedigree, and genotypic information. Purebred breeders and commercial cattle producers have been encouraged by breed associations and private companies to genotype their animals with one or more chips of various densities. Genotyping animals from purebred cattle operations that submit phenotypes, pedigree, and genotypes to breed associations conducting national genetic evaluations will likely enhance the ability of individual cattle breeders to identify superior animals. However, the potential usefulness of genotyping to enhance genetic selection within 1 Department of Animal Sciences, University of Florida, Gainesville, FL 2 Department of Animal Sciences, Colorado State University, Fort Collins, CO 3 North Florida Research and Education Center, University of Florida, Marianna, FL 4 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL Florida Beef Research Report

6 commercial cattle operations that do in-house genetic evaluations seems less clear. Increases in prediction accuracies will depend on the extent of genotyping (and density of genotyping chips), the availability of individual phenotypes, and the completeness of pedigree information. This research was aimed at comparing multibreed beef cattle evaluations for growth traits using four scenarios defined in terms of availability of phenotypic, pedigree, and genotypic information to represent genetic evaluations in purebred and in commercial cattle herds under subtropical conditions in Florida and the US southern region. Thus, the objectives of this research were: 1) to compare variance components and genetic parameters (heritabilities, genetic correlations) for birth weight direct and maternal, weaning weight direct and maternal, and postweaning gain direct; 2) to compare rankings of animals for birth weight direct and maternal, weaning weight direct and maternal, and postweaning gain direct; and 3) to evaluate EBV trends for each trait under four data scenarios (phenotypes, pedigree, and genotypes) as percentage Brahman increased from 0% to 100% in a multibreed Angus-Brahman population under subtropical environmental conditions. Materials and Methods Animals and Traits Animals belonged to the long-term multibreed Angus-Brahman (MAB) project of the University of Florida, Gainesville. The dataset included information on preweaning and postweaning growth from calves born between 1987 and There were 5,264 calves with birth weights (BW, lb; 2,689 bulls and 2,575 heifers), 5,262 calves with weaning weights adjusted to 205 d of age (WW, lb; 614 bulls, 2,573 heifers, and 2,075 steers), and 3,846 calves with postweaning gains from 205 d to 365 d of age (PWG, lb; 209 bulls, 1,784 heifers, and 1,853 steers). Number of calves per breed group, means, and SD for BW, WW, and PWG are shown in Table 1. Calves were the progeny of 293 sires (54 BG1, 37 BG2, 60 BG3, 35 BG4, 38 BG5, and 69 BG6) and 1,725 dams (291 BG1, 249 BG2, 254 BG3, 349 BG4, 200 BG5, and 282 BG6). Feeding and Management Calves resided at the Pine Acres Research Station (1987 to 1994) and at the Beef Research Unit (1995 to 2013) of the University of Florida from birth (December to March) to weaning (August, September). Preweaning, cows and calves were kept on bahiagrass pastures supplemented with bermudagrass hay and cottonseed meal during winter (mid-december to mid-march) and had access to a complete mineral supplement. Postweaning, calves remained at the Pine Acres Research Station or the Beef Research Unit, except from 2006 to 2010 when they were transported to the University of Florida Feed Efficiency Facility (UFFEF). When calves remained at their birth locations (1987 to 2005 and 2011 to 2013), they were kept on bahiagrass pastures supplemented with bahiagrass hay, concentrate (3.5 lb to 7.9 lb per day; 14.0 % crude protein; 488 Pellet Medicated Weaning Ration, Lakeland Animal Nutrition, Lakeland, Florida; soy hull pellets), and free access to a mineral supplement. Calves that went to UFFEF (2006 to 2010) were randomly allocated to pens within sire group (BG1 to BG6) by sex category (bull, heifer, and steer) and fed a diet of whole corn or corn gluten, cottonseed hulls, molasses, chopped grass hay, and a vitamin-mineral-protein supplement (FRM, Bainbridge, GA; mean crude protein=12.9%, mean dry matter=98.2%, mean net energy for maintenance=0.7 mcal/lb DM, and mean net energy for gain=0.5 mcal/lb DM). Tissue Sampling, Genotyping, and Imputation Tissue samples (blood, semen) from 1,232 animals from the MAB herd were collected at the Beef Research Unit of the University of Florida from 2006 to These samples included 161 parents (20 sires and 141 dams), and 1,071 progeny (109 bulls, 613 heifers, and 349 steers). Samples were processed and stored at -80 C at New Mexico State University. Subsamples were sent to GeneSeek (Gene Seek, Inc., Lincoln, NE, USA) in 2010 for genotyping with the Illumina3k chip. Animals genotyped with Illumina3k were imputed to Illumina50k using software findhap2 (VanRaden, 2011) and a reference population of 828 Brangus heifers previously genotyped with version 1 of the Illumina50k chip (Fortes et Florida Beef Research Report

7 al., 2012). The output file haplotypes from findhap2 was subsequently utilized as input file for an inhouse FORTRAN program used to construct phenotypic, genotypic, and pedigree files for the computation of variance components and genetic parameters with the BLUPF90 family of programs (Misztal et al., 2002). The genotype file contained 1,232 MAB animals, each with 46,768 SNP markers (2,639 actual Illumina3k SNP and 44,129 imputed Illumina50k SNP). Variance Components and Genetic Parameters Variance components, heritabilities, and genetic, environmental and phenotypic correlations for BW direct, BW maternal, WW direct, WW maternal, and PWG direct were computed using three multipletrait genomic-polygenic models (Aguilar et al., 2010) for scenarios 1, 2, and 3, and a multiple-trait polygenic model for scenario 4. The four scenarios represented genetic evaluations using: 1) all available phenotypic, pedigree, and genotypic data (genomic-polygenic model 1; GP1); 2) all available phenotypic data, pedigree from non-genotyped animals only, and all available genotypic data (genomic-polygenic model 2; GP2); 3) all available phenotypic and genotypic data, but no pedigree information (genomicpolygenic model 3; GP3); and 4) all available phenotypic and pedigree data and no genotypic information (polygenic model). Scenarios 1 and 4 represent purebred cattle breeders and commercial producers that keep all feasible records and scenarios 2 and 3 represent two cases of commercial operations with incomplete information. Variance components and genetic parameters from GP1, GP2, and GP3 were compared to those from the polygenic model. The fixed effects for the three genomic-polygenic and the polygenic models were: 1) contemporary group (location-year for BW and WW direct and maternal; location-year-pen for PWG); 2) age of dam (all traits); 3) sex of calf (males and females for BW, and bulls, heifers, and steers for WW and PWG; 4) direct heterosis for all traits as a function of calf heterozygosity; and 5) maternal heterosis for BW and WW as a function of dam heterozygosity. Random effects were direct additive genetic for BW, WW, and PWG, maternal additive genetic for BW and WW, and residual for all traits. Restricted maximum likelihood estimates of variance components, genetic parameters, and their standard errors were computed using the BLUPF90 family of programs (Misztal et al., 2002). Genomic-Polygenic and Polygenic Predictions Estimated breeding values (EBV) were computed for all traits (BW and WW direct and maternal, and PWG direct) for 5,190 animals (genotyped=1,232, non-genotyped=3,958) and genotyped animals using genomicpolygenic models 1, 2, and 3 and the polygenic model. Spearman rank correlations were used to compare rankings of animal EBV for each trait in the top 5%, 10%, 25%, and for all evaluated animals. Results Calves with Brahman fractions over 80% had higher BW and lower WW and PWG than calves with Brahman fractions 20% or lower (Table 1). Crossbred calves with Brahman fractions between 40% and 60% had the highest WW, whereas calves with Brahman fractions between 37.5% and 60% had the highest PWG. Variance Components and Genetic Parameters Estimates of additive genetic variances and covariances from genomic-polygenic model 1 were, on the average, slightly larger than those from the polygenic model (mean difference=15.8 lb 2 ), thus the inclusion of genotypic information had little effect on estimates of variance components for growth traits in this multibreed population. Contrarily, exclusion of pedigree information from genotyped animals (genomic-polygenic model 2) or from all animals (genomic-polygenic model 3) underestimated additive genetic variance and covariance components compared to those from the polygenic model (mean difference=-44.5 lb 2 for model 2 and -132,5 lb 2 for model 3). Estimates of environmental variances and covariances for BW, WW, and PWG were, on the average, slightly lower for genomic-polygenic model 1 (mean difference=-11.3 lb 2 ), and higher for genomic-polygenic models 2 (mean difference=61.0 lb 2 ) and 3 (mean difference=225.2 lb 2 ) than estimates from the polygenic model. Estimates of phenotypic Florida Beef Research Report

8 variances and covariances followed the same pattern across models as additive genetic variance components. Estimates of phenotypic variances and covariances for BW, WW, and GW from genomicpolygenic model 1 were slightly higher (mean difference=20.7 lb 2 ), whereas those from genomicpolygenic models 2 (mean difference=-57.9 lb 2 ) and 3 (mean difference=-96.8 lb 2 ) were lower than those from the polygenic model. The pattern for estimates of variance ratios across models mimicked the one for estimates of additive variance components. Estimates of heritabilities and genetic correlations (Table 2) from GP1 and the polygenic model were very similar (mean difference=0.01), while mostly lower estimates were obtained with GP2 (mean difference=-0.04) and GP3 (mean difference=-0.06). Environmental correlations (Table 3) from genomic-polygenic model 1 were nearly identical to those of the polygenic model (mean difference=-0.003), whereas those from genomic-polygenic models 2 (mean difference=0.05) and 3 (mean difference = 0.18) tended to be somewhat higher than estimates from the polygenic model. Nearly identical phenotypic correlations (Table 4) were obtained with GP1and the polygenic model (mean difference=0.003), but slightly lower estimates were computed with GP2 (mean difference=-0.013) and GP3 (mean difference=-0.020) than with the polygenic model. Rankings of Animals Evaluated with Genomic-Polygenic and Polygenic Models Rank correlations between EBV from the three genomic-polygenic and the polygenic models increased as the fraction of the population included in the computations increased from 5% to 10% to 25% to 100%. The highest rank correlations were between EBV from GP1 and the polygenic model (top 5% mean = 0.89; complete population mean=0.98). The second highest rank correlations were between EBV from GP1 and GP2 (top 5% mean=0.52; complete population mean=0.87), and between GP2 and the polygenic model (top 5% mean=0.53; complete population mean=0.87). The lowest rank correlations were between EBV from GP3 and EBV from any of the other models. Considering the cost of genotyping and the short time required for collecting phenotypes for growth traits, the close agreement between the polygenic model and GP1 would favor the use of the polygenic model for growth traits. However, genotypes here were a mixture of actual SNP from Illumina3k and imputed genotypes from Illumina50k. Imputation accuracy from Illumina3k to Illumina50k has ranged from 81% and 93% depending on the imputation procedure (Dassonneville et al., 2011; Huang et al., 2012). Thus, if animals had been genotyped with the Illumina50k, then perhaps larger differences between variance components, genetic parameters, and EBV from GP1 and the polygenic model could have been obtained. However, the issue of genotyping costs would have remained. High genotyping cost is still likely to be the main constraint to widespread use of genotyping for genomic-polygenic evaluation by purebred and commercial cattle producers. Acknowledgements Financial support provided by TSTAR Project number and by Florida Agricultural Experiment Station Hatch Project number FLA-ANS Literature Cited Aguilar, I., et al., J. Dairy Sci. 93: Dassonneville, R., et al., Interbull Bull. 44: Fortes, M. R. S., et al., J. Anim. Sci. 90: Huang, Y., et al., J. Anim. Sci. 90: Misztal, I., et al., Proc. 7 th World Cong. Genet. Appl. Livest. Prod., Communication VanRaden, P. M Florida Beef Research Report

9 Table 1. Numbers of calves, means and standard deviations per breed group and total Trait 1 BW, lb WW, lb PWG, lb Breed group 2 N Mean SD N Mean SD N Mean SD BG BG BG BG4 1, , BG BG Total 5, , , BW = Birth weight; WW = Weaning weight adjusted to 205 d of age; PWG = Postweaning gain from 205 d to 365 d of age. 2 Breed group: BG1 = 100% A to (80% A 20% B); 2) BG2 = (60% A 40% B) to (79% A 21% B); 3) BG3 = Brangus = (62.5% A 37.5% B); 4) BG4 = (40% A 60% B) to (59% A 41% B); 5) BG5 = (20% A 80% B) to (39% A 61%B); and 6) BG6 = (19% A 81% B) to 100% B; A = Angus, B = Brahman. Table 2. REML 1 estimates of direct and maternal heritabilities and additive genetic correlations for growth traits using genomic-polygenic and polygenic models Heritabilities and Additive Genetic Correlations Trait pair 2 GP1 SD GP2 SD GP3 SD PM SD BWD, BWD BWD,WWD BWD, PWGD BWD, BWM BWD, WWM WWD, WWD WWD, PWGD WWD, BWM WWD, WWM PWGD, PWGD PWGD, BWM PWGD, WWM BWM, BWM BWM, WWM WWM, WWM Restricted maximum likelihood. 2 BWD = birth weight direct, WWD = weaning weight direct, PWGD = postweaning gain direct, BWM = birth weight maternal, WWM = weaning weight maternal; GP1, GP2, GP3 = genomicpolygenic models 1, 2, and 3; PM = polygenic model; SD = standard deviation of 5,000 samples Florida Beef Research Report

10 Table 3. REML 1 estimates of environmental correlations for growth traits using genomic-polygenic and polygenic models Environmental correlations Trait pair 2 GP1 SD GP2 SD GP3 SD PM SD BWE,WWE BWE, PWGE WWE, PWGE Restricted maximum likelihood. 2 BWE = birth weight environmental, WWE = weaning weight environmental, PWGE = postweaning gain environmental; GP1, GP2, GP3 = genomic-polygenic models 1, 2, and 3; PM = polygenic model; SD = standard deviation of 5,000 samples. Table 4. REML 1 estimates of phenotypic correlations for growth traits using genomic-polygenic and polygenic models Phenotypic correlations Trait pair 2 GP1 SD GP2 SD GP3 SD PM SD BWP,WWP BWP, PWGP WWP, PWGP Restricted maximum likelihood. 2 BWP = birth weight phenotypic, WWP = weaning weight phenotypic, PWGP = postweaning gain phenotypic; GP1, GP2, GP3 = genomic-polygenic models 1, 2, and 3; PM = polygenic model; SD = standard deviation of 5,000 samples Florida Beef Research Report

11 Genomic Variability and Predictions for Postweaning Ultrasound Traits Using Actual and Imputed Illumina50k SNP Markers in Angus-Brahman Multibreed Cattle M. A. Elzo 1, M. G. Thomas 2, D. D. Johnson 1, C. A. Martinez 1, G. C. Lamb 3, D. O. Rae 4, J. G. Wasdin 1, and J. D. Driver 1 Synapsis Higher fractions of additive genomic variation for ultrasound ribeye area, backfat thickness, intramuscular fat, and weight were accounted for by 46,839 actual and imputed Illumina50k SNP compared to 2,899 SNP Illumina3k in a multibreed Angus-Brahman population. However, total genetic variation and heritabilities increased only for ultrasound back fat and ultrasound weight. Rank correlations between estimated breeding value from genomic-polygenic, genomic, and polygenic models were higher with actual-imputed Illumina50k than with the Illumina3k. Low regressions of estimated breeding value on Brahman fraction indicated that animals of comparable estimated breeding value for ultrasound and weight traits existed across all Angus-Brahman fractions. Summary The objectives were to estimate additive genetic variance fractions for 4 postweaning ultrasound and weight traits explained by 46,839 actual and imputed SNP genotypes, to compare rankings of calf additive genetic predictions from genomic-polygenic (GP), genomic (G), and polygenic (P) models, and to assess trends for GP, G, and P predicted additive genetic values as functions of calf Brahman fractions in a multibreed Angus-Brahman population. Traits were postweaning ultrasound ribeye area (UREA), backfat thickness (UBF), and percent intramuscular fat (UPIMF), and weight (UW). Phenotypes and Illumina3k genotypes were from 812 bulls, heifers, and steers housed at the Feed Efficiency Facility of the University of Florida from 2006 to Program findhap2 was used to impute from 2,899 Illumina3k SNP to 46,839 Illumina50k SNP using a reference population of 828 Brangus heifers. Fixed effects for all models were contemporary group (year-pen), age of dam, sex of calf, age of calf, Brahman fraction of calf, and heterozygosity of calf. Random effects were additive SNP (GP and G models), additive polygenic (GP and P models), and residual. Program GS3 was used to compute variance components, heritabilities, and additive genetic predictions. Additive genetic variance fractions explained by the 46,839 actual and imputed SNP were 0.17 for UREA, 0.32 for UBF, 0.25 for UPIMF, and 0.19 for UW. Heritabilities were 0.33 for UREA, 0.22 for UBF, 0.43 for UPIMF, and 0.54 for UW. These additive genetic variance fractions were 1.8, 1.0, 4.4, and 2.1 times greater and heritabilities were 1.0, 1.2, 1.0, and 1.2 times greater than those obtained with the 2,899 SNP from Illumina3k. Rank correlations between estimated breeding value (EBV) from GP and P models were the highest (0.93 to 0.96), followed by those between EBV from GP and G models (0.81 to 0.94), and by those between estimated breeding value from G and P models (0.66 to 0.81). Regression coefficients of EVB on Brahman fraction were small for all traits and models indicating that animals of comparable EBV existed in all breed groups. Imputation from Illumina3k to 50k increased the explained fraction of additive SNP variance resulting in higher rank correlations between additive genetic predictions from G and GP, and from G and P models for all ultrasound traits in this population. Introduction Brahman and Brahman-Bos taurus crossbred cattle are widely used in Florida and other subtropical regions of the United States because of their superior adaptability to hot and humid climatic conditions. However, Brahman and high-percent crossbred Brahman cattle tend to have smaller ribeye areas, less 1 Department of Animal Sciences, University of Florida, Gainesville, FL 2 Department of Animal Sciences, Colorado State University, Fort Collins, CO 3 North Florida Research and Education Center, University of Florida, Marianna, FL 4 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL Florida Beef Research Report

12 marbling, and lower tenderness than Bos taurus cattle, hence the pressing need for accurate genetic predictions for carcass traits in Brahman and Brahman-Bos taurus crossbred populations. Although high cost has restricted the availability of carcass data, ultrasound carcass measurements are widely used because they are cheaper, easier to measure, and closely associated with carcass traits (Houghton and Turlington, 1992). Another alternative to increase the accuracy of genetic predictions for carcass traits would be to genotype animals with high-density chips. A combination of low and high-density chips plus imputation (Dassonneville et al., 2011; VanRaden et al., 2013) may be a cost-effective alternative to the use of high-density chips throughout a population. Thus, the objectives of this research were: 1) to estimate fractions of additive genetic variances for postweaning ultrasound ribeye area (UREA), backfat thickness (UBF), percent intramuscular fat (UPIMF), and weight (UW) explained by 46,839 actual and imputed SNP genotypes, 2) to compare rankings of additive genetic predictions from genomic-polygenic (GP), genomic (G), and polygenic (P) models, and 3) to assess trends for GP, G, and P predicted additive genetic values as functions of Brahman fractions in a multibreed Angus-Brahman population. Materials and Methods Animals, Feeding, and Management The multibreed Angus-Brahman calves used in this study (n=812; 66 bulls, 413 heifers, and 333 steers) were born at the Beef Research Unit (BRU) of the University of Florida from 2006 to Calves were the offspring of 64 sires from 6 breed groups mated to 364 dams from these same 6 breed groups according to a diallel mating design (Elzo and Wakeman, 1998). Breed groups were: Angus=(1.0 to 0.80) A (0.0 to 0.20) B, ¾ A ¼ B=(0.79 to 0.60) A (0.21 to 0.40) B, Brangus=(0.625) A (0.375) B, ½ A ½ B=(0.59 to 0.40) A (0.41 to 0.60) B, ¼ A ¾ B=(0.39 to 0.20) A (0.61 to 0.80) B, and Brahman: (0.19 to 0.0) A (0.81 to 1.00) B. Numbers of calves per breed group are shown in Table 1. Calves stayed at the BRU from birth to weaning. Postweaning, calves received a preconditioning diet (3.5 lb to 7.9 lb concentrate per day; 14.0 % crude protein; 488 Pellet, Medicated Weaning Ration, Lakeland Animal Nutrition, Lakeland, FL; soy hull pellets, mineral supplement, and bahiagrass hay) for 3 to 4 weeks before moving them to the University of Florida Feed Efficiency Facility (UFFEF) in Marianna, Florida. At UFFEF, calves were randomly assigned to pens within sire group (Angus, ¾ A ¼ B, Brangus, ½ A ½ B, ¼ A ¾ B, and Brahman) by sex (bull, heifer, and steer) subclass and fed an ad libitum ration of corn or corn gluten, cottonseed hulls, molasses, chopped grass hay, and a vitamin-mineral-protein supplement. Average dry matter, crude protein, net energy for maintenance, and net energy for gain were 89.2%, 12.9%, 0.7 mcal/lb DM, and 0.5 mcal/lb DM from 2006 to 2010, respectively. Traits Traits were postweaning ultrasound ribeye area (UREA, in 2 ), ultrasound backfat thickness (UBF, in), ultrasound percent of intramuscular fat (UPIMF, %), and body weight on the day that ultrasound measurements were taken (UW, lb). Ultrasound traits were measured by a certified technician using an Aloka 500 ultrasound system (Hitachi Aloka Medical, Ltd., Wallinford, Connecticut, USA) at the end of the 70-d feed efficiency trial. Phenotypic data for UREA, UBF, and UPIMF were obtained by analyzing the ultrasonic images with UICS Scanning Software by Walter and Associates, LLC (Ames, Iowa, USA). Tissue sampling, Genotyping, and Imputation Blood samples were collected at weaning using 10 ml EDTA vacutainer tubes. Samples were processed at New Mexico State University (NMSU) and stored at -80 C. Genotyping with the Illumina3k was done at GeneSeek (Gene Seek, Inc., Lincoln, NE, USA). Imputation from Illumina3k to Illumina50k was done with program findhap2 (VanRaden, 2011) using a reference population of 828 registered Brangus heifers (Fortes et al., 2012) genotyped with version 1 of the Illumina50k chip. The output file haplotypes from findhap2 was matched with a file containing phenotypic data for UREA, UBF, UPIMF, and UW. Only calves with phenotypes for all 4 traits were kept (n=812). Lastly, SNP with minor allele frequencies lower than 0.04 were discarded (n=3,437). This resulted in a genotype file of 812 animals with SNP data on 46,839 loci (2,641 actual Illumina3k SNP plus 44,198 imputed Illumina50k SNP). These phenotype, genotype, and pedigree files were used as input files for the GS Florida Beef Research Report

13 program (Legarra et al., 2013) used to compute genomic-polygenic variance components and variance ratios, and genomic-polygenic, genomic, and polygenic predictions. Genomic-Polygenic Variance Components, Variance Ratios, and Predictions Single-trait genomic-polygenic mixed models (VanRaden, 2008; Legarra et al., 2008; Elzo et al., 2013) were used to obtain variance components for UREA, UBF, UPIMF, and UW. The mixed model contained: 1) contemporary group (year-pen), age of dam, sex of calf, age of calf, Brahman fraction of calf, and heterozygosity of calf as fixed effects; and 2) additive SNP marker locus effect as a function of the number of 2 alleles in each locus (mean zero; variance=additive SNP variance), calf additive polygenic effect (mean zero; variance=a*vg; A=additive relationship matrix, Vg=additive polygenic variance), and residual (mean zero, common variance) as random effects. The procedure used to estimate variance components and heritabilities was Markov Chain Monte Carlo (MCMC). Computations were carried out with program GS3, option VCE (Legarra et al., 2013). Additive genomic variances (VAGO), polygenic variances (VAPO), total additive genetic variances (VGTOT), phenotypic variances (PVAR), and heritabilities were computed for each MCMC sample as follows: 1) VAGO = SNP variance 46,839 i=1 2(1 q i )q i, where q i = frequency of allele 2 in locus i; 2) VGTOT = VAGO + VAPO; 3) PVAR = VAGO + VAPO + residual variance; and 4) heritability = VGTOT/PVAR. Posterior means and standard deviations for VAGO, VAPO, VGTOT, PVAR and heritabilities for UREA, UBF, UPIMF, and UW were computed using 1,000 MCMC samples following the burn-in period. Polygenic variances and heritabilities were also estimated with polygenic models for comparison purposes. Genomic-Polygenic, Genomic, and Polygenic Predictions Genomic-polygenic (GPEBV), genomic (GEBV), and polygenic predicted values (PEBV) for each trait were computed with option BLUP of program GS3 (Gauss-Seidel iteration; convergence criterion = 10-8 ) using genomic-polygenic, genomic, and polygenic models and posterior means of VAGO, VAPO, and VRES. Calf rankings across models were compared using Spearman s rank correlations. Linear regressions of GPEBV, GEBV, and PEBV on calf Brahman fraction were used to assess trends in predicted values as Brahman fraction increased. Results Overall means were 9.1 in 2 for UREA, 0.25 in for UBF, 2.78 % for UPIMF, and lb for UW (Table 1). The largest means were those of ¼ A ¾ B calves for UREA (9.6 in 2 ) and UBF (0.28 in), Angus calves for UPIMF (3.16 %), and ¾ A ¼ B for UW (784.2 lb). The smallest means were from Brahman calves for UREA (8.4 in 2 ), UBF (0.24 in), and UW (691.1 lb), and ¼ A ¾ B calves for UPIMF (2.40 %). The largest SD were in Angus for UREA (2.1 in 2 ), ¼ A ¾ B and Brahman for UBF (0.17 in), Brahman for UPIMF (1.62 %), and ¾ A ¼ B for UW (131.0 lb), and the smallest SD were those of Brahman calves for UREA (1.7 in 2 ) and UW (106.9 lb), ¾ A ¼ B, Brangus, and ½ A ½ B calves for UBF (0.15 in) and Brangus calves for UPIMF (1.47 %). Genomic and Polygenic Variance Components and Variance Ratios Table 2 contains posterior means and SD for VAGO, VAPO, VGTOT and PVAR from genomicpolygenic models and additive polygenic (VGPO) and phenotypic variances (PVARPO) from polygenic models for UREA, UBF, UPIMF, and UW. Table 3 presents posterior means and SD for variance ratios (VAGO/VGTOT and VAGO/PVAR) and heritabilities from genomic-polygenic and polygenic models for UREA, UBF, UPIMF, and UW. Estimates of VAGO with Illumina50k SNP markers were between 3% (UBF) to 342% (UPIMF) larger than estimates with Illumina3k SNP markers, whereas VAPO estimates with the Illumina50k were lower for UREA and UPIMF and higher for UBF and UW than with the Illumina3k (Table 4). Consequently, estimates of VGTOT with the Illumina50k were similar for UREA and UPIMF but larger for UBF (24%) and UW (16%) than with the Illumina3k. Thus, heritabilities with the Illumina50k were also similar for UREA and UPIMF and larger for UBF (22%) and UW (19%) than with the Illumina3k because PVAR estimates had similar values for all ultrasound traits with both Illumina chips (Table 4). Estimates of VGTOT from genomic-polygenic models were larger Florida Beef Research Report

14 than VGPO from polygenic models for all ultrasound traits (from 21% for UREA to 40% for UBF) indicating that the 46,839 actual-imputed SNP may have accounted for genetic variation beyond that explained by polygenic models. Lastly, because PVAR from genomic-polygenic and polygenic models were similar (Table 5), heritability estimates from genomic-polygenic models for all ultrasound traits were larger than estimates from polygenic models for all traits (from 18% for UREA to 41% for UBF). Rankings of Animals Evaluated with Genomic-Polygenic, Genomic, and Polygenic Models The highest rank correlations were between EBV from the GP and P models (from 0.93 for UBF to 0.96 for UW; P<0.0001), followed by those between EBV from the GP and G models (from 0.81 for UW to 0.94 for UPIMF; P<0.0001), and lastly by those between EBV from the G and P models (from 0.66 for UBF to 0.81 for UPIMF; P<0.0001). Rank correlations between EBV from GP and P models here were similar to rank correlations between GP and P models with Illumina3k (Elzo et al., 2013). Conversely, rank correlations between EBV from GP and G and from G and P models here were on the average 26% and 24% higher than corresponding values with Illumina3k SNP markers. These rank correlations suggested that some of the 44,198 imputed SNP from the Illumina50k chip were in linkage disequilibrium with QTL affecting UREA, UBF, UPIMF, and UW that provided additional additive genetic information for these traits, and increased the similarity among calf G, GP and P EBV in this population. Lastly, ultrasound trait EBV rankings from GP, G, and P models with actual-imputed Illumina50k SNP markers here and with Illumina3k SNP markers (Elzo et al., 2013) were compared using a subset of 615 calves present in both datasets. Higher rank correlations existed between EBV from Illumina50k and Illumina3k datasets with GP models (from 0.90 for UBF to 0.95 for UW), than rank correlations between EBV with P models (from 0.88 for UREA to 0.94 for UW) and G models (from 0.62 for UW to 0.78 for UBF). This indicated that the sets of actual-imputed Illumina50k and Illumina3k genotypes captured a substantially lower fraction of the additive genetic variation relative to polygenic effects and that their contribution to the EBV from GP models was small for all ultrasound traits. Thus, rank correlations between EBV from GP, P, and G models within and across Illumina50k and Illumina3k datasets suggested that polygenic models would be enough to rank animals appropriately for ultrasound and feed efficiency traits in this multibreed population. Trends of genomic-polygenic, genomic, and polygenic EBV from Angus to Brahman Regression coefficients of calf EVB on Brahman fraction were small for all ultrasound traits and GP, G, and P models. Significant regression values (P<0.04 to P<0.0001) existed for UREA (G model), UBF (GP and G models), and UW (all models). A similar pattern of significance was obtained with the Illumina3k MAB dataset (Elzo et al., 2013). Although EBV computed with GP, G, and P models tended to decrease as Brahman increased for all ultrasound traits in the actual-imputed Illumina50k and Illumina3k datasets, regression estimates were low for all traits indicating that this population contained animals with analogous EBV for UREA, UBF, UPIMF, and UW across all breed compositions. Acknowledgements Financial support provided by TSTAR Project number and by Florida Agricultural Experiment Station Hatch Project number FLA-ANS Literature Cited Dassonneville, R., et al., Interbull Bull. 44: Elzo, M. A., et al., Livest. Sci. 153, Elzo, M. A., and D. L. Wakeman J. Anim. Sci. 76: Fortes, M. R. S., et al., J. Anim. Sci. 90: Houghton, P. L., and L. M. Turlington J Anim. Sci. 70: Legarra, A., et al., Legarra, A., et al., Genetics 180: VanRaden, P. M VanRaden, P. M., et al., Genet. Sel. Evol. 43: Florida Beef Research Report

15 Table 1. Numbers of calves, means and standard deviations per breed group and total Trait a UREA, in 2 UBF, in UPIMF, % UW, lb Breed group N Mean SD Mean SD Mean SD Mean SD Angus ¾ A ¼ B Brangus ½ A ½ B ¼ A ¾ B Brahman Total a UREA = ultrasound rib eye area; UBF = ultrasound backfat; UPIMF = ultrasound percent intramuscular fat; UW = ultrasound weight. Table 2. Posterior means and standard deviations for additive genomic, polygenic, total genetic and phenotypic variances Trait a Variance b UREA, in 4 UBF, in 2 UPIMF, % 2 UW, lb 2 VAGO 0.09 ± ± ± ± VAPO 0.44 ± ± ± ± VGTOT 0.53 ± ± ± ± PVAR 1.34 ± ± ± ± VGPO 0.44 ± ± ± ± PVARPO 1.32 ± ± ± ± a UREA = ultrasound rib eye area; UBF = ultrasound backfat; UPIMF = ultrasound percent intramuscular fat; UW = ultrasound weight. b VAGO = additive genomic variance; VAPO = additive polygenic variance; VGTOT = total genetic variance = VAGO + VAPO; PVAR = phenotypic variance; VGPO = additive genetic variance from a polygenic model; PVARPO = phenotypic variance from a polygenic model. Table 3. Posterior means and standard deviations for additive genetic and genomic variance ratios Trait a Variance Ratios b UREA UBF UPIMF UW VAGO/VGTOT 0.17 ± ± ± ± 0.10 VAGO/PVAR 0.07 ± ± ± ± 0.07 Heritability 0.39 ± ± ± ± 0.10 HeritabilityPO 0.33 ± ± ± ± 0.09 a UREA = ultrasound rib eye area; UBF = ultrasound backfat; UPIMF = ultrasound percent intramuscular fat; UW = ultrasound weight. b VAGO = additive genomic variance; VGTOT = VAGO + VAPO; PVAR = phenotypic variance; HeritabilityPO = heritability from a polygenic model Florida Beef Research Report

16 Table 4. Ratios of posterior means of variances and variance ratios from actual-imputed Illumina50k a and Illumina3k b genomic-polygenic analyses Trait c Ratio 50k/3k d UREA UBF UPIMF UW VAGO VAPO VGTOT PVAR VAGO/VGTOT VAGO/PVAR Heritability a 2,641 actual Illumina3k SNP plus 44,198 imputed Illumina50k SNP. b 2,899 Illumina3k SNP (Elzo et al., 2013). c UREA = ultrasound rib eye area; UBF = ultrasound backfat; UPIMF = ultrasound percent intramuscular fat; UW = ultrasound weight. d VAGO = additive genomic variance; VAPO = additive polygenic variance; VGTOT = VAGO + VAPO; PVAR = phenotypic variance. Table 5. Ratios of posterior means of variances and variance ratios from genomic-polygenic and polygenic models Trait a Ratio b UREA UBF UPIMF UW VAPO/VGPO VGTOT/VGPO PVAR/PVARPO Heritability/HeritabilityPO a UREA = ultrasound rib eye area; UBF = ultrasound backfat; UPIMF = ultrasound percent intramuscular fat; UW = ultrasound weight. b VAPO = additive polygenic variance; VGTOT = total genetic variance; PVAR = phenotypic variance; VGPO = additive genetic variance from a polygenic model; PVARPO = phenotypic variance from a polygenic model; HeritabilityPO = heritability from a polygenic model Florida Beef Research Report

17 Effects of Administration of Prostaglandin F2α at Initiation of the 7-d CO- Synch+CIDR Ovulation Synchronization Protocol for Suckled Beef Cows and Replacement Beef Heifers V. R. G. Mercadante 1, L. E. Kozicki 2, F. M. Ciriaco 1, D. D. Henry 1, C. R. Dahlen 3, M. R. Crosswhite 3, J. E. Larson 4, B. E. Voelz 4, D. J. Patterson 5, G. A. Perry 6, R. N. Funston 7, T. L. Steckler 8, S. L. Hill 9, J. S. Stevenson 9, G. C. Lamb 1 Syopsis The objective of this study was to determine the effect of administering PGF 2α at the initiation of the 7-d CO-Synch+CIDR fixed-timed AI (TAI) protocol on pregnancy rates of suckled beef cows and replacement heifers. Addition of PGF 2α to the 7-d CO-Synch+CIDR protocol decreased concentrations of progesterone in cows and heifers and increased follicle diameter at CIDR removal in cows, but failed to increase TAI pregnancy rates. Summary Two experiments were conducted to determine the effect of administering PGF 2α at the initiation of the 7- d CO-Synch+CIDR fixed-timed AI (TAI) protocol on pregnancy rates of suckled beef cows and replacement heifers. Within location, cows were stratified by days postpartum (DPP), body condition score (BCS), and parity (Exp. 1; n=1,551) and heifers were stratified by BCS (Exp. 2; n=999) and assigned randomly to 1 of 2 treatments: 1) CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with a 25-mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and TAI on d 0); or 2) PG-CO-Synch+CIDR (a 25-mg injection of PGF 2α on d -10 of the CO- Synch+CIDR protocol). Follicle diameter and corpus luteum (CL) development were assessed on d -10 and -3, and pregnancy status determined on d 30 to 35. Blood was collected on d -20, -10, -3, and 0 relative to TAI to determine concentrations of progesterone (P4). In Exp. 1, TAI pregnancy rates did not differ (P=0.667) between treatments and were affected by BCS (P=0.003) and DPP (P=0.006). Concentrations of P4 were greater (P<0.0001) on d -3 for CO-Synch+CIDR than for PG-CO- Synch+CIDR (4.1±0.2 and 3.4±0.2 ng/ml, respectively). Follicle diameter on d -3 differed (P=0.05) between PG-CO-Synch+CIDR (13.4±0.3 mm) and CO-Synch+CIDR (12.5±0.3 mm) treatments. In Exp. 2, TAI pregnancy rates did not differ (P=0.32) between treatments. Concentrations of P4 differed (P<0.0001) on d -3 with greater concentrations of P4 for CO-Synch+CIDR than PG-CO-Synch+CIDR (3.75±0.20 ng/ml and 3.60±0.21 ng/ml, respectively). Follicle diameter was similar (P=0.75) between treatments on d -10 and d -3. Regardless of treatment, cyclic status tended (P=0.06) to improve pregnancy rates to TAI (55% vs. 45%, for cycling and noncycling heifers, respectively).we concluded that addition of PGF2α to the 7-d CO-Synch+CIDR protocol decreased concentrations of P4 in cows and heifers and increased follicle diameter at CIDR removal in cows, but failed to increase TAI pregnancy rates Florida Beef Research Report

18 Introduction Utilization of estrus or ovulation synchronization and fixed-timed artificial insemination (TAI) has facilitated the widespread utilization of AI with proven sires to desirable characteristics such as calving ease and birth weight, which can be especially important when breeding replacement heifers (Lamb et al., 2010). In addition, utilization of TAI protocols can also impact the economic viability of cow-calf systems by enhancing weaning weights per cow exposed (Rodgers et al., 2012). The majority of these TAI protocols depend largely on the use of exogenous progesterone (P4) supplemented by an intravaginal controlled internal drug release (CIDR) insert, GnRH-induced ovulation, and PGF 2α to induce luteolysis (Larson et al., 2006; Lamb et al., 2010). In addition, it has been shown that reduced concentrations of P4 can increase luteinizing hormone (LH) pulse frequency (Kojima et al., 1992) and enhance development and growth rates of ovarian follicles (Fortune, 1994), both of which have improved pregnancy to TAI (Lamb et al., 2001). Therefore, we hypothesized that the addition of an injection of PGF 2α at the initiation of the 7-d CO- Synch+CIDR ovulation synchronization protocol would decrease concentration of P4 and increase follicle diameter at the time of CIDR removal and enhance pregnancy rates to TAI in suckled beef cows and replacement beef heifers. Materials and Methods Experiment 1 A total of 1,551 primiparous and multiparous beef cows were enrolled at 10 different locations in 6 states (Florida, Illinois, Kansas, Mississippi, North Dakota and South Dakota). Within location, cows were stratified by days postpartum (DPP), BCS, and parity, and then assigned randomly to receive one of two treatments: 1) 100-μg injection of GnRH (2 ml Factrel; Zoetis Animal Health) at CIDR (1.38 g P4; Zoetis Animal Health) insertion [d -10] with 25-mg injection of PGF 2α (5 ml Lutalyse; Zoetis Animal Health) at CIDR removal [d -3], followed by injection of 100-μg GnRH and TAI [d 0] by 66 h after CIDR removal (CO-Synch+CIDR; n=773); or 2) same as CO-Synch+CIDR with an additional 25-mg injection of PGF 2α administered at CIDR insertion [d -10] (PG-CO-Synch+CIDR; n=778; Figure 1). Experiment 2 A total of 999 replacement beef heifers were enrolled at nine different locations in six states (Florida, Illinois, Mississippi, North Dakota, Nebraska and South Dakota). Within location heifers were stratified by BCS and then randomly assigned to receive the same treatments (CO-Synch+CIDR, n=498; PG-CO- Synch+CIDR, n=501; Figure 1) previously described in Exp. 1, with the modification of the timing of insemination (54 h after CIDR removal). Blood samples were collected on d -20, -10, -3 and 0 from a subset of cows (n=1,136) and heifers (n=273) randomly select within locations. Concentration of P4 was analyzed by radioimmune assay kits (Coat-A-Count; Siemens Healthcare Diagnostics, Los Angeles, CA, USA). Transrectal ultrasonography (5.0-MHz linear array transducer, Aloka 500V, Instrument of Science and Medicine, Vancouver, BC, Canada) was performed on d -10 and d 0 to determine the diameter of the dominant follicle and corpus luteum (CL) volume in a subset of cows (n=235) at the FL, ND-2 and KS-1 locations in Exp. 1 and in a subset of heifers (n=139) at the FL and ND locations in Exp. 2. The SAS (version 9.3; SAS/STAT, SAS Inst. Inc., Cary, NC, USA) statistical package was used for all analyses. The experiment was designed as a randomized block design. Experiment 1 pregnancy rates per TAI and final pregnancy rates at the end of the breeding season were analyzed using the GLIMMIX procedure. The model included the effects of treatment, and the fixed effects of location, parity (primiparous and multiparous), BCS (<5 5 to 5.75, and 6), DPP (<50 d, 50 d to <70 d, and 70 d) and the respective interactions. The GLIMMIX procedure also was used to analyze follicle diameter and presence and volume of CL on d -10 and -3. Follicle diameter on d -10 was used as a covariate in the analysis of follicle diameter on d - 3. The models included the effects of treatment, location, BCS, DPP, and respective interactions. Experiment 2 pregnancy rates per TAI and final pregnancy rate at the end of the breeding season were analyzed using the GLIMMIX procedure. The model included the effects of Florida Beef Research Report

19 treatment and the fixed effects of location, BCS (<5, 5.5 to 6, and 6.5), and the respective interactions. The GLIMMIX procedure was also used to analyze follicle diameter and CL volume on d - 10 and -3. The models included the effects of treatment, location, BCS and respective interactions. Results Experiment 1 Overall pregnancy rates to TAI (54.0±0.5% and 50.8±0.5%, for CO-Synch+CIDR and PG-CO- Synch+CIDR, respectively; Table 1) did not differ (P=0.84) between treatments, and no treatment location interaction was detected (P=0.17). In contrast, a location effect (P<0.0001) was detected for pregnancy rates to TAI being the greatest at the ND-2 location (67.0±0.3%) and the least at the KS-1 location (Table 1). Regardless of treatment, cows with BCS < 5 had the poorest pregnancy rate to TAI (43%) compared with cows with BCS between 5 and 5.75 (49%) and BCS to 6 (56%; P=0.003; Table 1). In addition, cows at less than 50 DPP had the poorest pregnancy rate to TAI (34%) compared with cows at > 50 DPP (46% for 50 to 69 DPP vs. 55% for >70 DPP; P=0.006; Table 1). Where cyclic status was assessed, 53% of cows were cyclic at the beginning of the synchronization protocols and cyclic status did not differ (P=0.88) between treatments. A location effect, however, was detected for cyclic status (P<0.0001; Table 1) with the ND-2 location having the fewest cyclic females (40%), whereas the SD-2 location had the greatest proportion of cyclic females (76%). Nonetheless, incidence of pregnancy to TAI was not associated with cyclic status at the initiation of the treatments (55.5% vs. 49.3%, for cyclic and anestrous cows, respectively; P=0.17) and no treatment cyclicity interaction was detected (P=0.87). Concentrations of P4 did not differ (P>0.10; Figure 2) between treatments on d -10 at CIDR insertion and on d 0 at TAI. Nevertheless, cows in the CO-Synch+CIDR treatment had greater (P<0.0001) concentrations of P4 at CIDR removal on d -3 compared with PG-CO- Synch+CIDR (Figure 2). When present in the ovary, the volume of the CL did not differ between treatments on d -10 (10.7±2.0 mm 3 and 13.9±2.4 mm 3, for CO-Synch+CIDR and PG-CO-Synch+CIDR cows, respectively; P=0.35) or on d -3 (17.5±2.3 mm 3 and 12.9±2.2 mm 3, for CO-Synch+CIDR and PG-CO-Synch+CIDR cows, respectively; P=0.18). Furthermore, diameter of the largest ovarian follicle present on d -10 at CIDR insertion did not differ (P=0.85) between treatments (Figure 3). In contrast, diameter of the largest ovarian follicle on d -3 at CIDR insert removal was greater (P=0.05) in the PG-CO-Synch+CIDR cows than in CO-Synch+CIDR cows (Figure 3). Experiment 2 Overall pregnancy rate by TAI (55.2±0.5% and 52.6±0.6%, for CO-Synch+CIDR and PG-CO- Synch+CIDR, respectively; Table 2) did not differ (P=0.32) between treatments, and no treatment location interaction was detected (P=0.47). However, there was a location effect (P<0.0001) with pregnancy rates to TAI being the greatest at the SD-2 location and the poorest at the MS location (Table 2). Where cyclic status was assessed, 80% of heifers were cyclic at the initiation of TAI protocols. Nonetheless, cyclic status differed (P<0.0001) among locations, with the FL location having the least proportion of cyclic heifers (48%) and SD-1 the most cyclic heifers (100%) at the beginning of the TAI protocols. Regardless of treatment, cyclic status tended (P=0.06) to improve pregnancy rates to TAI (55% vs. 45%, for cyclic and noncyclic heifers, respectively). Concentrations of P4 were similar (P>0.10; Figure 4) between treatments on d -10 at CIDR insertion and on d 0 at TAI. However, heifers in the CO-Synch+CIDR treatment had greater (P<0.0001) concentrations of P4 at CIDR removal on d -3 compared to PG-CO-Synch+CIDR (Figure 4). In addition, when present in the ovary, the volume of the CL did not differ between treatments on d -10 (P=0.75) or on d Florida Beef Research Report

20 (P=0.64). Also, diameter of the largest ovarian follicle was similar (P=0.75; Figure 5) between treatments on d -10 at CIDR insertion and on d -3 at CIDR removal. Conclusion In the present study, by administering PGF 2α at the initiation of the 7-d CO-Synch+CIDR protocol, we succeeded in reducing concentrations of P4 at the time of CIDR removal in both cows and heifers, and in enhancing follicle size at the time of CIDR removal in cows, but not heifers. Pregnancy rates to TAI, however, were not improved when PGF 2α was administered concurrently with GnRH at CIDR insertion. Further investigation into the ideal concentrations of P4 at CIDR insertion and removal, as well as the effects of follicle size and the presence of a CL at key stages of estrous synchronization protocols and their respective impacts on pregnancy outcomes in beef females need further investigation. Acknowledgements Sincere appreciation is expressed to P. Folsom, M. Foran, O. Helms, D. Jones, C. Nowell, T. Schulmeister, and D. Thomas for their assistance with data collection and laboratory analysis. The authors thank Zoetis Animal Health (Florham Park, NY) for their donation of PGF 2α (Lutalyse), GnRH (Fertagyl), and CIDR inserts (CIDR EAZI-Breed). Literature Cited Fortune, J. E Biol. Reprod. 50: Kojima, N., et al Biol. Reprod. 47: Lamb, G. C., et al J. Anim. Sci. 88:E Lamb, G. C., et al J. Anim. Sci. 79: Larson, J. E., et al J. Anim. Sci. 84: Rodgers, J. C., et al J. Anim. Sci. 10: Florida Beef Research Report

21 Table 1. Pregnancy rates to fixed-timed AI in suckled beef cows after treatment with CO-Synch+ CIDR or PG-CO-Synch+CIDR. Treatment 1 Item CO-Synch+CIDR PG-CO-Synch+CIDR Overall no. (%) Location FL 76/122 (62) 55/125 (44) 131/247 (53) xy IL-1 28/51 (55) 32/53 (60) 60/104 (58) xy IL-2 30/54 (55) 36/56 (64) 66/110 (60) xy KS-1 18/92 (19) 22/88 (25) 40/180 (22) z KS-2 71/105 (68) 60/105 (57) 131/210 (62) x MS 27/59 (46) 29/62 (47) 56/121 (46) y ND-1 37/61 (61) 33/60 (55) 70/121 (58) xy ND-2 16/27 (59) 21/28 (75) 37/55 (67) x SD-1 82/147 (56) 78/147 (53) 160/294 (55) xy SD-2 33/55 (60) 29/54 (54) 62/109 (57) xy Overall 418/773 (54) 395/778 (50) 813/1,551 (52) BCS 2 < 5 25/53 (47) 24/62 (39) 49/115 (43) x 5 to /398 (50) 196/409 (48) 395/807 (49) y 6 212/366 (58) 196/356 (55) 408/722 (56) y Days post-partum < 50 24/69 (35) 21/62 (34) 45/131 (34) x 50 to 69 68/141 (48) 57/132 (43) 125/273 (46) y /384 (57) 223/413 (54) 442/797 (55) y 1 CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with a 25-mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and fixed-timed AI on d 0). PG-CO-Synch+CIDR (25-mg injection of PGF 2α administered at CIDR insertion [d -10] of the CO-Synch+CIDR). 2 Body Condition Score in a scale of 1 (emaciate) to 9 (obese). x,y,z Percentages within item and column with differing superscripts differ (P<0.01). Effect of treatment (P=0.84); location (P<0.0001); treatment by location (P=0.17) Florida Beef Research Report

22 Table 2. Pregnancy rates to fixed-timed AI in replacement beef heifers after treatment with Co-Synch+CIDR or PG-CO-Synch+CIDR. Treatment 1 Location CO-Synch+CIDR PG-CO-Synch+CIDR Overall no. (%) FL 42/62 (68) 31/62 (50) 73/124 (59) x IL-1 13/32 (41) 10/33 (30) 33/65 (51) y IL-2 11/19 (58) 8/20 (40) 19/39 (49) xy MS 3/14 (21) 5/14 (36) 8/28 (29) z ND 14/25 (56) 14/26 (54) 28/51 (55) xy NE 50/118 (43) 54/115 (47) 104/233 (45) yz SD-1 11/21 (52) 13/20 (65) 24/41 (58) xy SD-2 63/101 (62) 69/108 (64) 132/209 (63) x SD-3 68/106 (64) 60/103 (58) 128/209 (61) x Overall 275/498 (55) 264/501 (53) 539/999 (54) 1 CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with 25 mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and fixed-timed AI on d 0). PG-CO-Synch+CIDR (a 25 mg injection of PGF 2α administered at CIDR insertion [d -10] of the CO-Synch+CIDR). x,y,z Percentages within item and column with differing superscripts differ. Effect of Treatment (P=0.61); Location (P<0.0001); Treatment*Location (P=0.47). Figure 1. Schematic of treatments. Blood samples (B) were collected on d -20, -10, -3, and 0. Individual body condition scores (BCS; 1=thin to 9=obese) were assigned at d -20. Ovarian ultrasonography (US) was performed on d -10 and -3. Pregnancy diagnosis was performed by US on d 35 and between d 30 to 45 after the end of the breeding season. The CO-Synch+CIDR females received an injection of GnRH on d -10 and CIDR was inserted, followed by injection of PGF 2α (PGF) and CIDR removal on d -3. All females received fixed-timed AI (TAI) followed by injection of GnRH on d 0 (by 66 h for cows, Exp.1; and by 54 h for heifers, Exp. 2) after CIDR insert removal. The PG-CO-Synch+CIDR females received the same treatment previously described with an additional injection of PGF on d Florida Beef Research Report

23 Progesterone, ng/ml CO-Synch+CIDR PG-CO-Synch+CIDR ** d -10 d -3 d 0 Day relative to TAI Figure 2. Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of suckled beef cows by treatment, Exp. 1. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P<0.0001). Follicle diameter, mm CO-Synch+CIDR PG-CO-Synch+CIDR ** d -10 d -3 Treatment Figure 3. Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-timed AI of suckled beef cows by treatment, Exp. 1. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P=0.05) Florida Beef Research Report

24 Progesterone, ng/ml CO-Synch+CIDR PG-CO-Synch+CIDR d -10 d -3 d 0 Day relative to TAI Figure 4. Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of replacement beef heifers by treatment, Exp. 2. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P<0.0001). 13 CO-Synch+CIDR PG-CO-Synch+CIDR Follicle diameter, mm d -10 d -3 Treatment Figure 5. Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-timed AI of replacement beef heifers by treatment, Exp. 2. CO-Synch+CIDR: an injection of GnRH on d - 10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. Effect of treatment (P=0.75) Florida Beef Research Report

25 Effects of Recombinant Bovine Somatotropin Administration at Breeding on the Cow, Conceptus, and Subsequent Offspring Performance of Beef Cattle V. R. G. Mercadante 1, F. M. Ciriaco 1, D. D. Henry 1, P. L. P. Fontes 1, N. Oosthuizen 1, T. Schulmeister 1, N. DiLorenzo 1, and G. C. Lamb 1. Synapsis The objective of this study was to determine if administration of recombinant bovine somatotropin (bst) to beef females at breeding has the potential to increase fertility, conceptus development, and subsequent offspring performance. Injection of 325 mg of bst around the time of breeding increased concentration of insulin-like growth factor-1 (IGF-1) and increased expression of myxovirus-2 on d 21 of suckled beef cows. However, it failed to alter fetal development and postnatal offspring performance. Summary To determine the effects of administration of a low dose of slow-release bst (Posilac, Elanco, Greenville, IN) on hormone concentration and conceptus development, a total of 190 suckled beef cows were exposed to the 7-d CO-Synch+CIDR fixed-time AI (TAI) protocol. Cows were blocked by days postpartum, BCS, breed, and randomly assigned to receive one of the following treatments: 1) two injections of 325 mg bst, one at TAI and a second injection 14 d after TAI (D-bST, n=40); 2) one injection of 325 mg bst at TAI and a placebo (saline) injection 14 d after TAI (TAI-bST, n=48); 3) a placebo injection at TAI and one injection of 325 mg bst 14 d later (14D-bST n=49); and 4) two injections of placebo, one at TAI and a second injection 14 d after TAI (Ctrl, n=53). Pregnancy was determined via transrectal ultrasonography 35 d after TAI and conceptus development was assessed by measuring crown to rump length (CRL) on d 35 and crown to nose length (CNL) on d 65 after TAI. Blood samples were collected on d 0, 7, 14, 21, 35 and 65 relative to TAI to determine concentrations of IGF-1, and on d 18 and 21 for isolation of peripheral blood leukocytes (PBL) and mrna expression of interferon stimulated genes. Plasma concentrations of pregnancy-specific protein B (PSPB) were also assessed on d 35 and 65 after TAI. Individual calf birth weight and sex were determined at birth. Liver biopsies were performed in a subset of calves at 8 ± 3 d of age, and expression of target genes related to the somatotropic axis were determined. There were no differences (P=0.77) among treatments on pregnancy to TAI (48.7±0.5%). Administration of bst at TAI increased (P<0.0001) plasma concentration of IGF-1 on d 7, 14, and 21. However, CRL and CNL (0.48±0.02 in and 0.67±0.001 in, respectively) did not differ (P=0.23) among treatments. Concentration of PSPB did not differ (P=0.18) among treatments and between days (P=0.30; 2.69±0.11 ng/ml), and gestation length (282±9 d) did not differ (P=0.49) among treatments. However, mrna expression of myxovirus-2 on d 21 differed (P=0.05) among treatments. Cows receiving an injection of bst at TAI had a greater fold increase in MX-2 mrna expression compared to cows receiving an injection of bst at TAI and 14 d later. Injection of bst did not alter calf liver mrna expression of insulin-like growth factor-1 (P=0.81), IGF-2 (P=0.29), and IGFBP-3 (P=0.66). However, injection of bst at TAI tended (P=0.06) to increase calf liver mrna expression of IGFR-1 compared to the calves born to cows in other treatments. In addition, calf birth weight was similar (P=0.52) among treatments. We conclude that administration of 325 mg bst during the time of TAI to suckled beef cows enhanced concentrations of IGF-1, but failed to improve pregnancy rates, fetal size, PSPB concentrations, and had no effect on calf birth weight Florida Beef Research Report

26 Introduction Developmental programming, also termed fetal programming, is the concept that perturbations during critical prenatal development stages may have lasting impacts on postnatal growth and adult function (Godfrey, 1998). Growth and development of the embryo and fetus are complex biological events influenced by genetic, epigenetic, maternal maturity, as well as environmental and other factors. These factors affect the size and functional capacity of the placenta, uteroplacental transfer of nutrients and oxygen from mother to fetus, conceptus nutrient availability, the fetal endocrine milieu, and metabolic pathways. The somatotropic axis and its major components growth hormone (GH) and IGF-1 are an essential constituent of multiple systems controlling growth and reproduction (LeRoith et al., 2001). In addition, the somatotropic axis plays a key role on embryonic and fetal development by acting directly on the oocyte, endometrium, placenta, and embryo. Early exposure of ovine embryos to increased concentrations of GH and IGF-1 has been linked to enhanced prenatal development and increased postnatal growth, with alterations in the somatotropic axis (Costine et al., 2005; Koch et al., 2010). In addition, strategies focusing on supplementation of GH and IGF-1, through administration of recombinant bovine somatotropin (bst), during the time of embryonic implantation and maternal recognition of pregnancy have been successful on enhancing conceptus development and increasing fertility of dairy cattle (Ribeiro et al., 2013). However, little information exists on the effects of bst treatment on beef females at breeding and in subsequent offspring performance. Therefore, we hypothesized that administration of bst to beef females at breeding has the potential to increase fertility, conceptus development and subsequent offspring performance. Materials and Methods A total of 190 multiparous suckled beef cows composed of Angus, Brangus, and Braford breeds were enrolled in the experiment. All cows were subjected to the 7-day CO-Synch + CIDR estrus synchronization protocol. In brief, cows received a 100-μg injection of GnRH (2 ml Factrel; Zoetis Animal Health) at CIDR (1.38 g P4; Zoetis Animal Health) insertion [d - 10] with a 25-mg injection of PGF 2α (5 ml Lutalyse; Zoetis Animal Health) at CIDR removal [d -3], followed by an injection of 100-μg GnRH and TAI [d 0] at 66 h after CIDR removal. Cows were blocked by days postpartum, body condition score (BCS), breed and randomly assigned to receive one of the following treatments (Figure 1): 1) two injections of placebo (1 ml of 0.9% saline), one at TAI and a second injection 14 d after TAI (CTRL, n=53); 2) two injections of 325 mg bst (Posilac, Elanco Animal Health, Greenville, IN, USA), one at TAI and a second injection 14 d after TAI (2bST, n=40); 3) one injection of 325 mg bst at TAI and a placebo injection 14 d after TAI (TAIbST, n=48); and 4) a placebo injection at TAI and one injection of 325 mg bst 14 d later (d14bst n=49). Blood samples from each cow were collected on d 0, 7, 14, 18, 21, 35 and 65. Concentrations of total IGF-1 were determined by a commercial ELISA kit (Quantikine ELISA Human IGF1 Immunoassay, R&D Systems, Inc., Minneapolis, MN, USA). Concentrations of PSPB were analyzed using a commercially available quantitative ELISA assay (BioPRYN, BioTracking LLC, Moscow, ID). Blood samples were collected on days 18 and 21 for isolation of peripheral blood leukocytes (PBL) and mrna expression of interferon stimulated genes (ISG). Pregnancy status was determined by transrectal ultrasonography on d 35 and 65 with an Ibex portable ultrasound equipped with a linear 5 MHz multi-frequency transducer (E.I. Medical Imaging, Loveland, CO, USA). Embryo crown to rump length (CRL) was measured on d 35 and fetal crown to nose length (CNL) was measured on d 65 post-tai. Images were measured twice using an image capturing software (ImageJ, National Institute of Health, Bethesda, Maryland, USA) for later measurements and final CRL and CNL values were calculated as the mean between the two measurements Florida Beef Research Report

27 Calf body weight was determined within 12 hr of birth and every 30 days until weaning using a scale (TRU-TEST, Mineral Wells, TX, USA). Cow-calf pairs were maintained together on pasture from birth until weaning with ad libitum access to bahiagrass hay, bahiagrass pasture, and water. A subset of 40 pregnant cows of Angus and Brangus breeds and 24 calves (12 males and 12 females; not necessarily born from the subset of 40 cows) equally distributed among treatments were selected for analysis of mrna expression of target genes. Liver biopsies were performed in a subset of calves at 8±3 d of age, and expression of target genes related to the somatotropic axis were determined. Extraction of RNA was conducted according to recommendations of the RNA-extraction kit manufacturer (PureLink RNA Mini Kit, Invitrogen, Carlsbad, CA). A total of seven genes were investigated, including five target genes, myxovirus 2 (MX2), 2-5 -oligoadenylate synthetase 1 (OAS1), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor 1 receptor (IGF1R), and insulin-like growth factor binding protein 3 (IGFBP3); and two reference genes, cyclophilin-1 and ribosomal protein S9 (RPS9). Cows were blocked by breed and stratified by days post-partum, and body condition score (BCS) and randomly assigned to treatments. All data was analyzed as a randomized block design using the SAS statistical package (SAS Inst. Inc., Cary, NC) with animal as the experimental unit. Concentrations of IGF-1, P4, PSPB, and the embryo/fetal measurements CRL and CNL were analyzed as repeated measures using the MIXED procedure. The models included the effects of day, treatment and its interactions, and the random effect of animal. Pregnancy rates were analyzed using the GLIMMIX procedure. The model included the effects of treatment, days post-partum, BCS, breed and its interactions. Calf body weight was analyzed as repeated measures using the MIXED procedure. The model included the effects of day, treatment, calf gender and its interactions, and the random effect of animal. Relative mrna expression was analyzed within day using the GLM procedure. The final model included the effects of sex, treatment and its interactions. Results Injection of 325 mg of bst to suckled beef cows increased plasma concentration of IGF-1 (Figure 2). Plasma concentrations of IGF-1 were similar (P=0.92) among treatments at TAI on d 0 (64.8±2.6 ng/ml). Cows receiving an injection of bst at TAI on d 0 had greater (P<0.0001) plasma concentrations of IGF-1 on d 7 and on d 14. Accordingly, cows receiving an injection of bst at TAI on d 14 had greater (P<0.0001) plasma concentrations of IGF-1 on d 21. Concentrations of progesterone (P4) of pregnant cows increased (P<0.0001) after TAI (0.25, 2.39, 5.37, 4.78 and 5.09±0.26 ng/ml, for d 0, 7, 14, 21 and 35, respectively). However, injection of 325 mg of bst did not alter concentration of P4 (P=0.49) among treatments and no treatment day interaction was detected (P=0.26). Concentrations of PSPB did not change (P=0.30) between d 35 (2.76±0.109 ng/ml) and 65 (2.62±0.110 ng/ml) post TAI, and did not differ among treatments (P=055). Pregnancy rates to TAI were determined by ultrasonography on d 35 post TAI and was similar among treatments (45.3, 45.0, 51.0 and 54.2 for CTRL, 2bST, TAIbST and d14dbst, respectively) and gestation length (282±17 d) was also similar (P=0.33) among treatments (Table 1). Fetal size was determined by ultrasonography on d 35 and 65 post TAI (Table 1) and was not altered by injection of bst. Crown to rump length on d 35 was similar (0.48±0.02 in; P=0.61) among treatments. In addition, crown to nose length on d 65 (0.67±0.001 in) did not differ (P=0.23) among treatments. Expression of ISG of pregnant cows was determined on d 18 and 21 post TAI. Relative mrna expression of OAS-1 was similar among treatments on d 18 (1.8, 1.0, 1.2 and 0.9-fold increase for TAIbST, d14dbst, 2bST and CTRL, respectively; P=0.22) and on d 21 (1.6, 1.0, 2.2 and 0.9-fold increase for TAIbST, d14bst, 2bST and CTRL, respectively; P=0.23). In addition, relative mrna expression of MX-2 (Figure 3) was similar among treatments on d 18 (1.2, 1.3, 0.9 and 1.5-fold increase for TAIbST, d14bst, 2bST and CTRL, respectively; Florida Beef Research Report

28 P=0.89). However, mrna expression of MX-2 on d 21 differed (P=0.05) among treatments. Cows receiving an injection of bst at TAI had a greater fold increase in MX-2 mrna expression compared to cows receiving an injection of bst at TAI and 14 d later (2.0 and 0.8, for TAIbST and 2bST, respectively), while d14bst and CTRL were intermediate (1.2 and 0.9, respectively). In our study, bst injection had no effect on calf performance from birth to weaning (Table 1). Birth weight was similar (P=0.65) among treatments (79.4, 72.9, 73.4 and 75.4±4.47 kg for TAIbST, d14bst, 2bST and CTRL, respectively). Furthermore, body weight did not differ (P>0.10) among treatments at 30, 60, 90, 120 and 150±17 d of age (163.8±5.59, 231.0±8.44, 303.3±9.36, 376.3±8.18, and 450.4±9.08 kg, respectively). Injection of bst did not alter calf liver mrna expression (Figure 4) of IGF-1 (P=0.81), IGF-2 (P=0.29), and IGFBP-3 (P=0.66). However, injection of bst at TAI tended (P=0.06) to increase calf liver mrna expression of IGFR-1 compared to the calves born to cows in other treatments (3.1-fold compared to 1.0, 1.2, and 1.5-fold for TAIbST, 2bST, d14bst, and CTRL, respectively). Conclusion In the present study, injection of 325 mg of bst around the time of breeding increased concentration of IGF-1 and increased expression of MX-2 on d 21 of suckled beef cows. However, it failed to alter fetal development and postnatal offspring performance. Further investigation is needed on the dose and timing of administration of bst and its effects on fetal development and postnatal offspring performance of beef cattle. Acknowledgements Sincere appreciation is expressed to P. Moriel, A. Ealy, S. Johnson, P. Folsom, M. Foran, O. Helms, D. Jones, C. Nowell, and D. Thomas for their assistance with data collection and laboratory analysis. The authors thank Zoetis Animal Health (Florham Park, NY) for their donation of PGF 2α (Lutalyse), GnRH (Fertagyl), and CIDR inserts (CIDR EAZI-Breed). Literature Cited Costine, B. A., et al J. Anim. Sci. 83: Godfrey, K. M Eur. J. Obstet. Gynecol. Reprod. Biol. 78: Koch, J. M.,et al J. Anim. Sci. 88: LeRoith, D., et al Endocr. Rev. 22: Lucy, M. C., et al Domest. Anim. Endocrinol. 12: Ribeiro, E. S., et al Biol. Reprod.: Florida Beef Research Report

29 Table 1. Fertility, gestation, fetal development and calf performance measurements from suckled beef cows previously treated with bst. Treatments 1 Item CTRL TAIbST d14bst 2bST SEM P-value Pregnancy rate, 24/53 25/49 26/48 18/40 (45) 0.77 no. (%) (45) (51) (54) Gestation length, d Fetal Size 2, in CRL d CNL d Calf age, d Body weight, lb ± ± ± ± ± bST, injection of 325 mg of bst on d 0 and 14; TAI-bST, injection of 325 mg of bst on d 0 and placebo injection on d 14; 14D-bST, placebo injection on d 0 and injection of 325 mg of bst on day 14; CTRL, placebo injection on d 0 and CRL crown-to-rump length; CNL crown-to-nose length Florida Beef Research Report

30 Figure 1. Experiment outline and schematic of treatments. All cows were estrous synchronized using a 7-Day CO-Synch + CIDR protocol where cows received an injection of GnRH on d -10 and CIDR was inserted, followed by injection of PGF 2α (PGF) and CIDR removal on d -3, followed by injection of GnRH and fixed-timed AI (TAI) on d 0. Individual body condition scores (BCS; 1=thin to 9=obese) were assigned on d -10. Blood samples (B) were collected on d 0, 7, 14, 18, 21, 35 and 65. Pregnancy diagnosis and fetal measurements were performed by ultrasonography (US) on d 35 and 65. Treatments: CTRL, placebo injection on d 0 and 14; 2bST, injection of 325 mg of bst on d 0 and 14; TAIbST, injection of 325 mg of bst on d 0 and placebo injection on d 14; d14bst, placebo injection on d 0 and injection of 325 mg of bst on day Florida Beef Research Report

31 Concentration of IGF-1, ng/ml a a b b a a b b a a b b CTRL TAIbST d14bst 2bST Day relative to TAI Figure 2. Concentrations of insulin-like growth factor 1 (IGF-1) of beef cows on days relative to fixed-timed AI (TAI) by treatment. Treatments: CTRL, placebo injection on d 0 and 14; 2bST, injection of 325 mg of bst on d 0 and 14; TAIbST, injection of 325 mg of bst on d 0 and placebo injection on d 14; d14bst, placebo injection on d 0 and injection of 325 mg of bst on day 14. a,b P< a Relative gene expression ab ab b CTRL TAIbST d14bst 2bST Day relative to TAI Figure 3. Expression of Myxovirus 2 (MX2) mrna on d 18 and 21 of gestation on suckled beef cows treated with bst. Treatments: CTRL, placebo injection on d 0 and 14; 2bST, injection of 325 mg of bst on d 0 and 14; TAIbST, injection of 325 mg of bst on d 0 and placebo injection on d 14; d14bst, placebo injection on d 0 and injection of 325 mg of bst on day 14. a,b P= Florida Beef Research Report

32 Relative gene expression CTRL TAIbST d14bst 2bST a b b IGF1 IGF2 IGFBP3 IGFR1 Target gene b Figure 4. Expression of hepatic insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor 1 receptor (IGF1R), and insulin-like binding protein 3 (IGFBP3) mrna of calves born to suckled beef cows treated with bst. Treatments: CTRL, placebo injection on d 0 and 14; 2bST, injection of 325 mg of bst on d 0 and 14; TAIbST, injection of 325 mg of bst on d 0 and placebo injection on d 14; d14bst, placebo injection on d 0 and injection of 325 mg of bst on day 14. a,b P= Florida Beef Research Report

33 Utilization of Fixed-time Artificial Insemination (TAI) to Reduce Breeding Season Length and its Effects on Subsequent Calf Value: A Case Study V. R. G. Mercadante, F. M. Ciriaco, D. D. Henry, P. L. P. Fontes, N. Oosthuizen, N. DiLorenzo, and G. C. Lamb. Synopsis During an 8-yr period we evaluated the impacts of TAI to reduce the length of the breeding season and its effects on subsequent calving distribution, calf value, and pregnancy rates at the North Florida Research and Education Center in Marianna, FL. We conclude that exposing beef females to TAI and reducing the breeding season length from 120 d to 70 d altered calving distribution, increased breeding season pregnancy rates, and increased calf value. Summary The development of TAI protocols has resulted in the opportunity for increased application of AI in commercial cattle operations. However, the long-term production and economic impact of implementing a TAI protocol in beef cattle operations has not been evaluated. Therefore, during an 8-yr period we evaluated the impacts of TAI to reduce the length of the breeding season and its effects on subsequent calving distribution, calf value, and breeding season pregnancy rates. The North Florida Research and Education Center consists of a beef herd containing 300 cows of Angus, Brangus, and Braford breed origin. During the 2006 and 2007 breeding seasons, the cows were exposed to a 120 d breeding seaspm (BS) by natural service. In 2008, and every subsequent BS to 2013, all females were exposed to TAI using either the 5- d or 7-d CO-Synch+CIDR protocols. Initially, calving season length resulted in cows being inseminated in 3 TAI groups (in 2008 and 2009), subsequently reduced to two TAI groups (in 2010 and 2011), and eventually to a single TAI group (in 2012 and 2013). Following the initial TAI for each group, females were detected for estrus and inseminated artificially after an observed estrus until d 23 after TAI. On d 23 after TAI, bulls were introduced and cows were naturally mated for the remainder of the breeding season. All bulls passed a breeding soundness examination prior to being introduced to cows. The breeding season length was reduced from 120 to 70 d between the 2008 and 2013 breeding seasons. Calving distribution and subsequent weaning performance were determined. Overall pregnancy rates increased from 81% and 86% in the 2006 and 2007 breeding seasons, respectively, to 94% and 93% in 2012 and 2013, respectively. Mean calving date from the first calf born during each calving season was reduced from 80.9 d in 2007 to 38.9 d in Utilizing a similar calf value across years of $2.00/lb, the mean value per calf increased by $86.8 per calf resulting from the 2008 breeding season to $168.8 per calf resulting from the 2013 breeding season. We conclude that exposing beef females to TAI and reducing the breeding season length for six years altered calving distribution, increased breeding season pregnancy rates, and increased calf value. Introduction Advances in reproductive biotechnologies and enhanced understanding of the dynamics of the bovine estrous cycle have made possible the development of protocols to manipulate the estrous cycle and control ovulation utilizing natural and/or artificially synthesized hormones Florida Beef Research Report

34 Utilization of estrus or ovulation synchronization and fixed-timed artificial insemination (TAI) has facilitated the widespread utilization of artificial insemination (AI). Currently only 7.6% of beef operations in the United Sates utilize AI as a reproductive management tool (NAHMS, 2009). When queried as to their reluctance to utilize AI, over 53% of operations cited labor concerns or complicated estrous synchronization protocols as primary reasons for not implementing this reproductive technology (NAHMS, 2009). During the past decade, TAI protocols have been developed that eliminate detecting estrus and yield satisfactory pregnancy rates. The majority of these TAI protocols depend largely on the use of exogenous progesterone (P4), gonadotropin release hormone (GnRH) to induce ovulation, and luteolysis via administration of prostaglandin F 2α (Lamb et al., 2010). Possible outcomes from the combined use of estrous synchronization and TAI include shortened calving season, increased calf uniformity, earlier births during the calving season. Together, these technologies can greatly impact the economic viability of cow-calf systems by enhancing pounds of calf weaned per cow exposed (Rodgers et al., 2012). Therefore, our object was to evaluate the long-term production and economic impact of implementing estrous synchronization and TAI protocols at the North Florida Research and Education Center (NFREC). Materials and Methods The NFREC consists of a beef herd containing 300 cows of Angus, Brangus, and Braford breed origin. During the 2006 and 2007 breeding seasons, the cows were exposed to a 120 d breeding season by natural service. In 2008, and every subsequent breeding season to 2013, all females were exposed to TAI using either the 5-d or 7-d CO-Synch+CIDR protocols with the goal of reducing the breeding season to 70 d, to expose every female in the herd to TAI, improve fertility and calf crop uniformity, and weaning weights. In order to achieve this, it was decided that all females in the operation were exposed to the following criteria: 1) Replacement heifers must become pregnant during the first 25 d of the breeding season. 2) Every cow will be exposed to estrous synchronization and TAI. 3) Each cow must produce a live calf every year and calve without assistance or they will be culled. 4) Every cow must provide the resources for the genetic potential of the calves and each calf she produces must be genetically capable of performing. 5) No supplemental feeding was offered to cows that failed to maintain body condition. 6) Any cow with an undesirable temperament or disposition was culled. Initially, calving season length resulted in cows being inseminated in 3 TAI groups (in the 2008 and 2009 breeding seasons), subsequently reduced to two TAI groups (in the 2010 and 2011 breeding seasons), and eventually to a single TAI group (in the 2012 and 2013 breeding seasons; Figure 1). Following the initial TAI for each group, females were detected for estrus and inseminated artificially after an observed estrus until d 23 after TAI. On d 23 after TAI, bulls were introduced and cows were naturally mated for the remainder of the breeding season. All bulls passed a breeding soundness examination prior to being introduced to females Florida Beef Research Report

35 Results As a result of incorporating estrous synchronization and TAI, in addition to other reproductive management practices, the breeding season was reduced from 120 to 70 d in the course of 5 yrs. Furthermore, currently almost all cows calve prior to initiation of the subsequent breeding season and are exposed to a single TAI on the first day of the breeding season. The effect of utilizing estrous synchronization and TAI on calving distribution can be observed in Figure 2. In 2006 and 2007, before initiation of TAI program, it took 90 d for 50% of the calves to be born. In 2013, however, it took less than 30 d for 50% of the calves to be born. Mean calving date from the first calf born during each calving season was reduced from 80.9 d for the 2007 breeding season to 38.7 d for the 2013 breeding season. In addition, overall pregnancy rates, including AI and natural service, increased from 81% and 86% in the 2006 and 2007 breeding seasons, respectively, to 92% and 93% in 2012 and 2013, respectively (Table 1). Assuming an average daily gain of 2.0 lb/d, a fixed calf value of $2.00/lb across yrs, the mean value per calf increased by $86.8 per calf resulting from the 2008 breeding season to $168.8 per calf resulting from the 2013 season. Overall, the net result of a more compact calving season with increased value of calves (in current dollars) by $168.8 per calf resulted in an increased net result of $47, per yr for the 300 head herd and 94% pregnancy rate at the NFREC (Table 1). Conclusion We conclude that exposing beef females to estrous synchronization and TAI, and reducing the breeding season length during a period of 6 yrs altered calving distribution, increased breeding season pregnancy rates, and increased calf value. Acknowledgements Sincere appreciation is expressed to P. Folsom, M. Foran, O. Helms, D. Jones, C. Nowell, D. Thomas, and all interns at the NFREC for their assistance with data collection. Literature Cited Lamb, G. C. et al J. Anim. Sci. 88:E Larson, J. E., et al J. Anim. Sci. 84: National Animal Health Management Service NAHMS Part II. Fort Collins, CO. Rodgers, J. C., et al J. Anim. Sci. 10: Florida Beef Research Report

36 Table 1. Breeding season length, final pregnancy rate, mean calving day, and change in calf value at weaning, after initiation of an estrous synchronization and TAI program at the North Florida Research and Education Center. Year Breeding season length, d Pregnancy rate, % Mean calving day Difference from 2006/2007, d Per calf increase in value, $ Herd increase in value, $1, Assuming an average daily gain of 2 lb/d, a fixed calf value of $2.00/lb 2 Assuming the pregnancy rate within each yr and a 300 head herd Figure 1. Timeline of events, and length of breeding seasons at the North Florida Research and Education Center from 2006 to Florida Beef Research Report

37 Percentage Calving day Figure 2. Cumulative calving percentage during each calving season at the North Florida Research and Education Center from 2006 to Florida Beef Research Report

38 Florida Beef Research Report

39 Resynchronization as a Strategy to Increase the Percentage of Replacement Beef Heifers Conceiving to Artificial Insemination (AI) After an Initial Fixed-time AI (TAI) P.L.P. Fontes, N. Oosthuizen, V.R.G. Mercadante, G.V. de Moraes, D.D. Henry, F.M. Ciriaco, N. DiLorenzo, and G.C. Lamb. Synopsis A study was conducted to develop a strategy to increase the percentage of replacement beef heifers conceiving to AI after an initial TAI. Our proposed protocol with estrus detection prior to TAI failed to increase pregnancies by AI. Summary Previously inseminated replacement beef heifers were randomly assigned to one of two treatments 12 d after an initial TAI: 1) on d 22 heifers received a 100-µg injection of GnRH and a CIDR containing 1.38 g of progesterone (P4), followed by CIDR removal and ultrasound for pregnancy diagnosis on d 29. Nonpregnant heifers received a 25-mg injection of prostaglandin F 2α (PGF) followed by TAI 54 h later (TRT1;n=678); 2) on d 12 all heifers received a CIDR, which was removed on d 19 when an estrus detection patch (Estrotect) was affixed to the tailhead of each heifer. On d 21 and 22, heifers with an activated patch received AI and all heifers without an activated patch by d 22 received GnRH and a CIDR, followed by the same protocol as described in TRT1 from d 29 onwards (TRT2; n=638). Ultrasound diagnosis occurred on d 60. Pregnancy rate to the initial TAI (43.4% and 41.1% for TRT1 and TRT2, respectively) and overall pregnancy rates to AI (58.4% and 59.1% for TRT1 and TRT2, respectively) did not differ (P>0.05) between treatments. In TRT2, of the 384 nonpregnant heifers exposed to a second TAI, 35.9% were pregnant. In TRT2, 226 heifers received AI as a result of activated patches with a resulting pregnancy rate of 36.7%, whereas the pregnancy rate of the 150 nonpregnant heifers that did not have activated patches and were exposed to a second TAI was 32.0%. The mean expected calving date of all heifers pregnant to AI did not differ (P>0.05) between treatments (10.6±2.0 d and 8.9±2.1 d, for TRT1 and TRT2, respectively). We conclude that resynchronization of estrus or ovulation of nonpregnant heifers previously exposed to TAI increased the percentage of heifers conceiving to AI, but inclusion of additional estrus detection prior to pregnancy diagnosis failed to increase AI pregnancy outcomes. Introduction Biotechnologies such as AI, can improve genetic gains in cow-calf operation systems. Increasing the number of females bred by AI allow producers to obtain genetic improvement at a faster rate. Protocols for the resynchronization of ovulation (Resynch) can increase the effective AI service rate and reduce the interval between AI services (Fricke, 2002). After TAI, supplementation with P4 via a CIDR insert showed no evidence of disruption in the establishment of pregnancy (Larson et al., 2009). Also, other previous studies have demonstrated that it is possible to resynchronize estrus of cows with unknown pregnancy status after a first TAI without compromising the pregnancies obtained by the first TAI (Thompson et al., 2010). Therefore, we developed and tested a breeding strategy utilizing detection of estrus and resynchronization of estrus in heifers of unknown pregnancy status. We hypothesized that this strategy would increase the percentage of pregnant heifers by AI compared to nonpregnant Florida Beef Research Report

40 heifers inseminated at a fixed-time. Materials and Methods This study was conducted during the summer breeding season in A total of 1,316 Bos taurus heifers at 15±4 mo of age were enrolled in the experiment. Animals were housed in a feedlot designed for developing replacement beef heifers and they all received ad libitum access to a total mixed ration (TMR). At the onset of the breading season, all heifers were submitted to a P4-based estrus synchronization protocol: on d -33 heifers received an intravaginal CIDR containing 1.38 g of P4. At d -19 the CIDR was removed and all heifers received 25 mg of PGF on d -3 followed by a TAI 66 h later. Heifers received a 100-µg injection of GnRH at the time of insemination (d 0). Heifers were randomly assigned to one of two treatments 12 d after initial TAI: 1) on d 22 heifers received a 100-µg injection of GnRH and a CIDR containing 1.38 g of P4, followed by CIDR removal and ultrasound for pregnancy diagnosis on d 29, with nonpregnant heifers receiving a 25-mg injection of PGF followed by TAI 54 h later (TRT1; n=638); 2) on d 12 all heifers received a CIDR, which was removed on d 19 when an estrus detection patch (Estrotect) was affixed to the tailhead of each heifer. On d 21 and 22, heifers with an activated patch received AI and all heifers without an activated patch by d 22 received GnRH and a CIDR, followed by the same protocol as described in TRT1 from d 29 onwards (TRT2; n=678). Ultrasound diagnosis occurred on d 60 (Figure 1). Heifers were assigned randomly to treatments. All data was analyzed as a completely randomized design using the SAS statistical package (SAS Inst. Inc., Cary, NC) with animal as the experimental unit. Mean expected calving date was analyzed as repeated measures using the MIXED procedure. Pregnancy rates were analyzed using the GLIMMIX procedure. Results Pregnancy rate to the initial TAI (43.4% and 41.1% for TRT1 and TRT2, respectively) and overall pregnancy rates to AI (58.4% and 59.1% for TRT1 and TRT2, respectively) did not differ (P>0.05) between treatments. In TRT1, of the 384 nonpregnant heifers exposed to a second TAI, 35.9% were pregnant. In TRT2, 226 heifers received AI as a result of activated patches with a resulting pregnancy rate of 36.7%, whereas the pregnancy rate of the 150 nonpregnant heifers that did not have activated patches and were exposed to a second TAI was 32.0%. The mean expected calving date of all heifers pregnant to AI did not differ (P>0.05) between treatments (10.6±2.0 d and 8.9±2.1 d, for TRT1 and TRT2 respectively). Conclusion We concluded that the proposed protocol utilizing detection of estrus of heifers of unknown pregnancy status coupled with estrus resynchronization failed to increase the percentage of heifers pregnant to AI compared with a TAI resynchronization protocol to inseminate heifers diagnosed nonpregnant 29 days after an initial TAI. In a recent study, our group (Lamb et al., 2015) demonstrated the potential of estrous synchronization in reducing the length of the breeding season and its positive impact in the subsequent calving distribution. Perhaps strategies such as resynchronization of estrous have the potential to increase the number of animals that become pregnant to AI and may increase the number of females pregnant at the beginning of the breeding season, increasing the Florida Beef Research Report

41 efficiency of cow-calf operations in Florida; however, further research is needed in order to develop better alternatives for resynchronization in beef females. Acknowledgements Sincere appreciation is expressed to Cole and John Hokana, Ellendale, ND for allowing us to use their heifers and Merlyn Sandbulte, Jim Marshall for assisting with detection of estrus, artificial insemination support, and collection of data. The authors thank Zoetis Animal Health (Florham Park, NY) for their donation of PGF 2α (Lutalyse), GnRH (Fertagyl), and CIDR inserts (CIDR EAZI-Breed) and Boyd Dingus (Estrotect) for donation of Estrotect estrus detection patches. Literature Cited Fricke J. Dairy Sci. 85: Larson et al., J. Anim Sci. 87:914. Thompson et al., J. Dairy Sci Lamb et al., J. Anim. Sci. 93(E-Suppl. s3):365. (Abstr) Florida Beef Research Report

42 Figure 1. Experimental Protocol. All heifers received a 14-d CIDR-PG protocol (CIDR insertion at d-3, followed by CIDR removal 14 days later [d-19]. At d-3, all heifers received 25 mg of PGF intramuscular (i.m.) and were TAI 66 h later together with 100 µg of GnRH i.m. [d 0]). TRT 1: On d 22, heifers received 100 µg of GnRH i.m. and CIDR for seven 7d. At CIDR removal [d 29], open heifers received 25 mg of PGF i.m. and 100 µg of GnRH i.m. together with TAI 55 h later. TRT 2: at d 12, heifers received a CIDR. 7 d later [d19] CIDR was removed and estrus detection patch was affixed. On d 21 and d 22, heifers with activated patch received AI. Heifers without an activated patch by d 22 received GnRH and a CIDR, followed by the same protocol described in TRT Florida Beef Research Report

43 Clover-annual Ryegrass Mixtures to Extend the Grazing Season in North Florida E.R.S. Santos 1, H.M.S. Silva 1, A.C.C. Melo 1, M. Ruiz-Moreno 1, A. Blount 1, C. Mackowiak 1, N. DiLorenzo, L. E. Sollenberger 2, J.C.B. Dubeux Jr 1 Synopsis Legumes can be a source of nitrogen for grasses when mixed in a system. The utilization of cool-season legumes in mixtures with annual ryegrass pastures can increase the total forage mass, improve animal performance, and extend the grazing season. Summary Symbiotic association between forage legumes and N 2 -fixing microorganisms reduces the need for pasture N fertilization and increases forage N concentration. Pastures of cool-season legumes mixed with annual ryegrass are an option to extend the grazing season in subtropical regions. We investigated the association of four cool-season legumes with annual ryegrass and contrasted these mixtures with annual ryegrass in monoculture. Clovers tested included: balansa, ball, berseem, and crimson. Treatments were replicated 4 times in a randomized complete block design. Response variables analyzed included total dry matter yield (DMY), clover DMY, annual ryegrass DMY, 15 N grass, 15 N legume, botanical composition, N concentration of grass and legume components, shoot N yield for grass and legume, percentage of N derived from atmosphere (%Ndfa), and N 2 -fixation by the legume. Legume-annual ryegrass mixtures yielded more biomass (average of 2,310 lb of DM/acre) compared with unfertilized annual ryegrass (870 lb DM/acre). Among mixtures, crimson clover-annual ryegrass was the most productive (4,090 lb DM/acre). Crimson (1,990 lb DM/acre) and berseem (1,530 lb DM/acre) clovers were the most productive legumes. Annual ryegrass yielded more biomass when mixed with crimson clover compared with other clovers. Clover percentage in the mixtures ranged from 26 to 74%, with berseem showing the highest proportion. Total shoot N yields were 26, 37, 44, 78, and 10 lb N/acre for balansa, ball, berseem, crimson, and unfertilized ryegrass, respectively. The %Ndfa for all clovers was high, varying from 67 to 98%. N 2 -fixation ranged from 10 to 46 lb N/acre, with crimson fixing the greatest amount. Crimson clover presented the best overall performance when mixed with annual ryegrass in North Florida. Introduction Legumes are known for their capability of associating with bacteria for fixing atmospheric N 2 (Russelle, 2008). Legumes provide carbon to bacteria and benefit from the nitrogen that these microorganisms can fix from the atmosphere. Fixed nitrogen can become available to associated grasses via nodule exudation, litter decomposition, and animal excreta, if used in a grazing system. Mixtures of legumes and grasses can increase farm profitability and provide environmental services. In addition to fixing atmospheric N 2, legumes have greater digestibility and crude protein concentration than grasses, which can improve animal diet and consequently animal production. Grass-legume mixtures have potential to mitigate greenhouse gas emissions if compared with industrial N fertilizer due to less CO 2 emission from fossil fuels in the former (Lal, 2004). In addition, methane emission by livestock might be reduced in diets containing forage legumes due to greater digestibility. Finally, nitrous and nitric oxide 1 University of Florida/IFAS North Florida Research and Education Center, 3925 Highway 71, Marianna, FL. 2 University of Florida/IFAS Agronomy Department, Gainesville, FL Florida Beef Research Report

44 emissions are greater after N fertilization (Bouwman et al., 2002; cited by Sollenberger, 2014). Refer to paper for comment. In Florida, agriculture plays an important role for the state income, and it is the state s second largest industry. Warm-season grasses such as bahiagrass (Paspalum notatum Flugge) and bermudagrass (Cynodon dactylon) are the forage crops most used for grazing and hay. However, these plants are sensitive to cold temperatures, short days, and they are dormant during the cool season. Ryegrass is a cool-season grass that is broadly used in Florida during the late winter and spring. Introduction of cool-season legumes into ryegrass pastures may be a tool to increase forage production, animal product, and provide environmental services while adopting a low input sustainable system. The genus Trifolium has a number of species that are well adapted to Florida environments and can be used for grazing. Therefore, a study was conducted to evaluate the association of balansa clover (Trifolium michelianum Savi), ball clover (Trifolium nigrescens Viv.), berseem clover (Trifolium alexandrinum L.), and crimson clover (Trifolium incarnatum L.) with ryegrass in contrast with ryegrass in monoculture. Materials and Methods The experiment was conducted at North Florida Research and Education Center, Marianna, FL. In order to stablish the trial, soil samples were collected and fertilization followed the UF/IFAS Standardized Fertilization Recommendations for Agronomic Crops (Mylavarapu, 1997). Treatments were the four species of clover planted in association with ryegrass. Tested clover species included balansa, ball, berseem, and crimson. In addition, a ryegrass monoculture was added as a control (Figure 1). Seeding rates were 30 lb/acre for ryegrass in monoculture and 20 lb/acre for ryegrass in the mixtures. Seeding rates for ball clover, balansa clover, berseem clover, and crimson clover were 3, 6, 15, and 20 lb/acre, respectively. The plots measured 6 x 20 ft and were established in a randomized complete block design with 5 treatments and 4 replicates. Yield samples were taken on April 10, 2014, by harvesting two 2.7 sq. ft. quadrats per plot at a 2-inch stubble height. Samples were dried at 131 F for 72 h after sampling. Response variables included total dry matter yield (DMY), clover DMY, annual ryegrass DMY, 15 N grass, 15 N legume, botanical composition, N concentration of grass and legume components, shoot N yield for grass and legume, percentage of N derived from atmosphere (%Ndfa), and N 2 -fixation by the legume. The proportion of nitrogen derived from atmosphere was determined by using the equation described by Unkovich et al. (2008). Data were analyzed by using the Proc Mixed from SAS and means were compared using PDIFF adjusted by Tukey (P<0.05). Treatments were considered fixed effects, and the random effects included blocks and its interaction with fixed effects. Results There was a significant difference among treatments for total DMY (P<0.0001). Annual ryegrass-crimson clover mixture yielded 4,090 lb DM/acre, and it was the treatment with the greatest production. The mixtures with balansa clover (1,550 lb DM/acre), ball clover (2,090 lb DM/acre) and berseem clover (2,030 lb DM/acre) did not differ from each other, however all of them were better when compared to the annual ryegrass monoculture (870 lb DM/acre, Table 1) Florida Beef Research Report

45 Crimson clover and berseem clover made the greatest contribution to the total DMY. They produced 1,990 and 1,530 lb DM/acre, respectively, and were significantly different (P<0.0001) from the contribution of balansa and ball clovers (420 and 540 lb DM/acre, respectively). Also, the annual ryegrass DMY (1,960 lb DM/acre) was influenced by the presence of crimson clover (P<0.0006), and ryegrass DMY was greater in association with crimson than with the other clovers or in monoculture (Table 1). Total N yield did not differ among clover varieties, and the values ranged from 78 lb/acre for crimson clover to 26 lb/acre for balansa clover. The %Ndfa was greater in annual ryegrass-balansa clover and annual ryegrass-crimson clover mixtures, 98% and 95%, respectively. Biological N 2 -fixation was greater for crimson clover (P<0.0001), with 46 lb/acre of N fixed, followed by berseem clover with 32 lb/acre. Balansa and ball clover did not differ from each other, fixing 12 and 10 lb/acre, respectively. Conclusions Annual ryegrass dry matter yield increased when it was grown in mixtures with cool-season clovers compared with when it grew alone with no N fertilizer. The N 2 -fixation by clovers ranged from 10 to 46 lb N/acre. Among the clovers, crimson clover presented the best overall performance when mixed with annual ryegrass in North Florida. The annual ryegrass-clover mixtures are an alternative to N-fertilized systems, adding N to the system and improving livestock nutrition. Literature Cited Bouwman, A.F., et al Global Biogeochemical Cycles 16: DOI: /2001GB Lal, R Environment International. 30: Mylavarapu, R., et al, G. UF/IFAS Standardized Fertilization Recommendations for Agronomic Crops. UF/IFAS extension, Russelle, M.P p In: Nitrogen in Agricultural Systems (Agronomy Monograph 49). American Society of Agronomy, Crop Science Society of America, Soil Science Society of America. Madison, WI. SAS Institute SAS statistics user s guide. Release version 6. SAS Inst., Cary, NC. Sollenberger, L.E In Proc. Brazilian Soc. Animal Sci. Mtg., 51st, Barra dos Coqueiros, Brazil. 29 July-1 Aug Sociedade Brasileira de Zootecnica, Brasilia, Brazil. Figure 1. Demonstration of experimental area at UF/IFAS NFREC, Marianna, FL Florida Beef Research Report

46 Table 1. Total dry matter yield (DMY), clover DMY, and annual ryegrass DMY in different ryegrassclover mixtures. Treatments Total DMY, lb/ac Clover DMY, lb/ac Ryegrass DMY, lb/ac Ryegrass-Balansa Clover 1,550 b 420 b 1,040 b Ryegrass-Ball Clover 2,090 b 540 b 1,490 b Ryegrass-Berseem Clover 2,030 b 1,530 a 4,80 b Ryegrass-Crimson Clover 4,090 a 1,990 a 1,960 a Unfertilized Annual Ryegrass 870 c b SE P < < a,b,c Means in a column with different superscripts differ, P<0.05 Table 2. Total nitrogen yield, % of nitrogen derived from atmosphere, and nitrogen fixation in different ryegrass-clover mixtures Florida Beef Research Report

47 Treatments Total N Yield, lb/ac Ndfa, % N Fixation, lb/ac Ryegrass-Balansa Clover 26 b 98 a 12 c Ryegrass-Ball Clover 36 b 67 c 10 c Ryegrass-Berseem Clover 44 a 86 b 32 b Ryegrass-Crimson Clover 78 ab 95 a 46 a Unfertilized Annual Ryegrass 10 c SE P < < < Nitrogen derived from atmosphere a,b,c Means in a column with different superscripts differ, P< Florida Beef Research Report

48 Florida Beef Research Report

49 Seeding Rates of Ball Clover in Mixtures with Annual Ryegrass in North Florida E.R.S. Santos 1, H.M.S. Silva 1, M. Ruiz-Moreno 1, A. Blount 1, C. Mackowiak 1, N. DiLorenzo, L. E. Sollenberger 2, J.C.B. Dubeux Jr 1 Synopsis Legumes have the capacity to associate with rhizobia strains and fix atmospheric N 2. Ball clover has reseeding ability and when planted in mixtures with annual ryegrass contributes by adding N to the system and extending the grazing season. Summary Legumes have the capacity to associate with rhizobia strains and fix atmospheric N 2. Ball clover has reseeding ability and when planted in mixtures with annual ryegrass contributes by adding N to the system and extending the grazing season. This experiment tested three seeding rates of ball clover (2, 4 and 6 lb/acre) in a mixture with annual ryegrass compared with annual ryegrass in monoculture, fertilized (45 lb N/acre), or not fertilized with N. Response variables included percentage of ball clover, dry matter yield (DMY) of annual ryegrass, DMY of ball clover, total DMY, percentage of plant N derived from atmosphere (%Ndfa) and N fixed contained in the shoot (Nfix). The study was performed in a randomized complete block design with 4 replicates. Ball clover proportion in the mixture increased from 32% (at 2 lb/acre seeding rate) to 47% (at 4 lb/acre seeding rate), with no difference observed between 4 and 6 lb/acre. Ball clover DMY increased linearly with increasing seeding rates, with DMY of 630, 910, and 1040 lb/acre for 2, 4, and 6 lb/acre seeding rates, respectively. Annual ryegrass DMY and total DMY were greater in the N fertilized treatment (3,400 lb DM/acre). Average %Ndfa was 95% for ball clover and there was no difference among treatments. The Nfix showed a linear increase with seeding rate (P=0.02) with values ranging from 16 to 25 lb N/acre. Treatments including seed rates of 4 and 6 lb/acre were similar in botanical composition, DMY of ball clover, DMY of ryegrass, total DMY, and Nfix. From the producer perspective, it is more economical to use 4 lb of ball clover seed/acre. Increasing seeding rates of ball clover in annual ryegrass mixtures allowed greater legume contribution in the pasture, improving forage N without decreasing yield. Introduction Clovers have the ability to naturally reseed because of seed dormancy during the warm-season. Proportion of hard seeds is an important trait driving natural reseeding. Reseeding ability also depends on grazing management. During flowering and seed maturation, it is important to manage livestock to allow plants to set seeds and let seeds mature before they are consumed by livestock. Mature seed consumed by livestock will likely pass through the gastrointestinal tract and return to the soil via feces. Hard seeds usually are not digested in the rumen and they have the ability to germinate when returned to the soil, after the dormancy period has passed. Seeds not ingested by grazing animals return directly to the soil. 1 University of Florida/IFAS North Florida Research and Education Center, 3925 Highway 71, Marianna, FL. 2 University of Florida/IFAS Agronomy Department, Gainesville, FL Florida Beef Research Report

50 Once again, the proportion of hard seeds and seed dormancy will define the ability of these seeds to germinate in the following cool-season. Ball clover (Trifolium nigrescens Viv.) has reseeding ability and when planted in mixtures with annual ryegrass contributes to add N to the system and to expand the grazing season. Seeding rates of ball clover for optimum growth of both forages in mixtures need to be investigated. Therefore, the overall objective of this experiment was to test three seeding rates of ball clover (2, 4, and 6 lb/acre in a mixture with annual ryegrass compared with annual ryegrass in monoculture, fertilized with 45 lb N/acre or not fertilized. Material and Methods The experiment was conducted at the North Florida Research and Education Center, Marianna, FL. In order to establish the trial, soil samples were collected and the plots fertilized according to UF/IFAS Standardized Fertilization Recommendations for Agronomic Crops (Mylavarapu, 1997). Treatments were three seeding rates (2, 4, and 6 lb/acre) of ball clover in a mixture with annual ryegrass compared with annual ryegrass in monoculture, fertilized with 45 lb N/acre or not fertilized. Seeding rate for annual ryegrass was 20 lb/acre. Nitrogen fertilizer was applied on March 3, 2015, using urea as a source of N. Plots measured 6 x 20 ft and were established in a randomized complete block design (Figure 1) with 4 replicates (n=20). Samples were taken on April 4, 2014, by harvesting two 2.7 sq. ft. quadrats per plot to a 2-inch stubble height. Samples were dried at 131 F for 72 h after sampling. Response variables included percentage of ball clover, total dry matter yield (DMY), clover DMY, shoot N mass, botanical composition, percentage of N derived from atmosphere (%Ndfa) and N fixed contained in the shoot (N fix ). The proportion of nitrogen derived from atmosphere was determined by using the equation described by Unkovich et al. (2008). Data were analyzed by using the Proc Mixed from SAS and means were compared using PDIFF adjusted by Tukey (P<0.05). Treatments were considered to be fixed effects and random effects included blocks and its interactions with fixed effects. Results Seeding rates of 4 and 6 lb/ac had greater clover contribution to total DMY (P=0.03). For these seeding rates, ball clover represented, respectively, 47 (1,000 lb/acre) and 46% (1,040 lb/acre) of the total dry weight, while at the rate of 2 lb/acre ball clover contributed only 32% of total DMY. There was no difference in the percentage of N derived from the atmosphere (%Ndfa) among clover treatments. The %Ndfa was 95, 97, and 94 for 2, 4, and 6 lb/acre, respectively. Nitrogen harvested in the shoot was 50% greater when seeding rate increased from 2 to 4 lb/acre, but no differences were observed between 4 and 6 lb/acre. Despite the contribution of clovers to the total DMY, annual ryegrass showed a greater yield (3,400 lb DM/acre) when fertilized with 45 lb N (P=0.0007), and there was no significant difference among treatments containing clovers (1,950, 2,030, 2,190 lb DM/acre for 2, 4, and 6 lb/acre, respectively) and the unfertilized ryegrass (1,620 lb DM/acre). In a grazing system, however, forage quality must be taken into account. Legumes generally have greater crude protein and digestibility than grasses, especially the cool-season legumes. Greater nutritive value might lead to better animal response. Shoot N yield in annual ryegrass was also greatest for the fertilized treatment (P<0.0001), and there was no difference among the treatments with clovers or the unfertilized annual ryegrass. However, no difference in total N yield was detected between the treatments containing clovers and the fertilized Florida Beef Research Report

51 ryegrass (28, 37, 46, 49 lb N/acre for the rates of 2, 4, 6 lb/acre, and the fertilized annual ryegrass, respectively), and they all differed (P=0.002) from the unfertilized annual ryegrass (15 lb/acre). Conclusions Total dry matter yield was not different among the different seeding rates of ball clover in mixture with annual ryegrass and the unfertilized annual ryegrass monoculture. Ball clover-annual ryegrass mixtures also yielded less compared with N-fertilized annual ryegrass. Clover contribution, however, has potential to improve animal performance and reduce off-farm N inputs via mineral fertilization. Because there were no significant differences in any variable between the seeding rates of 4 and 6 lb/acre, from the producer s perspective it is more economical to adopt the seeding rate of 4 lb/acre if considering planting the ball clover-annual ryegrass mixture. Literature Cited Mylavarapu, R., Wright, D., Kidder, G. UF/IFAS Standardized Fertilization Recommendations for Agronomic Crops. UF/IFAS extension, SAS Institute SAS Inst., Cary, NC. Sollenberger, L.E In Proc. Brazilian Soc. Animal Sci. Mtg., 51st, Barra dos Coqueiros, Brazil. 29 July-1 Aug Sociedade Brasileira de Zootecnica, Brasilia, Brazil Florida Beef Research Report

52 Figure 1. Ball clover seeding rate trial at UF/IFAS NFREC, Marianna, FL. Table 1. Ball clover %, total clover dry matter yield (DMY), nitrogen derived from atmosphere and nitrogen fixed, in mixtures of ball clover-annual ryegrass under different seeding rates of ball clover. Treatments Ball Clover, % Clover DMY, lb DM/acre Ndfa 1, % Nfix 2, lb/acre 2 lb/acre 32 b 634 b 95 a 16 b 4 lb/acre 47 a 996 a 97 a 24 a 6 lb/acre 46 a 1,039 a 94 a 25 a SE P Ndfa is N derived from atmosphere 2 Nfix is N fixed contained in the shoot a,b Means in a column with different superscripts differ P< Florida Beef Research Report

53 Table 2. Total dry matter yield (DMY), total shoot nitrogen mass, total nitrogen yield, and ryegrass percentage under different seeding rates of ball clover, in contrast with unfertilized annual ryegrass and fertilized annual ryegrass (45 lb N/acre). Total DMY, Shoot N yield Total N Yield Ryegrass, Treatments lb DM/acre Ryegrass, lb/acre lb/acre % 2 lb/acre 1,954 b 16 b 28 a 65 b 4 lb/acre 2,034 b 12 b 37 a 50 b 6 lb/acre 2,186 b 18 b 45 a 50 b Fertilized Ryegrass 3,399 a 49 a 49 a 97 a Unfertilized Ryegrass 1,624 b 15 b 15 b 96 a SE P < < a,b Means in a column with different superscripts differ P< Florida Beef Research Report

54 Florida Beef Research Report

55 Red Clover Varieties for North-Central Florida J.C.B. Dubeux, Jr. 1, P. Munoz 2, A.R.S. Blount 1, K.H. Quesenberry 2, L.E. Sollenberger, E.R.S. Santos 1 Synopsis Red clover varieties are an option for cool-season grazing. We tested nine red clover varieties in North (Marianna) and Central (Gainesville) Florida. Clover productivity varied with location, year, and harvest date, with maturity type being an important trait affecting productivity. Summary Red clover is an excellent option to increase species diversity in cool-season pastures, adding N to the systems via biological N 2 -fixation and providing high forage value. Two red clover variety trials were carried out, one at the North Florida Research and Education Center (NFREC) in Marianna during two seasons ( and ) and the other one at the Agronomy Forage Unit in Gainesville during one season ( ). Nine red clover varieties were tested: Barduro, Bulldog Red, Cinnamon Plus, FL24D, Freedom!, Kenland, Marathon, Red Ace, and Southern Belle. The trials were planted in a randomized complete block design, with four replications per treatment. Each plot measured 6 x 15 feet. In Marianna, clovers were harvested twice in the 2014 season (4/24 and 6/3/2014) and three times in the 2015 season (4/3, 5/7, and 6/11/2015), at a four-inch stubble height. Due to high deer pressure the trial was harvested only one time (April 2014) in the Gainesville location at a two-inch stubble height. In Marianna, red clover total dry matter yield ranged from 2,290 to 3,420 lb/acre in 2014 and from 1,730 to 2,840 lb/acre in In 2014, the top red clover varieties were Barduro (3,420 lb/acre), Red Ace (3,340 lb/acre), and FL 24D (3,280 lb/acre). In 2015, top varieties included Southern Belle (2,840 lb/acre), Cinnamon (2,790 lb/acre), Red Ace (2,760 lb/acre), and Barduro (2,740 lb/acre). In Gainesville, total dry matter ranged from 1,690 to 2,510 lb/acre. In this location the best varieties were Red Ace (2,510 lb/acre), FL24D (2,070 lb/acre), and Southern Belle (2,000 lb/acre). Red clover varieties can be used in North-Central Florida, adding value to the grazing systems in the region. Introduction Legumes are an option to add N via biological N 2 -fixation during the cool season. Nitrogen fixed during this time of the year will be available for grazing livestock, and recycled back to the grassland, helping to reduce N fertilizer costs. Cool-season legumes also have greater crude protein and digestibility than many other forages, improving cattle performance. Red clovers are traditionally planted in Florida during the cool-season. These clovers are adapted to Florida conditions and are usually used in mixtures with small grains (e.g., oat, rye, and triticale) and/or annual ryegrass (Lolium multiflorum Lam.). Red clover varieties vary in their time of flowering, with early-, mid-, and late-maturity types. Timing of flowering influences seasonal production and typically varies with environmental conditions such as photoperiod, temperature, and soil moisture. Therefore, varieties that perform better in one location may not necessarily be the best variety for another location with different environmental conditions. In this 1 North Florida Research and Education Center, University of Florida, Marianna, FL 2 Agronomy Department, University of Florida, Gainesville, FL Florida Beef Research Report

56 research, we tested nine red clover varieties under North and Central Florida conditions in order to recommend to producers the best varieties for these regions. Materials and Methods Site description The studies were conducted in Marianna and in Gainesville, FL (Figure 1). The trial in Marianna was carried at the North Florida Research and Education Center (NFREC), Marianna, FL (30 52 N, W, 34 m altitude). The soil at the research site in Marianna was an Orangeburg loamy sand (fine-loamy, kaolinitic, thermic Typic Kandiudults). Soil fertility results at the beginning of the trial at NFREC were ph=6.5, Mehlich-I P=48 ppm, K=78 ppm, Mg=157 ppm, Ca=625 ppm, S=29 ppm, B=0.22 ppm, Zn=3.3 ppm, Mn=52 ppm, Fe 13 ppm, Cu=1.8 ppm, SOM=1.36%, and CEC=7 meq/100 g. The trial in Gainesville was carried out at the Agronomy Forage Research Unit (AFRU), Hague, FL (29 48 N, W). The soil classification at this location was a loamy, siliceous, subactive, thermic, Arenic Endoaquult. Soil fertility results at the beginning of the trial were: ph=6.3, P>328 ppm (high), K=17 ppm (low), Mg=95 ppm (high), and Ca=674 ppm. Planting and harvesting Trials were established in 2013 and The soil was plowed and harrowed before planting. Plots measured 6 x 15 ft. and were allocated in a randomized complete block design, with four replications per treatment. Planting dates were Nov. 22, 2013 and Oct. 27, 2014 for Marianna and Nov. 15, 2013 for Gainesville. Before planting, plots were cultipacked, seeds were broadcasted, and plots were rolled thereafter. Harvests were performed when at least 20% of the plots had flowering plants. Harvests at a four-inch stubble height were performed on 4/24/2014, 6/3/2014, 4/3/2015, 5/7/2015, and 6/11/2015 for Marianna, while the Gainesville location was harvested at a two-inch stubble in 4/30/2014. Yield was estimated by harvesting a central strip of the plot (3 x 12 ft.) after trimming the borders (1.5 ft. each side). Total fresh weight was recorded and a subsample was taken to estimate the moisture content of the sample. After recording its fresh weight, the subsample was placed into a forced air-dryer at 131 F for 72 h. Fertilization, Weed and Pest Control Marianna In 2014, plots were fertilized on 5/16 with 300 lb/acre of (60 lb/acre of P 2 O 5 and 60 lb/acre of K 2 O) and 1 lb/acre of Boron. In 2015, at planting we applied 200 lb/acre of mixed with 10 lb/acre micronutrient mixture, followed by another application of 400 lb/acre of micronutrient mixture in 4/3/2015. On 1/13/2015, Pursuit was applied at 3 fl. oz. per acre to control weeds. Gainesville Plots were fertilized on 11/5/2013 with 320 lb/acre of using sulfate of potash. The trial at Gainesville had a high pressure of deer feeding on the clover plots. Even when a deer barrier (PlotSaver) was used under the recommended setups, it was not enough to keep the deer out of the plots and the plot yields were lower because of this. Statistics Data were analyzed using proc mixed from SAS (1996) using repeated measures. Fixed effects included variety, year, and harvest date. Random effects included blocks and its interactions with fixed effects. Lsmeans were compared using PDIFF procedure adjusted by Tukey (5%) Florida Beef Research Report

57 Results and Discussion Marianna Red clover varieties performed differently in the two years. Total dry matter yield ranged from 2,290 to 3,420 lb/acre in 2014 (Figure 2) and from 1,730 to 2,840 lb/acre in 2015 (Figure 3). In 2014, the top red clover varieties were Barduro (3,420 lb/acre), Red Ace (3,340 lb/acre), and FL24D (3,280 lb/acre). In 2015, top varieties included Southern Belle (2,840 lb/acre), Cinnamon Plus (2,790 lb/acre), Red Ace (2,760 lb/acre), and Barduro (2,740 lb/acre). Therefore, Barduro and Red Ace were consistently ranked among the top red clover varieties in both years and Southern Belle ranked well in both years. These three varieties are currently recommended by the UF/IFAS Forage Group (Blount et al., 2015). Marathon performance was inferior to that of the other varieties in both seasons. Consistent results of Barduro, Red Ace, and Southern Belle, in both seasons, place them among the recommended varieties for North Florida. Gainesville Total dry matter ranged from 1,690 to 2,510 lb/acre at this location (Figure 4). The top varieties were Red Ace (2,510 lb/acre), FL24D (2,070 lb/acre), and Southern Belle (2,000 lb/acre). However, yield of FL24D, Southern Belle, Kenland, Cinnamon Plus and Barduro did not differ by much. Marathon, Freedom! and Bulldog Red were lower performing varieties than Red Ace at the Gainesville site. Conclusions and implications Maturity type plays an important role in flowering, affecting as a result productivity and nutritive value. Early-maturing varieties will likely yield less than late-maturing ones, but harvest management might affect these responses. Since harvest timing was decided based on flowering of all varieties in the trial, the late maturing ones could probably yield more if they were harvested later in the season. These varieties, however, will likely be used under grazing conditions, where grazing frequency will likely be different than the cutting schedule used in this trial. In North Florida, Barduro, Red Ace, and Southern Belle performed consistently well, being the recommended varieties for North Florida. In Central Florida Red Ace and Southern Belle are the recommended varieties. FL24D is a new variety developed by University of Florida which will soon be in the seed market. Literature Cited Blount, A.R.S., et al Panhandle Ag e-news. Available online at SAS Institute SAS Inst., Cary, NC Florida Beef Research Report

58 Figure 1. Red clover variety trial at the North Florida Research and Education Center (NFREC), Marianna, FL Florida Beef Research Report

59 2014 Dry Matter Yield (lb/acre) /24/2014 6/3/2014 Figure 2. Biomass yield of red clover varieties measured in the season; NFREC, Marianna, FL. Standard error=175 lb/acre Florida Beef Research Report

60 Dry Matter Yield (lb/acre) /3/2015 5/7/2015 6/11/2015 Figure 3. Biomass yield of red clover varieties measured in the season; NFREC, Marianna, FL. Standard error=175 lb/acre Florida Beef Research Report

61 AFRU Dry Matter Yield (lb/acre) a ab ab ab ab ab b b b Cultivar Figure 4. Biomass yield of red clover varieties measured in the season; AFRU, Hague, FL. a,b Means with different superscripts differ, P< Florida Beef Research Report

62 Florida Beef Research Report

63 White Clover Varieties for North-Central Florida J.C.B. Dubeux, Jr. 1, P. Munoz 2, A.R.S. Blount 1, K.H. Quesenberry 2, L.E. Sollenberger, E.R.S. Santos 1 Synopsis White clover grows well in Florida environmental conditions, and is an option for cool-season grazing in North and Central Florida. We tested seven white clover varieties in North (Marianna) and Central (Gainesville) Florida. Top varieties were consistent across harvests and locations. Summary White clover is a cool-season legume that grows well in the Florida environment. White clover variety trials were carried out at two locations: North Florida Research and Education Center (NFREC) in Marianna during two seasons ( and ) and at the Agronomy Forage Unit in Gainesville, during one season ( ). White clover varieties tested included Barblanca, Crusade, Durana, FourLeaf, Ocoee, Osceola, and Regal Graze. Both trials were planted in a randomized complete block design, with four replications per treatment. Each plot measured 6 x 15 feet. In Marianna, clovers were harvested three times in each season (4/21, 5/16, 6/24/2014 and 4/3, 5/7, 6/11/2015) at a two-inch stubble height. In Gainesville, clovers were harvested two times in the 2014 season (4/30 and 6/10/2014) at a two-inch stubble height. In Marianna, white clover total dry matter yield ranged from 3,260 to 5,160 lb/acre in 2014 and from 1,490 to 3,060 lb/acre in In 2014, top white clover varieties included Regal Graze (5,160 lb/acre), FourLeaf (5,030 lb/acre), Osceola (4,740 lb/acre), and Ocoee (4,700 lb/acre). In 2015, top varieties were Osceola (3,060 lb/acre), Regal Graze (2,920 lb/acre), FourLeaf (2,700 lb/acre), and Ocoee (2,530 lb/acre). In Gainesville, total dry matter yields ranged from 2,870 to 1,440 lb/acre. At this location the top varieties were FourLeaf (2,870 lb/acre), Osceola (2,700 lb/acre), Regal Graze (2,670 lb/acre) and Ocoee (2,480 lb/acre). Choice of white clover variety must be based not only on yield, but also on tolerance to root-knot nematode. Ocoee ranked among the top white clover varieties and was selected for its tolerant to root-knot nematode, thus making it the best option for areas with nematode infestation. Introduction White clover is a cool-season legume that although a perennial in many areas of the US, often acts as an annual legume in Florida. Some varieties are excellent seed producers for natural reseeding, and occur naturally in some areas (Newman et al., 2014). It has high crude protein (>15%) and digestibility (approx. 70%), increasing forage nutritive value in grazed pastures. Biological N 2 -fixation is another added value of introducing white clover in livestock systems. Nitrogen fixed by clovers is recycled back to the soil via animal excreta or litter, benefiting associated grasses. Dry matter yield of white clover in Florida often ranges between 2,000 and 3,000 lb/acre, however, greater yields have been observed depending on the environment and inputs used. Assuming 15% crude protein, this represents 48 to 72 lb N per acre, with most of that originating from biological N 2 -fixation. 1 North Florida Research and Education Center, University of Florida, Marianna, FL 2 Agronomy Department, University of Florida, Gainesville, FL Florida Beef Research Report

64 White clover varieties perform differently in different regions of the state. Variety trials are necessary in order to recommend the best varieties for producers in the different regions. The goal of these studies was to assess the productive performance of seven white clover varieties in North- and Central-Florida. Materials and Methods Site description The studies were conducted in Marianna and in Gainesville, FL (Figure 1). The trial in Marianna was carried at the North Florida Research and Education Center (NFREC), Marianna, FL (30 52 N, W, 34 m altitude). The soil at the research site in Marianna was an Orangeburg loamy sand (fine-loamy, kaolinitic, thermic Typic Kandiudults). Soil fertility results at the beginning of the trial at NFREC were ph=6.5, Mehlich-I P=48 ppm, K=78 ppm, Mg=157 ppm, Ca=625 ppm, S=29 ppm, B=0.22 ppm, Zn=3.3 ppm, Mn=52 ppm, Fe 13 ppm, Cu=1.8 ppm, SOM=1.36%, and CEC=7 meq/100 g. The trial in Gainesville was carried out at the Agronomy Forage Research Unit (AFRU), Hague, FL (29 48 N, W). The soil classification at this location was a loamy, siliceous, subactive, thermic, Arenic Endoaquult. Soil fertility results at the beginning of the trial were: ph=6.3, P>328 ppm (high), K=17 ppm (low), Mg=95 ppm (high), and Ca=674 ppm. Planting and harvesting Trials were established in 2013 and 2014 in Marianna. The soil was plowed and harrowed before planting. Plots measured 6 x 15 ft. and treatments were allocated in a randomized complete block design, with four replications per treatment. Planting dates were Nov. 22, 2013 and Oct. 27, 2014 for Marianna and Nov. 15, 2013 for Gainesville. Before planting, plots were cultipacked, seeds were broadcasted, and the plots were rolled thereafter. Harvests were performed when at least 20% of the plots had flowering plants. White clover plots in Marianna were harvested on 4/21, 5/16, and 6/24/2014, and on 4/3, 5/7, and 6/11/2015, while the Gainesville location was harvested on 4/30 and 6/10/2014 at a two-inch stubble height. Yield was estimated by harvesting a central strip of the plot (3 x 12 ft.) after trimming the borders (1.5 ft. each side). Total fresh weight was recorded and a subsample was taken to estimate the moisture content of the sample. After recording its fresh weight, the subsample was placed into a forced air-dryer at 131 F for 72 h. Fertilization, Weed, and Pest Control Marianna In 2014, plots were fertilized on 5/16/2014 with 300 lb/acre of (60 lb/acre of P 2 O 5 and 60 lb/acre of K 2 O) and 1 lb/acre of Boron. In 2015, at planting we applied 200 lb/acre of mixed with 10 lb/acre micronutrient mixture, followed by another application of 400 lb/acre of micronutrient mixture in 4/3/2015. On 1/13/2015, Pursuit was applied at 3 fl. oz. per acre to control weeds. Gainesville In 2014, plots were fertilized in 11/5/2013 with 320 lb/acre of sulfate of potash. The trial at Gainesville had high pressure from deer feeding on the clover. Even when a deer barrier (PlotSaver) was used under the recommended setup, it was not enough to keep the deer out of the plots and the plot yields were lower because of this. Statistics Data were analyzed using proc mixed from SAS. Fixed effects included variety, year, and harvest date. Random effects included blocks and its interactions with fixed effects. Lsmeans were compared using PDIFF procedure adjusted by Tukey (5%) Florida Beef Research Report

65 Results and Discussion Marianna Total dry matter yields ranged from 3,260 to 5,160 lb/acre in 2014 and from 1,490 to 3,060 lb/acre in In 2014, top white clover varieties included Regal Graze (5,160 lb/acre), FourLeaf (5,030 lb/acre), Osceola (4,740 lb/acre), and Ocoee (4,700 lb/acre) (Figure 2). In 2015, top varieties were Osceola (3,060 lb/acre), Regal Graze (2,920 lb/acre), FourLeaf (2,700 lb/acre), and Ocoee (2,530 lb/acre) (Figure 3). In areas where there is a prevalence of root-knot nematode (RKN), Ocoee would be the best choice to plant because its tolerance to RKN. Yields observed in 2014 were above the average usually observed for white clover in Florida, likely due to weather conditions in the first year, where rainfall from planting to last harvest was 40.5 inches, compared with 35.1 inches deposited in the season (from planting to last harvest). Rainfall distribution and temperatures during the cool-season might have also contributed to better yields in the first season. Gainesville Total dry matter ranged from 2,870 to 1,440 lb/acre in this location (Figure 4). The top varieties were FourLeaf (2,870 lb/acre), Osceola (2,710 lb/acre), Regal Graze (2,670 lb/acre) and Ocoee (2,480 lb/acre). Crusade, Durana and Barblanca performed significantly worse than the other varieties mentioned above. Conclusions and Implications Top yielding varieties performed consistently across locations. White clover varieties that yielded better results included FourLeaf, Regal Graze, Osceola, and Ocoee. FourLeaf is an experimental line of the University of Florida that will be released in the near future. Areas with occurrence of root-knot nematode should be planted with Ocoee because of its tolerance to this pest. Literature Cited Blount, A.R.S., et al Available online at Newman, Y., et al Available online at SAS Institute SAS statistics user s guide. Release version 6. SAS Inst., Cary, NC Florida Beef Research Report

66 Figure 1. White clover variety trial at UF/IFAS NFREC, Marianna, FL Florida Beef Research Report

67 Dry Matter Yield (lb/acre) /21/2014 5/16/2014 6/24/2014 Figure 2. Biomass yield of white clover varieties measured in the season; NFREC, Marianna, FL. Standard error=169 lb/acre Florida Beef Research Report

68 Dry Matter Yield (lb/acre) Axis Title 4/3/2015 5/7/2015 6/11/2015 Figure 3. Biomass yield of white clover varieties measured in the season; NFREC, Marianna, FL. Standard error=169 lb/acre Florida Beef Research Report

69 Dry Matter (lb/acre) a a AFRU ab ab bc c c 0 Cultivar Figure 4. Biomass yield of white clover varieties measured in the season; AFRU, Hague, FL. a,b Means with different superscripts differ, P< Florida Beef Research Report

70 Florida Beef Research Report

71 Alfalfa Production in North-Central Florida J.C.B. Dubeux, Jr. 1, P. Munoz 2, A.R.S. Blount 1, K.H. Quesenberry 2, L.E. Sollenberger 2, B.F.X. Moraes 2, U. Rivero 1, E.R.S. Santos 1 Synopsis Alfalfa is a forage legume with high crude protein and digestibility. It requires high nutrient inputs and soil fertility. Warm weather and high humidity during the summer in the Florida Peninsula and in the Southern Coastal Plains increases disease pressure on the alfalfa crop. Adapted varieties to Florida are available for producers. Summary Alfalfa hay is a valued commodity used in Florida by the livestock industry. Most of the alfalfa hay purchased in Florida, however, comes from distant locations (e.g., Midwest US), increasing the final price. Pest and disease pressure, humidity, and warm temperatures prevailing in Florida during the summer require adapted alfalfa varieties. In this research, we studied six alfalfa varieties (Ameristand 855T RR, Ameristand 901 TS, Ameristand 915 TS RR, Bulldog 805, FL 77, and FL 99) in North Florida at UF/IFAS North Florida Research and Education Center in Marianna, FL and four varieties in Central Florida at the UF/IFAS Plant Science Research and Education Unit in Citra, FL. These varieties were tested under two harvest intensities, 2 and 4 inches. Alfalfa plots were established in November 2013 and harvested for two consecutive years. At both sites, plots were laid out in a split-plot arrangement in a randomized complete block design, with four blocks. Alfalfa varieties performed similarly in Citra and Marianna, with FL99, FL77, and Bulldog 805 being the best options. Productivity under irrigated conditions was greater than in the rainfed system. In North Florida, alfalfa productivity declined after the first spring cut. In Central Florida, alfalfa productivity declined from spring to summer, but recovered in the fall harvest. The trend was similar in both years. Shorter longevity of alfalfa stands observed in Florida compared with the Midwest production regions might be compensated for by the additional price paid in Florida for alfalfa hay. Introduction Alfalfa is considered the Queen of Forages because of its high nutritive value. Alfalfa also has high N- fixation capacity meaning that no nitrogen fertilization is needed for its production. Alfalfa s high dry matter yields (6-12 tons/acre), versatility of products (hay, greenchop, silage, haylage), and almost yearround forage production, from mid/late March to late November, makes it a great alternative for Florida. Because of its high nutritive value, alfalfa is often sold commercially as hay for the dairy and horse industries in Florida. Most of the alfalfa in the USA is produced in more northern environments; as a result, the transportation cost to bring alfalfa hay to Florida is high. At the same time, alfalfa hay has a strong market in the southeastern USA. Florida has approximately 119,000 dairy cows and a welldeveloped horse industry that generates $6.5 billion annually. Producing local alfalfa hay is an opportunity for growers in the southeastern USA. Reduced transportation costs compared with alfalfa 1 North Florida Research and Education Center, University of Florida, Marianna, FL 2 Agronomy Department, University of Florida, Gainesville, FL Florida Beef Research Report

72 transported from other areas in the USA provides local growers a potential advantage to supply the Florida market. Current alfalfa commercial varieties do not tolerate flooding, meaning that a well-drained site needs to be selected to establish an alfalfa stand. In order to establish a successful alfalfa stand, the ph needs to be maintained between 6.5 and 7.0. A soil test well in advance of the establishment day will indicate if ph corrections with lime are needed as well as the fertilization requirements with phosphorus, potassium and micronutrients. For Florida production, high-yield disease/pest resistant improved varieties with a fall dormancy >8 are preferred. There are many varieties in the market for the northern states, however, not many of them have been tested under Florida conditions. In North Florida, hay producers are expanding the alfalfa area (Figure 1). In North-Central Florida, alfalfa is also managed for hay on dairy farms or even under grazing conditions for pure-breed cattle producers (Figure 2). Warm weather during the summer associated with high relative humidity commonly found in the Florida Peninsula and in the Southern Coastal Plains increases disease pressure on the alfalfa crop. In many cases, soil fertility is also inadequate and cultivars used are not adapted to local conditions. Combinations of these factors reduce the potential for alfalfa production in the Southeast USA as compared to North- and Mid-west states. One way to overcome the pest and disease pressure is to breed alfalfa varieties adapted to local conditions. Fall dormancy, for example, is a critical trait that describes the alfalfa growth during the fall due to decreasing temperatures and day length. Fall dormancy scores range from 1 to 11, with lower numbers representing cultivars that exhibit less growth in the fall. Warmer regions should target non-dormant alfalfa varieties with fall dormancy >8. Currently, several non-dormant alfalfa varieties are being tested in North and Central Florida as part of the alfalfa breeding program of the University of Florida. This report presents alfalfa yield data collected from in Citra and Marianna, FL. Materials and Methods Experimental sites The studies were conducted in Marianna and in Citra, FL. The trial in Marianna was carried out at the North Florida Research and Education Center (NFREC) (30 52 N, W, 34 m altitude). The soil at the research site in Marianna was an Orangeburg loamy sand (fine-loamy, kaolinitic, thermic Typic Kandiudults). Soil fertility results at the beginning of the trial at NFREC were ph=6.5, Mehlich-I P=48 ppm, K=78 ppm, Mg=157 ppm, Ca=625 ppm, S=29 ppm, B=0.22 ppm, Zn=3.3 ppm, Mn=52 ppm, Fe=13 ppm, Cu=1.8 ppm, SOM=1.36%, and CEC=7 meq/100 g. In Citra, the trial was conducted at the University of Florida Plant Science Research and Education Unit (29 24' N; 82 10' W). The soil at the experimental site was Arredondo sand. Initial soil characterization showed an average soil ph of 6.8 and it was considered sufficient in P and Mg concentrations, but had a shortage of K before the current study, therefore, 100 lb/acre of K 2 O as muriate of potash was incorporated with irrigation and soil was firmed prior to planting. Treatments and design In Marianna, six alfalfa varieties were tested (Ameristand 855T RR, Ameristand 901 TS, Ameristand 915 TS RR, Bulldog 805, FL 77, and FL 99) under two harvesting intensities (two and four inches). Plots were laid out as a split-plot arrangement in a randomized complete block design, with four blocks. Harvest intensity was the main plot and alfalfa variety the split-plot. In Citra, the design was similar, except that only four varieties were tested (FL99, FL77, Bulldog805 and AmeriStand901TS). The varieties Bulldog 805 and AmeriStand 901TS are probably the most used varieties in this area and FL Florida Beef Research Report

73 and FL77 are discontinued varieties, however, they show better adaptation to the specific weather and soil conditions of Florida. Planting and harvesting In Marianna, the alfalfa variety trial was established on 11/22/2013. The soil was plowed and harrowed before planting. Plots measured 6 x 15 ft. and were allocated in a randomized complete block design, with four replications per treatment. Before planting, plots were cultipacked, seeds were broadcasted, and the plots were rolled thereafter. Harvests were performed when the plants presented approximately 10% of flowering. Six harvests occurred in 2014 (5/6, 6/24, 7/29, 9/3, 10/8, 11/18) and four in 2015 (3/18, 4/29, 8/5, 9/9). Yield was estimated by harvesting a central strip of the plot (3 x 12 ft.) after trimming the borders (1.5 ft. each side). Total fresh weight was recorded and a subsample was taken to estimate the moisture content of the sample. After recording its fresh weight, the subsample was placed into a forced air-dryer at 130 F for 72 h. In 2014, plots were fertilized on 5/16 (300 lb/acre of and 1 lb/acre of boron), 6/27 (200 lb/acre of ), 7/31 (200 lb/acre of and 1 lb/acre of boron), 9/17 (200 lbs./acre of and 10 lb/acre of micronutrient mixture: 5% B, 2.5% Fe, 5% Mn, 2.5% Zn, 1% Ca, 4% S), 10/27 (200 lb/acre of and 10 lb/acre of micronutrient mixture). In 2015, plots were fertilized on 4/3 (400 lb/acre of and 10 lb/acre of micronutrient mixture), 5/19 (300 lb/acre of Kmag), 6/11 (300 lb/acre of ), and 8/18 (300 lb/acre of ). Grass weeds were controlled with Cleanse (Clethodim) at 10 fl oz/acre on 8/1 and 9/10/2014 and 5/21/2015. Pursuit was applied at 3 fl oz/acre on 10/15/2014 and 1/13 and 5/25/2015. Paraquat (Gramoxone) was applied at 1 pt./acre right after harvest on 8/6/2015. Mustang Max was applied at 4 fl oz/acre on 7/31/2014 to control army worms. In Citra, plots also measured 6 x 15 ft. with 12 ft. alleyways between replications. Alfalfa varieties were seeded (20 lb/acre) using a Carter seed drill on November 12, Weed management and insect control were performed as recommended for the crop when necessary. Harvests were performed when the plants presented approximately 10% of flowering. The first harvest occurred in March 20, 2014, four months after planting. Subsequent harvests were performed once 10% of plants had flowers (32-40 day intervals). During 2014, nine harvests were performed and in 2015, this experiment was harvested seven times (Table 1). At harvest, only the central area of each plot was considered, and to minimize border effects, a 16-inch border from each side was not included in the yield sample. Harvests were performed with a sickle bar with a cutting blade of 32 inches. The harvested area in each plot was 32 inches x 12 ft. All material from the harvested area was weighed fresh in the field and subsampled (approximately 1 lb fresh weight subsample) to determine dry matter (DM) concentration. After harvesting, the remaining area of the plot was also cut in order to make it uniform for the subsequent harvest and all cut material was removed from the plots. We fertilized with 60 lb/acre of K 2 O and 30 lb/acre of P 2 O 5 after each harvest. Statistics Data were analyzed using Proc Mixed from SAS. Fixed effects included variety, year, and harvest date. Random effects included blocks and its interactions with fixed effects. Means were compared using the PDIFF procedure adjusted by Tukey (5%). Results At both sites, FL99 was ranked among the top-yielding varieties followed by FL77 and Bulldog 805. Ameristand varieties in general yielded less. Because FL99 and FL77 are discontinued varieties, there are no seeds currently available in the market. Bulldog 805 is the only alfalfa variety currently recommended Florida Beef Research Report

74 for Florida (Blount et al., 2015). These three varieties (FL99, FL77, and Bulldog 805) presented the best stand after two years of harvest in Marianna (Figure 3). In Marianna, alfalfa productivity declined after the first spring cut in 2014 and did not recover after that (Table 3). No irrigation was provided in Marianna and this likely explains the differences between the two sites. In Citra, alfalfa productivity showed a different trend, declining in productivity from spring to summer, but recovering in the fall harvest (Figure 4). This trend was similar in both years. Presence of irrigation might be one of the factors. Photoperiod and temperature might also be part of the difference among sites. In general, alfalfa in Citra produced 50% more than in Marianna, likely due to different environmental and management conditions. In both locations, harvesting at a two-inch stubble height resulted in greater alfalfa productivity than four inches (Table 4). Harvesting intensity affects tillering and regrowth after harvest by not only modifying residual leaf area and carbohydrates in the roots, but also by creating a light environment that favors plant regrowth. Conclusions Alfalfa production is an option for North and Central Florida. Alfalfa varieties performed similarly in Citra and Marianna, with FL99, FL77, and Bulldog 805 being the best options. Productivity under irrigated conditions was greater than in the rainfed system. In North Florida, alfalfa productivity declined after the first spring cut. In Central Florida, alfalfa productivity declined from spring to summer, but recovered in the fall harvest. The trend was similar in both years. Shorter longevity of alfalfa stands observed in Florida compared to the Midwest production regions might be compensated for by the additional price paid in Florida for alfalfa hay. Literature Cited Blount, A.R.S., et al Available online at Newman, Y., et al Available online at SAS Institute SAS statistics user s guide. Release version 6. SAS Inst., Cary, NC Florida Beef Research Report

75 Figure 1. Commercial field of Alfalfa in Jackson County, FL. Photo credit: Jose Dubeux, UF/IFAS NFREC. Figure 2. Livestock grazing bulldog 805 alfalfa in High Springs, FL. Photo credit: Dan Marvel Florida Beef Research Report

76 Figure 3. Alfalfa varieties after two years of harvests at UF/IFAS NFREC, Marianna, FL. Photo credit: Jose Dubeux Table 1. Harvest dates for the alfalfa experiment in 2014 and 2015; Citra, FL. Harvest 2014 Harvest st 03/20/14 10th 02/23/15 2nd 04/28/14 11th 03/26/15 3th 06/02/14 12th 04/23/15 4th 07/01/14 13th 05/21/15 5th 07/31/14 14th 06/16/15 6th 08/28/14 15th 07/09/15 7th 09/29/14 16th 08/13/15 8th 11/05/14 9th 12/11/ Florida Beef Research Report

77 Table 2. Dry matter yield of alfalfa varieties at UF/IFAS NFREC, Marianna, FL (average of seasons) Treatment Marianna Citra lb/acre/harvest Ameristand 901 TS 695 ab 1,145 b Bulldog ab 1,214 ab FL ab 1,198 ab FL a 1,325 a Ameristand 855T RR 693 ab Ameristand 915 TS RR 654 b Standard error ab Means followed by the same letter in the same column do not differ (P>0.05) by the Pdiff procedure from SAS adjusted by Tukey. Table 3. Dry matter yield of alfalfa varieties at UF/IFAS NFREC, Marianna, FL at different harvest dates Harvest date Dry matter yield (lb/acre/harvest) 5/6/2014 2,735 6/24/2014 1,600 7/29/ /3/ /8/ /18/ /18/ /29/ /5/ /9/ Standard error Florida Beef Research Report

78 Dry Matter Yield (lb/acre) /20/2014 4/20/2014 5/20/2014 6/20/2014 7/20/2014 8/20/2014 9/20/2014 Citra, FL 10/20/ /20/ /20/2014 1/20/2015 Harvest Date 2/20/2015 3/20/2015 4/20/2015 5/20/2015 6/20/2015 7/20/2015 8/20/2015 Figure 4. Dry matter yield of alfalfa varieties at the University of Florida Plant Science Research and Education Unit (Citra, FL) at different harvest dates. Table 4. Dry matter yield of alfalfa varieties at UF/IFAS NFREC, Marianna, FL at different harvest intensities Harvest intensity Marianna, lb/acre/harvest Citra, lb/acre/harvest 2 inches 783 a 1,278 a 4 inches 666 b 1,163 b Standard error ab Means followed by the same letter in the same column do not differ (P>0.05) by the Pdiff procedure from SAS adjusted by Tukey Florida Beef Research Report

79 Prevalence of Shiga-Toxin Producing Escherichia Coli in Two Cohorts of Beef Cattle is Associated with Diversity of Microflora and Animal Age Raies A. Mir 1,2, Sarah M. Markland 1,2, Mauricio Elzo 1, Soohyoun Ahn 3, Nicolas DiLorenzo 1, G. Cliff Lamb 1, Jerry Wasdin 1, Danny Driver 1, K. Casey Jeong 1,2 Synopsis In the first cohort of cattle, with ages ranging from 0 to 12 months, Shiga-toxin producing Escherichia Coli (STEC) shedding was greater in young calves of 1-3 months age and as the microflora of calves developed, STEC shedding decreased. In the second cohort of cattle, aged 1 to 11 years, where STEC prevalence peaked at 2 years of age then decreased as animals became older. Summary Many of the factors that modulate the colonization and persistence of Shiga-toxin producing Escherichia Coli (STEC) in cattle remain unknown thus causing a challenge for reducing STEC in this host. The objectives of this study were to better understand the effects of animal age and role of natural microflora present in the cattle intestinal tract on the prevalence of STEC in beef cattle. The prevalence of STEC in calves 1-3 months of age was 60%, which was significantly greater than calves older than 4 months of age. We detected greater STEC prevalence and lower microflora diversity in early age, and microflora of STEC shedding animals was different from the non-stec animals. During sample collection in the second cohort, beginning after 1 year of age, heifers had significantly lower STEC prevalence than cows (37.5% vs. 70%). After 2 years of age, STEC prevalence peaked and tended to decrease as animals became older. Introduction The Centers for Disease Control and Prevention (CDC) has estimated that pathogenic Shiga-toxin producing Escherichia coli (STEC) cause about 269,000 cases of illness (including approximately 3,700 hospitalizations and 30 deaths) in the United States every year (Scallan et al., 2011). Among STEC strains the E. coli O157:H7 serogroup is the best-known and can cause symptoms in affected individuals that include hemorrhagic colitis, bloody diarrhea, and hemolytic uremic syndrome (HUS) in humans (Kaper et al., 2004). Non-O157 STEC serogroups, such as O26, O45, O103, O111, O121, and O145, commonly called the Big Six serogroups, accounted for about 71% of non-o157 STEC isolates between 1983 and 2002 and have also been associated with human disease outbreaks in the United States (Brooks et al., 2005). In 2012, the Big Six non-o157 STEC were declared to be food adulterants by the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA, 2012). Controlling the prevalence of STEC in cattle to reduce outbreaks of this pathogen in humans has proved to be a challenge because of the multiple factors that modulate the colonization and persistence of STEC in cattle. Reducing the prevalence of STEC in cattle at the pre-harvest level has been recently highlighted as an intervention point (Arthur et al., 2011; Jeong et al., 2011), and it has been suggested that lowering the STEC prevalence on farms may reduce the total number of E. coli O157:H7 outbreaks in humans (Matthews et al., 2013). Several studies indicated that diet and management practices may affect the prevalence of STEC, especially E. coli O157:H7 in beef cattle (Fox et al., 2007; Jacob et al., 2010; Zhao et al., 2014). For example, it has been reported that cattle being fed a sorghum-based diet have a high prevalence of E. coli O157:H7, and fecal shedding of E. coli O157:H7 associated with the inclusion of distiller s grain in the feed (Fox et al., 2007; Jacob et al., 2010). However, information at the pre-harvest level, including animal factors and management practices that may modulate the non-o157 STEC prevalence is limited Florida Beef Research Report

80 Procedure Animal management and sample collection Calves and heifers Fecal samples were collected from the recto-anal junction (RAJ) of calves and heifers using sterile cotton swabs in March (n=259), June (n=263), August (n=261) and December (n=193), representing the time at which calves were 1-3, 4-6, 7-9, and months of age, respectively. Due to culling of calves for reasons unrelated to the study only 193 calves were available for the December sample. The sampling scheme resulted in 188 animals that had four matched samples, which were used to assess colonization dynamics. All samples were transported on ice and processed the same day using the protocol described below. Identification of Shiga toxin-producing Escherichia coli A combination of culture-based and nucleic acid-based methods for the detection and enumeration of Shiga toxin-producing Escherichia coli (STEC) were used. We used MacConkey agar (Becton Dickinson Company, MD, USA) to culture Gram-negative enteric bacteria in fecal samples. Purified isolates were subjected to colony PCR for the detection of stx1and stx2 genes. Metagenomic Analyses To understand the association of microflora with animal age and its effect on the STEC dynamics, we did metagenomic analyses of the fecal samples from the claves using the latest technology of 454 pyrosequencing (Macrogen, South Korea). Statistical Analyses of the Prevalence and Concentration of STEC Statistical analysis of the microbiological findings with animal factors was conducted using the STATA software package (STATA MP 11.2, StataCorp, College Station, Texas, USA) with a significance threshold of α < Results Association between Animal Factors and the STEC Prevalence in Beef Calves The main finding from this study is that beef calves shed STEC at an early age and the shedding of STEC decreases as the animal ages until it reaches a low level of ~20% in the beef herd at 12 months of age (Figure 1). The concentration of STEC (log CFU/swab) also decreased as calves matured. Our results indicate that females shed higher STEC when they are ~12 months old. There was no consistent pattern of STEC shedding across calf breed groups over the four samplings during their first year of life. This study found that STEC shedding was greater in young calves 1-3 months of age and that STEC shedding decreased as the microflora of calves developed. The microflora of STEC shedding animals was shown to be different from the non-shedding animals (Figure 2). Further studies are needed to determine the transmission routes and factors affecting early STEC colonization in calves. Association between Animal Factors and the STEC Prevalence in Beef Heifers and Cows In this study we showed that STEC shedding is less prevalent in heifers than in cows (Figure 3A). The lower concentration of normal microflora (Enterobacteriaceae) leads to greater STEC concentrations (Figure 3B). It is also shown that animal age is a critical factor for the prevalence and dynamics of STEC in cattle. Heifers younger than two years of age without any previous live births had significantly lower STEC prevalence compared to cows that had previously given birth. Association between Microflora and the STEC Shedding Our results indicate that Shannon index is linearly correlated with F:B ratio and we speculate that older calves have diverse microflora which reduces STEC prevalence and concentration of STEC shed by the calves. These findings have clear implications for the development of on-farm mitigation strategies. By segregating animals with the greatest risk of infection and greatest rates of shedding, it could be possible Florida Beef Research Report

81 to lower the concentrations of STEC at the pre-harvest level and prevent transmission from cattle to humans. This information could also be used to develop dynamic transmission models that use different rates of colonization with respect to age to simulate the prevalence in herds over time and evaluate the efficacy of potential interventions. Since findings here indicated that when animals became two years old they were more likely to be colonized and were more likely to transmit STEC to other animals through increased shedding, further understanding of factors that increase the STEC prevalence at this stage of development would be critically important to provide interventions to reduce STEC levels in animals. Acknowledgements This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number Literature Cited Arthur, T. M. et al Appl. Environ. Microb. 77: Brooks, J. T. et al J. Infect. Dis. 192: Fox, J. T. et al J. Anim. Sci. 85: Jacob, M. E. et al Appl. Environ. Microb. 76: Jeong, K. C. et al Appl. Environ. Microb. 77: Kaper, J. B. et al Nat. Rev. Microbiol. 2: Matthews, L. et al PNAS. 110: Scallan, E. et al Emerg. Infect. Dis. 17: USDA USDA News Release Release No Zhao, L. et al Foodborne Pathog. Dis. 11: Florida Beef Research Report

82 Figures Figure 1: Prevalence of STEC in beef calves. Figure 2: Difference in microflora of STEC versus non-stec animal Florida Beef Research Report

83 Figure 3: Prevalence and concentration (log CFU/swab) of STEC in heifers and cows Florida Beef Research Report

84 Florida Beef Research Report

85 Prevalence of Cefotaxime Resistant Enterobacteriaceae in Beef Cattle in Florida Raies A. Mir 1,2, Sarah M. Markland 1,2, Thomas A. Weppelmann 2, Mauricio Elzo 1, K. Casey Jeong 1,2 Synopsis The animal factors that affect antibiotic resistance, specifically cefotaxime resistance, have yet to be studied in detail. The prevalence of cefotaxime (a third-generation cephalosporin) resistance in beef claves and adult cattle in Florida was tracked over a period of one year. The findings of this study indicate that environmental factors may influence the prevalence of cefotaxime resistance in beef cattle. Summary Third-generation cephalosporins are used extensively in human medicine and, to some extent, as a therapeutic agent in veterinary medicine. The animal factors that affect antibiotic resistance in general and cefotaxime resistance specifically have not been studied in detail. We tracked cefotaxime resistance in adult cattle from eleven different farms and followed a cohort of beef calves for one year at one particular farm. Calves and adult cattle had never been exposed to any prophylactic antibiotic. The prevalence of cefotaxime resistant bacteria in calves was 60%, 50%, 68% and 6% for March, June, August, and December sampling, respectively. Animal factors of age, breed group, and husbandry management practices were not significantly associated with cefotaxime resistance in calves. The prevalence of cefotaxime resistant Enterobacteriaceae bacteria in adult cattle varied among farms, ranging from 5.2% to 100%. The bacteria isolated from adult cattle were resistant to high concentrations of cefotaxime and demonstrated multi-drug resistance against ten different antibiotics. The findings of this study suggest that antibiotic resistance develops in nature and may be transmitted to food animals from the environment. The basic mechanism in the development of antibiotic resistance is not yet understood. Introduction Antibiotic resistance causes more than 23,000 deaths and $55 billion in the US (overall societal costs). Natural bacterial resistance plays a vital role in the evolution and spread of antibiotic resistance (Walsh and Duffy, 2013). It appears the acquisition of microbial resistance is independent of antibiotic usage in human and veterinary medicine (Call et al., 2008). Previous studies have reported the presence of antibiotic resistance in food animals. However, the origin of resistance remains unknown (Johnson et al., 2009; Hiroi et al., 2012; Mollenkopf et al., 2012). The US is an intensive user of antibiotics and cephalosporins account for 14% of total dispensed antibiotics in the US (Braykov et al., 2013; Laxminarayan et al., 2013). Cefotaxime is a third generation cephalosporin widely used to treat infections and surgical care (Page et al., 1993). Resistance to antibiotics including cephalosporins has been reported from hospitals in more than 100 countries around the world (WHO, 2014). The resistance is usually mediated by production of extended spectrum β- lactamase (ESBL) enzymes by bacteria (Bush and Fisher, 2011). Knowledge involving the factors that influence the dynamics of resistance can be useful in controlling antibiotic resistance (Alekshun and Levy, 2007). This study provides insight to understanding the prevalence of cefotaxime resistance in food animals. Further environmental assessment is needed to better understand the dynamics of antibiotic resistance in pre-harvest animal production Florida Beef Research Report

86 Animal management and sample collection The fecal samples were collected in four different seasons of the growing period, March (n=259), June (n=263), August (n=261), and December (n=193). Due to culling of calves for reasons unrelated to this study, only 193 calves were available for the December sample. The sampling scheme resulted in 188 animals that had all four sampling time points. Fecal samples were collected directly from the rectal-anal junction (RAJ) of animals. Identification and characterization of Cefotaxime resistant bacteria This study utilized a combination of culture-based and nucleic acid-based methods for the detection and enumeration of cefotaxime resistant bacteria from the fecal samples. We screened fecal samples on Tryptic Soy Agar and MacConkey agar both supplemented with Cefotaxime (4 mg/l). For the genetic characterization, the DNA of cefotaxime resistant isolates was used as a template for the multiplex PCR to amplify nine ESBL genes. Antibiotic resistance profiling and identification of resistance genes The isolates were tested against 10 antimicrobials by the standard Kirby Bauer disk diffusion method following the Clinical and Laboratory Standards Institute guidelines (CLSI, 2011). Resistant bacterial colonies on MacConkey plus cefotaxime agar were purified and then bla TEM and bla CTX-M genes were amplified using specific primers. The PCR products were eluted and sent for sequencing to the Cancer and Genetics Research Center (CGRC) at UF. Statistical analyses All statistical analysis was conducted using the STATA software package (STATA MP 11.2, StataCorp, College Station, Texas, USA) with a significance threshold of α<0.05. Results Prevalence of Cefotaxime resistant bacteria in beef calves over one year We found high prevalence of cefotaxime resistance in young 1-3 month old beef calves even if they had no history of antibiotic usage (Figure 1). Our results indicate that cefotaxime resistance levels are higher in warmer climates (June and August) than in December (Figure 1), indicating there might be influence of climate on cefotaxime resistance. No significant association between animal breed or sex and the occurrence of cefotaxime resistance could be established. Cefotaxime resistance in beef heifers and cows All 1,365 samples showed growth on the Tryptic Soy Agar (TSA) containing cefotaxime. Cefotaxime resistance ranged from 6% (farm # 5) to 100% (farm # 7) on MacConkey agar (Figure 2A and 2B). MacConkey agar is selective for Enterobacteriaceae members which have the highest capability to transmit the resistance to other bacteria. MacConkey positive isolates will be utilized for our future experiments. Results here indicated that, although cephalosporins were not used for prophylactic treatment in these farms, cefotaxime resistance was widely prevalent and resistance had developed irrespective of anthropogenic selection pressure (Figure 2B). As shown in Figure 3, most of the bacterial isolates tested in this study had a minimum inhibitory concentration (MIC) of more than 20 µg/ml, confirming that they were intrinsically resistant to the therapeutic treatment of cefotaxime at a clinical level. Nucleotide Blast search of our CTX-M positive isolates showed that the isolates in this study were carrying genes which resembled previously reported bla CTXM-15 and bla CTXM-1 genes. These results indicate that cefotaxime resistance in these isolates is genetically related to clinical isolates and thereby pose severe public health risks Florida Beef Research Report

87 Acknowledgements This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number Literature Cited Alekshun, M. N., and S. B. Levy Cell 128: Braykov, N. P. et al Infection control and hospital epidemiology: the official journal of the Society of Hospital Epidemiologists of America 34: Bush, K., and J. F. Fisher Annu. Rev. Microbiol. 65: Call, D.R. et al Anim. Health Res. Rev. 9: Checkley, S. L. et al La revue veterinaire canadienne 51: Clinical and Laboratory Standards Institute CLSI Document M02-A11/M07-A9. 32:1-53. Hiroi, M. et al J. Vet. Med. Sci. 74: Johnson, J. R. et al J. Clin. Microbiol. 47: Laxminarayan, R. et al Lancet Infect. Dis. 13: Mollenkopf, D. F. et al Appl. Environ. Microbiol. 78: Page, C. P. et al Arch. Surg-Chicago. 128: Walsh, F., and B. Duffy PLoS One 8: e WHO WHO Library Cataloguing-in-Publication Data: Florida Beef Research Report

88 Figures Figure 1. Prevalence of Cefotaxime resistance in beef calves. Figure 2. Number of fecal samples (A) and prevalence of cefotaxime resistance in 11 cattle farms (B) Florida Beef Research Report

89 Figure 3. Minimum inhibitory concentration (MIC) test for the 23 cefotaxime resistant isolates Florida Beef Research Report

90 Florida Beef Research Report

91 Effect of Prenatal Trace Mineral Source on Neonatal and Growing Calf Liver and Serum Mineral Status D. M. Price* 1, A. F. Swain 1, J. M. Guevera 2, C. R. Trcalek 2, M. M. O'Neil 1, M. Irsik 2, O. Rae 2, M. J. Hersom 1, J. V. Yelich 1 ; Synopsis Calf trace mineral status varies across days after calving and is influenced by trace mineral source and breed. In this particular year, mineral source did affect calf performance. Summary The effect of cow prenatal trace mineral (TM) supplement source on calf TM status from birth through 3, 115 days of age, and weaning was examined. Factorial treatment (TRT) arrangements (Angus=AN, n=95 and Brangus=BN, n=96 cows; Inorganic=ING, n=98, and Organic=ORG yeast, n=93) utilized calves born to cows supplemented (3 day/week at a rate of 1 lb 1 1,000 lb BW 1 day in a pellet) with TM 90 d before expected parturition. Calf BW were collected at birth (n=191). A subset of calves (n=43) had bodyweight, serum and plasma collected by jugular vein puncture at birth (0 hour, before colostrum intake), 12 hour, 24 hour, 30, 115 days of age, and weaning. A subset of calves (ING, n=12, 6/breed, ORG, n=14, 7/breed) had liver biopsies performed and serum collected by jugular vein puncture on day 115 and weaning. Processed samples were frozen at 20 C and plasma was refrigerated until analyzed for TM (serum: Co, Cu, Fe, Mn, Mo, Se, Zn; plasma: Se). Time affected (P<0.02) all 0-24 hour serum and plasma TM except for Co (P=0.60). Calf Mn concentrations were undetectable at 0-24 hour. Copper status was deficient, whereas other minerals were marginal to adequate at the 0-24 hour period. On day 30, Se concentrations (whole blood and serum) were greater (P<0.05) for ORG than ING, Co concentration was greater for ING compared to ORG. At weaning, ORG had greater (P 0.02) body weight and ADG than ING. Serum Co and Zn were affected (P<0.001) by day, while all liver TM except for Co were affected (P<0.03) by day. The ORG calves had greater (P 0.01) serum Se, liver Se and Mn than ING. The AN calves had greater (P 0.04) serum Co, Se, and liver Co, Fe, and Zn, while BN had greater (P<0.05) serum Mo and liver Mn. These data demonstrate calf TM status varies by time, prenatal TM source and breed. Introduction Recent research has shown that altering the environment the fetus is exposed to during gestation can have significant effects on the performance of that animal in its postnatal life, a concept termed fetal programming. This originally started with the nutrient restriction model, which resulted in increased adipose tissue deposition as well as decreased muscle development (Zhu et al., 2006; Du et al., 2010) leading to alterations in carcass composition (Larson et al., 2009; Underwood et al., 2010). Trace minerals have traditionally been supplemented to cattle diets as inorganic salts. In spite of this tradition, recent attention has been placed on the use of organic or chelated trace mineral supplementation in the rumen diets. Organic trace minerals differ from inorganic forms as a result of their chemical association with an organic ligand. Numerous groups of these organic trace minerals are formed from this mineral-organic ligand combination, which are available in the animal feeding industry and include chelates, proteinates, and complexes (AAFCO, 2000). The recent attention towards organic mineral Florida Beef Research Report

92 supplementation in ruminant diets has been fueled by numerous studies that have shown an increase in overall performance in cattle supplemented with organic minerals. It is generally accepted that this association is due to the increased bioavailability of organic minerals. Minerals play a significant role in many metabolic processes that affect growth performance, reproductive efficiency and immune function. Selecting the correct mineral supplement is crucial for maintaining these processes. With research clearly demonstrating across animal species that organic minerals are move bioavailable to the animal further investigation is imperative to determine whether the greater bioavailability of these organic mineral will have a more positive effect on the overall performance of beef cattle reproduction. Materials and Methods Pregnant Angus (AN, n=95) and Brangus (BN, n=96) cows were allotted to two mineral supplements containing either inorganic (ING, n=98) or organic (ORG, n=93) trace minerals in a two by two factorial design. Mineral supplementation began approximately 82±2 days for all cows (range 35 to 147 days) and 72±1 day for cows whose calves had blood and liver biopsies collected for trace mineral status prior to calving and continued through weaning. From the initiation of the experiment to the end of the breeding season cows grazed on dormant bahiagrass pastures, fed large round bales of hay for ad libitum consumption, and supplemented with soybean hulls to maintain a body condition score of 5 on a 9 point scale. Cows received the trace mineral supplement in a pelleted supplement fed at a rate of 1 lb per 100 lbs of cow body weight. Trace mineral and soybean hull supplements were offered three days per week. After the breeding season, cows grazed bahiagrass pastures in large mineral treatment groups. Trace mineral supplement was provided as a loose mineral for consumption based on formulated guidelines. Mineral was distributed on a weekly basis plus 10% to ensure appropriate consumption. The ING trace mineral supplement was formulated to meet the mature beef cow mineral requirements based on NRC recommendations (Table 1). The ORG trace mineral supplement was formulated to meet beef cow NRC requirements based on the assumption of greater bioavailability for organic sources of minerals. Calves (n=43) had body weight and blood collected at birth (prior to suckling), 12 hours post parturition, and 30 days of age. A subset of calves that were evaluated at calving were used to collect serum and liver samples at 115±1.5 days of age and at weaning (205±1.5 days of age). Blood was collected from calves via jugular venipuncture and processed for serum collection. Serum trace mineral analysis for cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) was carried out using ICP-Spectroscopy-MS (DCPAH, Lansing, MI). All calves (n=191) were used to calculate weaning average daily gain. Calf mineral status data for 0 to 24 hour post-partum was analyzed using the Mixed model of SAS. Repeated measures analysis was conducted using mineral source treatment, breed, time, and their interactions were included in the model. Means for the 0 to 24 hour period are presented. Mineral status data for day 30 were analyzed using the Mixed model with treatment and breed as fixed effects in the model. Day 115 and weaning data were analyzed using repeated measures in the Mixed procedure of SAS. The fixed effects of mineral source treatment, breed, day, and appropriate interactions were used. Some minerals data required log transformation for analysis. Pearson correlations were determined for Florida Beef Research Report

93 serum and liver trace mineral concentrations. Significance was determined at P 0.05 and a trend Results Time affected (P<0.02) calf serum trace mineral status during the 0 to 24 hour sampling period; with the exception of Co which was not affected by time and Mn which was undetectable. Table 2 presents the 0 to 24 hour mean calf serum trace mineral status. During the 0 to 24 hour period there were no treatment x breed interactions for any of the trace minerals analyzed. Selenium concentration in either whole blood or serum was greater (P<0.001) for ORG compared to ING calves. Concentrations of Co and Mo were not affected (P 0.12) by treatment or breed. In contrast, Cu and Zn concentrations were greater (P<0.05) in BN than AN, whereas AN calves tended to have greater (P=0.09) Fe concentration than BN calves after birth. The mean mineral status across all calves indicated calves were deficient in Mn and Cu, but marginal to adequate for the remaining trace minerals. Similar to the 0 to 24 hour sampling period, day 30 Se concentration (Table 3) in whole blood and serum was greater (P<0.001) for ORG compared ING calves. Additionally, serum Se concentration was greater (P=0.05) in AN compared BN calves. Calves from dams supplemented with ING had greater (P=0.02) Co concentration than ORG calves. Concentrations of Cu, Fe, Mn, Mo, and Zn on day 30 did not differ between treatments (P 0.37) or breed (P 0.42). On day 30 all trace mineral were indicated to be marginal to adequate, Fe concentrations were determined to be high in status across all treatments. Serum Co and Zn were affected (P<0.01) for day 115 and weaning, Co concentrations increased from day 115 to weaning whereas Zn concentrations decreased with time. Organic calves had greater (P<0.001) serum Se concentrations (29.8 ng/ml) compared to ING calves (18.53 ng/ml), while AN calves had greater (P<0.001, ng/ml) Se concentration compared to BN calves (19.99 ng/ml). Calf Co and Mo concentrations demonstrated breed differences, primarily driven by greater concentrations at weaning for Co for ING-AN and Mo for ORG-BN. Liver concentrations of trace minerals (Table 5) were affected (P 0.01) day for all minerals. Liver concentrations increased for Co, Cu, Mn, Mo, Se, and Zn; but decreased for Fe. Angus calves had greater (P 0.05) Co, Fe, and Zn liver concentrations than BN, whereas BN calves had greater (P<0.05) Mn concentration than AN calves. Mineral source treatment increased (P 0.01) Mn and Se concentrations in ORG calves compared to ING calves. Mineral source did not affect the liver concentration of Co, Cu, Mo, or Zn in calves at day 115 or weaning. Correlations between serum and liver concentrations were generally poor except for Co and Se (Co, r 2 =0.88; Cu, r 2 =0.00; Fe, r 2 =0.12; Mn, r 2 =0.08; Mo, r 2 =0.04; Se, r 2 =0.58; Zn, r 2 =-0.12). Serum and liver concentrations are both valid means to assess trace mineral status, however for most trace minerals there are poor correlations between measures implying that one or the other assessment should be used, but comparisons between means are not valid. Literature Cited AAFCO Official Publication. Association of American Feed Control Officials, Inc. Du, M., et al J. Anim. Sci. 88:E51-E60. Larson, D. M., et al J. Anim. Sci. 87: Underwood, K. R., et al : Zhu, M. J, et al J. Pysiol. 575: Florida Beef Research Report

94 Table 1. Composition of trace mineral supplement provided to cows Component Inorganic Organic Dry matter, % Crude protein, % Total digestible nutrients, % Ca, % P, % Mg,% K, % S, % Co, ppm Cu, ppm I, ppm Fe, ppm Mn, ppm Se, ppm Zn, ppm Vitamin A, IU/lb 31,606 31,606 Vitamin D 3, IU/lb 2,835 2,835 Vitamin E, IU/lb Table 2. Effect of maternal trace mineral supplement source on 0 to 24 hour post-partum calf trace mineral serum and whole blood (WB) concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Trt x Breed Mean Status 3 Co, μg/ml M-A Cu, μg/ml D Iron, μg/ml A Mn, μg/ml Mo, ng/ml A Se-WB, ng/ml < A Se, ng/ml < A Zn, μg/ml M 1 ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. 3 Trace mineral status, A = adequate, M = marginal, D = deficient. Reference ranges for TM status from Herdt and Hoff, Vet. Clin. Food Anim. 27: Florida Beef Research Report

95 Table 3. Effect of maternal trace mineral supplement source on 30-day post-partum calf trace mineral serum and whole blood (WB) concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Trt x Breed Mean Status 3 Co, μg/ml A Cu, μg/ml A Fe, μg/ml H Mn, μg/ml A Mo, μg/ml M-A Se-WB, ng/ml < M Se, ng/ml < M-A Zn, μg/ml M 1 ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. 3 Trace mineral status, A = adequate, M = marginal, D = deficient. Reference ranges for TM status from Herdt and Hoff, Vet. Clin. Food Anim. 27: Table 4. Effect of maternal trace mineral supplement source on 115-day post-partum and weaning calf serum trace mineral concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Day Trt x Breed x Day Co, ng/ml d < weaning Cu, ng/ml d weaning Fe, ng/ml d weaning Mn, ng/ml d weaning Mo, ng/ml d <0.001 weaning Se, ng/ml d <0.001 < weaning Zn, ng/ml d < weaning ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source Florida Beef Research Report

96 Table 5. Effect of maternal trace mineral supplement source on 115-day post-partum and weaning calf liver trace mineral concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Day Trt x Breed x Day Co, μg/g d weaning Cu, μg/g d weaning Fe, μg/g d weaning Mn, μg/g d weaning Mo, μg/g d < weaning Se, μg/g d weaning Zn, μg/g d weaning ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. Table 6. Effect of maternal trace mineral supplement source on calf body weight Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Day Trt x Breed x Day Birth weight, lb d ADG, lb/d Day 115, lb <0.01 <0.01 Weaning, lb ADG, lb/day ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. 3 Treatment x breed interaction only Florida Beef Research Report

97 Effects of Trace Mineral Source on Cow Performance and Mineral Status During a Production Cycle D. M. Price 1, K. M. Havill 1, A. F. Swain 1, J. M. Guevera 2, C. R. Trcalek 2, M. Irsik 2, O. Rae 2, M. J. Hersom 1, Joel V. Yelich 1 Synopsis The source of trace mineral did affect trace mineral status of cows. Cow trace mineral status varied over the production cycle and was affected by cow breed. Summary No 3-way interactions were detected except for serum Cu, Mn and Mo. At PostC, BN were 0.17 BCS greater (P=0.03) than AN. Cow BW and BCS did not differ (P>0.21) by TRT at each period. The AN had greater (P 0.03) BW change from TMst to PreC (49 vs. 40±2 kg) and from PostC to Pbrd ( 44±4 vs. 33±3 kg) than BN. Change in BCS from PreC and Pbrd to weaning were greater (P<0.05) in ING ( 0.15 vs ±0.05 and 0.36 vs. 0.10±0.08, respectively) than ORG. Time (P 0.02) affected all serum and liver TM concentrations except for serum Fe (P=0.13). Serum Co and liver Cu concentrations were greater (P<0.05) in ING (1.7 vs. 1.3±0.1 ng/ml and 266.4±15.0 vs ±14.1 µg/g, respectively), while serum Mo was greater (P=0.006), in ORG (2.8 vs. 1.7±0.3 vs. ng/ml). Serum Fe and liver Cu and Mn concentrations in BN (149.1±5.5 µg/dl, 280.9±15.2 and 11.7±0.5 µg/g) were greater (P<0.03) than AN (132.4±5.3 µg/dl, 186.7±13.9 and 9.5±0.5 µg/g). Serum Se was greater (P=0.003) in AN than BN (73.5±1.7 vs. 65.4±1.8 ng/ml). Cow TM status varies over time and is affected by breed and TM source; indicating development of nutritional management strategies can be based on cattle breed. Introduction Mineral nutrition for beef cows, replacement heifers, and bulls is a key component of the total nutritional profile for the cattle herd. Trace minerals are key components to many proteins and enzymes that are important to ruminant growth-performance, reproduction, health, and carcass characteristics. Improvements in any category of cattle performance could have a positive outcome on beef enterprise profitability. Trace minerals have traditionally been supplemented to cattle diets as inorganic salts. In spite of this tradition, recent attention has been placed on the use of organic or chelated trace mineral supplementation in the rumen diets. Organic trace minerals differ from inorganic forms as a result of their chemical association with an organic ligand. Numerous groups of these organic trace minerals are formed from this mineral-organic ligand combination, which are available in the animal feeding industry and include chelates, proteinates, and complexes (AAFCO, 2000). Previous research in beef cattle using organic sources of trace minerals has demonstrated some improvement in reproductive parameters important to whole herd efficiency; although, most of this research has focused on the postpartum cow. Minerals play a significant role in many metabolic processes that affect growth performance, reproductive efficiency and immune function. Selecting the correct mineral supplement is crucial for maintaining these processes. Mineral source in the form of organic minerals provided during critical stages of the production cycles of gestating and suckled beef cows will enhance the overall cow herd mineral status. The objective of this research was to determine the effect of trace mineral supplement source starting prepartum and cow breed on cow trace mineral concentration in serum and liver samples across a production cycle Florida Beef Research Report

98 Materials and Methods Pregnant Angus (AN, n=95) and Brangus (BN, n=96) cows were allotted to two mineral supplements containing either inorganic (ING, n=98) or organic (ORG, n=93) trace minerals in a two by two factorial design. Mineral supplementation began approximately 82±2 days for all cows (range 35 to 147 days) and 72±1 day for cows whose calves had blood and liver biopsies collected for trace mineral status prior to calving and continued through weaning. From the initiation of the experiment to the end of the breeding season cows grazed on dormant bahiagrass pastures, fed large round bales of hay for ad libitum consumption, and supplemented with soybean hulls to maintain a body condition score (BCS) of 5 on a 9 point scale. Cows received the trace mineral supplement in a pelleted supplement fed at a rate of 1 lb per 100 lbs of cow body weight. Trace mineral and soybean hull supplements were offered three days per week. After the breeding season, cows grazed bahiagrass pastures in large mineral treatment groups. Trace mineral supplement was provided as a loose mineral for consumption based on formulated guidelines. Mineral was distributed on a weekly basis plus 10% to ensure appropriate consumption. The ING trace mineral supplement was formulated to meet the mature beef cow mineral requirements based on NRC recommendations (Table 1). The ORG trace mineral supplement was formulated to meet beef cow NRC requirements based on the assumption of greater bioavailability for organic sources of minerals. A subset of cows was used to collect liver biopsies and blood samples from cows via jugular venipuncture and blood processed for serum collection. Serum trace mineral analysis for cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) was carried out using ICP- Spectroscopy-MS (DCPAH, Lansing, MI) at 4 and 6 periods, respectively: start of TM (TMst), precalving (PreC), calving (C, serum only), post-calving (PostC, serum only), prebreeding (Pbrd) and weaning. Cow body weight and BCS were recorded at each period. Data were analyzed with the Mixed procedure of SAS using a repeated measures analysis. Fixed effects included mineral source treatment, breed, time, and appropriate interactions. Covariates of cow body weight and body condition score were used and mineral concentration data transformed when appropriate. Pearson correlations were determined for serum and liver mineral concentrations. Significance was determined at P 0.05 and a trend Results Day of sampling affected serum (Table 2) and liver (Table 3) mineral concentrations (P 0.01) for all minerals except serum Co. Cobalt concentration in serum and liver did exhibit a treatment x time interaction (P<0.05) reflected by ING cows having greater concentrations of Co at rebreeding and weaning sampling times compared to ORG. A significant correlation (P=0.01, r 2 =0.55) was evident for mean serum and liver Co. Mean serum and liver Co concentrations were deemed adequate. Serum Cu exhibited a treatment x breed x time interaction (P<0.05) that can be summarized simply by ING cows had greater concentration than ORG, BN had greater concentration than AN, and concentrations decreased over time. Liver Cu concentrations were affected by treatment, breed, and day (P 0.001), but had no correlation with serum Cu (P=0.34, r 2 =-0.10). Mean serum Cu was deemed deficient to marginal, while liver concentration were deemed adequate. Serum Fe was greatest in ORG-BN at weaning which resulted in a treatment x breed x time interaction (P<0.05). No samples were available for analysis at the pre-mineral time due to sampling error. Liver Fe was affected by day (P<0.001), with greatest concentrations occurring at weaning. Iron demonstrated no correlation (P=0.15, r 2 =0.17) between serum and liver concentrations. Mean serum Fe concentration was deemed adequate while liver concentration was high Florida Beef Research Report

99 Serum Mn concentrations exhibited a treatment x breed x time interaction (P<0.05), whereas liver Mn demonstrated breed and day effects (P<0.001 and 0.002, respectively). There was no correlation (P=0.69, r 2 =0.04) between serum and liver Mn concentrations. Mean serum Mn concentrations were indicated to be deficient and liver concentrations were marginal. Concentrations of Mo in serum were variable which resulted in a treatment x breed x time interaction (P<0.001), and largely influenced by ORG-BN value at weaning. Liver Mo concentration exhibited a breed x time (P=0.002) in that BN had greater concentration and concentrations were greater for ING cows at pre-calving whereas ORG cows had greater values at differing points. Mean serum and liver Mo concentrations were correlated (P<0.01, r 2 =0.32) for cows. Mean Mo concentrations were marginal whereas liver concentrations were adequate. Selenium serum and liver concentrations exhibited a treatment x time interaction (P<0.05) and serum concentration of Se in AN cows were greater (P=0.02) across sampling days compared to BN. Both serum and liver concentrations were greater at pre-calving and rebreeding (P<0.001) than pre-mineral or at weaning. A significant correlation (P<0.01, r 2 =0.55) was evident between serum and liver Se concentrations. Mean serum Se concentrations were classified as deficient, but liver concentrations were classified as adequate. Both serum and liver Zn concentrations varied between sampling times and increased across days of supplementation (P 0.01), however there was no correlation (P=0.48, r 2 =0.07) between serum and liver concentrations. Mean serum Zn concentrations were marginal to adequate whereas liver concentrations were adequate. Cow serum and liver trace mineral concentrations varied across the production cycle. Although cows were supplemented continuously serum and liver status across the production cycle was still deficient to marginal for some trace minerals. Likely the demands of the cow s physiology over the production cycle influenced measured circulating and stored trace mineral concentration and status. Literature Cited AAFCO Official Publication. Association of American Feed Control Officials, Inc., Oxford, IN Florida Beef Research Report

100 Table 1. Composition of trace mineral supplement provided to cows Component Inorganic Organic Dry matter, % Crude protein, % Total digestible nutrients, % Ca, % P, % Mg,% K, % S, % Co, ppm Cu, ppm I, ppm Fe, ppm Mn, ppm Se, ppm Zn, ppm Vitamin A, IU/lb 31,606 31,606 Vitamin D 3, IU/lb 2,835 2,835 Vitamin E, IU/lb Florida Beef Research Report

101 Table 2. Effect of trace mineral supplement source on cow serum trace mineral concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Day Inter 3 Co, ng/ml Pre-Min Pre-Calve Rebreed Wean Cu, μg/ml < ,4 Pre-Min Pre-Calve Rebreed Wean Fe, μg/ml Pre-Min..... Pre-Calve Rebreed Wean Mn, ng/ml < ,4 Pre-Min Pre-Calve Rebreed Wean Mo, ng/ml < ,2,3,4 Pre-Min Pre-Calve Rebreed Wean Se, ng/ml < Pre-Min Pre-Calve Rebreed Wean Zn, μg/ml Pre-Min Pre-Calve Rebreed Wean ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. 3 Interaction 1=treatment x breed, 2=treatment x time, 3= breed x time, 4 = treatment x breed x time; P< Serum sample collected prior to initiation of mineral supplementation. 5 Serum sample collected prior to parturition. 6 Serum sample collected prior to initiation of breeding season. 7 Serum sample collected prior to weaning of the calf Florida Beef Research Report

102 Table 3. Effect of trace mineral supplement source on cow liver trace mineral concentrations Mineral source breed combination 1 P-value Item ING- AN ING- BN ORG- AN ORG- BN SEM Trt 2 Breed Day Inter 3 Co, μg/g < Pre-Min Pre-Calve Rebreed Wean Cu, μg/g <0.001 < Pre-Min Pre-Calve Rebreed Wean Fe, μg/g < Pre-Min Pre-Calve Rebreed Wean Mn, μg/g 0.40 < Pre-Min Pre-Calve Rebreed Wean Mo, μg/g Pre-Min Pre-Calve Rebreed Wean Se, μg/g <0.001 Pre-Min Pre-Calve Rebreed Wean Zn, μg/g <0.001 Pre-Min Pre-Calve Rebreed Wean ING = inorganic, ORG = organic, AN = Angus, BN = Brangus. 2 Treatment of mineral source. 3 Interaction of 1=treatment x breed, 2=treatment x time, 3= breed x time, 4 = treatment x breed x time; P< Serum sample collected prior to initiation of mineral supplementation. 5 Serum sample collected prior to parturition. 6 Serum sample collected prior to initiation of breeding season. 7 Serum sample collected prior to weaning of the calf Florida Beef Research Report

103 Effects of Prepartum Mineral Supplement Source and Cow Breed on Cow Colostrum Composition and Neonatal Calf Serum Immunoglobulin Concentration D. M. Price 1, J. M. Guevera 2, C. R. Trcalek 2, K. K. Arellano 2, M. Irsik 2, O. Rae 2, M. J. Hersom 1, J. V. Yelich 1 Synopsis Organic mineral supplementation provided to gestating cows increased calf Ig concentrations after birth and at 30 d of age. However, breed had more effect on colostrum and milk composition than prepartum mineral supplement treatment. Summary Treatment and breed did not affect colostrum fat, total protein, lactose, dry matter or somatic cell count. Colostrum total solids were greater (P 0.05) for Angus (27.10±0.89 %) than Brangus (24.56±0.86 %). Colostrum had no detectable concentrations of Co, Cu, Fe, Mn, or Mo. However, Zn was greater (P>0.05) for Angus (90.80±2.15 µg/g) than Brangus (79.30±2.15 µg/g). A treatment by breed interaction (P=0.03) was observed for Se colostrum (Angus-Organic, ; Brangus-Inorganic, 0.88±0.06; Angus-Inorganic, 0.74±0.07; Brangus-Organic, 0.72±0.07 µg/g). Treatment and breed did not affect (P>0.05) milk fat, true protein, total solids, lactose, and somatic cell count. Colostrum immunoglobulins (Ig) were not affected by breed or treatment with mean concentrations of 11,112±491, 415±30, 536±41, and 12,063±515 mg/dl for IgG, IgM, IgA and total Ig, respectively. At 0 h, calf serum had no detectable IgG, IgM, and IgA values. Treatment or breed did not affect calf serum IgG and IgM concentrations at 12 h and 24 h. However, calves from organic supplemented cows had greater IgA concentrations than inorganic calves at 12 (P=0.04; organic=287.9 mg/dl, inorganic=202.8 mg/dl) and 24 h (P=0.03; organic=290.3 mg/dl, inorganic=195.2 mg/dl). Treatment had no effect on 12 h calf total Ig concentrations; but at 24 h, organic calves tended to have greater (P=0.06) total Igs than inorganic calves. There was no breed effect or interaction for total Igs at 12 or 24 h. At 30 d of age, organic calves (1,653 mg/dl) had greater (P=0.04) IgG concentrations than inorganic (1,273 mg/dl) calves. Introduction Mineral nutrition for beef cows, replacement heifers, and bulls is a key component of the total nutritional profile for the cattle herd. Trace minerals are key components to many proteins and enzymes that are important to ruminant growth-performance, reproduction, health, and carcass characteristics. Improvements in any category of cattle performance could have a positive outcome on beef enterprise profitability. Trace minerals have traditionally been supplemented to cattle diets as inorganic salts. In spite of this tradition, recent attention has been placed on the use of organic or chelated trace mineral supplementation in the rumen diets. Organic trace minerals differ from inorganic forms as a result of their chemical association with an organic ligand. Numerous groups of these organic trace minerals are formed from this mineral-organic ligand combination, which are available in the animal feeding industry and include chelates, proteinates, and complexes (AAFCO, 2000). Previous research in beef cattle using organic sources of trace minerals has demonstrated some improvement in reproductive parameters important to whole herd efficiency; although, most of this research has focused on the postpartum cow. Minerals play a significant role in many metabolic processes that affect growth performance, reproductive efficiency and immune function. Selecting the correct mineral supplement is crucial for maintaining these processes. Mineral source in the form of organic minerals provided during critical stages of the Florida Beef Research Report

104 production cycles of gestating and suckled beef cows will enhance the overall cowherd mineral status and calf health status. The objective of this research was to determine the effect of prepartum trace mineral supplement source and cow breed on colostrum and milk composition, mineral components, and somatic cell count (SSC) and immunoglobulins (Ig) in colostrum and calf serum. Materials and Methods Forty mature pregnant Angus (AN, n=20) and Brangus (BN, n=20) cows were allotted to two mineral supplements containing either inorganic (ING) or organic (ORG) trace minerals in a two by two factorial design. Mineral supplementation began approximately 90 d prior to expected parturition and continued through weaning. Cows grazed on dormant bahiagrass pastures, fed large round bales of hay for ad libitum consumption, and supplemented with soybean hulls to maintain a body condition score of 5 on a 9 point scale. Cows received the trace mineral supplement in a pelleted supplement fed at a rate of 1 lb per 1000 lbs of cow body weight. The ING trace mineral supplement was formulated to meet the mature beef cow mineral requirements based on NRC recommendations (Table 1). The ORG trace mineral supplement was formulated to meet beef cow NRC requirements based on the assumption of greater bioavailability for organic sources of minerals. Trace mineral and soybean hull supplements were offered three days per week. Colostrum (prior to calves suckling) and milk (30 d post-parturition) samples were collected by handmilking and frozen at -20 C for eventual mineral and milk composition analysis. Colostrum analysis (percentage basis) included fat, total protein, total solids, lactose, as-fed ash, moisture, and DM, and somatic cell count (SCC). Centrifuged colostrum was analyzed for trace minerals (Co, Cu, Fe, Mn, Mo, Se and Zn). Trace mineral analysis was carried out using ICP-Spectroscopy-MS (DCPAH, Lansing, MI). Milk analysis (percentage basis; Dairy One, Ithaca, NY) included fat, true protein, total solids, and lactose, plus milk urea nitrogen (MUN) and SCC. Data were analyzed using PROC GLM of SAS with treatment and breed as fixed effects. Somatic cell counts were transformed using the log of SCC and data were back calculated to obtain least squares means and errors. Calf serum was collected via venipuncture at 0 (pre-suckling), 12, and 24 h and 30 d of age. Calf serum and colostrum samples were frozen at -20 C until quantification of concentrations of IgG, IgM, and IgA were determined by single radial immunodiffusion. Total Ig (TIg) concentrations for calf serum and colostrum were calculated by summing of all Ig concentrations. Data were analyzed using PROC GLM of SAS for each sample time with treatment and breed as fixed effects. Results Angus cows had greater (P=0.05, 27.1%) colostrum total solids than BN cows (24.6%), but mineral source did not affect total solids (Table 2). Mineral source and breed had no effect (P>0.05) on colostrum total protein (16.7%), fat (5.06%), lactose (2.97%), dry matter (4.38%), or SCC (7.4 x 10 6 cells/ml). Colostrum as-fed ash tended (P=0.07) to be greater for AN (1.15 %) than BN (1.06 %). Colostrum had no detectable concentrations of Co, Cu, or Fe trace minerals. Not all colostrum samples had detectable Mn or Mo concentrations. For the samples that did contain adequate concentrations of Mn and Mo there was no effect (P>0.05) of breed or mineral source. However, colostrum Zn was greater (P>0.05) for AN (90.8 µg/g) than BN (79.3 µg/g). A treatment by breed interaction (P=0.03) was observed for Se colostrum in that BN-ING had the greatest concentration followed in order by AN-ORG, ANG-ING and Florida Beef Research Report

105 BN-ORG. Mineral source and breed did not affect (P>0.05) 30-d milk (Table 3) true protein (3.07 %), fat (1.98 %), lactose (5.11 %), total solids (10.89 %), and SCC (3.2 x 10 5 cells/ml). Clinical mastitis is considered to occur at SCC of 2.0 x 10 5 cells/ml. Milk urea nitrogen was greater (P=0.02) for BN (12.12 mg/dl) than AN (9.40 mg/dl), but not different between mineral sources. Neither breed nor mineral source (P 0.05) affected colostrum Igs (Table 4). Mean colostrum concentrations of Ig were 11,112, 415, 536, and 12,063 mg/dl for IgG, IgM, IgA and total Ig, respectively. At 0 hour, calf serum had no detectable IgG, IgM, and IgA values as would be expected because blood samples were collected prior to calves nursing. Mineral source or breed (P 0.05) did not affect calf serum IgG and IgM concentrations at 12 hour and 24 hours after birth. However, ORG calves had greater IgA concentrations than ING calves at 12 hour (P=0.04; ORG=287.9 mg/dl, ING=202.8 mg/dl) and 24 hour (P=0.03; ORG=290.3 mg/dl, ING=195.2 mg/dl). The increase in Ig occurred because calves were allowed to consume colostrum after the colostrum and blood samples were collected at birth. Mineral source had no effect (P>0.05) on 12 hour calf total Ig concentrations; but at 24 hour after birth, ORG calves tended to have greater (P=0.06) total Igs than ING calves. There was no breed effect or breed x mineral source for 12 hour total Igs at (P>0.05) or 24 hour total Ig (P>0.05). At 30 day of age, ORG calves (1,653 mg/dl) had greater (P=0.04) IgG concentrations than ING (1,273 mg/dl) calves. Mineral source alone did not affect colostrum or milk compositional analysis or SCC. Mineral source tended to affect Zn concentration and Se exhibited a breed x mineral source interaction. Breed type did affect colostrum and milk component concentrations. Organic mineral supplements increased calf serum Igs in the first 24 hours after birth. Colosturm and serum IgA was greater in Angus than Brangus animals. Literature Cited AAFCO Official Publication. Association of American Feed Control Officials, Inc., Oxford, IN Florida Beef Research Report

106 Table 1. Composition of trace mineral supplement provided to cows Component Inorganic Organic Dry matter, % Crude protein, % Total digestible nutrients, % Ca, % P, % Mg,% K, % S, % Co, ppm Cu, ppm I, ppm Fe, ppm Mn, ppm Se, ppm Zn, ppm Vitamin A, IU/lb 31,606 31,606 Vitamin D 3, IU/lb 2,835 2,835 Vitamin E, IU/lb Table 2. Effect of trace mineral source and cow breed on colostrum characteristics Inorganic- Angus Inorganic- Brangus Organic -Angus Organic -Brangus SEM Source x Breed P-value Total Solids, % Total Protein, % Fat, % Lactose, % As fed ash, % Dry matter, % Somatic cell count, 10 5 cells/ml Manganese, ug/g Molybdenum, ug/g Selenium, ug/g Zinc, ug/g Florida Beef Research Report

107 Table 3. Effect of trace mineral source and cow breed on 30-d milk characteristics Inorganic- Inorganic- Organic Organic Source x Breed SEM Angus Brangus -Angus -Brangus P-value True Protein, % Fat, % Lactose, % Total Solids, % Somatic cell count, 10 3 cells/ml Milk urea nitrogen, mg/dl Table 4. Effect of trace mineral source and cow breed on colostrum and calf immunoglobulin concentrations Inorganic- Angus Inorganic- Brangus Organic -Angus Organic -Brangus SEM Source x Breed P-value Colostrum IgG, mg/dl 10,196 11,048 11,638 11, IgM, mg/dl IgA, mg/dl Total Ig, mg/dl 11,180 11,764 12,693 12, Calf Serum 12 h post-partum IgG, mg/dl 3,565 3,071 3,815 3, IgM, mg/dl IgA, mg/dl Total Ig, mg/dl 4,297 3,559 4,310 4, h post-partum IgG, mg/dl 3,934 3,802 4,652 4, IgM, mg/dl IgA, mg/dl Total Ig, mg/dl 4,342 4,110 5,183 5, Florida Beef Research Report

108 Florida Beef Research Report

109 Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Ruminal Fermentation of Beef Steers Consuming Bermudagrass Hay F. M. Ciriaco 1, D. D. Henry 1, V. R. G. Mercadante 1, T. Schulmeister 1, M. Ruiz-Moreno 1, G. C. Lamb 1, and N. DiLorenzo 1 Synopsis Supplementation with energy and protein sources is crucial during critical periods of the year and molasses and crude glycerol combined in a liquid mixture have the potential of serving as an energy supplement for beef cattle consuming forage-based diets. Summary A study was conducted to evaluate the effects of molasses and crude glycerol in a 50:50 liquid mixture on ruminal fermentation of beef steers consuming Tifton 85 bermudagrass hay. Steers were provided Tifton 85 bermudagrass hay ad libitum and four increasing amounts of the 50:50 mixture were compared: 1) CTRL=0 lb/d; 2) SUP1=1 lb/d; 3) SUP3=3 lb/d; and 4) SUP5=5 lb/d. Eight ruminally cannulated Angus crossbred steers were used in a duplicated 4 4 Latin Square design. Steers were housed at the North Florida Research and Education Center (NFREC) Feed Efficiency Facility (FEF) for four periods of 28 d each and intake of hay was monitored using the GrowSafe system. The liquid supplement was weighed and offered daily to each steer. Ruminal fluid and blood were collected every 3 h postfeeding for 24 h for measurement of average ruminal ph, analyses of volatile fatty acids (VFA) profile, and ammonianitrogen (NH 3 -N) and blood urea nitrogen (BUN) concentrations. Concentrations of NH 3 -N and BUN were influenced by liquid supplementation, decreasing linearly as the amount of supplementation increased. Average ruminal ph was also influenced by liquid supplementation and decreased linearly as the amount of liquid supplement increased. A treatment time interaction was observed for ruminal ph. Molar proportions of branched chain VFA (BCVFA) and acetate linearly decreased whereas molar proportions of propionate, butyrate, and valerate linearly increased as the amount of liquid supplemented increased. Total VFA concentration was affected cubically by liquid supplementation where it decreased from CTRL to SUP1 and SUP5, however, SUP3 was not different when compared to the others. There was an effect of liquid supplementation on acetate to propionate ratio (A:P) where it decreased linearly as the amount of liquid supplemented increased. The liquid mixture of molasses and crude glycerol has the potential of improving animal performance due to increase in propionate production. Introduction The Southeastern region of the United States has several characteristics that make it unique when compared to other beef cattle regions. The states that represent the southeast and Gulf Coast regions account for 48% of all beef cows in the United States. In the current context of increasing feed input costs, the abundant forage production in the Southeastern U.S. provides an opportunity to decrease the cost of production, considering that feed is the largest cost in a cattle operation. However, the predominant forages in this region can be of limited nutritive and often not sufficient to support high levels of production. As a result, there are some critical periods during the year in which there is a need for supplementation with energy and/or protein in cow/calf operations in the Southeast (Hersom et al., 2011). Combined with the abundance of forages, another advantage of beef production in the Southeastern U.S. is the availability of several byproducts from diverse industries, which can have great nutritional value for cattle and can provide an excellent opportunity to correct nutritional imbalances Florida Beef Research Report

110 through strategic supplementation. The sugar industry is strong in the state of Florida and as a result, byproducts such as molasses have been fed to cattle for decades (Pate and Kunkle, 1989; Kunkle et al., 2000). Crude glycerin (or crude glycerol) is generally recognized as safe (GRAS) animal food ingredient and it can be produced as a coproduct of the soap making industry; however, more recently, the rapid expansion of the biodiesel industry has generated large amounts of crude glycerol as a byproduct, resulting in reduction of prices and increased concerns related to its disposal in the environment, which makes crude glycerol a potential high-energy feed source for cattle (Tan et al., 2013). The objective of this study was to evaluate the effects of different amounts of supplementation with a 50:50 mixture of molasses and crude glycerol on Tifton 85 bermudagrass hay intake and digestibility of nutrients in the total tract of beef heifers. Materials and Methods The study was initiated in October, 2013 and was conducted at the FEF located at the NFREC in Marianna, FL. Liquid supplement was provided by Westway Feed Products (New Orleans, LA). Eight ruminally cannulated Angus crossbred steers (712±93 lb BW) were used in a 4 4 duplicated Latin square design. In each of the four 28-d periods, d 0 to 14 were for adaptation to the diet, d 14 for collection of ruminal fluid, blood and ruminal ph, and d 21 to 28 for washout, when all the animals were consuming only hay. All procedures described were performed similarly for all four periods. Steers were randomly assigned to 1 of 4 treatments: 1. CTRL = no supplementation 2. SUP1 = 1 lb/d of the 50:50 molasses and crude glycerol mixture 3. SUP3 = 3 lb/d of the 50:50 molasses and crude glycerol mixture 4. SUP5 = 5 lb/d of the 50:50 molasses and crude glycerol mixture On d 0 steers were housed in individual pens at the FEF and had ad libitum access to water and Tifton 85 bermudagrass hay, which was chopped using a Tub Grinder (Haybuster, Jamestown, ND), placed in the feed bunk and sampled for nutrient analysis and further use in the in vitro experiment. The amount of liquid supplement corresponding to each treatment was weighed and offered daily to each animal in a plastic container inside the pen, separately from hay. Any unconsumed supplement was weighed and recorded for the first 7 d. By d 8, all steers were consuming the entire amount of liquid supplement daily, thus no orts recording was necessary. Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, Alberta, Canada) to record hay intake by weight change measured to the nearest gram. Ruminal fluid and blood were collected before liquid feeding (0 h) and every 3 h postfeeding for 24 h. Ruminal fluid was strained from a representative sample of digesta through four layers of cheesecloth and ph was immediately measured using a manual ph meter (Corning Pinnacle M530, Corning Inc., Corning, NY). A 10-mL sample was taken and 0.1 ml of a 20% H 2 SO 4 solution was added to stop fermentation. Ruminal fluid samples were stored frozen at -20 C for further analysis. Blood samples were collected from jugular venipuncture in 10-mL evacuated tubes containing sodium heparin, placed on ice following collection, and centrifuged for 15 min at 4,000 g at 4 C. After centrifugation, plasma was transferred Florida Beef Research Report

111 into polypropylene vials (12 mm 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at -20 C for further analysis. Data were analyzed as a duplicated 4 4 Latin square with repeated measures using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The model included the fixed effects of treatment, time, the treatment time interactions, square, period within square, and animal within square. Animal within period was the subject and the covariance structure used for all the parameters was unstructured, with the exception for total VFA, which was analyzed using compound symmetry. Unstructured and compound symmetry were the best covariance structures based upon the smallest Akaike Information Criterion (AIC) values. Orthogonal polynomial contrasts were conducted to determine the linear, quadratic, and cubic effects of supplementation level on animal performance or nutrient digestibility. Differences between treatment means were identified by Tukey s least squares means comparison and significance was declared at P<0.05 and tendencies considered when 0.05<P<0.10. Results Chemical composition of Tifton 85 bermudagrass hay and liquid mixture fed to heifers is presented in Table 1. Ruminal fermentation parameters and BUN are presented in Table 2. Concentrations of NH 3 -N and BUN were influenced by liquid supplementation, decreasing linearly (P<0.001) as the amount of supplementation increased. Average ruminal ph was also influenced by liquid supplementation and decreased linearly (P=0.03) as the amount of liquid supplement increased. A treatment time interaction (P=0.005) was observed for ruminal ph and is presented in Figure 2. The greatest drop in ruminal ph was observed at 3 h after supplementation for steers on SUP5 (from 6.81 at 0 h to 6.32 at 3 h postfeeding), followed by steers on SUP3 (from 6.81 at 0 h to 6.58). Ruminal VFA profile is presented in Table 3. Molar proportions of acetate (P<0.001) and branched chain VFA (BCVFA; P<0.001) linearly decreased whereas molar proportions of propionate (P<0.001), butyrate (P=0.007), and valerate (P=0.002) linearly increased as the amount of liquid being supplemented increased. Total VFA concentration was affected cubically (P=0.005) by liquid supplementation where it decreased from CTRL to SUP1 and SUP5, however, SUP3 was not different when compared to the others. There was an effect of liquid supplementation on A:P where it decreased linearly (P=0.004) as the amount of liquid supplemented increased which reflects the decrease in acetate and increase in propionate molar proportions. Liquid supplementation is clearly shifting VFA production in the rumen towards more propionate and butyrate production at the expense of acetate. Although ruminal ph was reduced with liquid supplementation in the current study, even with the greatest amount of supplementation (SUP5), ph never dropped below 6.0, which is the threshold known to affect fiber digestion. Thus, we conclude that providing the 50:50 liquid mixture of molasses and crude glycerol to beef steers consuming Tifton 85 bermudagrass hay might be beneficial to improve animal performance due to increase in propionate production Florida Beef Research Report

112 Table 1. Analyzed 1 chemical composition of Tifton 85 bermudagrass hay and liquid supplement fed to beef steers. Item Hay Liquid Supplement 2 Dry matter (DM), % Organic matter, % DM Crude protein, % DM Neutral detergent fiber, % DM Acid detergent fiber, % DM Total digestible nutrients, % DM Calcium, % DM Phosphorus, % DM Magnesium, % DM Potassium, % DM Sodium, % DM Sulfur, % DM Methanol, ppm 3 - <100 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 2 50:50 mixture (as-fed) of molasses:crude glycerol (Westway Feed Products, New Orleans, LA.) 3 Analyzed by SDK Laboratories (Hutchinson, KS). Table 2. Effects of supplementing increasing levels of a 50:50 mixture of molasses:crude glycerol on daily average ruminal fermentation parameters and blood urea nitrogen (BUN) of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment 1 Item 2 CTRL SUP1 SUP3 SUP5 SEM 3 Contrast 4 NH 3 -N, mm L BUN, mg/dl L Ruminal ph L 1 All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTR: no supplementation; SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture. 2 Ruminal fluid and blood samples were collected every 3 h for 24 h. 3 SE of treatment means, n = 8 steers/treatment. 4 Orthogonal polynomial contrast: L = Linear effect of liquid feed supplementation amounts, P Florida Beef Research Report

113 Table 3. Effects of supplementing increasing amounts of a 50:50 mixture of molasses:crude glycerol on ruminal volatile fatty acid (VFA) profile of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment 1 Item CTRL SUP1 SUP3 SUP5 SEM 2 Contrast 3 VFA, mol/100 mol Acetate L Propionate L Butyrate L BCVFA L Valerate L Caproate NS Total VFA, mm C A:P L 1 All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTR: no supplementation; SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture. 2 SE of treatment means, n = 8 steers/treatment 3 Orthogonal polynomial contrasts: L or C = Linear or cubic effect of liquid feed supplementation amounts, P 0.05; NS = not significant, P> BCVFA = Branched chain VFAs: isobutyrate + isovalerate + 2 methylbutyrate ph CTRL SUP1 SUP3 SUP Time postfeeding, h Figure 2. Effects of supplementing increasing amounts of a 50:50 mixture of molasses:crude glycerol on ruminal ph of steers fed Tifton 85 bermudagrass hay ad libitum. Treatment time interaction observed (P=0.005). All treatments contained Tifton 85 bermudagrass hay fed ad libitum. CTRL: no supplementation SUP1: 1 lb/d of a 50:50 molasses:crude glycerol mixture; SUP3: + 3 lb/d of a 50:50 molasses:crude glycerol mixture; SUP5: 5 lb/d of a 50:50 molasses:crude glycerol mixture (n=8 steers/treatment) Florida Beef Research Report

114 Florida Beef Research Report

115 Effects of Supplementation with a Mixture of Molasses and Crude Glycerol on Performance and Total Tract Digestibility of Beef Heifers Consuming Bermudagrass Hay F. M. Ciriaco, D. D. Henry, V. R. G. Mercadante, T. Schulmeister, M. Ruiz-Moreno, G. C. Lamb, N. DiLorenzo Synopsis Supplementation with energy and protein sources is crucial during critical periods of the year and molasses and crude glycerol combined in a liquid mixture have the potential of serving as an energy supplement for beef cattle consuming forage-based diets. Summary A study was conducted to evaluate the effects of molasses and crude glycerol in a 50:50 liquid mixture on performance and total tract digestibility of beef heifers consuming Tifton 85 bermudagrass hay. Heifers were provided Tifton 85 bermudagrass hay ad libitum and four increasing amounts of the 50:50 mixture were compared: 1) CTRL=0 lb/d; 2) SUP1=1 lb/d; 3) SUP3=3 lb/d; and 4) SUP5=5 lb/d. Twenty-four Angus crossbred heifers were housed at the North Florida Research and Education Center (NFREC) Feed Efficiency Facility (FEF) for 28 d and intake of hay was monitored using the GrowSafe system. The liquid supplement was weighed and offered daily to each animal. Total dry matter intake (DMI) increased linearly as the level of supplementation increased. Hay intake ranged from 1.36 (CTRL) to 1.23% (SUP5) of body weight (BW), and along with final BW was not affected by liquid supplementation. As the liquid supplementation amounts increased, there was a linear increase in average daily gain (ADG; 2.89, 3.02, 3.06, and 3.44 lb for 0, 1, 3, and 5 lb/d of 50:50 mixture, respectively), while feed to gain (F:G) was not affected. Apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) increased linearly, while crude protein (CP) total tract digestibility decreased linearly as the level of supplementation increased. Increased fiber digestion, along with energy supplementation, led to increased ADG in heifers consuming Tifton 85 bermudagrass hay. Introduction The Southeastern region of the United States has several characteristics that make it unique when compared with other beef cattle regions. The states that represent the southeast and Gulf Coast regions account for 48% of all beef cows in the United States. In the current context of increasing feed input costs, the abundant forage production in the Southeastern U.S. provides an opportunity to decrease the cost of production, considering that feed is the largest cost in a cattle operation. However the predominant forages in this region can be of limited nutritive and often not sufficient to support high levels of production. As a result, there are some critical periods during the year in which there is a need for supplementation with energy and/or protein in cow/calf operations in the Southeast (Hersom et al., 2011). Combined with the abundance of forages, another advantage of beef production in the Southeastern U.S. is the availability of several byproducts from diverse industries, which can have great nutritional value for cattle and can provide an excellent opportunity to correct nutritional imbalances through strategic supplementation Florida Beef Research Report

116 The sugar industry is strong in the state of Florida and as a result, byproducts such as molasses have been fed to cattle for decades (Pate and Kunkle, 1989; Kunkle et al., 2000). Crude glycerin (or crude glycerol) is generally recognized as safe (GRAS) animal food ingredient and it can be produced as a coproduct of the soap making industry; however, more recently, the rapid expansion of the biodiesel industry has generated large amounts of crude glycerol as a byproduct, resulting in reduction of prices and increased concerns related to its disposal in the environment, which makes crude glycerol a potential high-energy feed source for cattle (Tan et al., 2013). The objective of this study was to evaluate the effects of different amounts of supplementation with a 50:50 mixture of molasses and crude glycerol on Tifton 85 bermudagrass hay intake, animal performance, and digestibility of nutrients in the total tract of beef heifers. Materials and Methods The study was conducted in April 2013 at the FEF located at the NFREC in Marianna, FL. Liquid supplement was provided by Westway Feed Products (New Orleans, LA). A total of 24 Angus crossbred heifers (weighing average 838±68 lb) were used in the study in a generalized randomized block design. On d 0, heifers were weighed after 16-h feed withdrawal, stratified, and blocked by initial BW (2 blocks: lightest and heaviest), and randomly assigned to one of four treatments on as fed basis: 1. CTRL = no supplementation 2. SUP1 = 1 lb/d of the 50:50 molasses and crude glycerol mixture 3. SUP3 = 3 lb/d of the 50:50 molasses and crude glycerol mixture 4. SUP5 = 5 lb/d of the 50:50 molasses and crude glycerol mixture All heifers were housed in individual pens at the FEF for 28 d and had ad libitum access to water and Tifton 85 bermudagrass hay, which was ground and placed in the feed bunk. The amount of liquid supplement corresponding to each treatment was weighed and offered daily in a plastic container inside the pen to each individual animal. Any unconsumed amount of supplement was weighed and recorded for the first 7 d. By d 8, all heifers were consuming the entire amount of liquid supplement daily, thus no orts recording was needed. After the 14-d adaptation to diets and facility, heifers were weighed and collection of daily feed intake data started. Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, Alberta, Canada) to record intake by weight change measured to the nearest gram. Beginning on d 22 and d 23, feed (hay and liquid) and fecal samples were collected, respectively, for four consecutive d to determine apparent total tract digestibility of DM, OM, CP, NDF, and ADF. Feed samples were collected daily immediately after delivery of liquid supplement. Fecal samples were collected twice daily at 0800 h and 1600 h from the ground, inside the pen, right after the animal defecated. Feed and fecal samples were pooled within heifer and indigestible NDF (indf) was used as an internal indigestible marker. On d 28 heifers were weighed again after withholding feed for 16 h, for evaluation of performance during the entire period. Concentrations of indf in feed and feces were determined as described by Cole et al. (2011) with the following modification: in vitro incubations were conducted for 288 h instead of 96 h using the Daisy II incubator (Ankom Technology, Macedon, NY) to ensure complete digestion of potentially Florida Beef Research Report

117 digestible NDF in hay as reported by Krizsan and Huhtanen (2013). Data was analyzed as a generalized randomized block design using the MIXED Procedure of SAS (SAS Institute Inc., Cary, NC). Heifer was considered the experimental unit and the model included the fixed effects of treatment, and the random effect of block. Orthogonal polynomial contrasts were conducted to determine the linear, quadratic, and cubic effects of supplementation level on animal performance or nutrient digestibility. Significance will be determined at P 0.05 and tendencies were considered when 0.05<P Results After ad libitum hay intake was recorded, the liquid supplement represented 0%, 6%, 16%, and 26% of the diet daily DMI (CTRL, SUP1, SUP3, and SUP5, respectively). Chemical composition of Tifton 85 bermudagrass hay and liquid mixture fed to heifers is presented in Table 1. Animal performance is presented in Table 2. Final BW was not affected by liquid supplementation (P 0.10). Total DMI was influenced by liquid supplementation, increasing linearly (P=0.005) as the level of supplementation increased; however, liquid supplementation did not affect (P 0.10) hay DMI. Because hay DMI was not affected by treatment, total DMI was expected to increase as the liquid supplementation increased, considering that the heifers consumed the entire amount of liquid supplement in each treatment. There was a linear increase in ADG (P=0.03) as liquid supplementation amounts increased; however, liquid supplementation did not affect F:G (P 0.10). Nutrient intake and apparent total tract digestibility of nutrients of heifers are presented in Table 3. Liquid supplementation linearly decreased (P 0.05) CP, NDF, and ADF intake, whereas intake of DM and OM were increased linearly (P 0.05). Apparent total tract digestibility of DM, OM, NDF, and ADF increased linearly (P<0.001), whereas apparent total tract digestibility of CP decreased linearly (P=0.002) as the level of supplementation increased. Although it is difficult to separate the contribution of the added supplemental energy to the ADG response from that of the increased fiber digestion, it is likely that the latter contributed significantly considering that hay ranged from 74% (SUP5) to 94% (SUP1) of the total dietary DM consumed by the heifers. We conclude that a 50:50 mixture of molasses:crude glycerol provides a rapidly fermentable source of carbohydrates in the rumen, possibly stimulating microbial growth, consequently increasing fiber digestibility in the total tract. The increase in fiber digestibility, along with energy supplementation, led to an increase in animal ADG. Literature Cited Cole, N. A. et al Prof. Anim. Sci. 27: Hersom, M. et al Proceedings of the 22 nd Florida Ruminant Nutrition Symposium, p Kunkle, W. E. et al J. Anim. Sci. 77:1-11. Krizsan, S. J. and P. Huhtanen J. Dairy Sci. 96: Pate, F. M. and W. E. Kunkle Florida Agricultural Experiment Station. Circular S-365. Tan, H. W. et al Renew. Sustain. Energy Rev. 27: Florida Beef Research Report

118 Table 1. Analyzed 1 chemical composition of Tifton 85 bermudagrass hay and liquid supplement fed to beef heifers. Item Hay Liquid Supplement 2 Dry matter (DM), % Organic matter (OM), % DM Crude protein (CP), % DM Neutral detergent fiber (NDF), % DM Acid detergent fiber (ADF), % DM Total digestible nutrients (TDN), % DM Calcium, % DM Phosphorus, % DM Magnesium, % DM Potassium, % DM Sodium, % DM Sulfur, % DM Methanol 3, ppm - <100 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 2 50:50 mixture (as-fed) of molasses:crude glycerol (Westway Feed Products, New Orleans, LA.) 3 Analyzed by SDK Laboratories (Hutchinson, KS). Table 2. Effects of supplementing increasing amounts of a 50:50 mixture of molasses and crude glycerol on performance of beef heifers fed Tifton 85 bermudagrass hay ad libitum. Treatment 1 Item CTRL SUP1 SUP3 SUP5 SEM 2 Contrast 3 Initial BW, lb NS Final BW, lb NS ADG, lb L Total DMI, lb/d L Hay DMI, lb/d NS Hay DMI, % BW NS F:G NS 1 CTRL: Tifton 85 bermudagrass hay fed ad libitum; SUP1: Tifton 85 bermudagrass hay fed ad libitum + 1 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture; SUP3: Tifton 85 bermudagrass hay fed ad libitum + 3 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture; SUP5: Tifton 85 bermudagrass hay fed ad libitum + 5 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture. 2 Pooled standard error of treatment means, n = 6 heifers/treatment 3 Orthogonal contrast: L = Linear effect of liquid feed supplementation, P 0.05; NS, P Florida Beef Research Report

119 Table 3. Effects of supplementing increasing amounts of a 50:50 mixture of molasses and crude glycerol on nutrient intake and apparent total tract digestibility of nutrients of heifers fed bermudagrass hay ad libitum. Treatment 1 Item CTRL SUP1 SUP3 SUP5 SEM 2 Contrast 3 4-d Intake, lb/d DM L OM NS CP NS NDF L ADF L Digestibility, % DM L OM L CP L NDF L ADF L 1 CTRL: Tifton 85 bermudagrass hay fed ad libitum; SUP1: Tifton 85 bermudagrass hay fed ad libitum + 1 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture; SUP3: Tifton 85 bermudagrass hay fed ad libitum + 3 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture; SUP5: Tifton 85 bermudagrass hay fed ad libitum + 5 lb/d (asfed) of a 50:50 molasses:crude glycerol mixture. 2 Pooled standard error of treatment means, n = 6 heifers/treatment. 3 Orthogonal contrast: L = Linear effect of liquid feed supplementation, P 0.05; NS, P Florida Beef Research Report

120 Florida Beef Research Report

121 Effect of Frequency of Supplementation with Megalac-R on Non-esterified Fatty Acids and Blood Urea Nitrogen Concentration in Lactating Beef Cows M. E. Garcia-Ascolani 1, T. M. Schulmeister 1, M. Ruiz-Moreno 1, D. D. Henry 1, F. M Ciriaco 1, P. L P. Fontes 1, G. C. Lamb 1, N. M. Long 2, N. DiLorenzo 1 Synapsis Beef cows in early lactation, consuming similar overall quantities of an energy supplement, but supplemented 3, 5, or 7 days a week had similar concentrations of non-esterified fatty acid (NEFA) and blood urea nitrogen (BUN); therefore, indicating similar energy balance levels. Reducing the frequency of supplementation may help decrease the costs associated with feeding management such as labor and fuel, without negatively affecting animal performance. Summary During the spring of 2015, an experiment was conducted to determine the effects of supplementing a ruminally protected lipid (Megalac-R) either 3, 5, or 7 days per week on concentrations of serum nonesterified fatty acid (NEFA) and blood urea nitrogen (BUN) of beef cows in early lactation. For two weeks (Phase 1, adaptation period, d 0-14), eighteen Angus crossbred cows (first 90 d of lactation) were individually supplemented with 10 lb/wk (as is) of corn gluten feed (CGF) pellets, at three different frequencies: 3, 5, or 7 d/wk. During the last three days of the adaptation phase, blood samples were collected from each cow before supplementation (h 0), and 8 and 16 h after supplementation. On d 14 to d 34 (Phase 2) Megalac-R was added to the CGF supplement at a rate of 3.5 lb/wk (as is). On the final three days of Phase 2 blood samples were collected from each cow immediately before supplementation (h 0), and 8 and 14 h after supplementation. Serum NEFA and BUN concentration were analyzed. Concentrations of NEFA and BUN were similar among treatments. A treatment x sampling day interaction was observed for NEFA concentrations (P<0.001); however, within each sampling day, there were no differences among treatments, except on d 13, when cows receiving supplement 3 per week had reduced (P=0.03) concentrations compared to cows receiving supplement 5 per week. Concentrations of BUN were similar among treatments, but there was a difference in the concentrations among sampling days (P<0.001). Therefore, supplementing a ruminally protected lipid 3, 5, or 7 d/wk did not alter serum NEFA and BUN concentration in lactating beef cows, resulting in no significant differences in energetic balance of the cows, measured by these two parameters, remained similar among treatments. Supplementing lactating cows less than 7 days per week is a feasible option to reduce feeding costs without affecting animal performance. Introduction In North Florida, the breading season of cow/calf operations usually occurs between December and May, which also occurs during the dormant stage of the warm season forages. This situation makes it necessary to apply feeding strategies to provide sufficient energy and protein to the lactating beef cows, in order to assure their weight recovery to support a subsequent pregnancy. Offering a feed supplement may offset the negative energy balance that the cow is experiencing during lactation; however, it may also increase the costs associated with management and feeding of the herd. Heifers receiving a high protein supplement 1 per week did not show differences in performance when compared with heifers receiving supplement 3 per week (Mathis, 2003). In addition, the authors reported a decrease of 60% in associated feed transport and labor costs. However, when they reduced the frequency of supplementation of an energy supplement, weight gain and conception rate were reduced (Mathis et al., 2003). When the performance of mid-to late gestation beef cows supplemented with hay only, distillers grain plus solubles (DDGS) offered 7 d/wk or 3 d/wk, plus hay every day, and alternating supplements; 4 d/wk hay only, and DDGS every other day was evaluated (Klein et al., 2014), cows receiving supplement 4 d/wk with hay and DDGS every other day had the least dry matter intake and hay intake, but had similar total BW gain Florida Beef Research Report

122 and gain to feed ratio to the supplementation strategies that included feeding DDGS. Megalac-R (Church and Dwight Co., Princeton, NJ) is a rumen protected fat and a source of concentrated energy that has been reported to reduce the incidence of metabolic ailments during the transition period of dairy cows. Our objective was to measure non-esterified fatty acids (NEFA) and blood urea nitrogen (BUN) concentrations in blood serum, to determine differences in fat tissue mobilization and nitrogen utilization in lactating beef cows supplemented 3, 5, or 7 d/wk. Materials and Methods Eighteen early lactation beef cows (Average BW=1,100 ± 47 lb) in their first 90 d of lactation, were used at the UF - North Florida Research and Education Center (NFREC) Beef Unit in a completely randomized design study. The experiment was divided into two phases. Phase 1 (d 0-14) occurred when cows were individually supplemented corn gluten feed (CGF) pellets at a rate of 10 lb/wk (as is). The supplement was provided to the cows at 0700 h, at three different frequencies: 3 d/wk (F3), 5 d/wk (F5), or 7 d/wk (F7). Cows in F3 received 3.3 lb/d of CGF on Monday, Wednesday and Friday; cows in F5 received 2 lb/d of CGF from Monday to Friday, and cows in F7 received 1.4 lb/d daily. During the entire duration of the study, cows and calves had access to a ryegrass (Lolium multiflorum) pasture from where a sample was collected weekly and analyzed for nutritional value. During the last 3 days of Phase 1 (d 11-13), 10- ml blood samples were obtained via jugular venipuncture into vacuum tubes with no additives, before supplementation (0 h), 8 h and 16 h after supplementation. Phase 2 (d 14-34) had a similar supplementation system, with the only difference that Megalac-R (M-R) was added to the CGF pellets supplement at a rate of 3.5 lb/wk (as is). Cows in F3 received 1.2 lb/d M-R on Monday Wednesday and Friday, whereas cows in F5 received 0.7 lb/d M-R from Monday to Friday, and cows in F7 received 0.5 lb/d M-R daily. During the final 3 d of this period (d 32-34), blood samples were taken pre and post supplementation, in a similar fashion to Phase 1. For both phases, immediately after finishing blood collection, blood tubes were allowed to stand for 1 h at room temperature in the laboratory, and then were placed in the refrigerator for 24 h. After 24 h refrigeration, samples were centrifuged for 15 min at 4,000 g at 4 C. After centrifugation, serum was transferred into polypropylene vials (12 mm 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and stored at -20 C for further analysis. Serum samples were thawed at room temperature before analysis. Serum NEFA was determined using the acyl-coa synthetase, acyl-coa oxidase method (NEFA-HR, Wako Pure Chemical Industries, Richmond, VA). Blood urea nitrogen was determined using a modification to the urease and glutamate dehydrogenase method (Liquid Urea Nitrogen BUN, Pointe Scientific Inc., Canton, MI). Data were analyzed using the MIXED procedure of SAS, using cow as the experimental unit considering double repeated measures (day and hour postfeeding within day). The model included the fixed effects of treatment, day, hour postfeeding, and their interactions. Results Mean concentrations of NEFA and BUN, per frequency of supplementation, are summarized in Table 1. There was no difference in NEFA and BUN concentration among treatments (P>0.10). A treatment sampling day interaction occurred for NEFA concentration (P<0.001), as it is showed in Fig. 1. On d 13 (Sunday), which corresponds to the second consecutive day when only F7 cows were supplemented with CGF, NEFA concentrations of F3 cows were less than those in F5 (P=0.03). Perhaps the greater quantity of supplement received by cows in the F3 treatment on d 11 resulted in differences between the two treatments (i.e., 3.3 lb for F3 vs. 2 lb for F5). At the end of Phase 2, no differences between treatments were observed in blood NEFA concentrations in any of the days sampled (i.e., d 32-34). Concentrations of BUN did not differ (P=0.74) among treatments, and no treatment d or 3-way interactions were observed (P>0.10). However, concentrations of BUN differed across days of sampling. Concentrations of BUN were greater (P<0.05) during Phase 2 than Phase 1. In conclusion, reducing the Florida Beef Research Report

123 frequency of supplementation does not exert a negative effect on blood metabolites of lactating beef cows. Adding M-R to the supplement decreased blood NEFA, but increased concentrations of BUN. Literature cited Klein, S.I.et al J. Anim. Sci. 92: Mathis, C New Mexico State University Cooperative Extension Service. Circular 564. College of Agriculture and Home Economics Florida Beef Research Report

124 Table 1. Mean non-esterified fatty acid (NEFA) and blood urea nitrogen (BUN) concentration in the serum of beef lactating cows per frequency of supplementation. Frequency of supplementation 1 F3 F5 F7 SEM P-value NEFA, meq/l BUN, mg/dl F3 = 3 d/wk supplementation; F5 = 5 d/wk supplementation; F7 = 7 d/wk supplementation. Within a row, means without a common superscript differ (P<0.05) NEFA, meq/l * F3 F5 F Day of sampling Figure 1. Concentration of serum non-esterified fatty acids (NEFA) on d 11, 12, and 13 for lactating beef cows on a ryegrass pasture and different frequencies of supplementation. F3 = 3 d/wk; F5 = 5 d/wk; F7 = 7 d/wk. * F3 differs from F5 (P=0.03) Florida Beef Research Report

125 Effects of Prepartum Supplementation of a Rumen Fermentation Enhancer on Subsequent Beef Cow Performance D. D. Henry 1, F. M. Ciriaco 1, D. Demeterco 2, V. R. G. Mercadante 1, P. L. P. Fontes 1, T. M. Schulmeister 1, M. E. Garcia-Ascolani 1, N. Michael 3, E. Block 3, N. DiLorenzo 1 G. C. Lamb 1. Synopsis Added pounds at weaning is one of the most efficient strategies to increase profits for beef cattle producers. Reducing the dietary cation-anion difference (DCAD) of diets can potentially increase milk production and, thereby, calf weaning weight. Summary We determined the effects of a prepartum negative dietary cation-anion difference (DCAD) supplement on subsequent performance of beef cows. Forty-three multiparous cows (1,252±161 lb; average body weight (BW) ± standard deviation) were used in a completely randomized design. Prepartum cows were stratified by breed, body condition score (BCS), and the previous year s calving date and assigned to one of two treatments: control (CTRL; 3.75 lb/d of 50:50 corn gluten feed [CGF]:soybean meal [SBM] mixture, dry matter (DM) basis) and treatment (BCLR; 1.25 lb/d of an anion source and 2.5 lb/d of 50:50 CGF:SBM mixture, DM basis). Cows had ad libitum access to bermudagrass hay and water. Daily, cows were individually penned and received supplement (CTRL and BCLR were fed for 21.8±9.3 and 24±9.5 d, respectively) until calving. After calving, cows and calves were weighed within 12 h of parturition and blood samples were collected from the cow. Weekly, blood samples were collected from cows, and BW of cows and calves and BCS of cows were recorded. On d 28, 84, and 140 postpartum, milk yield was recorded and milk samples were analyzed to determine energy corrected milk (ECM). Calculated DCAD of CTRL was meq/lb DM whereas BCLR was meq/lb. Cow weight and calf weight was not affected by treatment. The average daily gain (ADG) of cows and calves, and BCS of cows were not different between treatments. Similarly, ECM did not differ between CTRL and BCLR. Supplemental DMI was greater for CTRL than for BCLR Concentrations of plasma calcium and β-hydroxybutyrate (BHBA) did not differ between treatments. Prepartum supplementation of 1.25 lb/d of an anion source to beef cows did not enhance subsequent cow or calf performance. Introduction Dairy producers regularly have to consider the calcium concentrations of cows during the transition period. Often, cows mobilize calcium from the bones to provide for milk production. Many researchers have considered decreasing the DCAD of the diets to improve calcium metabolism and, thereby, decrease incidence of calcium related diseases, such as milk fever (Rezac et al., 2014). Extensive research on DCAD in dairy cows has been performed; however, research is limited in beef cattle. In beef cattle, one of the most financially important aspects to consider is weaning weight. Milk yield of cows is highly related to weaning weight. In dairy cattle, research has shown that reducing the DCAD of diets can increase milk production (DeGroot et al., 2010). Therefore, there is a potential to increase weaning weight of calves, indirectly, by increasing milk yield of cows fed a negative DCAD diet. Diets with reduced DCAD often have negative effects on DMI (Spears et al., 2011). This leads to issues around the time of parturition whenever the cow is most likely to be in a negative energy balance Florida Beef Research Report

126 However, providing cattle with acidified co-products to reduce DCAD has shown promising results. When acidified co-products, such as Bio-Chlor, have been fed to dairy cattle, DMI was not altered and milk production was increased (DeGroot et al., 2010; Weich et al., 2013). The objective of this study was to evaluate the effects of a negative DCAD supplement (Bio-Chlor) on beef cow performance, milk production and subsequent calf performance. Materials and Methods The study was conducted at the North Florida Research and Education Center in Marianna, FL. The DCAD balancer (Bio-Chlor) was provided by Church & Dwight Co. (Princeton, NJ). A total of 43 primiparous cows (944±93; lb avg. BW±standard deviation) were used in a completely randomized design. All cows had ad libitum access to bahiagrass hay and water. Cows were stratified by breed, BCS and the previous year s calving date, and randomly assigned to treatments. Cows were gathered daily to receive one of two treatments: 1. CTRL=3.75 lb/d of a 50:50 corn gluten feed (CGF): soybean meal (SBM) mixture 2. BCLR=2.5 lb/d of a 50:50 CGF:SBM mixture and 1.25 lb/d of Bio-Chlor A timeline of events can be found in Figure 1. Briefly, cows were weighed and evaluated for BCS for assignment to treatments and cows were fed their respective treatments starting 30 d before their expected calving dates (CTRL and BCLR were fed for 21.8±9.3 and 24±9.5 d, respectively). On the day of parturition (d 0), cows and calves were weighed, cow BCS was recorded, and a blood sample was taken from each cow. Body weight of cows and calves, and blood samples from cows were collected weekly for 140 d post-parturition. Milk yield and samples of the cows was recorded on d 28, 84, and 140. Blood was analyzed, colorimetrically, for calcium and BHBA. Utilization of BHBA was utilized to evaluate energy balance between treatments. Progesterone was analyzed in the plasma to determine postpartum cycling status. Milk samples were analyzed for protein, fat (to determine ECM), and somatic cell count. Data were analyzed as a completely randomized design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with cow as the experimental unit. Repeated measures were used to analyze cow and calf BW, ECM, and BHBA. For these variables, the model included the fixed effects of treatment, day, and treatment day interactions. Cow was considered subject and the covariance structures were determined by the lowest Akaike Information Criterion. For all other variables, the model included the fixed effect of treatment. Significance was declared at P Results Supplemental DMI was greater (P<0.001) for CTRL (3.53±0.068 lb/d) than for BCLR (2.71±0.073 lb/d). There was no treatment day interaction (P=0.39) on cow BW throughout the experiment; however, an effect of treatment (P=0.02) was observed, with BCLR weighing less than CTRL (Figure 2) cows. No interaction (P=0.59) nor treatment effect (P=0.90) occurred for calf BW (Figure 3). Average daily gain of cows (0.22±0.095 lb) and calves (2.21±0.088 lb) were not different between treatments (P>0.05). Body condition score of cows (4.97±0.05; 1 to 9 scale) was not affected by treatment (P=0.89). Concentrations of plasma Ca (10.67±0.51 mg dl -1 ; Table 1) and BHBA (173.0±9.18 mmol L -1 ; Figure 4) did not differ (P>0.05) between treatments. Similarly, ECM did not differ between CTRL and BCLR (P>0.05; Figure 5). There was no effect (P=0.54) of treatment on ECM. Prepartum supplementation of Florida Beef Research Report

127 1.25 lb/d of an anion source to beef cows did not enhance subsequent cow or calf performance. In general, reduction of DCAD in the diets of dairy cattle increases milk production and prevents disorders such as milk fever. The results observed in this study do not imply the same implications in beef cattle. When beef cows were provided 1.25 lb/d of an anion source (Bio-Chlor), no effects on milk production or subsequent calf production were evident. Literature Cited DeGroot, M. A., et al J. Dairy Sci. 93: Rezac, D. J., et al J. Anim. Sci. 92: Spears, J. W., et al J. Anim. Sci. 89: Weich, W., J. Dairy Sci. 96: Florida Beef Research Report

128 Figure 1. Timeline of events. BW, lb Day CTRL BCLR Figure 2. Effect of Bio-Chlor on cow body weight. Treatment, P=0.02; day, P<0.0001; Treatment day, P= Florida Beef Research Report

129 BW, lb Day CTRL BCLR Figure 3. Effect of Bio-Chlor on calf body weight. Treatment, P=0.90; Day, P<0.0001; Treatment day, P= BHBA, mmol/l CTRL BCLR 50 0 d 7 d 14 Figure 4. Effect of Bio-Chlor on plasma beta-hydroxybutyrate (BHBA) concentration. Treatment, P=0.29; Treatment day, P= Florida Beef Research Report

130 ECM, lb CTRL BCLR Day Figure 5. Effects of Bio-Chlor on Energy corrected milk (ECM). Treatment, P=0.54; Day, P<0.0001; Treatment day, P=0.55. Table 1. Effect of Bio-Chlor on DCAD 1 and plasma calcium concentration of beef cows Treatment 2 Item CTRL BCLR SEM 3 P-value DCAD Plasma calcium, mg/dl DCAD=Dietary cation-anion difference. 2 CTRL=3.75 lb/d of a 50:50 corn gluten feed (CGF): Soybean meal (SBM) mixture; BCLR=2.5 lb/d of a 50:50 CGF:SBM mixture and 1.25 lb/d of Bio-Chlor. 3 Standard error of the mean Florida Beef Research Report

131 Effects of Pre- and Post-Breeding Supplementation of a Ruminally Protected Lipid on Subsequent Beef Cow Performance D. D. Henry 1, F. M. Ciriaco 1, D. Demeterco 2, V. R. G. Mercadante 1, P. L. P. Fontes 1, T. M. Schulmeister 1, M. E. Garcia-Ascolani 1, N. Michael 3, E. Block 3, N. DiLorenzo 1, G. C. Lamb 1. Synopsis To enhance production of beef it is important that beef cows have a calf every 365 d. It has been hypothesized that providing ruminally protected lipids could improve cow reproductive performance. Summary We determined the effects of supplementation of a ruminally protected lipid (on subsequent beef cow reproductive performance. Sixty primiparous cows [944±93lb; average body weight (BW) ± standard deviation] were used in a completely randomized design. Cows were stratified by breed, body condition score (BCS) and the previous year s calving date, and assigned to one of two treatments: control (CTRL; 3.0 lb/d of corn gluten feed) and treatment (MLAC; 3.0 lb/d of corn gluten feed and 0.5 lb/d of Megalac- R). Cows grazed a mixed winter forage pasture of triticale and ryegrass and had ad libitum access to water. Supplementation of CTRL and MLAC occurred 30 d prior to artificial insemination (AI) until 7 d post-ai. Daily, cows were individually penned and received their respective supplementation. Cow BW and BCS were recorded 35 and 28 d prior to AI and 35 and 60 d after AI. At initiation of the breeding season cows were exposed to the 7-d CO-Synch+CIDR ovulation synchronization protocol. Pregnancy was diagnosed on d 30, 60, and 90 after AI. Cows pregnant to AI were monitored until calving to determine length of gestation and calf birth weight. Treatment did not affect mean cow BW (1,083±6 lb). There was no effect of treatment on pregnancy rates to AI (53±0.5%) or at 90 d after AI (94.5±0.5%). Length of gestation was greater for MLAC (285±1.3 d) than for CTRL (281±1.1 d). No effect of treatment was observed for calf birth weight (75±7lb). Supplementation of 0.5 lb/d of a ruminally protected lipid to primiparous cows did not enhance subsequent cow reproductive performance. Introduction The dairy industry has used added fat to diets of lactating cows to improve milk production. In recent years, protected long-chain fatty acids have been utilized along with high-forage diets to provide added energy without negatively affecting fiber digestion, and consequently milk fat. However, there has not been extensive feeding of protected fats to beef cows. Researchers and producers have known for many decades the most important aspect of cow-calf enterprises is producing a calf every 365 d. It may be possible to improve the efficiency of beef cow reproduction by providing ruminally protected fats. Fats can enhance reproduction performance (Lopes et al., 2011) by increasing circulating progesterone concentrations (Lopes et al., 2009) in cattle. Furthermore, researchers have reported that protected fats, such as those found in Megalac-R (omega-3 and -6 fatty acids; Church & Dwight Co., Princeton, NJ), do not inhibit ruminal fiber digestion in beef cattle (Cooke et al., 2011). When provided to feeder cattle, protected fats decreased negative effects of transportation (Araujo et al., 2010) and increased performance and carcass quality (Cooke et al., 2011). Protected fats are more expensive than other energy sources and their inclusion in the diet of a beef cow would only be justified if supplementation increased performance beyond the simple increase in energy Florida Beef Research Report

132 Therefore, the objective of this study was to evaluate the effects of supplementation of a ruminally protected fat source on subsequent beef cow reproductive performance. Materials and Methods The study was conducted at the North Florida Research and Education Center in Marianna, FL. The protected fat (Megalac-R) was provided by Church & Dwight Co. (Princeton, NJ). A total of 60 primiparous cows [944±93lb; average BW±standard deviation] were used in a completely randomized design. All cows were grazing a mixed winter forage pasture of triticale (X triticosercale wittmack) and ryegrass (Lolium multiflorum) and had ad libitum access to water. Cows were stratified by breed, BCS and the previous year s calving date, and randomly assigned to treatments. Cows were gathered daily to receive one of two treatments: 1. CTRL = 3.0 lb/d of corn gluten feed 2. MLAC = 3.0 lb/d of corn gluten feed and 0.5 lb/d of Megalac-R A timeline of events can be found in Figure 1. Briefly, 31 d prior to fixed-time AI, BW, BCS and blood samples were collected from all cows to provide data for stratification. From 21 d prior to breed until 7 d post breeding, cows were supplemented with their respective treatments. Determination of BW, BCS and pregnancy status was recorded 30, 60 and 90 d post-ai. Blood was analyzed for progesterone to determine estrous in cows. Fetal measurements were taken on d 30 post-ai. Cows were followed until the 2015 calving season to determine gestation length and calf birth weight. Data were analyzed as a completely randomized design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with cow as the experimental unit. Repeated measures were used to analyze cow BW and BCS. For these variables, the model included the fixed effects of treatment, day and treatment day interactions. Cow was considered subject and the covariance structures were determined by the lowest Akaike Information Criterion. For all other variables, the model included the fixed effects of treatment. Significance was declared at P Results There was no effect (P=0.13) of treatment on pregnancy rates to AI (53±0.5%; Table 1). Final pregnancy rate at d 90 (94.5±0.5%) did not differ (P=0.56) between treatments (Table 1). Days pregnant at final pregnancy diagnosis did not differ between CTRL and MLAC (89.7 vs d; P=0.13; Table 1). Length of gestation was greater (P=0.02) for MLAC (285±1.3 d) than for CTRL (281±1.1 d; Table 1). No difference (P=0.26) was observed for fetal measurements at d 30 after AI between treatments (Table 1). No effect of treatment was observed for calf birth weight (75±6.6 lb; P>0.05). Treatment did not affect (P>0.05) mean cow BW (1,083±6.4 lb; Table 2). Similarly, BCS of cows was not different (P=0.42) for CTRL vs. MLAC (Table 2). Providing primiparous cows with 0.5 lb/d around the time of fixed time AI did improve reproductive performance. Further research is needed to determine the effects of supplementing protected fats during gestation to evaluate subsequent calf performance Florida Beef Research Report

133 Literature Cited Araujo, D. B., et al J. Anim. Sci. 88: Cooke, R. F., et al J. Anim. Sci. 89: Lopes, C. N., et al J. Anim. Sci. 89: Lopes, C. N., et al J. Anim. Sci. 87: Florida Beef Research Report

134 Figure 1. Timeline of events. Table 1. Effect of protected fat supplementation on reproductive performance of primiparous cows Treatment 1 Item CTRL MLAC SEM 4 P-value Pregnancy rate, % 30 d d Days pregnant at d Gestation length, d Fetus crown-rump length at d 35, mm Calf birth weight, lb CTRL=3.0 lb/d of corn gluten feed; MLAC=3.0 lb/d of corn gluten feed and 0.5 lb/d of Megalac-R. 2 Pregnant to artificial insemination. 3 Final pregnancy rate to artificial insemination and clean-up bulls. 4 Standard error of the means; 30 cows per treatment Florida Beef Research Report

135 Table 2. Effect of protected fat supplementation on body weight (BW) and body condition score (BCS) of primiparous cows Treatments 1 and day 2 CTRL MLAC P-value Item d -28 d 35 d 60 d -28 d 35 d 60 SEM 3 TRT TRT DAY Cow BW, lb 917 1,061 1, ,087 1, Cow BCS CTRL=3.0 lb/d of corn gluten feed; MLAC=3.0 lb/d of corn gluten feed and 0.5 lb/d of Megalac-R 2 Day of study in relation to fixed time artificial insemination. 3 Standard error of the mean, n=30 cows per treatment Florida Beef Research Report

136 Florida Beef Research Report

137 Effect of Rate of Inclusion of Fermenten in a Backgrounding Diet on Performance and Carcass Characteristics in Growing Angus Crossbred Steers M. E. Garcia-Ascolani 1, T. M. Schulmeister 1, M. Ruiz-Moreno 1, D. D. Henry 1, F. M Ciriaco 1, G. Medeiros 1, G. C. Lamb 1, N. M. Long 2, N. DiLorenzo 1 Synopsis Inclusion of a commercial protein supplement, Fermenten, in up to 4% of the diet DM did not improve performance in growing Angus crossbred steers fed a backgrounding diet. Summary Fermenten is a commercial protein supplement made with dried condensed corn fermentation solubles and processed grain by-products. Because Fermenten provides amino acids, peptides, and non-protein nitrogen, it has been hypothesized that could improve animal performance in growing cattle with high protein requirements. During the summer of 2015, an experiment was conducted at the University of Florida (UF) North Florida Research and Education Center (NFREC), in Marianna, to determine the effects of including Fermenten in a typical backgrounding diet (59% concentrate), on the performance and carcass characteristics of 81 growing Angus crossbred steers. The experiment included a 14-d adaptation period and a 56-d collection period, during which individual intake data was recorded using the Grow Safe System. Fermenten was included at 0, 2 or 4% of the diet dry matter (DM) in a total mixed ration (TMR). Diets were formulated to contain similar degradable intake protein (DIP) concentrations. Every 14 d, growth performance was assessed, and carcass ultrasound was performed every 28 d to measure ribeye area and fat depth at the 12 th rib. Steers receiving 4% Fermenten had decreased body weight (BW) and average daily gain (ADG) when compared with diets containing 0 and 2% Fermenten. The same pattern was observed for dry matter intake (DMI), and gain to feed ratio (G:F). However, when DMI was expressed as a percentage of BW, no differences were observed among treatments. Including up to 4% of Fermenten in a backgrounding diet DM did not improve animal performance or carcass characteristics in growing beef steers. Introduction Protein supplementation is critical during the growing phase of beef cattle, because protein accretion is needed to form muscle and to enlarge organs, such as those in the gastrointestinal tract. When feeding ruminants, protein supplementation must be directed not only to meet the animal needs, but also the nitrogen requirements of the rumen microbes (Dryden, 2008). Ruminal microorganisms are able to synthetize their own proteins and amino acids if nitrogen, a rapidly available source of energy and carbon skeletons are accessible. These proteins synthesized in the rumen are named microbial crude protein (MCP) and greatly contribute to the protein supply of the animal. The efficiency of the MCP synthesis relies on the inclusion of adequate amounts of degradable intake protein (DIP) in the diet, which is the fraction of protein readily available to ruminal microorganisms. Fermenten (Church and Dwight Co., Princeton, NJ) is a feed supplement which provides amino acids, peptides, and non-protein nitrogen and has been hypothesized to increase the microbial population, therefore enhancing ruminal fermentation. Fermenten has been included to dairy diets with satisfactory results where it enhanced growth rate in heifers reaching calving weight and size earlier and entering the milk herd earlier (Arm & Hammer Animal Nutrition, 2011). The objective of this study was to determine the effects of Fermenten inclusion in a backgrounding diet, on growth performance, and carcass characteristics in Angus crossbred steers Florida Beef Research Report

138 Materials and Methods Eighty one Angus crossbred steers were used at the UF NFREC Feed Efficiency Facility in a generalized randomized block design. Body weight at weaning was used as the block factor (light steers BW=335±2.1 lb; medium steers BW 375±2.2 lb; heavy steers BW=435±4.8 lb). Treatments consisted of different inclusion rates of Fermenten in a typical backgrounding diet. Inclusion rates of Fermenten were 0, 2, and 4% of the diet DM. Diets were formulated to be similar in DIP and their nutritional value is shown in Table 1. Steers were assigned to 9 pens of 9 steers each and had ad libitum access to water and feed. The experiment included an adaptation period of 14 d, to adjust steers to pens and feeding system, followed by 56 d of a data collection period. On d -1 and 0, unshrunk BW of the steers was recorded and the average was used as the initial BW. Carcass ultrasound was performed to measure ribeye area and fat depth at the 12 th rib of the right flank. Unshrunk BW was recorded every 14 d and carcass ultrasound was performed every 28 d. Final BW was recorded as the average of the unshrunk weights on d 55 and d 56. Individual feed intake data was collected by the GrowSafe system. Feed samples were collected weekly, dried at 55 C for 48 h, ground to pass a 2 mm screen, composited by treatment, and analyzed for nutritional value. Data were analyzed using the MIXED procedure of SAS, with steer as the experimental unit. The model included the random effect of steer, and fixed effect of treatment, block, and pen. Results Values of mean BW at d 56, ADG from d 0 to d 56, DMI, DMI as a percentage of the BW and G:F are shown in Table 2. The inclusion of 4% of Fermenten negatively affected the performance of the steers. Steers fed 4% of Fermenten had a decrease (P<0.05) in BW of 6% at d 56, when compared to the means of 0 and 2% Fermenten inclusion. Similarly, a decrease of 23% in ADG was observed (P<0.05) when comparing 4% Fermenten inclusion with 0 and 2% inclusion. The results of decreased BW and ADG are largely explained by the decrease in DMI observed in steers consuming the 4% Fermenten inclusion diet. Steers fed the 4% Fermenten inclusion consumed 1.5 lb of DM less (P<0.05) than those consuming 0 or 2% Fermenten (no difference between them, P=0.86). Despite the decreased intake, G:F was decreased (P<0.05) in steers fed 4% Fermenten when compared with the other two treatments. No effects of treatment (P>0.10) were observed on ribeye area growth or fat depth at 12 th rib. In conclusion, including Fermenten at 4% of the diet DM in growing Angus crossbred steers fed a backgrounding diet had a negative effect on feed intake and resulting animal performance. Literature cited Arm & Hammer Animal Nutrition Case study: New York Dairy Feeding FERMENTEN. Dryden, G. McL Animal Nutrition Science. Cambridge University Press, UK Florida Beef Research Report

139 Table 1. Nutritional composition of backgrounding diets including increasing levels of Fermenten. Inclusion of Fermenten, % of diet DM Item 0% 2% 4% DM, % CP, % DIP 1, % ADF 2, % andf 3, % TDN 4, % NEm 5, Mcal/lb NEg 6, Mcal/lb Calcium, % Phosphorus, % Magnesium, % Potassium, % Sodium, % Iron, ppm Zinc, ppm Copper, ppm Manganese, ppm Molybdenum, ppm Calculated using book values for degradable intake protein (DIP). 2 Acid detergent fiber. 3 Neutral detergent fiber (analyzed using α-amylase). 4 Total digestible nutrients. 5 Net energy of maintenance. 6 Net energy of gain Florida Beef Research Report

140 Table 2. Performance of Angus crossbred steers fed a backgrounding diet with different inclusion rates of Fermenten. Inclusion of Fermenten, % of diet DM Parameter 0% 2% 4% SEM P-value BW on d 0, lb BW on d 56, lb 524 a 532 a 498 b ADG, lb/d 1.94 a 1.97 a 1.50 b DMI, lb/d 14.0 a 13.9 a 12.4 b DMI as percentage of body weight, % G:F a a b Ribeye area 1 on d 0, inches >0.05 Ribeye area 1 on d 28, inches >0.05 Ribeye area 1 on d 56, inches >0.05 Fat thickness 1 on d 0, inches >0.05 Fat thickness 1 on d 28, inches >0.05 Fat thickness 1 on d 56, inches >0.05 a,b Within a row, means with a different superscript differ (P<0.05). 1 Treatment day interaction, P<0.05. No effect of treatment (P>0.05) within measurement day Florida Beef Research Report

141 Evaluation of Brassica Carinata as a Protein Supplement for Growing Beef Heifers T. M. Schulmeister, M. Ruiz-Moreno, J. Benitez, M. Garcia-Ascolani, F. M. Ciriaco, D. D. Henry, G. C. Lamb, J. C. B. Dubeux, N. DiLorenzo Synopsis Brassica carinata is a new oilseed crop in Florida with the potential of producing high-quality biodiesel for use as a biojet fuel. A high-protein meal is obtained as a byproduct of oil extraction; however, this meal has not been tested as a potential supplement for growing beef cattle. Summary An experiment was conducted from January to April of 2015, to determine the effects of the B. carinata meal on performance, time to attainment of puberty, and blood profile of growing beef heifers consuming bahiagrass hay. Thirty-two Angus crossbred heifers (597±93 lb of initial body weight (BW)), were blocked by initial BW and randomly allocated to 10 pens. All heifers had ad libitum access to water and bahiagrass hay (Paspalum notatum). Pelleted B. carinata meal was provided daily to treatment pens. Body weight and blood samples were collected every 7 d for a period of 70 d, before the daily supplementation. Plasma was collected for the analyses of progesterone (P4), blood urea nitrogen (BUN), glucose, and acute phase proteins. Based on preliminary results from yr 1 of the study, supplementing B. carinata meal improved (P=0.03) average daily gain (ADG) in heifers consuming bahiagrass hay. Time to attainment of puberty in heifers, as evidenced by concentrations of P4, was not affected (P=0.36) by the inclusion of B. carinata meal at 0.3% of initial BW. The experiment will be repeated in January Introduction The Southeastern and Gulf Coast regions account for 48% of all beef cows in the U.S. Beef heifer development is a highly profitable market in Florida, in which there is typically a 30% percent replacement of heifers annually, and although there is an abundant production of forage in the Southeast, feed is the largest cost in a cattle operation. With the most common forages in this region being of limited nutritive value and often not sufficient to support high levels of production, there are some critical periods during the year in which there is a need for supplementation with protein in cattle operations in the Southeast (Hersom et al., 2011). Combined with the abundance of forages, another advantage of beef production in the Southeastern U.S. is the availability of several byproducts from diverse industries, which can have great nutritional value for cattle and can therefore provide an opportunity to correct nutritional imbalances through supplementation. Brassica carinata is a new oilseed crop in Florida with the potential of producing high-quality biodiesel for use as a biojet fuel. A high-protein meal (approximately 37-43% crude protein (CP)) is obtained as a byproduct of oil extraction, however, this meal has not been tested as a potential supplement for growing beef cattle. Brassica carinata has been grown commercially for several years in Canada as a summer crop, however, cultivation is currently underway in Florida as the climate is excellent due to the many intrinsic characteristics of B. carinata, ranging from drought tolerance and resistance to extreme changes in temperature, as well as the ability to be planted on fallow lands during the winter to prevent erosion and depletion of essential nutrients from Florida Beef Research Report

142 the soil (Bliss et al., 2014). Feeding growing heifers in the winter is a substantial cost to the producer, however appropriate nutrition is vital to both the heifer s health and reproductive status as nutritional requirements are greater at this stage (Kunkle et al., 2001). Considering the poor quality of hay typically available in the winter in Florida, B. carinata meal could be a viable source of protein supplementation to meet the nutritional requirements of growing heifers and provide economic benefit to the producer as well. The objective of this study was to evaluate the effect of B. carinata meal as a protein supplement on animal performance and time to attainment of puberty in growing beef heifers. Materials and Methods The present study was conducted from January to April 2015 at the North Florida Research and Education Center (NFREC) in Marianna, FL. Pelleted B. carinata meal was provided by Agrisoma Biosciences, Inc. (Gatineau, Quebec). Thirty-two Angus crossbred heifers were used in the study in a generalized randomized block design. On d -1 and 0, heifers were weighed to obtain a two-day average weight, stratified and blocked by initial BW (2 blocks: lightest and heaviest), and randomly assigned to either BCM (0.3% of live BW of B. carinata meal pellets) or CTL (0.0% supplementation of live BW, bahiagrass hay only). All heifers were randomly allocated to one of ten pens in which they had ad libitum access to water and bahiagrass hay. The protein supplement was provided daily, in the morning, to the heifers in feed bunks. Refusals were weighed and recorded for the first 4 d, however by d 5 all heifers were consuming the entire amount of meal supplemented. Heifers were weighed and bled every 7 d for 70 d. Blood was collected, centrifuged, and plasma was stored at -20 C for further analyses. On d 69 and 70 heifers were weighed again for a 2-d average weight. Plasma samples were analyzed for concentrations of P4 using the DPC Immulite 1000 chemiluminescent immunoassay system and results were analyzed with SAS using the LIFETEST procedure to determine the effect of treatment on time of attainment of puberty. Data were analyzed as a generalized randomized block design using the MIXED Procedure of SAS (SAS Institute Inc., Cary, NC). Pen was considered the experimental unit and the model included the fixed effects of treatment, and the random effect of block. Significance was determined at P 0.05 and tendencies were considered when 0.05<P Results Chemical composition of bahiagrass hay and protein supplement fed to heifers is presented in Table 1. Time to attainment of puberty was not affected (P=0.36) in heifers receiving BCM, compared with the CTL, as presented in Figure 1. Average daily grain for yr 1 was significantly greater (P=0.03) for the BCM, compared with the CTL, as presented in Figure 2. Current results indicate that feeding B. carinata meal as a protein supplement at 0.3% of BW/d is a viable option for increasing ADG of growing beef heifers Florida Beef Research Report

143 Acknowledgements The authors would like to thank the staff and interns at the NFREC Marianna, for all of their assistance with this project. Literature Cited Bliss, C. M. et al University of Florida, IFAS, (EDIS) Publication SS-AGR-284. Cole, N. A. et al Prof. Anim. Sci. 27: Hersom, M. et al Proceedings of the 22 nd Florida Ruminant Nutrition Symposium, p Kunkle, W. E. et al J. Anim. Sci. 77:1-11. Kunkle, W. E. et al University of Florida, IFAS (EDIS) Publication AN117. Krizsan, S. J. and P. Huhtanen J. Dairy Sci. 96: Florida Beef Research Report

144 Table 1. Chemical composition of bahiagrass hay and B. carinata meal (BCM) fed to beef heifers 1 Item Hay BCM 2 DM 3, % CP, % DM NDF, % DM ADF 4, % DM TDN 5, % DM Sulfur, % DM Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY) 2 BCM, 0.3% of BW of B. carinata meal (Agrisoma Biosciences Inc., Gatineau, Quebec) 3 DM, Dry matter 4 Crude Protein 5 Acid Detergent Fiber 6 ADF, Acid detergent fiber 7 TDN, Total digestible nutrients BW, lb Heifer body weight over 70 days Days of experiment % of BW CTL = Attainment of puberty (mean = d 56) Treatment effect for time to attainment of puberty, P = 0.36 Figure 1. Evolution of body weight (BW) and time to attainment of puberty in heifers fed bahiagrass hay as libitum and supplemented with B. carinata meal at 0.3% of BW Florida Beef Research Report

145 1.4 ADG from day 0-70 ADG, lb/d % of BW CTL Treatment Figure 2. Effect of supplementing B. carinata meal at 0.3% of body weight (BW) on performance of beef heifers fed bahiagrass hay ad libitum Florida Beef Research Report

146 Florida Beef Research Report

147 Effects of Chitosan on Ruminal Fermentation and In Situ Nutrient Degradability D. D. Henry, F. M. Ciriaco, M. E. Garcia-Ascolani, V. R. G. Mercadante, T. M. Schulmeister, P. L. P. Fontes, G. C. Lamb, and N. DiLorenzo Synopsis Global impact of cattle production is a growing concern for policy makers and the general public and chitosan has the potential of reducing the carbon footprint of beef production by enhancing energetics of beef cattle consuming high-roughage diets. Summary A study was conducted to evaluate the effects of chitosan on in situ nutrient degradability and ruminal fermentation of beef steers consuming bermudagrass hay. Eight ruminally-cannulated steers were provided Tifton-85 bermudagrass hay ad libitum while housed in the University of Florida Feed Efficiency Facility at the North Florida Research and Education Center (NFREC) in Marianna for two 21-d periods. Steers were placed in a cross-over design where they either received only bermudagrass hay (CTRL) or 80 g/d of chitosan dosed directly into the rumen via cannula (CHIT). In situ ruminal degradability of dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was not affected by treatment (P>0.05). There was a treatment time-point interaction for ruminal molar proportions of acetate and propionate, where propionate molar proportions were greater for CHIT at 18, 21, and 24 h compared to CTRL. Similarly, the acetate to propionate ratio (A:P) was affected by an interaction of treatment time-point and reduced for CHIT as compared to CTRL at 18, 21, and 24 h. Chitosan did not have effects on in situ degradability of nutrients; however, energetics of fermentation were improved when steers received 80 g/d of chitosan along with bermudagrass hay. Introduction American agriculture produces approximately 6% of total U.S. greenhouse gas emissions (EPA, 2012). Livestock production accounts for 3.2% of the approximately 430 teragrams (Tg) of carbon dioxide equivalents (CO 2eq) produced by agriculture in the US each year. Enteric methane (CH 4) production is responsible for nearly 55% of livestock s contribution to greenhouse gas emissions (Pitesky et al., 2009). A strategy to reduce the carbon footprint of animal agriculture is to decrease the emission intensity of animals. Emission intensity is defined as the amount of greenhouse gas (i.e., CH 4) produced per unit of product (i.e., beef, milk, wool, etc.). Therefore, efficiency of beef production is inversely related to the carbon footprint. Not only is it imperative to the environment to increase efficiency of beef production, it is also financially beneficial for producers. In the southeast U.S., especially FL, beef producers rely on readily available, yet low-quality forages, such as bahiagrass. The single greatest cost that producers incur are feed costs, accounting for nearly 55% of total costs. This becomes important when considering that 70% of energy requirements of cattle are used prior to weaning (Shike, 2013). Therefore, by increasing efficiency of cattle by increasing digestibility of diets, producers can potentially alter their profit margins. Chitosan (N-acetyl-D-glucosamine polymer) is a natural biopolymer formed from the deacetylation of Florida Beef Research Report

148 chitin. Chitin, the second most abundant organic compound on earth, can be found in the cell walls of lower plants (e.g., fungi) and the exoskeletons of some arthropods and crustaceans (e.g., crab and shrimp). Chitosan has been studied for various applications in food preservation and medicine due to its antimicrobial actions (Cuero, 1999; Shahidi et al., 1999). In the U.S., chitosan has been deemed generally recognized as safe (GRAS), which ultimately allows for chitosan to be used as an alternative to antibiotics. Chitosan should not be considered a single compound, but rather a series of compounds with differing levels of deacetylation and other physic-chemical characteristics (Goiri et al., 2009). Chitosan, as an in vivo CH 4 inhibitor, is a novel product for ruminants; however, there have been studies with monogastrics (i.e., poultry and swine) showing that it can alter protein fermentation in the lower gastrointestinal tract (O Shea et al., 2011; Han et al., 2013). The objective of this study was to determine the effects of chitosan on in situ ruminal degradation of nutrients and ruminal fermentation. Materials and Methods The study was conducted at the Feed Efficiency Facility located at the NFREC in Marianna, FL. Chitosan was provided by PharmaNutrients Inc. (Lake Forest, IL). Eight crossbred, ruminally-cannulated steers (844±86 lb; avg body weight ± standard deviation) were used in a cross-over design. Steers were treated with either only bermudagrass hay (CTRL) or 80 g/d of chitosan dosed directly into the rumen via cannula (CHIT). Steers were monitored for two 21-d periods and had ad libitum access to water and Tifton-85 bermudagrass hay (Table 1). After a 14-d adaptation to treatments and facility, steers were subjected to a 24-h collection of ruminal fluid where samples were collected every 3 h to evaluate ruminal ph, volatile fatty acid (VFA) profile, and ammonia-nitrogen (NH 3-N). On d 15, nylon in situ bags, containing 5 g (as is) of the same bermudagrass hay the steers were consuming, were placed in the rumen to determine in situ degradability of DM, OM, CP, NDF, and ADF. On d 16, 17, and 18, in situ bags were removed to determine degradability at 24, 48, and 72 h. On d 18 steers were placed on a washout diet of bermudagrass hay only until d 21 before the next period began. Data were analyzed as a cross-over design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with steer as the experimental unit. The model for in situ degradability of nutrients included the fixed effects of treatment, period and order, and the random effect of steer(order). To evaluate ruminal fermentation parameters, repeated measures were used with steer(period) as the subject and the model included the fixed effects of treatment, time-point, treatment time-point interactions and order. Covariance structure was determined by fit statistics using the lowest Akaike Information Criterion. Significance was declared at P Results No effects of CHIT were observed for ruminal degradability of DM at any hour of incubation (P>0.10; data not shown). Likewise, OM, NDF, ADF and CP had similar values between treatments (P>0.10; data not shown). There was a treatment time-point interaction (P=0.0002; data not shown) for ruminal ph. Ruminal concentrations of NH 3-N were similar among treatments (P=0.97; data not shown). There was a tendency (P=0.07; data not shown) for a treatment time-point interaction for total VFA. Treatment time-point interactions (P<0.05) were observed for ruminal molar proportions of acetate, propionate, isobutyrate and isovalerate + 2-methylbutyrate (Table 2). There were no time-points in which acetate molar proportions Florida Beef Research Report

149 were significantly affected by treatment; however, propionate molar proportions were greater (P<0.05) for CHIT compared to CTRL at 18, 21 and 24 h. Since acetate molar proportions did not change and propionate molar proportions increased, A:P was reduced (P<0.05) at 18, 21, and 24 h for CHIT vs. CTRL. In conclusion, when provided to ruminally-cannulated steers consuming Tifton-85 bermudagrass hay, 80 g/d of chitosan did not affect ruminal degradability of nutrients at 24, 48 and 72 h of incubation. The VFA profile of the steers was altered with augmentation of propionate molar proportions leading to a reduction of the A:P. This data implies that chitosan may have the potential to increase production of cattle consuming high-roughage diets by shifting ruminal fermentation. Literature Cited Appuhamy, J. A. D. R. N., et al J. Dairy Sci. 96: Cuero, R. G Chitin and Chitinases EPA Goiri, I., et al Anim. Feed Sci. Technol. 148: Han, W., et al J. Anim. Sci. 91: Johnson, K. A. and D. E. Johnson J. Anim. Sci. 73: O Shea, C. J., et al J. Anim. Sci. 89: Pitesky, M. E., et al Clearing the Air. Livestock s Contribution to Climate Change. Shahidi, F. J., et al Trends Food Sci. Technol. 10: Shike, D. W Driftless Region Beef Conference Proceedings Florida Beef Research Report

150 Table 1. Chemical composition 1 of the Tifton-85 bermudagrass hay Item Tifton-85 bermudagrass hay Dry matter (DM), % 91.2 Crude protein, % DM 13.0 Neutral detergent fiber, % DM 71.5 Acid detergent fiber, % DM 40.6 Total digestible nutrients, % DM 56.0 Calcium, % DM 0.41 Phosphorus, % DM Analyzed by a commercial laboratory using a wet chemistry package (Dairy One; Ithaca, NY). Figure 1. Timeline of events during experimental period Florida Beef Research Report

151 Table 2. Effects of chitosan ruminal volitle fatty acid (VFA) molar proportions (mol/100 mol) and the acetate:propionate with Tifton-85 bermudagrass hay Treatments 1 P-value 3 Item CTRL CHIT SEM 2 TRT TRT TP VFA, mol/100 mol Acetate Propionate Butyrate Isobutyrate Isovalerate + 2 MB Valerate Caproate Acetate:Propionate CTRL = Control (ad libitum access to Tifton-85 bermudagrass hay) and CHIT = Chitosan treatment (ad libitum access to Tifton-85 bermudagrass hay plus the addition of 80 g/d of chitosan directly into the rumen via cannula). 2 SE of treatment means, n = 8 steers/treatment. 3 Observed significance levels for treatment effects and treatment time-point interactions; TRT = treatment; TP = time-point. 4 MB = Methylbutyrate Florida Beef Research Report

152 Florida Beef Research Report

153 Effects of Chitosan on Enteric Methane Production and Nutrient Digestibility of Beef Heifers D. D. Henry, F. M. Ciriaco, M. Ruiz-Moreno, V. R. G. Mercadante, T. M. Schulmeister, G. C. Lamb, N. DiLorenzo Synopsis Global impact of cattle production is a growing concern for policy makers and the general public and chitosan has the potential of reducing the carbon footprint of beef production by enhancing total tract digestibility of high-roughage diets. Summary A study was conducted to evaluate the effects of chitosan on enteric methane (CH 4) and total tract nutrient digestibility of beef heifers consuming either a high-concentrate (HC; 85% concentrate) or a low-concentrate (LC; 36% concentrate) diet. Heifers were provided their respective diets ad libitum along with one of three dietary inclusion rates of chitosan: 1) CTRL=0.0% of chitosan in the diet dry matter (DM); 2) CHI-0.5=0.5% of chitosan in the diet DM; and 3) CHI-1=1.0% of chitosan in the diet DM. Twenty-four crossbred heifers were housed in the University of Florida Feed Efficiency Facility (FEF) at the North Florida Research and Education Center (NFREC) for two 22-d periods where daily intake was monitored using the GrowSafe system. Neither chitosan nor diet had any effect on dry matter intake (DMI). Average daily gain (ADG) was not affected by chitosan; however, as expected, heifers consuming HC had greater gains compared to those consuming LC. Enteric CH 4 production was similar among chitosan treatments. When comparing CH 4 production between HC and LC, heifers consuming LC produced nearly three times as much CH 4 per day compared to HC. There was a chitosan diet interaction for digestibility of DM, organic matter (OM), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Heifers consuming LC with CHI-1 had greater digestibility of DM, OM and ADF with a tendency for enhanced digestibility of NDF. Chitosan did not have an effect on enteric production of CH 4; however, total tract digestibility of nutrients was improved when heifers received 1.0% of chitosan in the diet DM along with a high-roughage diet. Introduction American agriculture produces approximately 6% of total U.S. greenhouse gas emissions (EPA, 2012). Livestock production accounts for 3.2% of the approximately 430 teragrams (Tg) CO 2eq produced by agriculture in the US each year. Enteric CH 4 productions is responsible for nearly 55% of livestock s contribution to greenhouse gas emissions (Pitesky et al., 2009). Production of CH 4 warranted of review not only because it has about 25 times the global warming potential of CO 2, but also due to its effects on energy expenditures. In cattle, CH 4 can account for up to 12% of gross energy losses; thereby, it is possible that for every 100 calories taken in, 12 calories could be lost to CH 4 production (Johnson and Johnson, 1995). For several years, researchers have sought strategies to mitigate enteric CH 4 production in ruminants through various approaches, such as lipid supplementation (Beauchemin et al., 2007), alternative hydrogen sinks (e.g., nitrates and sulfates; van Zijderveld et al., 2010) and ionophores (Appuhamy et al., 2013). It is crucial to continue investigating current strategies as well as novel techniques of CH 4 emission abatement Florida Beef Research Report

154 Chitosan (N-acetyl-D-glucosamine polymer) is a natural biopolymer formed from the deacetylation of chitin. Chitin, the second most abundant organic compound on earth, can be found in the cell walls of lower plants and the exoskeletons of some arthropods and crustaceans (e.g., crab and shrimp). Chitosan has been studied for various applications in food preservation and medicine due to its antimicrobial actions (Cuero, 1999; Shahidi et al., 1999). In the U.S., chitosan has been deemed generally recognized as safe (GRAS) status, which ultimately allows for chitosan to be used as an alternative to antibiotics. Chitosan should not be considered a single compound, but rather a series of compounds with differing levels of deacetylation and other physic-chemical characteristics (Goiri et al., 2009). Chitosan, as an in vivo CH 4 inhibitor, is a novel product for ruminants; however, there have been studies with monogastrics (i.e., poultry and swine) showing it can alter protein fermentation in the lower gastrointestinal tract (O Shea et al., 2011; Han et al., 2013). The objective of this study was to determine the effects of increasing inclusion rates of chitosan on enteric CH 4 production and total tract digestibility of nutrients in beef heifers. Materials and Methods The study was conducted at the FEF in Marianna, FL. Chitosan was provided by PharmaNutrients Inc. (Lake Forest, IL). A total of 24 crossbred heifers (838±68 lb; average body weight ± standard deviation) were used in a randomized block design with a 2 3 factorial arrangement of treatments. Factors included diet, a HC (85% concentrate) and LC (36% concentrate), and chitosan inclusion rate, 0.0, 0.5, and 1.0% of diet DM. On d 0, heifers were weighed after 16-h feed withdrawal and stratified by initial body weight (BW), and randomly assigned to one of three inclusion rates of chitosan (DM basis): CTRL = no supplementation CHI-0.5 = 1 lb/d of the 50:50 molasses and crude glycerol mixture CHI-1.0 = 3 lb/d of the 50:50 molasses and crude glycerol mixture Heifers were housed two per pen for 22 d and had ad libitum access to water and their respective diets (Table 1). After the 14-d adaptation to diets and facility, heifers were weighed and collection of daily feed intake data began. Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, Alberta, Canada) to record intake by weight change measured to the nearest gram. Heifers were adapted to CH 4 collection canisters (Figure 1) for three days prior to collection. Collection and analysis of CH 4 was performed for five consecutive days (d 13 to 18). Beginning on d 17 and d 18, feed and fecal samples were collected, respectively, for four consecutive days to determine apparent total tract digestibility of DM, OM, CP, NDF, and ADF. Fecal samples were collected twice daily at 0800 and 1600 h via rectal grab. Feed samples were pooled within pen and fecal samples were pooled within heifer. Indigestible NDF (indf) was used as an internal indigestible marker. On d 22 heifers were weighed again after withholding feed for 16 h, for evaluation of performance during the entire period. Concentrations of indf in feed and feces were determined as described by Cole et al. (2011) with the following modification: in vitro incubations were conducted for 288 h instead of 96 h using the Daisy II incubator (Ankom Technology, Macedon, NY) to ensure complete digestion of potentially digestible NDF in hay as reported by Krizsan and Huhtanen (2013) Florida Beef Research Report

155 Data were analyzed as a randomized block design with a 2 3 factorial arrangement using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with heifer as the experimental unit. The model included the fixed effects of diet and chitosan inclusion rate and the random effect of period (block). Orthogonal polynomial contrasts were conducted to determine linear and quadratic effects of chitosan inclusion rate. Significance was declared at P Results Inclusion of chitosan had no effect (P>0.05) on ADG (data not shown) or DMI. No diet chitosan interactions occurred for CH 4 production (Table 2). There was no effect of chitosan on CH 4 production as g/d (P=0.71), g/kg of DMI (P=0.65), g/kg of DM digested (P=0.77), or g/kg of metabolic BW (P=0.79). However, diet affected (P<0.01) enteric CH 4 production where animals consuming LC produced nearly three times more CH 4 than animals consuming HC. A diet chitosan interaction was observed for nutrient digestibility of DM (P=0.05), OM (P=0.03) and ADF (P=0.05) with a tendency for an interaction on NDF (P=0.08) digestibility (Table 3). When chitosan was included up to 1.0% of the diet DM in the LC diet, total tract digestibility of DM and OM was increased by 21 and 19%, respectively. The increase on DM and OM digestibility was likely due to a 21% increase in ADF digestibility and a tendency for a 21% increase in NDF digestibility. With the results observed from this study, it appears that chitosan may alter ruminal fermentation to improve total tract digestibility of low-quality diets. It may be beneficial to provide cattle on low-quality pastures, such as cow-calf systems in the SE U.S., with chitosan to improve digestibility of the forage; however, further research is needed to test this hypothesis. Literature Cited Appuhamy, J. A. D. R. N., et al J. Dairy Sci. 96: Beauchemin, K. A., et al Can. J. Anim. Sci. 87: Cuero, R. G Chitin and Chitinases EPA Goiri, I., et al Anim. Feed Sci. Technol. 148: Han, W., et al J. Anim. Sci. 91: Johnson, K. A. and D. E. Johnson J. Anim. Sci. 73: O Shea, C. J., et al J. Anim. Sci. 89: Pitesky, M. E., et al Clearing the Air. Livestock s Contribution to Climate Change. Shahidi, F. J., et al Trends Food Sci. Technol. 10: Florida Beef Research Report

156 van Zijderveld, S. M., et al J. Dairy Sci. 93: Table 1. Ingredients and chemical composition of the basal diets Item 36% concentrate (LC) 85% concentrate (HC) Ingredient composition, % DM Corn gluten feed, pelleted Soybean hulls, pelleted Peanut hulls Liquid supplement Meal supplement Chitosan supplement Chemical composition DM, % OM, % DM CP, % DM NDF, % DM ADF, % DM Ether extract, % DM Calcium, % DM Phosphorus, % DM Westway Converter SR Southeast 32 (Westway Feed Products Inc., New Orleans, LA). Chemical composition of the supplement (as fed basis): 62% DM; 32.0% CP; 0.1% fat; contained trace minerals (cobalt, copper, iron, manganese, zinc, iodine, and selenium). 2 Supplied vitamins (A, D) and minerals (Beef Four Plus, W.B. Fleming Co. Tifton, GA). 3 Chitosan supplement was comprised of (DM basis) 100% soybean meal for control heifers, 75% soybean meal and 25% chitosan for 0.5% chitosan inclusion rate, and 50% soybean meal and 50% chitosan for 1.0% chitosan inclusion rate Florida Beef Research Report

157 Table 2. Effect of diet and inclusion level of chitosan on DMI and CH 4 emissions in beef heifers HC diet 1 LC diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P-value 3 Item SEM 4 DIET CHIT CHIT DIET DMI, lb/d CH 4 emissions g/d < g/kg DMI < g/kg DM digested < g/kg MBW < Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and minerals supplement; DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period. 6 Apparent total tract digestibility measured using indf as indigestible marker. 7 MBW = Metabolic body weight Florida Beef Research Report

158 Table 3. Effect of diet and inclusion level of chitosan on nutrient digestibility when indf was utilized as an internal marker HC diet 1 LC diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P-value 3 Item SEM 4 DIET CHIT CHIT DIET Digestibility, % DM 62.1 c 62.8 c 60.9 c 33.2 a 35.7 ab 40.3 b 1.73 < OM 63.0 c 63.5 c 61.9 c 34.5 a 37.7 ab 41.1 b 1.46 < CP < NDF 46.3 c 46.7 c 43.7 c 21.3 a 23.8 ab 25.7 b 1.53 < ADF 41.0 bc 41.3 c 36.3 b 18.4 a 21.6 a 22.3 a 1.68 < a,b,c Within a row, means with different superscripts differ, P < Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and minerals supplement; DM basis. 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and minerals supplement; DM basis. 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment Florida Beef Research Report

159 Effect of Residual Feed Intake Classification on Maintenance Energy Requirements and Efficiency of Energy Use in Growing Heifers T. P. Vining 1, P. A. Lancaster 1, N. DiLorenzo 2, G. C. Lamb 2, J. M. B. Vendramini 1 Synopsis Residual feed intake was not an effective selection tool for reducing maintenance energy requirements or improving efficiency of energy use in growing heifers. Summary The objective of this study was to determine energy expenditure in low and high residual feed effect (RFI) heifers. Angus crossbred heifers (n=60; initial BW=60 lb) were fed a growing diet (ME=0.98 Mcal/lb DM) for 70 d. Feed intake was recorded daily using the GrowSafe system, and BW was recorded every 14 d. Non-pregnant heifers (n=15) were removed from data analysis. Residual feed intake was calculated as the residual from the regression of DMI on mid-test BW 0.75 and ADG (R 2 =0.31). Heifers were separated into low, medium, and high RFI groups based on 0.5 SD above and below the mean RFI (0.00±4.07 lb/d). Low (n=8) and high (n=8) RFI heifers were fed at ad-libitum, and 1.0X and 0.5X expected maintenance energy requirement to measure diet digestibility, methane production, and energy requirements. Low RFI heifers consumed 31% less feed than high RFI heifers during the performance experiment, but there was no difference in ad libitum DMI during the metabolism experiment. There was no difference in dry matter digestibility of the diet or methane production between low and high RFI heifers at any amount of feed intake most likely due to similar DMI. Low RFI (more feed efficient) heifers had greater maintenance energy requirements compared with their high RFI counterparts. There was no difference in efficiency of metabolizable energy use for maintenance or gain between low and high RFI heifers. These data indicate that selection based on RFI may not lead to improved energy efficiency in growing heifers. Introduction Feed accounts for the largest variable cost to livestock production. It follows, that a reduction in the amount of feed per unit of production (i.e. feed efficiency) would result in increased profitability. Feed conversion ratio (FCR; feed:gain) is the typical trait used to measure feed efficiency in growing cattle; however, FCR is strongly negatively correlated with ADG and mature BW such that selection based on FCR would indirectly result in larger mature cows. Residual feed intake is the difference between an animal s actual feed intake and expected feed intake based on body weight (BW) and BW gain over a specific feeding period so that more feed efficient cattle eat less than expected (low RFI). Residual feed intake is not related to ADG or mature BW such that selection based on RFI will not result in larger mature cows. An ideal feed efficiency trait would identify cattle with superior energy utilization resulting in a more energy efficient beef cattle herd. Early reports (Basarab et al., 2003, Nkrumah et al., 2006) indicated that low RFI (more feed efficient) cattle produced less heat even when fed at similar levels of intake, indicating that low RFI cattle possess a lesser maintenance energy requirement. However, more recent research (Lancaster, 2008; Lines et al., 2014) indicates no difference in maintenance energy requirement between low and high RFI cattle. The hypothesis of the current study was that maintenance energy requirement and efficiency of metabolizable energy (ME) use are similar for low and high RFI cattle. The Florida Beef Research Report

160 objective of this study was to determine differences in energy partitioning in growing heifers with low and high RFI. Research Protocol The study was conducted at the North Florida Research and Education Center (NFREC) in Marianna, FL. Sixty Angus crossbred heifers were housed in group pens in the NFREC feed efficiency facility for 70 days beginning January 22, The GrowSafe feed intake system (GrowSafe Ltd., Airdrie, Alberta, Canada) was used to record daily feed intake and full body weight was recorded every 14 days. Heifers were inseminated via fixed-time A.I. (TAI) on March 14, 2014 using the 7d-CO-Synch + CIDR protocol (Lamb et al. 2006). Of the 60 heifers, 45 were confirmed pregnant and used in the data analysis, and to select the 16 heifers (8 low and 8 high RFI) used in the metabolism experiment. The 16 heifers were weighed and randomly assigned to individual pens at the NFREC feed efficiency facility on May 6, Heifers were fed at ad-libitum, and at 1.0X maintenance and 0.5X expected maintenance energy requirement for three consecutive 11 day feeding periods following a two week diet adaptation period. The same diet was fed during the metabolism experiment as was used during the performance experiment. During the ad-libitum feeding period of the metabolism experiment, the GrowSafe system was used to record daily feed intake, whereas heifers consumed the entire quantity of feed offered during the 1.0X and 0.5X maintenance periods. Dry matter intake (DMI) was computed from dry matter percentage of weekly feed samples and the average daily recorded feed intake from the GrowSafe system. Average daily gain (ADG) was calculated by regression of 14-d BW on day of experiment for each heifer. Feed conversion ratio (FCR) was calculated as the ratio of DMI to ADG. Residual feed intake (RFI) was calculated as actual DMI minus expected DMI from regression of DMI on ADG and average BW Residual gain efficiency (RGE) was calculated as actual ADG minus expected ADG from regression of ADG on DMI and average BW The diet fed throughout the duration of the study was composed of 51% peanut hulls, 22% soybean hull pellets, 22% corn gluten feed pellets and 5% molasses based liquid supplement on as-fed basis with monensin supplied at 35 mg/kg DM of the diet. The nutrient composition of the diet was 84.36% dry matter, 12.02% crude protein, 3.10% ether extract, 66.12% neutral detergent fiber, 50.31% acid detergent fiber, and 0.98 Mcal ME/lb DM. Indigestible neutral detergent fiber (indf) was used as an internal marker to determine diet digestibility of dry matter, crude protein, and neutral detergent fiber. The sulfur hexafluoride (SF 6) tracer technique (Johnson, 1994) was used to quantify methane emissions. Heat production (HP) was determined using the oxygen pulse method (Brosh et al. 1998; 2002). Metabolizable energy required for maintenance (MEm) and fasting HP (FHP) were then calculated from regression of log HP on metabolizable energy intake. Efficiencies of ME used for maintenance (k m) and growth (k g) were calculated from FHP, ad-libitum retained energy, and MEm. Heifers were separated into low, medium, and high RFI groups based on 0.5 SD above and below the mean RFI (0.00±4.07 lb/d). Data were analyzed using the MIXED procedure of SAS version 9.4 (SAS Institute, Cary, NC) to evaluate the fixed effect of RFI group. The CORR procedure of SAS was used to generate phenotypic correlation coefficients among growth, feed intake, and feed efficiency traits, and to generate phenotypic correlation coefficients of feed efficiency traits with energy requirements and Florida Beef Research Report

161 efficiencies of ME use. Residual feed intake groups were considered different when P 0.05 and tendencies were determined if P>0.05 and Results The phenotypic correlations among growth and feed efficiency traits in the performance experiment are presented in Table 1. There was a moderate positive correlation (0.43) between DMI and ADG. Feed conversion ratio was moderately negatively correlated with ADG (-0.33), but strongly positively correlated with DMI (0.70). Residual gain efficiency was strongly positively correlated with ADG (0.87), but not DMI. Residual feed intake was independent of ADG, but strongly correlated with DMI (0.83). During the performance experiment, low RFI heifers consumed 31% less (P=0.01) feed and had 31% less (P=0.01) FCR than high RFI heifers even though final BW and ADG were similar (Table 2). These data indicate that selection based on FCR or RGE would lead to larger mature cattle, requiring greater feed inputs in order to meet maintenance energy requirements. During the ad libitum period of the experiment no difference (P=0.51) was observed in DMI (26.28 vs lb/d for low and high RFI heifers, respectively). Average digestibility of dry matter was 40.96, 43.91, and 48.35%, crude protein was 55.93, 58.42, and 66.05%, and neutral detergent fiber was 30.48, 33.23, and 36.24% for the ad libitum, 1.0X expected MEm, and 0.5X expected MEm periods, respectively, but there were no differences between low and high RFI heifers likely because there was no difference in DMI. There were no differences (P>0.20) in fecal energy loss, urinary energy loss, or methane energy loss between low and high RFI heifers at any amount of feed intake, which also is likely due to the lack of differences in DMI. Low RFI heifers tended (P=0.08) to have greater HP during the ad libitum intake period (76.0 vs kcal/lb BW.75 ), and had a greater (P<0.05) HP during the 0.5X maintenance intake period (49.1 vs kcal/lb BW.75 ) compared with high RFI heifers, but no difference (P=0.18) in HP was observed between low and high RFI heifers during the 1.0X maintenance intake period. Heifers with low RFI had 16% greater (P<0.05) MEm and tended (P=0.09) to have 10% greater FHP compared with high RFI heifers (Table 3), which is contradictory to the generally accepted theory that low RFI cattle have lower maintenance energy requirements. No differences were observed between low and high RFI heifers for the efficiency of energy use for maintenance and gain. Feed conversion ratio and RFI were strongly negatively correlated (P<0.05) with FHP (-0.51 and -0.52) and MEm (-0.69 and -0.64) and RGE was strongly positively correlated (P<0.05) with MEm (0.78) such that more feed efficient heifers would have greater maintenance energy requirements. Additionally, RGE was strongly negatively correlated (P<0.05) with efficiency of energy use for maintenance (-0.65) and gain (-0.57) such that the more feed efficient heifers would have lower efficiencies of energy use. In conclusion, feed efficiency based on RFI did not equate to energy efficiency in growing heifers. Feed efficiency may differ from energy efficiency because of differences in composition of gain that affect weight gain gaining muscle mass is more efficient on a live weight basis because muscle is 70% water, but less efficient on an energy basis because muscle protein turnover has a large energy cost. Therefore, evaluating energy efficiency is the best method to determine the true efficiency of nutrient utilization of cattle Florida Beef Research Report

162 LITERATURE CITED Basarab, J. A., et al Can. J. Anim. Sci. 83: Brosh, A., Y., et al J. Anim. Sci. 76: Brosh, A., Y., et al Livest. Prod. Sci. 77: Johnson, K., M. et al Environ. Sci. Technol. 28: Lamb, G. C., et al J. Anim. Sci. 84: Lancaster, P. A Ph.D. Dissertation, Texas A&M University, College Station Lines, D. S., et al Anim Prod Sci-. Nkrumah, J. D., et al J. Anim. Sci. 84: Florida Beef Research Report

163 Table 1. Phenotypic correlations among performance and feed efficiency traits in Angus crossbred heifers Trait 1 ADG DMI FCR RFI RGE FBW 0.52* 0.53* ADG 0.43* -0.33* * DMI 0.70* 0.83* 0.00 FCR 0.86* -0.67* RFI -0.29* 1 ADG=average daily gain; DMI=dry matter intake; FCR=feed conversion ratio; RFI=Residual feed intake; RGE=residual gain efficiency; FBW=final body weight. *Correlations are different from zero at P Table 2. Growth, feed intake, and feed efficiency traits of Angus crossbred heifers with low (< 0.5 SD), medium (± 0.5 SD), and high (> 0.5 SD) residual feed intake Low RFI Med RFI High RFI SEM 2 P-value No. heifers Initial BW, lb Final BW, lb ADG, lb/d DMI, lb/d a b c FCR, lb/lb 8.66 a b c RFI, lb/d a b 4.89 c RGE, lb/d FCR=feed conversion ratio, RFI=residual feed intake; RGE=residual gain efficiency 2 SEM=standard error of the mean. abc Within a row, means without a common superscript differ (P 0.05). Table 3. Effect of residual feed intake group on fasting heat production, metabolizable energy required for maintenance, and efficiency of ME use for maintenance and gain in Angus crossbred heifers (n=16) Trait 1 Low RFI High RFI SEM 2 P-value FHP, kcal/lb of BW MEm, kcal/lb of BW k m k g FHP=fasting heat production; MEm=metabolizable energy required for maintenance; k m=efficiency of metabolizable energy use for maintenance; k g=efficiency of metabolizable energy use for gain. 2 SEM=standard error of the mean Florida Beef Research Report

164 Table 4. Phenotypic correlations among energy requirements and feed efficiency traits in Angus crossbred heifers (n=16) Trait 1 FHP MEm k m k g FCR * * RFI * * RGE * * * 1 FHP=fasting heat production; MEm=metabolizable energy required for maintenance; k m=efficiency of metabolizable energy use for maintenance; k g=efficiency of metabolizable energy use for gain; FCR=feed conversion ratio; RFI=residual feed intake; RGE=residual gain efficiency. * Correlations are different from zero at P <P Florida Beef Research Report

165 Research and Graduate Programs The Department has 38 faculty members working in the various disciplines of Nutrition, Breeding and Genetics, Physiology, Molecular Biology, Meat Science and Management. Additionally, there are several faculty members at the outlying Research and Education Centers that participate in our research and graduate programs. The Department offers programs in Master of Science, (thesis and non-thesis) and Doctor of Philosophy. Undergraduate Programs The Department offers three undergraduate degree options; Animal Biology Specialization which is the pre-professional track, Equine Specialization, and Food Animal Specialization. Resources Students have access to an on-campus beef teaching cow herd in addition to two research and production oriented herds close to campus. There are also two additional out-lying Research and Education Centers with nearly 1,000 beef cows of both Bos indicus and Bos taurus breeding available for research and to provide hands-on experience for our students. Extension and the Beef Industry The Department plays an active role in facilitating communication and dissemination of research and production-oriented material to Florida cow-calf producers. Beef producers and state and county faculty work cooperatively in an effort to improve the production, efficiency and marketability of Florida beef cattle. Florida is in a unique position of having more large-scale cow-calf operations than any other state in the United States.