M. D. MacNeil

Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.

Genetic evaluation of Angus cattle for carcass marbling using ultrasound and genomic indicators

The objectives were to estimate genetic parameters needed to elucidate the relationships of a molecular breeding value (MBV) for marbling, intramuscular fat (IMF) of yearling bulls measured with ultrasound, and marbling score (MRB) of slaughtered steers, and to assess the utility of MBV and IMF in predicting the breeding value for MRB. Records for MRB (n = 38,296) and IMF (n = 6,594) were from the American Angus Association database used for national cattle evaluation. A total of 1,006 records of MBV were used in this study. (Co)variance components were estimated with ASREML, fitting an animal model with fixed contemporary groups for MRB and IMF similar to those used in the Angus national genetic evaluation. The overall mean was the only fixed effect included in the model for MBV. Heritability estimates for carcass measures were 0.48 +/- 0.03, 0.31 +/- 0.03, and 0.98 +/- 0.05 for MRB, IMF, and MBV, respectively. Genetic correlations of IMF and MBV with MRB were 0.56 +/- 0.09 and 0.38 +/- 0.10, respectively. The genetic correlation between IMF and MBV was 0.80 +/- 0.22. These results indicate the MBV evaluated may yield a greater genetic advance of approximately 20% when used as an indicator trait for genetic prediction of MRB compared with IMF. However, neither of these indicators alone provides sufficient information to produce highly accurate prediction of breeding value for the economically relevant trait MRB. Given that the goal is a highly accurate prediction of true breeding value for MRB, results of this work point to the need to 1) continue progeny testing, and 2) continue increasing the genetic correlation between the MBV and MRB.

Reproductive performance of heifers offered ad libitum or restricted access to feed for a one hundred forty-day period after weaning.

Reproductive performance was evaluated in composite heifers born over a 3-yr period that were randomly assigned to control (fed to appetite; n = 205) or restricted (fed at 80% of that consumed by controls adjusted to a common BW basis; n = 192) feeding for a 140-d period, beginning about 2 mo after weaning at 6 mo of age and ending at about 12.5 mo of age. Heifers were fed a diet of 67% corn silage, 18% alfalfa, and 9% of a protein-mineral supplement (DM basis). Restricted heifers consumed 27% less feed over the 140 d and had less ADG (0.53 +/- 0.01 vs. 0.65 +/- 0.01 kg/d; P < 0.001) than control heifers. After 140 d, all heifers were placed in common pens and subjected to an estrous synchronization protocol to facilitate AI at about 14 mo of age. Heifers were then exposed to bulls for the remainder of a 51-d breeding season. Average BW of heifers diverged within 28-d after initiation of feed restriction, and differences (P < 0.001) persisted through the prebreeding period (309 +/- 1 vs. 326 +/- 1 kg at approximately 13.5 mo of age) and subsequent grazing season (410 +/- 2 vs. 418 +/- 2 kg at about 19.5 mo of age). From the end of the 140-d restriction at about 12.5 to 19.5 mo of age, ADG was greater (P < 0.001) in restricted heifers than control heifers (0.51 +/- 0.01 vs. 0.47 +/- 0.01 kg/d). Proportion of heifers attaining puberty by 14 mo of age tended to be less (P = 0.1) in restricted (60 +/- 3%) than control-fed heifers (68 +/- 3%). Mean BW at puberty was less (P < 0.01) in restricted (309 kg) than control (327 kg) heifers. Pregnancy rate from AI tended to be less (P = 0.08) in restricted (48 +/- 4%) than control heifers (57 +/- 3%). Proportion of animals that were pubertal at breeding and pregnant from AI were positively associated (P < 0.1) with heifer age and ADG from birth to beginning of study. Final pregnancy rates were 87 and 91% for restricted and control heifers, respectively (P = 0.27). Day of breeding season that conception occurred was negatively associated with ADG from birth to weaning (P = 0.005), but was not associated with ADG within treatment (P = 0.60). Economic analysis revealed a

Preovulatory estradiol and the establishment and maintenance of pregnancy in suckled beef cows.

33 reduction in cost to produce a pregnant heifer under the restricted protocol when accounting for pregnancy rates and differences in BW and market prices between selection at weaning and marketing as open heifers at l.5 yr of age. A potential economic advantage exists for rearing replacement heifers on a restricted level of feeding during the postweaning period.

National cattle evaluation system for combined analysis of carcass characteristics and indicator traits recorded by using ultrasound in Angus cattle

In postpartum beef cows, GnRH-induced ovulation of small dominant follicles decreased pregnancy rates and increased late embryonic/fetal mortality. In Exp. 1, single ovulation reciprocal embryo transfer (ET) was used to examine the relationship between preovulatory serum concentrations of estradiol at GnRH-induced ovulation in donor and recipient cows and establishment and maintenance of pregnancy. Suckled beef cows (n = 1,164) were administered GnRH (GnRH1, 100 μg) on d -9 (GnRH1), PGF(2α) on d -2, and GnRH2 (GnRH2, 100 μg) on d 0 (CO-Synch protocol) either with (donors; n = 810) or without (recipients; n = 354) AI. Single embryos (n = 394) or oocytes (n = 45) were recovered from the donor cows (d 7; ET) and all live embryos were transferred into recipients. Serum concentration of estradiol at GnRH2 was positively correlated with follicle size at GnRH2 (r = 0.45, P < 0.01) and progesterone at ET (r =0.34, P < 0.01). Donor cows with greater estradiol at GnRH2 were more likely to yield an embryo than an unfertilized oocyte (P < 0.01). Donor and recipient cows were retrospectively divided into 4 groups [low estradiol (<8.4 pg/mL) or high estradiol (≥8.4 pg/mL)] based on serum concentration of estradiol at GnRH2. Pregnancy rate at d 27 for low-low (n = 78), low-high (n = 80), high-low (n = 91), and high-high (n = 101) groups (donor-recipient, respectively) was 45, 65, 43, and 61% respectively (P < 0.02). Because recipient cows with greater estradiol concentration at GnRH2 had greater pregnancy rates in Exp. 1, the objective of Exp. 2 was to evaluate the effect of estradiol supplementation on pregnancy rate. Ovulation was synchronized in suckled beef cows (n = 600) using the CO-Synch protocol with the insertion of a controlled internal drug release (CIDR; intravaginal progesterone supplement) from d -9 until d -2. Approximately one-half of the cows (n = 297) received an injection of estradiol cypionate (ECP; 0.5 mg intramuscularly) 24 h before AI. Compared with the no treatment (Control) cows, ECP treatment increased (P < 0.01) pregnancy rates of cows induced to ovulate smaller dominant follicles (<12.2 mm). In conclusion, GnRH-induced ovulation of small dominant follicles was associated with reduced serum estradiol, fertilization rate (donor cows), and pregnancy establishment (recipient cows). Furthermore, ECP supplementation during the preovulatory period increased pregnancy rates in cows induced to ovulate smaller dominant follicles.

Pregnancy establishment and maintenance in cattle.

The objectives were to 1) evaluate genetic relationships of sex-specific indicators of carcass merit obtained by using ultrasound with carcass traits of steers; 2) estimate genetic parameters needed to implement combined analyses of carcass and indicator traits to produce unified national cattle evaluations for LM area, subcutaneous fat depth (SQF), and marbling (MRB), with the ultimate goal of publishing only EPD for the carcass traits; and 3) compare resulting evaluations with previous ones. Four data sets were extracted from the records of the American Angus Association from 33,857 bulls, 33,737 heifers, and 1,805 steers that had measures of intramuscular fat content (IMF), LM area (uLMA), and SQF derived from interpretation of ultrasonic imagery, and BW recorded at the time of scanning. Also used were 38,296 records from steers with MRB, fat depth at the 12th to 13th rib interface (FD), carcass weight, and carcass LM area (cLMA) recorded on slaughter. (Co)variance components were estimated with ASREML by using the same models as used for national cattle evaluations by the American Angus Association. Heritability estimates for carcass measures were 0.45 +/- 0.03, 0.34 +/- 0.02, 0.40 +/- 0.02, and 0.33 +/- 0.02 for MRB, FD, carcass weight, and cLMA, respectively. Genetic correlations of carcass measures from steers with ultrasonic measures from bulls and heifers indicated sex-specific relationships for IMF (0.66 +/- 0.05 vs. 0.52 +/- 0.06) and uLMA (0.63 +/- 0.06 vs. 0.78 +/- 0.05), but not for BW at scanning (0.46 +/- 0.07 vs. 0.40 +/- 0.07) or SQF (0.53 +/- 0.06 vs. 0.55 +/- 0.06). For each trait, estimates of genetic correlations between bulls and heifers measured by using ultrasound were greater than 0.8. Prototype national cattle evaluations were conducted by using the estimated genetic parameters, resulting in some reranking of sires relative to previous analyses. Rank correlations of high-impact sires were 0.91 and 0.84 for the joint analysis of MRB and IMF with previous separate analyses of MRB and IMF, respectively. Corresponding results for FD and SQF were 0.90 and 0.90, and for cLMA and uLMA were 0.79 and 0.89. The unified national cattle evaluation for carcass traits using measurements from slaughtered animals and ultrasonic imagery of seed stock in a combined analysis appropriately weights information from these sources and provides breeders estimates of genetic merit consistent with traits in their breeding objectives on which to base selection decisions.

Evaluation of candidate gene effects for beef backfat via Bayesian model selection

A single ovulation, reciprocal embryo transfer study was used to investigate effects of oocyte competence and maternal environment on pregnancy establishment and maintenance in beef cows. Estrous cycles were synchronized in suckled beef cows and embryo donors were inseminated on d 0 (n = 810). Cows were classified on d 0 as having a small (<12.5 mm) or large (≥12.5 mm) ovulatory follicle and randomly chosen as donors or recipients to remove confounding effects of ovulatory follicle size on fertility. Embryos (n = 393) or oocytes (n = 44) were recovered on d 7, and all viable embryos were transferred into recipients (n = 354). All statistical analyses were conducted using the GLM procedure of SAS. Path analysis (with significance set at P < 0.10) was used to examine potential cause-effect relationships among the measured variables. Greater donor cow BW, circulating estradiol concentration at insemination, postpartum interval, and ovulatory follicle size directly increased (P < 0.10) fertilization success. Greater donor cow age was the only factor that directly decreased (P < 0.10) fertilization success. Viability of d-7 embryos was directly inhibited (P < 0.10) by rapid follicular growth rate from d -2 to 0 and heavier BW. Direct beneficial effects to embryo viability were increased serum progesterone concentration on d -2 and ovulatory follicle size. Pregnancy maintenance from d 7 to 27 was enhanced (P < 0.10) by increased serum estradiol concentration on d 0 and progesterone concentration on d 7 in the recipient cow. Increased follicular diameter in the recipient cow on d 0 was detrimental to pregnancy maintenance from d 7 to 27. This manuscript defines the complex interplay and relative contributions of endocrine and physical factors both prior and subsequent to fertilization that influence both oocyte competence and maternal environment and their roles in establishment and maintenance of pregnancy.

Effects of flunixin meglumine on pregnancy establishment in beef cattle.

Candidate gene approaches provide tools for exploring and localizing causative genes affecting quantitative traits and the underlying variation may be better understood by determining the relative magnitudes of effects of their polymorphisms. Diacyglycerol O-acyltransferase 1 (DGAT1), fatty acid binding protein (heart) 3 (FABP3), growth hormone 1 (GH1), leptin (LEP) and thyroglobulin (TG) have been previously identified as genes contributing to genetic control of subcutaneous fat thickness (SFT) in beef cattle. In the present research, Bayesian model selection was used to evaluate effects of these five candidate genes by comparing competing non-nested models and treating candidate gene effects as either random or fixed. The analyses were implemented in SAS to simplify the programming and computation. Phenotypic data were gathered from a F2 population of Wagyu × Limousin cattle. The five candidate genes had significant but varied effects on SFT in this population. Bayesian model selection identified the DGAT1 model as the one with the greatest model probability, whether candidate gene effects were considered random or fixed, and DGAT1 had the greatest additive effect on SFT. The SAS codes developed in the study are freely available and can be downloaded at: http://www.ansci.wsu.edu/programs/.

Triennial Reproduction Symposium: influence of follicular characteristics at ovulation on early embryonic survival.

The objective of this research was to determine effects of a single injection of the PG synthesis inhibitor flunixin meglumine (FM; 1.1 mg/kg of BW, intramuscularly) approximately 13 d (range 10 to 15 d) after AI on pregnancy establishment. Three experiments were conducted using estrus-synchronized heifers and cows. Technicians and AI sires were equally represented across treatments within locations and experiments. Bulls were introduced on the day of FM treatment. Pregnancy to AI was diagnosed 28 to 50 d after AI using ultrasonography. In Exp. 1, beef heifers (n = 1,221) were divided within 5 locations to receive FM or no further treatment (control). At insemination, heifers were divided into 2 similar pastures or pens, and approximately 13 d later, 1 group of heifers within each location was processed through an animal handling facility to administer FM treatment. There was no location x treatment interaction (P = 0.62) on AI pregnancy rates, so data were pooled. Pregnancy rates to AI were reduced (P = 0.02) among heifers receiving the FM treatment procedure (66%) compared with control heifers (72%). In Exp. 2, suckled beef cows (n = 719) were assigned within 2 locations to receive FM or no further treatment (control) approximately 13 d after AI. At insemination, control and FM cows were divided into separate pastures, and only FM cows were handled after AI for the FM treatment procedure. There was no location x treatment interaction (P = 0.75), so data were pooled. Pregnancy rates to AI did not differ (P = 0.80) between FM (57%) and control cows (59%). In Exp 3, beef heifers (n = 247) and suckled beef cows (n = 335) from 1 location received no injection (control) or injection of FM approximately 13 d after AI when all cows and heifers were processed through a working facility. Pregnancy rates to AI were not different (P = 0.37) between FM (45%) and control (42%) cows or between FM (56%) and control (55%) heifers. We conclude FM administration at 1.1 mg/kg of BW approximately 13 d after AI did not improve pregnancy establishment in beef cows and heifers and that the effects of handling heifers at this time may decrease pregnancy establishment.

Circulating bovine pregnancy associated glycoproteins are associated with late embryonic/fetal survival but not ovulatory follicle size in suckled beef cows1

Reproductive failure in livestock can result from failure to fertilize the oocyte or embryonic loss during gestation. Although fertilization failure occurs, embryonic mortality represents a greater contribution to reproductive failure. Reproductive success varies among species and production goals but is measured as a binomial trait (i.e., pregnancy), derived by the success or failure of multiple biological steps. This review focuses primarily on follicular characteristics affecting oocyte quality, fertilization, and embryonic health that lead to pregnancy establishment in beef cattle. When estrous cycles are manipulated with assisted reproductive technologies and ovulation is induced, duration of proestrus (i.e., interval from induced luteolysis to induced ovulation), ovulatory follicle growth rate, and ovulatory follicle size are factors that affect the maturation of the follicle and oocyte at induced ovulation. The most critical maturational component of the ovulatory follicle is the production of sufficient estradiol to prepare follicular cells for luteinization and progesterone synthesis and prepare the uterus for pregnancy. The exact roles of estradiol in oocyte maturation remain unclear, but cows that have lesser serum concentrations of estradiol have decreased fertilization rates and decreased embryo survival on d 7 after induced ovulation. When length of proestrus is held constant, perhaps the most practical follicular measure of fertility is ovulatory follicle size because it is an easily measured attribute of the follicle that is highly associated with its ability to produce estradiol.