Single Sire Research Articles

PS-6 Nutrition During Early Pregnancy Impacts Offspring Ovarian Characteristics

Abstract During early pregnancy offspring are directly exposed to nutrients consumed by their mother, and the development of their own reproductive tract is underway. The objective of this research was to determine how characteristics of offspring ovaries were affected by different maternal rates of gain during the first trimester of gestation in beef heifers. Before breeding antral follicle counts were determined via ultrasound. Beginning at breeding Angus heifers were managed to achieve one of two rates of gain: low (0.20 kg/d, n = 8; LG) or moderate (0.75 kg/d, n = 8; MG) for the first trimester of pregnancy, after which they were managed as a single group through F1 calving. Calves remained with their dams until weaning and were managed as a single group through breeding. These F1 heifer calves were then synchronized and bred to a single sire using female sexed semen. On the 84th day of gestation (approximate heifer age 17 months) ovaries were removed from the reproductive tract, weighed, and visible antral follicles were counted. Cross-sections from each ovary were post-fixed and embedded in paraffin, then each of 3 sections (5 µm, with 10 sections between to avoid counting follicles more than once) were stained with Hematoxylin and Eosin for histological evaluation. Number of primordial, primary, secondary, and antral follicles were determined for each section. Data were analyzed using the GLM procedure of SAS with significance declared at P ≤ 0.05. The CORR procedure of SAS was used to calculate correlations between pre-breeding antral follicle counts, surface follicle counts, and histological follicle counts. Though no differences were observed in number of visible follicles (P = 0.45), the corpus luteum (CL) was heavier (P = 0.03) and average ovarian length was greater (P = 0.04) in offspring from LG dams compared with those from MG dams. No differences were observed in the number of primordial, primary, secondary, or antral follicles between treatments (P ≤ 0.18). There was a correlation between the number of histological follicles and surface follicles (r = 0.73; P = 0.002) and pre-breeding antral follicles and surface follicles (r = 0.60; P = 0.014), but there was no correlation between pre-breeding antral follicles and histological follicles (P = 0.10). Heavier CLs have a positive correlation with amount of progesterone released, which is essential for pregnancy maintenance. Longer lengths of ovaries suggest more area for follicles to develop, which is indicative of future reproductive success. Data corroborates previous reports regarding positive correlations between antral follicle count determined via ultrasound and counts of surface follicles on extracted ovaries. These findings demonstrate that nutrition during the first trimester of gestation impacts offspring reproductive tract development.

303 Overview of Paternal Programming in Livestock Species

Abstract The concept of developmental programming from a maternal standpoint is well established. Perhaps even more impactful, however, is developmental programming that may arise from paternal contributions when single sires are responsible for breeding multiple females. Research in rodent models has shown that paternal obesity, high fat or low protein diets, exercise, stress, and drug and alcohol exposure can lead to epigenetic modifications in sperm and seminal plasma. Some of these changes withstand the extensive remodeling associated with fertilization and early embryo development, culminating in discernable offspring phenotypes that remain present for multiple generations. Certainly, the observations in rodent models related to alterations in offspring body weight, adiposity, metabolic responses, bone density, and reproductive outcomes and function have implications for the livestock industries if similar paternal programming phenomena exist. Livestock production systems are robust and dynamic and many paradigms exist with potential to alter male germ line epigenome, with specific changes likely related to timing of aberrant conditions relative to spermatogenesis and sperm residence in the epididymis. Within the context of routine management systems, our livestock patriarchs can experience major diet and weight changes, transportation and handling stress, environmental extremes, and/or changes in exercise duration, frequency, and intensity. In addition, livestock managers may embrace the concept of delivering specific quantities and types of feedstuffs, and apply other interventions if proven beneficial. Several research groups using livestock models have revealed that epigenetic machinery delivered in sperm and seminal plasma can be altered. A sheep model demonstrated the feeding rumen-protected methionine to rams from weaning to puberty resulted in epigenetic and phenotypic alterations still present in the F2 generation. This presentation will review the status of literature regarding paternal programming, identify knowledge gaps, and suggest areas for future research efforts with potential to enhance production efficiencies of livestock operations.

298 Late Gestational Nutrient Restriction of Primiparous Beef Heifers Decreases Neonatal Calf Body Weight and Organ Mass

Abstract We hypothesized that late gestational nutrient restriction (NR) would decrease organ mass in neonatal calves. Fall-calving crossbred beef heifers (BW: 466 ± 30 [SD] kg; BCS: 5.4 ± 0.5) bred to a single sire were individually fed 100% (control; n = 10) or 70% (nutrient restricted; NR; n = 12) of NASEM metabolizable energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Diets were based on chopped sorghum sudan hay and supplemented to meet targeted nutritional planes. Calves were removed immediately post-calving and fed colostrum and milk replacer relative to BW until euthanasia (48.7 ± 0.4 h of age) for organ mass determination. Nutritional plane was a fixed effect, and maternal sire, calf sex (when P < 0.25), and date of study initiation were covariates. Post-calving, NR dams were 62 kg BW and 2.1 BCS less (P < 0.001) than controls. Calf BW, liver, large intestine, omental/mesenteric fat, kidney, and heart weights were less (P ≤ 0.05) and carcass, rumen/reticulum/omasum, stomach complex, and total gastrointestinal tract weights tended to be less (P ≤ 0.09) in offspring from NR dams. Maternal nutrient restriction did not affect (P ≥ 0.11) weights of the small intestine, pancreas, abomasum, spleen, perirenal fat, adrenals, lungs, femur, and brain. Expressed per BW, lung weight was greater (P = 0.05), and brain and femur weight tended to be greater (P ≤ 0.10) in calves from NR dams, but other organ masses were unaffected (P ≥ 0.15). Expressed relative to brain weight, liver and large intestine weights were less (P ≤ 0.04) and carcass, total gastrointestinal tract, omental/mesenteric fat, and kidney weights tended to be less (P ≤ 0.08) in NR. In summary, late gestational NR decreased some organ masses in neonatal calves while sparing other tissues via asymmetric growth.

Form of dietary selenium affects mRNA encoding interferon-stimulated and progesterone-induced genes in the bovine endometrium and conceptus length at maternal recognition of pregnancy.

Widespread regions of the southeast United States have soils, and hence forages, deficient in selenium (Se), necessitating Se supplementation to grazing cattle for optimal immune function, growth, and fertility. We have reported that supplementation with an isomolar 1:1 mix (MIX) of inorganic (ISe) and organic (OSe) forms of Se increases early luteal phase (LP) concentrations of progesterone (P4) above that in cows on ISe or OSe alone. Increased early LP P4 advances embryonic development. Our objective was to determine the effects of the form of Se on the development of the bovine conceptus and the endometrium using targeted real-time PCR (qPCR) on day 17 of gestation, the time of maternal recognition of pregnancy (MRP). Angus-cross yearling heifers underwent 45-d Se-depletion then repletion periods, then at least 90 d of supplementation (TRT) with 35 ppm Se per day as either ISe (n = 10) or MIX (n = 10). Heifers were inseminated to a single sire after detected estrus (day 0). On day 17 of gestation, caruncular (CAR) and intercaruncular (ICAR) endometrial samples and the developing conceptus were recovered from pregnant heifers (ISe, n = 6 and MIX, n = 6). qPCR was performed to determine the relative abundance of targeted transcripts in CAR and ICAR samples, with the expression data subjected to one-way ANOVA to determine TRT effects. The effect of TRT on conceptus development was analyzed using a one-tailed Student's t-test. When compared with ISe-treated heifers, MIX heifers had decreased (P < 0.05) abundance of several P4-induced and interferon-stimulated mRNA transcripts, including IFIT3, ISG15, MX1, OAS2, RSAD2, DGAT2, FGF2 in CAR and DKK1 in ICAR samples and tended (P ≤ 0.10) to have decreased mRNA abundance of IRF1, IRF2, FOXL2, and PGR in CAR samples, and HOXA10 and PAQR7 in ICAR samples. In contrast, MIX-supplemented heifers had increased (P < 0.05) mRNA abundance of MSTN in ICAR samples and an increase in conceptus length (ISe: 17.45 ± 3.08 cm vs. MIX: 25.96 ± 3.95 cm; P = 0.05). Notably, myostatin increases glucose secretion into histotroph and contributes to advanced conceptus development. This advancement in conceptus development occurred in the presence of similar concentrations of serum P4 (P = 0.88) and whole blood Se (P = 0.07) at MRP.

180 Supplementation of One-Carbon Metabolites to Beef Heifers During Early Gestation Increases Fetal Organ Weight at Day 63 of Gestation

Abstract We hypothesized a low maternal plane of nutrition will impair fetal growth in early gestation and that supplementation of one-carbon metabolites (OCM) will rescue the growth restriction. The experimental design was a 2 × 2 factorial with 2 levels of gain, each with or without supplementation of OCM. Angus-cross heifers were bred with female-sexed semen from a single sire. At breeding (d 0), beef heifers were individually fed using a Calan gate system and assigned to treatment (n = 8 per treatment): Control intake (targeted: 0.45 kg/d, actual: 0.60 kg/d ADG) without OCM (CON-OCM), CON with OCM (CON+OCM), Restricted intake (targeted/actual: -0.23 kg/d ADG) without OCM (RES-OCM), or RES with OCM (RES+OCM). The OCM supplement consisted of ruminal-protected choline (60 g/d) and methionine (10 g/d) in a fine-ground corn carrier fed daily, and weekly injections of folate (320 mg) and vitamin B12 (10 mg). The -OCM heifers received the corn carrier and saline injections. Heifers were slaughtered and fetal tissues collected on d 63. Data were analyzed using the MIXED procedures of SAS. Fetal body weight tended (P = 0.07) to be lighter and stomach complex weight was lighter (P = 0.03) in RES than CON. When organ weights were normalized to total brain weight to assess allometric growth, OCM × gain interactions were observed in heart (P = 0.04), left longissimus dorsi (P = 0.04), and right hemisphere (P = 0.01) with CON+OCM greater than CON–OCM. The normalized left hemisphere trend to be greater in RES+OCM than RES-OCM (P = 0.08). These data suggest that OCM supplementation during early gestation increases allometric growth of fetal cardiac and skeletal muscle tissue. Day 63 is within the period of peak primary myogenesis in cattle, therefore differences in muscle growth during this time could have lasting implications in post-natal calf growth and performance.

208 Nutrient Restriction During Late Gestation Alters Beef Heifer Colostrum Composition

Abstract We hypothesized that late gestational nutrient restriction of beef heifers would decrease nutrient partitioning to the mammary gland, resulting in less colostrum nutrient availability for neonatal calves. Fall-calving Hereford-SimAngus heifers (single-sired; BW: 451 ± 28 [SD] kg; BCS: 5.4 ± 0.7) bred to a single sire were individually fed 100% (control; n = 12) or 70% (nutrient restricted; NR; n = 13) of NASEM net energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Diets were based on chopped sorghum sudan hay (1.74 Mcal ME, 6.66% CP, 72.0% NDF; DM basis) supplemented to meet targeted nutritional planes. Calving was observed for all females, and total colostrum from one rear quarter was collected pre-suckling (50.5 ± 17 min [SD] postpartum). Colostrum nutrient composition was determined by colorimetric assays run on a UV-visible light microplate reader. Data were analyzed with treatment, calving date, and calf sex (when P &amp;lt; 0.25) as fixed effects. We have previously reported that single-quarter colostrum weight and volume were 40% less (P = 0.04) in NR compared with control heifers. Colostrum lactose concentration was not affected (P = 0.32) by treatment, but yield differences resulted in 46% less (P = 0.03) total single-quarter colostrum lactose in NR than control. Colostrum from NR heifers had 36% greater (P = 0.004) protein concentration than control, but greater colostrum yield of control heifers allowed for total protein to be unaffected (P = 0.57) by treatment. Nutrient restricted heifers had 23% less (P = 0.04) colostrum urea N concentration and 60% less (P = 0.01) total colostrum urea N than control. Late gestational nutrient restriction (P ≥ 0.59) did not affect colostrum triglyceride concentration or total. In summary, energy and protein restriction had disparate effects on nutrient partitioning to colostrum in primiparous beef females.

68 Late Gestational Nutrient Restriction Decreases Blood Flow to the Mammary Gland in Lactating Primiparous Beef Females

Abstract To determine the effects of late gestational nutrient restriction on mammary blood flow during lactation, fall-calving Hereford-SimAngus heifers (single-sired; BW: 451 ± 28 [SD] kg; BCS: 5.4 ± 0.7) bred to a single sire were individually-fed 100% (control; CON; n = 10) or 70% (NR; n = 13) of NASEM net energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Post-calving, all females were individually fed chopped tall fescue hay supplemented to meet lactational nutrient requirements in Calan gates. Transrectal color Doppler ultrasonography of both external pudendal arteries was conducted every 21 d from d 23 to 107 of lactation. Total mammary blood flow was calculated and hemodynamics from both sides averaged. Data were analyzed with fixed effects of nutritional plane, day of lactation, and their interaction, using repeated measures. Calving date and calf sex (if P &amp;lt; 0.25) were fixed effects, and Doppler angle of insonation was included in the model for flow-related variables. We have previously reported that CON cows had greater (P ≤ 0.003) BW, BCS, and backfat from calving to d 147 of lactation, and NR cows had 15% less (P &amp;lt; 0.04) milk yield. External pudendal artery pulsatility index tended to be greater (P = 0.06) and resistance index, peak systolic velocity, and cross-sectional area were greater (P ≤ 0.04) in CON cows. Heart rate was greater (P = 0.008), yet mammary blood flow was 19% less (P = 0.004) in NR cows. Mammary blood flow/cow BW and blood flow/milk yield were not affected (P ≥ 0.13) by nutritional plane. All hemodynamic variables were affected by day (P ≤ 0.04) except resistance index (P = 0.11). In summary, late gestational nutrient restriction decreased mammary blood flow similarly to the reduction observed in milk yield.

179 Sire Distribution of Calves in a Herd with use of Fixed Time Artificial Insemination Followed by Immediate Bull Exposure for Natural Service

Abstract Use of fixed time AI followed by immediate exposure of females to bulls for natural service can be a useful management strategy for commercial cow-calf producers to limit labor and time related to bull turnout as well as increase pregnancy rates earlier in the breeding season. Considering potential influence of factors such as bull fertility and time to and length of estrus in females, expectations for outcomes in natural service sire versus AI sire parentage is relatively unknown. We hypothesized that of calves born in the first 14 d of the calving season, less than 5% would be sired by natural service bulls exposed to females immediately following fixed time AI. Heifers (n = 191) and cows (n = 156) were synchronized and inseminated using the 7-d CO-Synch + CIDR fixed time AI protocol. All females were inseminated by the same AI technician using a single sire for heifers and a different single sire for cows. All females were exposed to bulls immediately following insemination. DNA was collected from a random subset of calves (Calves born from heifers n = 59; Calves born from cows n = 89) born in the first 14 d of the calving season for parentage analysis to estimate the overall percentage of calves sired by AI and natural service. Chi-Square tests were used to determine if the percentage of calves actually sired by natural service differed from the hypothesized 5% or less. Among calves born from heifers, the actual percentage sired by natural service (5.1%; n = 3/59) was similar (P = 0.98) to our hypothesized percentage. Among calves born from cows, the actual percentage sired by natural service (14.6%; n = 13/89) was greater (P &amp;lt; 0.001) than our hypothesized percentage of 5%. If commercial producers use fixed time AI followed by immediate bull exposure in cows, natural service bulls may sire more calves early in the calving season than expected.

Screening for phenotypic outliers identifies an unusually low concentration of a \u03b2-lactoglobulin B protein isoform in bovine milk caused by a synonymous SNP

BackgroundMilk samples from 10,641 dairy cattle were screened by a mass spectrometry method for extreme concentrations of the A or B isoforms of the whey protein, β-lactoglobulin (BLG), to identify causative genetic variation driving changes in BLG concentration.ResultsA cohort of cows, from a single sire family, was identified that produced milk containing a low concentration of the BLG B protein isoform. A genome-wide association study (GWAS) of BLG B protein isoform concentration in milk from AB heterozygous cows, detected a group of highly significant single nucleotide polymorphisms (SNPs) within or close to the BLG gene. Among these was a synonymous G/A variation at position + 78 bp in exon 1 of the BLG gene (chr11:103256256G > A). The effect of the A allele of this SNP (which we named B’) on BLG expression was evaluated in a luciferase reporter assay in transfected CHO-K1 and MCF-7 cells. In both cell types, the presence of the B’ allele in a plasmid containing the bovine BLG gene from -922 to + 898 bp (relative to the transcription initiation site) resulted in a 60% relative reduction in mRNA expression, compared to the plasmid containing the wild-type B sequence allele. Examination of a mammary RNAseq dataset (n = 391) identified 14 heterozygous carriers of the B’ allele which were homozygous for the BLG B protein isoform (BB’). The level of expression of the BLG B’ allele was 41.9 ± 1.0% of that of the wild-type BLG B allele. Milk samples from three cows, homozygous for the A allele at chr11:103,256,256 (B’B’), were analysed (HPLC) and showed BLG concentrations of 1.04, 1.26 and 1.83 g/L relative to a mean of 4.84 g/L in milk from 16 herd contemporaries of mixed (A and B) BLG genotypes. The mechanism by which B’ downregulates milk BLG concentration remains to be determined.ConclusionsHigh-throughput screening and identification of outliers, enabled the discovery of a synonymous G > A mutation in exon 1 of the B allele of the BLG gene (B’), which reduced the milk concentration of β-lactoglobulin B protein isoform, by more than 50%. Milk from cows carrying the B’ allele is expected to have improved processing characteristics, particularly for cheese-making.

525 Late-Breaking: Late Gestational Nutrient Restriction of Primiparous Beef Heifers Decreases Milk Yield and Pre-weaning Calf Growth

Abstract Fall-calving Hereford-SimAngus heifers (single-sired; BW: 451 ± 28 [SD] kg; BCS: 5.4 ± 0.7) bred to a single sire were individually-fed 100% (control; CON; n = 12) or 70% (nutrient restricted; NR; n = 13) of NASEM net energy and metabolizable protein requirements for maintenance, pregnancy, and growth from d 160 of gestation to calving. Post-calving, all females were limit-fed chopped tall-fescue hay supplemented to meet lactational nutrient requirements in Calan gates to constrain calves’ diets to milk only until d 147. Four-hour milk yields were measured 5 times; calf size was determined every 21 d. Data were analyzed with treatment, calving date, and calf sex (when P &amp;lt; 0.25) as fixed effects; day and day x treatment were included for milk yield. From calving to d 147, CON cows had greater (P ≤ 0.003) BW, BCS, and backfat, but NR cows gained more (P ≤ 0.004) BW, BCS, and backfat. Milk weight and volume was 15% less (main effect; P &amp;lt; 0.04) for NR cows. Calf size did not differ at birth (P ≥ 0.27). From d 42 to 147, calves born to NR dams had lower (P ≤ 0.02) BW. Heart, abdominal, and flank girths were less (P ≤ 0.07) for NR calves through d 147. Calf ribeye area tended to be less (P ≤ 0.11) in NR calves at d 42, 84, and 126. Nutrient restricted calves had less shoulder to rump length (d 63 to 147; P ≤ 0.08), shoulder height (d 63 to 126; P ≤ 0.02), cannon length (d 42 to 105 and 147; P ≤ 0.09), and cannon circumference (d 63 and 105 to 147; P ≤ 0.06). These data indicate that even when dams are fed to meet lactational requirements, milk yield and calf growth are reduced by late gestational nutrient restriction.

PSXII-29 Evaluation of Barbados Blackbelly and St. Croix sheep under accelerated lambing using purebred and terminal sire mating: Lamb survival and body weight

Abstract Objectives were to evaluate lamb survival and body weight (BW) under an accelerated, pasture-lambing system. Lambs were born to Barbados Blackbelly (BB) or St. Croix dams (SC) mated within breed (purebred) or to Dorset (terminal) in single sire mating groups. Lambs were born in April (2013 and 2015), December (2013 and 2015), and August (2014 and 2016) and weaned at 9 weeks of age. Traits considered were BW at birth (BW0; n = 959), 65-d adjusted weaning BW (BW65; n = 788), and survival to weaning (n = 959). Data were analyzed with fixed effects of dam age, sex, birth type, dam breed (BB vs SC), dam mating system (purebred vs terminal), birth month-year, and significant two-way interactions and a random dam effect. Dam breed did not impact BW65 (P = 0.09). While BW0 was greater for lambs born to SC than BB dams (3.44 vs 3.23 kg; P &amp;lt; 0.01), survival to weaning was greater for lambs born to BB than SC dams (0.93 vs. 0.87; P = 0.02). The dam mating system x birth month-year interaction was significant for both BW traits (P ≤ 0.01). Within birth month-year, BW0 was 18 to 24% greater and BW65 was 12 to 24% greater for terminal than purebred lambs (P &amp;lt; 0.01). However, BW0 differences between dam mating systems were greatest for August born lambs whereas BW65 differences were greatest for April born lambs. Lamb survival was not affected by dam mating system or birth month-year (P ≥ 0.27). Using a terminal sire improved BW without affecting survival of lambs reared by landrace hair sheep dams, making this a viable management tool to increase productivity of forage-based production systems.

342 Effects of Maternal Nutrient Restriction During Late Gestation on Primiparous Dam Performance and Fetal Growth

Abstract To determine the effects of late gestational nutrient restriction (NR) on heifer performance, fetal growth, and calving difficulty, single-sired fall-calving Hereford-SimAngus heifers (BW: 451 ± 28 [SD] kg; BCS: 5.4 ± 0.7) bred to a single sire were allocated by fetal sex and expected calving date to either 100% (control; CON; n = 12) or 70% (n = 13) of NASEM net energy and metabolizable protein requirements for maintenance, pregnancy, and growth. Beginning on d 160 of gestation, heifers were individually fed chopped sorghum sudan hay (1.74 Mcal ME, 6.66% CP, 72.0% NDF; DM basis) and based on individual intakes, supplemented to meet targeted nutritional planes. Dam BW, BCS, and backfat (BF) were determined pre-treatment, every 21 d (BW) or 42 d (BCS and BF) during treatments, and post-calving. At birth, calf BW and size (length, heart girth, abdominal girth, flank girth, cannon circumference, cannon length, shoulder height, and ribeye area) were measured. Data were analyzed with treatment, treatment initiation date, and calf sex (when P &amp;lt; 0.25) as fixed effects. Dam BW tended to be less (P = 0.09) and BCS was less (P = 0.04) at d 202 of gestation for NR dams. Dam BW, BCS, and BF were less (P ≤ 0.01) in NR dams for the remainder of gestation. Post-calving, NR dams weighed 64 kg less (P ≤ 0.01) than CON, with a BCS of 3.6 ± 0.1. Nutrient restriction did not affect (P ≥ 0.27) calf gestation length, BW, or size. Calf BW as % post-calving dam BW tended to be greater (P = 0.09) for NR calves. Fetal presentation was normal for all CON births, while 23.1% of NR dams had fetal malpresentation (P = 0.12). In the current study, NR dam BW and BCS were sacrificed during late gestation without altering fetal growth trajectory.

Genome-Wide SNP Analysis Reveals Multiple Paternity in Burmese Pythons Invasive to the Greater Florida Everglades

Reproductive strategies are an essential component of invasion ecology that influence invasion success and rates of population growth. Burmese Pythons (Python bivittatus) are large constrictor snakes that were introduced to the Greater Everglades Ecosystem of southern Florida, USA, from Asia. Since their introduction, these giant constrictors have spread throughout wetlands of southern Florida while increasing in abundance and causing declines in the native species upon which they prey. Multiple paternity in reproduction could facilitate invasion success by increasing the genetic diversity produced within each reproductive event. We used Diversity Arrays Technology genome-wide genotyping to assess multiple paternity in the progeny of wild Burmese Pythons in Florida. We analyzed >4,000 single nucleotide polymorphisms from 153 neonates belonging to 4 clutches collected in southwestern Florida. Complementary hierarchical and K-means clustering analyses of the genetic distances within clutches revealed that three clutches were each fertilized by two sires, with a fourth fertilized by a single sire. The proportions of offspring attributable to each sire within multiple paternity clutches ranged from nearly even to highly skewed. Analysis of multivariate dispersion showed significantly increased genetic variability in the multiple paternity clutches. These results improve our understanding of the reproductive strategy and invasion potential of a giant constrictor with significant ecological impacts.

A retrospective analysis of bull:cow ratio effects on pregnancy rates of beef cows previously enrolled in fixed-time artificial insemination protocols.

This retrospective study aimed to determine if the number of cows exposed per bull affects pregnancy rates of cows returning to estrus after fixed-time artificial insemination (FTAI). Data were compiled over the course of 13 breeding seasons (six fall and seven spring seasons) between 2010 and 2017 from the Virginia Department of Corrections herd. Available records contained data from 17 farms and 324 groups of cows (average 47 cows/group). Multiparous cows and heifers (average age per group: 5.11 ± 0.14 yr; n = 14,868) were exposed to FTAI. After FTAI, animals were placed on pasture with bulls diagnosed as fertile by a breeding soundness exam for natural service of cows who did not become pregnant to FTAI (n = 7,248; average 22 cows/group). Animals were classified as pregnant to FTAI, to natural service on first return to estrus, or to natural service on second or subsequent estrus determined by fetal aging at pregnancy diagnosis. The bull:cow ratio for the total number of cows exposed ranged from 1:9 to 1:73 with an average of 1:31. The bull:cow ratio considering only open cows exposed after FTAI ranged from 1:2 to 1:44 with an average of 1:14. There was significant negative, small correlation between the bull:cow ratio for total number of cows exposed and return to estrus pregnancy rate in fall breeding seasons (P = 0.01, r2 = 0.04) but not in spring (P = 0.90). There was a significant negative, small correlation between bull:cow ratio of open cows exposed and pregnancy rates to first return to estrus in fall herds with a single sire (P < 0.001, r2 = 0.11). There was no correlation in fall herds using multiple sires or spring herds (P ≥ 0.12). Bull:cow ratio accounted for only 1–11% of variation in the pregnancy rates, thus we conclude that a decreased bull:cow ratio (up to 1:73) did not affect natural service return to estrus pregnancy rate. Cattlemen may consider a reduced number of bulls needed for natural service breeding after FTAI, which can decrease bull related costs and increase the economic feasibility of adopting FTAI protocols.

Effects of Maternal Nutrient Restriction During Gestation on LH Production in the Female Bovine Fetus

Abstract Gestational nutrient restriction causes epigenetic and phenotypic changes that affect multiple physiological processes in the offspring. Gonadotropes, the cells in the anterior pituitary that secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are particularly sensitive to nutritional changes during fetal development. Our objective herein was to investigate the effects of gestational nutrient restriction on LH protein content and number of gonadotropes in the fetal bovine pituitary. We hypothesized that moderate nutrient restriction during mid to late gestation decreases pituitary LH production, which is associated with a reduced number of gonadotropes. Embryos were produced in vitro with X-bearing semen from a single sire then split to generate monozygotic twins. Each identical twin was transferred to a virgin dam yielding four sets of female twins. At gestational d 158, the dams were randomly assigned into two groups, one fed 100% NRC requirements (control) and the other fed 70% of NRC requirements (restricted) during the last trimester of gestation, ensuring each pair of twins had one twin in each group. At gestational d 265, the fetuses (n = 4/group) were euthanized by barbiturate overdose, and the pituitaries were collected. Western blots were performed using an ovine LH-specific antibody (Dr. A.F. Parlow, NIDDK). The total LH protein content in the pituitary tended to be decreased in the restricted fetuses compared to controls (P &amp;lt; 0.10). However, immunohistochemistry analysis of the pituitary did not reveal any significant changes in the total number of LH-positive cells (control = 460±23 cells/0.5 mm2; restricted = 496±45 cells/0.5 mm2, P = 0.58). In conclusion, while maternal nutrient restriction during gestation resulted in a trend of reduced LH content in the fetal pituitary, immunohistological findings suggest that these changes are likely related to the individual potential of each gonadotrope to produce LH, rather than alterations in cell differentiation during fetal development.

14 Opening the Black Box of Fertility Prediction

Abstract Analysis of the U.S. swine herd shows variation in pregnancy rate is more attributable to male-factor subfertility than the dam. To date, a limited degree of correlations has been observed between conventional semen analysis parameters and actual fertility after standard quality cutoffs are met. Thus, a clear ability to predict male-factor fertility is lacking. Knowledge of what makes fertilization competent spermatozoa has been long sought after for centuries. It was only in the last half-century that we understood spermatozoa undergo a biological process after ejaculation to acquire the capacity to fertilize. Since then, work has been done to elucidate the molecular pathways involved in sperm capacitation. Recent technological advances in flow cytometry, namely image-based flow cytometry, allows for high-throughput, single-cell phenotyping. Single-cell phenotyping with biomarkers reflecting significant sperm capacitation events, mitochondrial status, cell health, and more, allows multi-million bioimage data sets to be easily attained. These datasets can then be analyzed utilizing machine and deep learning analytic methods and correlated with single sire field fertility data to open the black box of boar fertility prediction. Our findings establish a new paradigm in sperm function and pave the way for accurate fertility prediction in future precision agriculture applications. This work was supported by the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture (USDA) award number 2019-67012-29714.

Hiphop: Improved paternity assignment among close relatives using a simple exclusion method for biallelic markers.

Assignment of parentage with molecular markers is most difficult when the true parents have close relatives in the adult population. Here, we present an efficient solution to that problem by extending simple exclusion approaches to parentage analysis with single nucleotide polymorphic markers (SNPs). We augmented the previously published homozygote opposite test (hot), which counts mismatches due to the offspring and candidate parent having different homozygous genotypes, with an additional test. In this case, parents homozygous for the same SNP are incompatible with heterozygous offspring (i.e., "Homozygous Identical Parents, Heterozygous Offspring are Precluded": hiphop). We tested this approach in a cooperatively breeding bird, the superb fairy-wren, Malurus cyaneus, where rates of extra-pair paternity are exceptionally high, and where paternity assignment is challenging because breeding males typically have first-order adult relatives in their neighbourhood. Combining the tests and conditioning on the maternal genotype with a set of 1376 autosomal SNPs always allowed us to distinguish a single most likely sire from his relatives, and also to identify cases where the true sire must have been unsampled. In contrast, if just the hot test was used, we failed to identify a single most-likely sire in 2.5% of cases. Resampling enabled us to create guidelines for the number of SNPs required when first-order relatives coexist in the mating pool. Our method, implemented in the R package hiphop, therefore provides unambiguous parentage assignments even in systems with complex social organisation. We also identified a suite of Z- and W-linked SNPs that always identified sex correctly.

101 Heifer plane of nutrition during the prepubertal phase modifies reproductive development in male fetuses

Maternal nutritional perturbation during gestation influences fetal development which, in turn, can affect postnatal growth, gonad development, gamete quality, and health of the offspring. The objective of this study was to determine whether maternal diet during the pre-pubertal phase would induce modifications in future male fetal reproductive development. Angus×Holstein-Friesian heifers (n=80) with a mean (±s.d.) age of 141±8d and bodyweight (BW) of 119±23kg had unrestricted access to a perennial ryegrass dominated sward supplemented with either 0.5kg of concentrate daily to achieve an average daily gain (ADG) of 0.50kg (moderate, MOD) or concentrate ad libitum to achieve an ADG of &amp;gt;1.0kg (high, HI). Heifers were offered these divergent dietary regimens from 4.5 to 8.5 months of age, after which they were all offered a moderate plane of nutrition. Heifers were bred to a synchronised oestrus at a mean age of 502±8.2d and mean BW of 360±46.7kg using semen from a single proven sire. Transrectal uterine ultrasonography was carried out 63 days post-AI to determine pregnancy and fetal sex; a cohort of heifers reflective of group average BW and age and carrying male fetuses were slaughtered on Day 100 of gestation (HI: n=10; MOD: n=12). Fetal crown–rump length and weight were recorded. One testis was snap frozen for RNA sequencing and the other was processed for stereology. Mean (±s.d.) fetal crown–rump length and weight were 19.5±0.69cm and 314.6±33.6g, respectively, and did not differ between groups. RNA sequencing revealed 45 differentially expressed genes (DEGs) between groups, 44 of which were upregulated in the HI group. A total of 29 biochemical pathways were identified as significantly enriched (P&amp;lt;0.05), six of which were predicted to be involved in the development of the reproductive tract based on several DEGs. Histological analysis revealed greater (P&amp;lt;0.05) fetal testicular interstitial tissue density in the MOD group and a strong tendency towards increased seminiferous tubule density (P=0.06) in the HI group. In conclusion, offering a high plane of nutrition during the prepubertal period not only increases the rate of sexual development in the heifer but also induces latent in utero modifications to the testicular development of their subsequent male progeny. The consequences for postnatal development of male progeny remain to be elucidated. This research was supported by Science Foundation Ireland (16/IA/4474).

201 Effects of feeding vitamin and mineral and (or) energy supplements to beef heifers during the first 83 days of gestation on progesterone concentrations, corpus luteum size, and fetal body measurements

Abstract Our objectives were to determine the effects of feeding vitamin and mineral (VTM) and (or) energy (NRG) supplements to beef heifers during the first 83 days of gestation on concentrations of progesterone (P4), corpus luteum (CL) size, and fetal body measurements. Crossbred beef heifers (n = 35; initial BW = 359.5 ± 7.1 kg) were blocked by weight and assigned to one of four treatments (n = 8 or 9/group) in a 2 × 2 factorial arrangement with main effects of vitamin and mineral (NoVTM or VTM) and energy (NoNRG or NRG) supplements. A basal total mixed ration was fed once daily with VTM and NRG top-dressed. The VTM treatment (113 g•heifer-1•d-1) was initiated 71 to 148 d before AI. At AI, heifers were bred to a single sire, and NRG was initiated with target gains of 0.28 kg/d for NoNRG and 0.79 kg/d for NRG. Serum was collected on d 14, 28, 42, 56, 70, and 83 after AI and analyzed for. Gravid reproductive tracts were collected by ovariohysterectomy on d 83 ± 0.27 of gestation, and fetuses were dissected. An NRG × day interaction (P = 0.006) was observed for P4, with concentrations similar on d 14 to 56, a tendency (P = 0.09) for divergence at d 70, and greater (P = 0.002) concentrations for NRG on d 83 compared with NoNRG heifers. Furthermore, NRG heifers had greater CL weights than NoNRG (P = 0.003). Fetuses from NRG dams had greater (P = 0.009) femur weights than fetuses from NoNRG. Fetal liver weight was greater (P = 0.05) from dams fed VTM than NoVTM. Overall, providing NRG supplements during early gestation resulted in heavier CLs, that produced more P4, and greater fetal femur growth, whereas providing VTM supplements enhanced fetal liver growth.

37 Early pregnancy detection in cattle by analysis of maternal circulating microRNA expression

Abstract Pregnancy detection in cattle is performed by rectal palpation, transrectal ultrasonography or detection of pregnancy associated glycoproteins at approximately d 28 of gestation. Majority of embryonic loss in cattle occurs much earlier. MicroRNAs (miRNAs) are excellent markers for physiological states including pregnancy. The objective of this experiment was to assess expression of miRNAs in maternal serum to serve as markers for pregnancy detection at d 18 post-artificial insemination. Cows of varying age and parity (n = 80) were synchronized using a 7-d Co Synch and CIDR estrous synchronization protocol. One insemination by a single sire was performed 60 to 66 h following CIDR removal. Blood was collected via jugular venipuncture on d 0 and 18 post-insemination. Total RNA isolation was performed using PAXgene Blood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) or by miRNeasy (Qiagen, Hilden, Germany). Ultrasound was performed d 30 of gestation. Taqman microRNA assay utilizing primers for Let7a, mir-16,15b, 25, and 26 was performed on RNA from whole blood and serum in pregnant (n = 10) and open (n = 10) cows. Analysis of variance and pair-wise student’s t-test among LSMeans for relative abundance were performed with fixed effects for pregnancy, day, isolation, and their interactions. MicroRNA abundance did not differ due to pregnancy. Relative abundance for miR-16 increased in d 18 (1.86 ± 0.49) compared to d 0 samples (3.42 ± 0.48), regardless of pregnancy status or isolation method (P = 0.03). Let-7a (serum, 0.99 ± 0.98; PAXgene, 6.25 ± 0.87), miR-15b (serum, 0.59 ± 1.67; PAXgene, 12.44 ± 1.48) and miR-26 (serum, 1.44 ± 1.2; PAXgene, 7.64 ± 1.06) relative abundance was higher in serum compared to PAXgene isolated samples (P &amp;lt; 0.05). These results disagree with previous reports and indicate that these candidate miRNAs are not a reliable indicator for early pregnancy detection in cattle.