Antral follicle count (AFC) and anti-Müllerian hormone (AMH) concentrations are reflective for ovarian reserve and have been associated with improved reproductive performance in cattle. Key events for regulation of uterine receptivity are orchestrated by progesterone. As progesterone concentrations are greater in animals with high than low AFC, we tested the hypothesis, if the resulting improved uterine environment will lead to improved conceptus elongation and endometrial response to interferon tau. For four years, 10 heifers with lowest and highest AFC, respectively, were selected from 120 heifers. Reproductive tracts and blood samples for progesterone and AMH analysis were collected after synchronization and insemination. For a recovered conceptus, length was determined, and interferon tau (IFNT) transcript abundance was analyzed. Endometrial transcript abundance of interferon-stimulated gene 15 (ISG15) and oxytocin receptor (OXTR) were analyzed. Progesterone concentrations did not differ between low and high AFC groups (P = 0.1). A difference in conceptus length was not observed. Endometrial abundance of ISG15 did not differ between pregnant low and high AFC heifers. Abundance of OXTR was greater in open low AFC than open high AFC heifers (P < 0.01). Interaction of AMH and pregnancy status was determined, with greater AMH in pregnant than open high AFC heifers (P < 0.05). Improved uterine environment in high vs. low AFC heifers did not result in longer conceptuses or improved endometrial response. As the increase in OXTR transcript abundance was only detected in low AFC heifers, reported differences in reproductive performance might be associated with earlier initiation of luteolysis.

Transrectal ultrasonography is known as the gold standard for pregnancy detection, but requires costly equipment and technical skills; therefore, access to an inexpensive and more user-friendly method with similar accuracy could benefit cattle producers. Detection of pregnancy-associated glycoproteins can accurately determine pregnancy in ruminants; however, usually requires specialized equipment for the assay. Thus, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. In study 1, blood samples from previously identified pregnant Bos taurus females from six different herds (nulliparous n = 1,205 and multiparous n = 1,539; samples collected between d 27 to 285 of gestation over a three-year period) were utilized. In study 2, postpartum females (primiparous n = 48 and multiparous n = 66) from one herd were utilized: (n = 1,066; samples collected weekly for up to 12 weeks postpartum). In study 1, level of agreement between different methods of pregnancy detection was determined by Pearson’s correlation and Kappa scores. In study 2, data were analyzed as a repeated measure using the MIXED procedure of SAS with main effects of parity, days postpartum (dpp), and parity by days postpartum, then data were analyzed further using the REG procedure of SAS. In study 1, transrectal ultrasonography and lateral flow were positively correlated (r = 0.77; P <0.01), with 92.4% agreement. In study 2, the abundance of absorbance of PAGs rapidly decreased from 0 to 50 days postpartum, then continued to gradually decrease (P <0.01; r = 0.90). Prior to 42 days postpartum, PAG concentrations were sufficiently elevated resulting in false positive readings in all assays. In conclusion, there is very good agreement between transrectal ultrasonography and PAG assays, but likelihood of false positive results are highif assays are performed fewer than 42 days postpartum.

Vaccines are created to help the immune system fight pathogens faster and more effectively, but little is known on how season of the year impacts the immune system response to vaccination. This research was conducted to evaluate how ambient temperature (summer vs fall) impacted antibody production and immune cell type changes in Brahman and Brahman-influenced cows. Cows (n = 18) were immunized (2 mL I.M.) with a combination Modified Live Vaccine for Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBR) on d 0 in either July 2022 (Summer; n = 12) or November 2022 (Fall; n = 6). Blood samples (30 mL/cow) were collected on d -3, 0, 2, 4, 6, 8, 10, 14 post vaccination and transferred to 50 mL conical tubes containing EDTA to separate plasma and peripheral blood mononuclear cells (PBMC). Density gradient centrifugation was used to isolate PBMC. Serum samples were also collected (10 mL/cow) on d -3, 0, 14. Cells were incubated with propidium iodide and antibody surface cell markers for CD4, CD8, CD25, CD14, CD86, and CD335. An Amnis FlowSight flow cytometer was used to determine the percentage of each cell type in the PBMC. Concentrations of antibodies for BVDV and IBR were determined in serum samples collected pre-vaccination (d -3 and 0) and on d 14. Differences in antibody titers and leukocyte populations were analyzed as repeated measures using the MIXED model procedure in SAS. There were no differences in IBR (P = 0.2) or BVDV (P = 0.47) antibody concentrations between seasons. Percentage of antigen presenting cells (CD14+) was greater in the Summer compared with the Fall (P = 0.01; Summer=11.7 ± 1%; Fall = 7.3 ± 1.1%), but natural killer cells (CD335+) were greater in the Fall compared with the Summer (P = 0.002; Summer = 0.6 ± 0.6%; Fall = 4.4±0.7%). There was a significant treatment by time interaction for antigen presenting cells (P = 0.04), T-helper cells (CD4+; P = 0.01), activated T-helper cells (CD4+CD25+; P ≤ 0.0001), activated cytotoxic T-cells (CD8+CD25+; P = 0.03), activated T-cells (CD25+; P ≤ 0.0001), and natural killer cells (P ≤ 0.0001), but there was no difference between seasons in activated antigen presenting cells (CD14+CD86+; P = 0.19). From pre-vaccination to d 14, natural killer cells were elevated during the Fall compared with the Summer on all days (P = 0.04). Antigen presenting cells were greater in the Summer compared with the Fall on d 4, 10 and 14 (P < 0.02). T-helper cells, activated T-helper cells, activated cytotoxic T-cells, and activated T-cells increased post-vaccination in the Fall, but did not change during the Summer. Thus, ambient temperature (summer vs fall) did not directly impact production of antibodies for IBR or BVDV but did induce changes in specific immune cells following vaccination.

This experiment aimed to compare synchronization cost associated with two protocols: one utilizing estrus detection (6d; PG 6-d CIDR+TAI) and one utilizing strictly fixed-time artificial insemination (FTAI; 7d; 7-d CO-Synch+CIDR). Cows and heifers (n = 657) from 10 herds in three states were grouped by days postpartum and age, and randomly assigned to one of two protocols. Cows and heifers assigned to the 6 d protocol were administered prostaglandin F2α (PGF) on d-9, CIDR insertion and administration of gonadotropin-releasing hormone (GnRH1) on d -6, and CIDR removal and PGF administration on d 0. Cows and heifers that were assigned to the 6-d protocol were subsequently grouped into three treatments based on presence and/or timing of estrus and insemination (PG1: Insemination before CIDR insertion, n = 136; PG2: Insemination after CIDR removal, n = 26; T6: Insemination at TAI, n = 166). Cows and heifers assigned to the 7-d protocol (T7: Insemination at FTAI, n = 329) were administered GnRH1 and CIDR insertion on d -7, followed by CIDR removal and PGF administration on d 0. For all animals, FTAI and GnRH2 administration occurred at 54 h (heifers) or 66 h (cows) after CIDR removal. Pregnancy was determined from d 30 to d 40 after FTAI by transrectal ultrasonography. Total cost and profit per pregnancy was calculated for herds of 30, 100, 300, and 500 females with varying estrus response and pregnancy rates. Expenditures were calculated assuming synchronization costs (PG1 = $46.15, PG2 = $75.05, T6 = $77.91, T7 = $73.72), general labor cost considering herd size and handling days, as well as estrus detection labor cost assuming $10.00 per h, a 95% overall herd pregnancy rate, a 1:40 bull:cow ratio, and average bull purchase price of $3,000.00. Revenue was estimated assuming a maximum 205-d weaning age, 1.09 kg/d gain, $200.00/45.4 kg, and 85% weaning rate. Differences in estrus response and pregnancy rate were determined using the GLIMMIX procedure of SAS (v9.4), including herd as a random variable. Estrus response was greater (P < 0.0001) in females assigned to the 6d (73 ± 4%) compared with the 7d (53 ± 4%) protocol; however, pregnancy success was similar among treatment groups (P = 0.32; PG1 = 45 ± 4%, PG2 = 58 ± 10%, T6 = 46±4%, T7 = 52±3%). At the observed estrus response and pregnancy rate for each treatment group, the 6d protocol resulted in a decreased cost per pregnancy compared with the 7d protocol [Table 1; herds of: 30 = ($3.53), 100 = ($5.16), 300 = ($6.58), 500 = ($7.00) females]. Additionally, using the same variables, the 6d protocol resulted in an increased profit per pregnancy compared with the 7d protocol (herds of: 30 = $3.50, 100 = $6.41, 300 = $17.27, 500 = $12.06 females). These results suggest that labor associated with estrus detection may be offset by the proportion of calves subsequently born earlier within the calving season, allowing for improvements to profitability, especially in larger herds.

Introduction Sperm interacts with the female reproductive tract and oocyte through proteins, and these cell-to-cell interactions may play a role in sperm fertility. For consideration of a protein as a potential marker of fertility, there must be variability expressed among animals. The proteins dystroglycan (DAG1) and plasma serine protease inhibitor (SERPINA5) have been reported to play a role in cell-to-cell interactions. Thus, the objectives of this study were to characterize the localization and abundance variability of DAG1 and SERPINA5 in bovine sperm, and to investigate the relationship of DAG1 and SERPINA5 with field fertility (i.e., sire conception rate; SCR), in vitro embryo production (IVP), and sperm parameters. Material and methods Dairy bulls (n = 22) were classified as high-SCR (SCR > 1.0) or low-SCR (SCR < –4.0), and good [blastocyst (BL)-by-cleavage (CL) ratio (BL/CL) > 39%] or poor (BL/CL < 38%) BL/CL. Sperm was evaluated for DAG1 and SERPINA5 immunolocalization, and concentration in two separate ejaculates. Variance between bulls compared with within bulls was evaluated using a generalized linear model (GLM) procedure. The relationship of SCR and IVP classification on DAG1 and SERPINA5 concentrations, percentage of tail labeled for SERPINA5, SCR, sperm total and progressive motility, sperm plasma membrane integrity (PMI), CL, BL, and BL/CL were evaluated with the GLIMMIX procedure, and the correlations between these variables were evaluated. Results Both proteins were localized on the sperm head; however, SERPINA5 was also localized on the sperm tail. There was greater variance in concentration among bulls than within bulls for DAG1 ( P < 0.0001; 69.4 vs . 49.1, respectively) and SERPINA5 ( P < 0.0001; 325.8 vs . 285.4, respectively). There was a positive correlation between the concentrations of DAG1 and of SERPINA5 ( P = 0.01; r = 0.54). In addition, the percentage of tail labeled for SERPINA5 was correlated with PMI ( P = 0.05; r = 0.44). There was no relationship between SCR and IVP classifications and DAG1 (P ≥ 0.55), SERPINA5 (P ≥ 0.54), or the percentage of sperm tail labeled for SERPINA5 (P ≥ 0.22). Discussion In conclusion, DAG1 and SERPINA5 were localized to the sperm head, and SERPINA 5 was also localized to the tail. Concentrations of DAG1 and SERPINA5 on the sperm head were correlated with each other. The percentage of tail labeled for SERPINA5 was correlated with sperm PMI; however, neither protein was associated with SCR or IVP. Thus, when evaluated by immunofluorescent microscopy, DAG1 and SERPINA5 concentrations are variable and are not good fertility markers for bull sperm.

Assisted reproductive technologies are used to propagate desirable genetics in a shortened timeframe. Selected females undergo ovarian stimulation with the use of follicle stimulating hormone (FSH) to increase embryo recovery for subsequent transfer programs. The FSH receptor (FSHR) c.337 C > G variant was reported to have a reduction in viable embryo numbers in an ovarian stimulation protocol. We, therefore, hypothesized that FSHR c.337 C > G would result in reduced in-vitro blastocyst development. Beef heifers were genotyped and selected based on the c.337 C > G FSHR genotype (CC, CG, GG; n = 15-16/genotype). Estrus was synchronized with a Select Synch protocol and heifers were slaughtered 5 days after induced ovulation. Anterior pituitaries, serum and reproductive tracts were collected at slaughter for analysis. Cumulus oocyte complexes (COCs) were collected and pooled within genotype for in-vitro fertilization (IVF) and subsequent blastocyst development. No differences were observed in carcass weights, anterior pituitary weights, serum progesterone, corpus lutea weight, surface follicle counts, histological follicle counts or follicular fluid estradiol concentration (P > 0.1) due to FSHR genotype. Differences were observed for ovulation rates in the GG FSHR genotype group (P < 0.01). However, cleavage and blastocyst rates were not affected due to FSHR genotype (P > 0.1), following standard IVF protocols. The FSHR variant does not influence antral follicle counts, estradiol production, or in-vitro blastocyst development in beef heifers. The GG FSHR genotype had an increased ovulation rate, which may indicate a greater potential for twinning, but research with a larger population is warranted to support this hypothesis.

The objective of this study was to determine if prenatal stress impacted semen characteristics during pubescent maturation of Brahman bulls (n = 23). Prenatal stress was achieved by transporting Brahman dams for 2 h on d 60, 80, 100, 120, and 140 (± 5 d) of gestation. Semen from sexually maturing males born to transported (PNS; n = 11) or non-transported (CON; n = 12) dams was classified based on motility and concentration as prepubertal (< 10% and < 50 million), peripubertal (≥ 10% or ≥ 50 million), pubertal (≥ 10% and ≥ 50 million), or sexually mature (≥ 30% or ≥ 500 million). Sperm characteristics were determined by staining sperm with specific dyes [viability - Propidium Iodide and SYBR-14; Mitochondrial membrane potential (MMP) – JC-1; DNA damage – Aracidine orange; ROS –conversion of H2DCFDA to DCF and conversion of hydroethidine to ethidium] and analysis on an Amnis FlowSight flow cytometer. Treatment differences were tested by SAS procedures. Analysis of repeated measures (PROC MIXED) and chi-square (PROC FREQ) compared semen characteristics and percent pubertal versus mature, respectively. There was a weak tendency (P = 0.14) for more control bulls to reach puberty (100%) compared with PNS bulls (82%), but no difference (P = 1.0) in percentage reaching sexual maturity by 22 months of age (82% and 82%). Scrotal circumference (P < 0.01) and motility [at collection (P < 0.01) and post-thaw (P = 0.05)] increased as bulls matured but was not impacted by treatment (P > 0.86) or treatment by time (P > 0.60). Sperm viability (at collection, post-thawing, and post-stress test) was not impacted by treatment (P > 0.20), time (P > 0.53), or treatment by time (P > 0.50). Proportion of viable sperm with high MMP was not impacted by treatment (P = 0.70) or treatment by time (P = 0.49), but was increased (P = 0.03) in early peripubertal collections compared with pubertal or mature collections. Proportion of sperm with damaged DNA was decreased in PNS (1.6 ± 0.3%; P = 0.04) versus CON (2.4 ± 0.2%) but not impacted by time (P = 0.47) or treatment by time (P = 0.41). Percentage of sperm with elevated ROS (H2O2 and •O2) was not impacted by treatment (P > 0.33), time (P > 0.17), or treatment by time (P > 0.41). Prenatal stress tended to impact the proportion of bulls that reached puberty but had no impact on the percentage that reached sexual maturity by 22 months of age. Prenatal stress also impacted the proportion of sperm with DNA damage, but did not impact scrotal circumference, motility, MMP, or ROS. Stage of sexual maturity impacted scrotal circumference, motility, and MMP, but did not impact ROS. In summary, PNS may impact the ability to reach puberty, but did not impact sperm characteristics from prepuberty to sexual maturity.

Objective: Studies evaluating single and multiple gonadotropin-releasing hormone (GnRH) treatments at time of prostaglandin (PG) administration using the 7-d CO-Synch + controlled internal drug-release device (CIDR) protocol reported decreased interval to estrus and in-creased estrus expression, or increased conception rates, respectively. Therefore, the objectives of these studies were to determine effects of supplementing GnRH at PG on estrus expression and conception rates in beef females synchronized with different protocols.Materials and Methods: In Exp 1 and 2, heifers (n = 1,626) and cows (n = 1,409), respectively, were synchronized using the 7-d CO-Synch + CIDR fixed-time AI (FTAI) protocol. In Exp 3, heifers (n = 3,270) were synchronized using the melengestrol acetate-PG FTAI heifer protocol; all animals were randomly assigned to receive a single dose of GnRH (5 mu g) at PG or no GnRH (control). Estrus and pregnancy rates were analyzed using the GLIMMIX procedure of SAS.Results and Discussion: In all experiments, there was no effect of treatment on estrus (P >= 0.21), but there was an effect of estrus (P < 0.0001) on FTAI pregnancy rates. Estrual females had greater FTAI pregnancy rates compared with nonestrual females. In Exp 1 there was a treatment x estrus interaction (P = 0.01) on FTAI pregnancy rates. Heifers in the control group that exhibited estrus had greater (P <= 0.04) FTAI pregnancy rates (72.8 +/- 3.2%) compared with estrual heifers that received 5 mu g of GnRH (67.2 +/- 3.4%) and compared with heifers that did not exhibit estrus in the control (54.4 +/- 4.7%) or 5 mu g of GnRH (60.8 +/- 4.6%) groups. There was no effect (P >= 0.17) of treatment on breeding-season pregnancy rates in any of the experiments.Implications and Applications: Utilization of a supplementary dose of 5 mu g of GnRH may be a useful tool to address differences in fertility of FTAI in short-term progestin synchronization protocols among animals that do not exhibit estrus.

Nutritional changes immediately after insemination cause increased embryonic mortality, but the mechanisms controlling this are not well known. Our objective was to evaluate the impact of nutritional change on estrus expression, steroid concentrations, peripheral and uterine luminal fluid metabolites, and embryo quality in beef heifers. Heifers (n = 139) were assigned to one of two pre-artificial insemination (AI) dietary treatments: LOW (≤ 90% NEm) or HIGH (≥ 139% NEm). Heifers were on treatment for 33–36 days before AI (d0) when half of the heifers in each treatment were randomly reassigned to generate four treatments; HIGH-HIGH, HIGH-LOW, LOW-HIGH, and LOW-LOW. Heifers remained on treatments until embryo collection (d 6–8). Negative energy balance was achieved among LOW heifers as demonstrated by body weight loss and increased NEFA concentrations (P < 0.05). Pre-AI treatment influenced expression of estrus (P = 0.05; HIGH 80.4 ± 4.0% vs. LOW 69.4 ± 4.2%). Estradiol concentrations and interval to estrus were not affected by treatment (P > 0.55); however, progesterone concentrations were reduced among LOW compared to HIGH (3.57 ± 0.27, 4.64 ± 0.26 ng/mL, respectively; P = 0.004), and heifers maintained on the HIGH pre-AI diet had consistently greater concentrations of progesterone from d 0 to d 8 (P = 0.014). Pre-AI treatment influenced embryo stage (P = 0.05; HIGH 3.61 ± 0.32 vs. LOW 2.72 ± 0.30). Post-AI treatment affected embryo grade (P = 0.02; HIGH 1.78 ± 0.23 vs. LOW 2.64 ± 0.27). In summary, pre-AI nutrient restriction caused decreased expression of estrus, reduced progesterone concentrations after AI, and negatively impacted embryo development, while post-AI restriction hindered embryo quality.

Damage to the bovine corpus luteum (CL) has been reported following modified-live virus vaccination (MLV). The objective of this study was to investigate the degree of luteal apoptosis after MLV or inactivated vaccine (IV) administration at time of artificial insemination. Beef females were estrous synchronized and on d 0 were vaccinated with a MLV or IV after being transrectally ultrasounded to record presence and location of dominant follicles and CL. Thirteen cows (MLV, n=7; IV, n=6) were selected on d 10-13 across treatment based on dominant follicle size and estrus expression for ovariectomy. Ovaries were obtained and CL were frozen in OCT and sectioned for immunohistochemistry analysis. At the time of ovary collection, some MLV animals had two CL. These were characterized into old or new CL, relative to if ovulation had occurred in response to the synchronization protocol or afterward due to an abnormal cycle, respectively. For immunohistochemistry analysis, two nonconsecutive sections were evaluated for apoptosis using a TUNEL staining kit (ab66110) according to manufacturer’s instructions, with the modification of DAPI being used as a cell marker. Two random fields of view from each section were photographed with a Keyence BZ-X810 microscope and were evaluated for number of total and apoptotic cells. The GLIMMIX procedure of SAS was utilized to analyze percentage of apoptotic cells with image included as a random effect. Percentage of apoptotic cells were affected by treatment (P < 0.0001). MLV-new CL had the greatest percentage (35.56 ± 5.5%) of luteal cell apoptosis, while MLV-old CL (7.05 ± 4.6%) and IV (4.24 ± 4.9%) were similarly decreased. In summary, a greater degree of apoptosis in new CL which formed after an abnormal cycle following MLV administration indicates the ability of MLV to induce estrous cycle dysfunction and extend its effects to luteal cell development and function.

The objective of this study was to determine factors impacting abundance of pregnancy-associated glycoproteins (PAGs) after embryo transfer. Cows were synchronized using the 7-d CO-Synch+CIDR® protocol and grouped by estrus expression 48h after prostaglandin (d0) [Estrual (PC): n = 198; Non-estrual: n = 406]. Non-estrual cows were administered GnRH and randomly assigned to Estradiol treatment (E2: n=202; 0.1mg estradiol 17-β) or no treatment (NC: n = 204). In vivo produced embryos were transferred on d7, with grade, stage, and flush balanced across treatments. Circulating concentrations of estradiol and progesterone were determined on d0 and d7, respectively. Abundance of PAGs was determined on d24 and d26. Cows that were pregnant on d30 were grouped by age (H: 2, C: >2 years) and subjected to analysis. Correlations were analyzed using PROC CORR in SAS, while PAGs were analyzed using PROC GLIMMIX with treatment, age, year, sire, and embryo grade and stage as fixed effects. Abundance of PAGs on d24 was influenced by treatment (P = 0.01; PC=0.75±0.09, E2=0.59±0.09, NC=0.56±0.08), age (P < 0.01; H=0.75±0.09, C=0.52±0.08), year (P < 0.0001; 2019=0.42±0.08, 2020=0.84±0.09), and sire (P = 0.02; A=0.49±0.12, B=0.62±0.09, C=0.47±0.11, D=0.77±0.08, E=0.83±0.21), but not embryo grade (P = 0.89) or stage (P = 0.40). There was a negative correlation between d0 estradiol and d24 PAGs (P < 0.0001, r=-0.34), and a positive correlation between d7 progesterone and d24 PAGs (P < 0.0001, r=0.36). On d26, PAGs tended to differ by treatment (P = 0.09; PC=2.43±0.20, E2=2.33±0.21, NC=2.03±0.20). Age (P = 0.02; H=2.48±0.22, C=2.05±0.16), and sire (P = 0.0007; B=2.01±0.20, D=2.52±0.17) influenced PAGs, but embryo grade (P = 0.33) and stage (P = 0.32) did not on d26. Sire was associated with PAGs, and presumably embryo attachment, but not pregnancy rate. Increased progesterone concentrations on d7 were correlated with earlier attachment as measured by PAGs. Estrus expression prior to d0 may enhance earlier attachment of the fetal/maternal interface, and supplemental estradiol on d0 may improve attachment by d26 when ovulation is induced. Funded by NIFA 2019-67015-29411.

Presynchronization was evaluated as a method to improve estrus response before fixed-time AI (FTAI). The objective was to compare FTAI results in beef cows from two different presynchronization approaches. Blood samples were collected on Day-14 (Day 0 = CIDR removal) to determine progesterone concentration (>= 1 ng/ mL = high, <1 ng/mL = low). In a subset (n = 1289), an additional blood sample was collected between Day -21 and-29 to determine cyclicity (if both the Day-14 and Day-21 to-29 samples were classified as low progesterone cows were classified as noncycling). Cows (n = 1388) from 30 herds were grouped by days postpartum (DPP) and age, and randomly assigned to either of two protocols. Cows assigned to the PG 6-day CIDR & FTAI protocol (PG6d) received prostaglandin F2 alpha (PG) on Day-9, CIDR insertion and GnRH on Day -6, and CIDR removal and PG on Day 0. Cows assigned to the 7&7 Synch protocol (7&7) were administered PG and CIDR insertion on Day-14, GnRH on Day-7, and CIDR removal and PG on Day 0. For both protocols, FTAI occurred concurrently with GnRH 66 h after second PG. Pregnancy was determined by transrectal ultrasonog-raphy 30-40 d after FTAI. The GLIMMIX procedure of SAS was used to detect differences in estrus response and pregnancy success with herd as a random variable. Estrus response (0-66 h) was analyzed with two models, one included cyclicity and another replaced cyclicity with progesterone concentration at Day-14. In both models, cows assigned to the 7&7 protocol had greater (P < 0.01) estrus response than cows assigned to the PG6d protocol. The model including cyclicity, estrus response was impacted by the cyclicity by DPP interaction (P = 0.03), cyclicity by protocol interaction (P = 0.04), and the tendency of BCS by protocol interaction (P = 0.08). In the estrus response model that included progesterone concentration at Day-14, significant variables included the protocol by progesterone concentration at Day-14 (P = 0.01), and BCS (P < 0.01), while DPP (P = 0.08) and progesterone concentration at Day-14 (P = 0.07) were tendencies. Pregnancy success was influenced by estrual status (P < 0.01), body condition score (P = 0.04), and cycling status (P = 0.02), but was not influenced by protocol (P = 0.75; PG6d = 38 +/- 5% and 7&7 = 37 +/- 5%). In conclusion, effectiveness of presynchronization method depended on a cows' physiological status, and the 7&7 protocol increased estrus response compared with PG6d, but there was no difference in pregnancy success.

Control of follicular development is essential for estrous synchronization. This experiment determined differences in follicular development during synchronization in Brahman females. Cows (n=219) and heifers (n=90) in Fall (n=170) and Spring (n=139) were synchronized with a CIDR insert and administration of GnRH or PG treatment on d0 (1/2GnRH: n=106, GnRH: n=103, PG: n=100), and CIDR removal and PG administration on d6 followed by estrus detection and AI for 96h. Blood samples were collected on d0, d6, and d8 and/or AI to determine circulating progesterone (P4) concentrations. Transrectal ovarian ultrasonography occurred daily. Cows were grouped by CL presence (d0CL, d6CL) and P4 concentrations (d0P4, d6P4, d8P4, Low< 1 ng/mL, High≥1 ng/mL) on d0 and d6. The GLIMMIX procedure of SAS was used to analyze follicular wave initiation, wave emergence day, estrus expression, and pregnancy success. There were significant interactions of treatment by d0CL (P=0.0237), and season by d0P4 (P=0.0118) on wave initiation. Cows receiving 1/2GnRH with a CL on d0 (97±2%) and Fall cows having High d0P4 (96±3%) had the greatest probability of wave initiation. Age significantly impacted wave initiation (P=0.0213; heifers: 93±4%, cows: 79±5%). Interactions of treatment by season (P=0.0405) and treatment by d0CL (P=0.0379) significantly impacted wave emergence timing. Season (P< 0.0001; Fall: 23±5%, Spring: 65±6%), and the interactions of treatment by d0P4 (P=0.0291) and new wave by d0P4 (P=0.0357) influenced estrus expression. Cows receiving 1/2GnRH with High d0P4 and cows with High d0P4 that initiated a new wave had the greatest probability of estrus expression. Pregnancy rate differed by estrus expression (P=0.0055; no estrus=25±6%, estrus=54±8%) and semen type (P=0.0105; conventional=52±9%, sexed=26±6%), and tended to differ by d8P4 (P=0.0570; Low=30±7%, High=48±8%). These results suggest that differences in follicular development and behavioral estrus in synchronized Brahman females may be due to seasonality and endogenous progesterone production.

It has been reported that plasma serine protease inhibitor (SERPINA5) and dystroglycan (DAG1) are loosely attached to the sperm and SERPINA5 can also be present on sperm tails. The objective of this study was to evaluate whether SERPINA5 and/or DAG1 could be used as a fertility marker in dairy bulls. Frozen semen from dairy bulls (n=19) were evaluated for concentration of SERPINA5 and DAG1 and percentage of sperm tail labeled for SERPINA5 (SERPINA5-tail) by immunofluorescence in a minimum of 200 sperm cells per bull in two different ejaculates. Semen was evaluated for total motility, progressive motility, and viability. Semen was used for in vitro embryo production (~150 oocytes/bull). Bull fertility was classified by two methods, sire conception rates [SCR - High-SCR ( >1.0) or Low-SCR (< -4.0)] and the ratio of embryos that developed into a blastocyst (BL) from those that had cleaved (CL) as Good (BL/CL≥38.5%) or Poor (BL/CL< 38.5%). The GLIMMIX procedure of SAS was used to evaluate fertility classification with SCR, BL/CL, and the interaction in the model with bull as a random effect. Progressive motility, CL, SERPINA5 and DAG1 concentration, and SERPINA5-tail did not differ (P≥0.14) among SCR and BL/CL classifications or their interaction. There was no difference (P>0.33) in total motility between High-SCR and Low-SCR or BL/CL classification; however, the interaction was significant (P=0.02; High-SCR/Good 54.9±2.8%, High-SCR/Poor 39.4±5.4%, Low-SCR/Good 44.8±4.5%, Low-SCR/Poor 52.03±3.2%). High-SCR bulls had decreased BL (P=0.03; 30.6±1.6%, 35.8±1.4%, respectively) and tended to have decreased BL/CL ratio (P=0.10; 38.1±2.0%, 42.9±1.8%, respectively) compared with Low-SCR bulls. In summary, DAG1 and SERPINA5 do not seem to be a putative fertility marker. In vitro embryo production is not a good predictor of SCR, since High-SCR and Low-SCR bulls may have Good or Poor in vitro embryo developments.

Increased antral follicles are associated with greater fertility and a uterine environment that is more supportive of early embryonic development in beef heifers. Glucose is a primary energy source for embryos, and glucose concentrations are elevated in uterine luminal fluid (ULF) of pregnant heifers. We hypothesized that ULF glucose concentrations and endometrial transcript abundance for glucose transporters on d16 after insemination would be greater in heifers with increased numbers of antral follicles. Heifers classified with either increased or diminished antral follicle counts were artificially inseminated following the CO-Synch protocol (d0). On d16 after insemination, reproductive tracts of heifers were collected at an abattoir to retrieve conceptuses to determine pregnancy. Uterine luminal fluid was collected, endometrium was biopsied, total RNA was extracted and glucose transporter transcript abundances were determined. Data were analyzed using the MIXED procedure of SAS with antral follicle group, pregnancy status, and the interaction as fixed effects. Glucose concentrations in ULF were greater in heifers with increased antral follicle numbers. Glucose ULF concentrations increased in pregnant heifers. Facilitated glucose transporter member 1 (SLC2A1) transcript abundance was increased in the endometrium of pregnant heifers but was not different due to antral follicle number or the interaction. Differences in uterine concentrations of glucose associated with antral follicle number could be due to another mechanism, since glucose transporters were not different between antral follicle numbers. Therefore, heifers with increased number of antral follicles have increased energy availability in the uterus to support trophoblast proliferation and function.

Antiparasitic resistance has become a major concern in the livestock industry and can have a tremendous impact on calf performance. The objective of this study was to determine resistance to different anthelmintic treatments in Brahman and Brahman/Hereford (F1) calves at weaning. Purebred fall weaned Brahman calves (n=95) and spring weaned F1 calves (n=45) were assigned to one of six treatment groups at weaning: Control (received no anthelmintic), received a benzimidazole (Synanthic 22.5% or Safeguard), a macrocyclic lactone (Dectomax or LongRange), or a combination of the two (LongRange and Synanthic). Fecal samples were collected on d 0, 14 or 28 (dependent on treatment), and 42 to determine fecal egg count (eggs/g; EPG). Body weights were collected at all time points. Data were analyzed as an ANOVA using the GLM procedure, or as an ANOVA with repeated measures using the MIXED procedure in SAS. There tended to be an effect of treatment (P = 0.0865), and there was an effect of breed (P < 0.0001), time (P < 0.0001), treatment by breed (P = 0.0458), treatment by time (P = 0.0067), and treatment by breed by time (P = 0.0214) on EPG. Overall, Brahmans had increased EPG compared with F1s (P < 0.0001; 178.83±11.73 vs. 79.81±17.08). Benzimidazoles had a greater reduction in EPG (99.29%; P = 0.0011) compared with macrocyclic lactones (45.36%). There was no effect of treatment (P = 0.5184), or treatment by time (P = 1.0) on body weight. Body weight increased similarly among all treatment groups throughout the study. Furthermore, there was no difference in ADG during the study among treatments (P = 0.73). These data indicate that antiparasitic resistance occurs in all breeds, and resistance varies between benzimidazoles and macrocyclic lactones. Furthermore, there were differences between breeds and/or seasons among treatments even within a single operation.

Impairment of bovine reproductive function and decreased pregnancy rates have been reported in modified-live vaccinated females. The objective of this study was to determine if virus could be found in various areas of the bovine reproductive tract following vaccination. Previously vaccinated beef females (n = 50) were administered a single dose of one of three treatments on d0 of the estrous cycle; BoviShield (n = 20), Titanium 5 (n = 20), or ViraShield (n = 10) based on estrus expression. Blood samples were collected on d0, 15, and 30 to evaluate Infectious Bovine-Rhinotracheitis (IBR) and Bovine Viral Diarrhea Virus (BVDV) titers and progesterone concentrations. On d15/16, half the animals from each treatment were non-surgically flushed with sterile saline (Uterine Fluid), and uterine biopsies (Uterine Tissue) were collected. On d17, follicular fluid and granulosa cells were collected by follicle aspirations. Remaining animals had uteri flushed and uterine tissue collected on d29, and follicles aspirated on d31. All samples were evaluated for presence of virus. Data were analyzed using the MIXED procedure of SAS. Sub-luteal progesterone concentrations were observed in 15% of the BoviShield and Titanium treatments on d15 and d30, versus 0% in the ViraShield treatment. Virus isolation and RT-PCR results were negative in all samples for all animals. Titers for BVDV-1 did not differ between treatments (P = 0.99) or treatment by time (P = 0.48). Titers for IBR tended to differ by treatment (P = 0.09) and treatment by time (P = 0.06), while BVDV-2 titers were affected by treatment (P < 0.01) and treatment x time (P < 0.01). Sub-luteal progesterone animals had a rapid rise in IBR and BVDV-2 titers from d0 to d15 and then tended (P < 0.07) to decrease to d30, compared to animals that had normal cycles where titers increased from d0 to d15 and remained elevated on d30. Collectively, these results indicate an alternative mechanism of impacting reproductive efficiency, possibly through an immune response.

Abstract Blood pregnancy tests have gained popularity as there is no need for a costly ultrasound machine or special training; however, blood pregnancy tests only provide an answer of pregnant or open. Conversely, palpation and transrectal ultrasonography can determine gestational age. The objective of this study was to determine if a commercially available blood pregnancy test could detect differences in pregnancy-associated glycoprotein (PAG) concentrations indicative of gestational age. Previously identified pregnant females were grouped by age (heifers n=173, cows n=512); blood samples were collected between d 27 and 190 of gestation. Serum was tested in duplicate using a commercially available blood pregnancy test, IDEXX Alertys Ruminant Pregnancy Test. Procedures were adapted to allow concentrations to be within detectible range of the assay. Data was analyzed using MIXED procedure of SAS with age and gestational age (animals grouped into four gestational groups 1;< 30, 2;30–90, 3;91–178, and 4; >178 d) in the model. There was an effect of age, gestational age, and age by gestational age interaction (P< 0.01). Heifers had greater PAG concentrations compared to cows. Among heifers, PAG concentrations did not differ between gestational groups 1, 2, and 3 (P>0.37), but group 4 had greater PAG concentrations than all other groups (P< 0.01). Among cows, PAG concentrations decreased from group 1 to 2 (P< 0.01), and then increased throughout gestation (P< 0.01). Within age, group data were analyzed using REG procedure of SAS. There was a positive correlation between gestational age and PAG concentrations among both heifers (P< 0.01; r2=0.25) and cows (gestational age 30 and greater P< 0.01; r2=0.64). In summary, among heifers circulating PAG concentrations increased with gestational age, but gestational age only accounted for 25% of the variation. Among cows, gestational age (d 30–190) accounted for 64% of variation in PAG concentrations, thus a modified blood pregnancy test may allow for determining gestational age.