Trace minerals are known to play important roles in early embryo development. The study objective was to determine effects of trace mineral source on heifer reproductive performance. Beef heifers (n = 129) were randomly assigned to one of two treatments. From weaning through breeding, all heifers were individually fed a basal diet supplemented with Co, Cu, Mn, and Zn either from organic sources (COMP; Cu, Mn, and Zn amino acid complexes and Co glucoheptonate; Availa-4, Zinpro Corporation, Eden Prairie, MN) or inorganic sources (INORG; Cu, Mn, and Zn hydroxychlorides; Intellibond C, M, and Z, Micronutrients, Indianapolis, IN) and Co as CoSO4. Blood samples and a reproductive tract score (RTS) were collected to determine pubertal status. All animals were synchronized and artificially inseminated (AI). Pregnancy status was determined by lymphocyte gene expression, circulating concentrations of pregnancy associated glycoproteins (PAG), and by transrectal ultrasonography after AI. Embryonic loss was defined as when a previously pregnant animal was subsequently diagnosed not pregnant. Data were analyzed using the MIXED procedure in SAS. Puberty (P = 0.44), pelvic area (P = 0.74), RTS (P = 0.49), and estrus expression (P = 0.82) were not influenced by treatment. There was no effect of treatment (P = 0.37) or treatment by time (P = 0.19) on pregnancy, but there was a tendency (P = 0.13) for decreased embryonic loss among COMP heifers (27 ± 6%) compared to INORG heifers (38 ± 6%). There was a treatment by pregnancy status by time interaction (P < 0.01) on circulating PAG concentrations with PAG concentrations tending (P = 0.08) to be greater on d 25 among heifers in the COMP treatment compared to heifers in the INORG group. In summary, source of trace mineral did not affect puberty, RTS, pelvic area, or overall pregnancy success, but feeding complexed trace minerals tended to increase circulating PAG concentrations and embryo survival.

IntroductionSperm 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 methodsDairy 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.ResultsBoth 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).DiscussionIn 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.

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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.

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.