IDENTIFICATION OF AQUAPORIN 7 GENE POLYMORPHISM AND IN-VIVO FERTILITY OF EGYPTIAN BUFFALO BULLS

This work aimed to investigate the genetic polymorphism of Aquaporin 7 (AQP7) gene and its association with the in-vivo fertility of Egyptian buffalo bulls. A total of 188 fresh semen ejaculates were collected from 47 buffalo bulls and subjected to semen evaluation. The bulls were grouped according to in-vivo fertility relying on sire conception rate (SCR) into high (SCR>50%, n=41) and low (SCR<50%, n=6) fertile bulls. DNA was extracted from semen and a 200 bp fragment of the AQP7 gene was amplified by PCR. The genetic polymorphism of AQP7 gene was detected by restriction fragment length polymorphism, the single strand conformation polymorphism and the nucleotide sequencing. The results revealed that SCR was significantly (p<0.001) increased in high (71.4±1.3) than low (44.7±2.8) fertile bulls while the ejaculate volume, individual motility%, lives sperm%, sperm concentration and sperm abnormalities showed a non-significant difference in both groups. The AQP7 gene showed no genetic variation in all bulls. The alignment of the resultant sequence with buffalo AQP7 gene sequence (ID: XM_006066699.2) showed 100% identity (122/122), however, deletion of A49 was discovered when the sequence was aligned with ID: XM_006066700.2 of buffalo in GenBank. In conclusion, the sire conception rate is not associated with any genetic variation in Aquaporin 7 gene. The Aquaporin 7 gene could be a highly conserved sequence in Egyptian buffalo bulls. Future research on large herd populations in different regions of AQP7 gene is required for evaluating the AQP7 gene polymorphism and its association with the fertility of Egyptian buffalo bulls.

PSV-5 Relationship between sire conception rate, sperm motility, sperm SERPINA5 relative concentration, and in vitro produced embryos in dairy bulls

Sire conception rate (SCR) is a field measure of fertility among bulls, but it can be influenced by several factors (Sperm transport, sperm-egg binding, early embryo development, etc). The objective of this study was to evaluate the relationship between SCR, sperm motility, SERPINA5 concentrations, and in vitro embryo development. Measurements were performed in 19 bulls with SCR values ranging from -7.7 to 4.45. For each bull, an aliquot of frozen-thawed semen was used for analyses of total (TMOT) and progressive (PROG) motility. Remaining semen was fixed with 2% formaldehyde, and concentration of SERPINA5 was determined by immunolocalization (antibody SERPINA5/Dylight405; PA5-79976-Invitrogen / ab201798-Abcam). Mean fluorescence intensity was determined in ~200 sperm heads/bull. Approximately 149 oocytes/bull were fertilized in vitro for embryo development analysis (cleavage and blastocyst rates). Statistical procedures were performed in SAS (9.4) using the procedures CORR for correlations (SCR, TMOT, PROG, SERPINA5, cleavage and blastocyst) and GLIMMIX for comparison of “field-fertility” (SCR divided in HIGH or LOW) and “field-embryo-fertility” (LOW-SCR sires were divided based on blastocyst rate (HIGH or LOW) resulting in two classifications; LOW-HIGH≥31% and LOW-LOW≤26%, respectively). There were positive correlations (P &lt; 0.05) between cleavage-blastocyst (r=0.50), SERPINA5-cleavage (r=0.48), and TMOT-PROG (r=0.76). Sire SCR was not associated with SERPINA5, TMOT, PROG, cleavage and blastocyst rate (P &gt; 0.52). Among LOW-SCR sires, LOW-LOW sires (-4.83±0.60) tended to have a better SCR score than LOW-HIGH (-6.18±0.42) sires (P = 0.08), but there were no differences (P &gt; 0.43) between LOW-HIGH, LOW-LOW, and HIGH sires for SERPINA5, TMOT, PROG, and cleavage. In conclusion, some LOW SCR sires have good embryo development indicating a different mechanism for their low SCR; however, these differences in SCR could not be explained by TMOT, PROG, SERPINA5, cleavage and blastocyst. There were, however, positive correlations between cleavage-blastocyst rate, and SERPINA5-cleavage rate.

PSV-8 Sire field fertility does not correlate with sire conception rate (SCR) score

Accurate sire fertility evaluation remains a significant challenge in the livestock industry. Sire conception rate (SCR) has been used since 2008 in the dairy industry to rank sire fertility. The objective was to characterize individual sire pregnancy rate, pregnancy loss, and determine the correlation with the sire’s respective SCR. Our main hypothesis is that large variances in fertility parameters by sire cannot be explained by variance of the SCR values. Data from 6,570 timed artificial inseminations (AI) and 25,287 timed embryo transfers (ET) from 55 dairy farms were retrospectively analyzed to assess: pregnancy rate at day 30 (P30), pregnancy rate at day 60 (P60) and pregnancy loss (PL) during this interval. The effect of sire (AI: n = 39; ET: n = 81) for each reproductive parameter was evaluated. SCR records published prior to individual service were obtained to correlate with fertility parameters. For AI mating, pregnancy rate at P30 was 33.13% (2,177/6,570), and 27.6 % for P60 (1,815/6,570) and PL occurred in 16.6% (362/2,177) of pregnancies. Large variance was observed between sires for each parameter but no or negligible correlation (r &lt; 0.2) with published SCR was observed. For ET, pregnancy rate at P30 was 47.8% (12,082/25,287) and 40.5% for P60 (10,246/25,287) with an overall PL of 15.2% (1,836/12,082). Similar to AI, all fertility parameters were highly variable among sires but no or negligible correlation (r &lt; 0.03) with respective SCR was observed. In summary, the current method to evaluate sire fertility using SCR does not truly represent the field fertility status. Large variance in pregnancy loss between days 30 and 60 of gestation were observed among sires and these phenotypes should be considered when evaluating sire fertility to increase the score reliability.

63 Effect of sire conception rate on bovine early embryo development

Currently, the only measure of sire fertility in the bovine is sire conception rate (SCR), which is determined by Day 70 pregnancy diagnosis and not reflective of early embryo development. Therefore, this study aimed to establish the relationship between SCR and early embryo development. In the first experiment, 65 sires of negative (&lt;−1, n=25), average (−1 to 1, n=19), and high (&gt; +1, n=21) SCR were characterised for their ability to produce embryos using an invitro embryo production (IVP) system. For each sire, 100 cumulus–oocyte complexes (COCs) were used. COCs were matured for 22h, fertilized by co-incubation with sperm selected from density gradient centrifugation for 18h, and then placed in culture medium. A sire of known IVP performance was used as a control in each run. Cleavage and blastocyst rates (BL) were measured on Days 3 and 8 post-insemination, respectively. Photographs were taken on Days 3, 5, and 8 to identify arrest stages of non-blastocyst embryos. Sires were ranked based on their blastocyst rate and grouped into quartiles for statistical analysis. Differences in BL were determined by ANOVA using sire, IVP run, and a sire×IVP run interaction. In addition, the correlation between SCR and BL was determined. All data were analysed using SAS software version 9.4 (SAS Institute Inc.). Mean BL between each quartile was significant (P&lt;0.05), with rates ranging from 8 to 22% and 32 to 62% for the lowest and highest quartile, respectively. There was no correlation (P=0.90) between SCR and BL. Arrest stage was measured by subtracting the number of Day-8 blastocysts from, first, embryos that were morulas on Day 5, and then embryos that were 8- to 16-cell stage embryos on Day 5. This method is based on the assumption that embryos closer to the blastocyst stage on Day 5 are more likely to contribute to the Day 8 blastocyst population. The most frequent arrest stage was the 4- to 6-cell stage (39/52 sires). It has been shown that decreased rates of autophagy are associated with embryonic arrest at the 4- to 8-cell stage in humans, leading us to investigate this mechanism in the second experiment. Select high (n=3) and low (n=4) performing sires identified in experiment 1 were used to generate 4- to 6-cell embryos, and autophagy rates were measured using live immunofluorescence with CYTO-ID autophagy dye (n=20 embryos/sire). The mean fluorescent intensity of each embryo was divided by the number of cells within the embryo. Differences in autophagy between high and low sires were determined by ANOVA using SAS. Interestingly, low-performing sires had a significantly higher autophagy rates than high-performing sires (77.8±3.1 vs. 50.0±3.5). This could indicate that embryos produced with low-performing sires had higher levels of stress than their counterparts. In summary, the effect of sire on embryonic development seems to be independent of the SCR classification. The most common arrest stage observed is the 4- to 6-cell stage, right before embryonic genome activation. Further research is required to elucidate the mechanisms by which sires influence pre-implantation development. This research was supported by USDA-NIFA AFRI Competitive Grant No. 2019-67015-28998.

Evaluation of bull fertility of Sahiwal breeding bulls

In the present investigation, bull fertility of Sahiwal breeding bulls has been studied. The study was conducted on records of 43 Sahiwal bulls maintained under 8 sets of Sahiwal breeding project at ICAR- National Dairy Research Institute, Karnal (India). The data on bull fertility of Sahiwal breeding bulls during 27 years (1987-2013) were analysed. The presented study revealed that the average conception rate based on first AI and overall conception rate of Sahiwal breeding bulls were estimated as 45.95% and 46.38 %. Average sire conception rate of Sahiwal breeding bulls range from – 2% to + 3% and – 2% to + 4 % for conception rate based on first AI and overall conception rate. It has been observed that the average conception rate based on first AI was lower as compare to overall conception rate and higher conception rate of Sahiwal breeding bulls is having higher sire conception rate.

256 GENOMIC EVALUATION OF FERTILITY TRAITS AND DISCOVERY OF HAPLOTYPES THAT AFFECT FERTILITY OF US DAIRY CATTLE

Genomic evaluations of dairy cattle became official in the United States in January 2009 for Holsteins and Jerseys, and later for Brown Swiss, Ayrshires, and Guernseys. Up to 33 yield, fitness, calving, and conformation traits are evaluated, and the fertility traits included daughter pregnancy rate and heifer and cow conception rates. Additional fertility traits, such as age at first calving and days from calving to first insemination, also are being studied. Male fertility (sire conception rate) is evaluated phenotypically rather than through genomics. Over 1 million animals have genotypes in the national database, which reflects collaboration with Canada and Europe. Most of the genotypes are from females and are from genotyping chips with <30 000 single nucleotide polymorphisms (SNP). To combine data across chips, genotypes are imputed to a set of >77 000 SNP. The imputation process involves dividing the chromosome into segments of approximately equal length and determining the paternal or maternal origin of the alleles. Because some segments were never homozygous, they were assumed to contain an abnormality that resulted in early embryonic death. If a decrease in sire conception rate could be associated with a bull that was a carrier of such a chromosomal segment, the haplotype was designated as affecting fertility. Once the region was identified, bioinformatic analysis was used to discover the causative variant for many of those haplotypes. Accuracy of genomic evaluations is determined by size of the reference population and heritability of the trait. The reference population for Holsteins includes >180 000 bulls and cows. Because fertility traits have low heritabilities, genomic information is particularly useful in improving evaluation accuracy. Accuracy of fertility evaluations is expected to increase further by discovering causative variants for various aspects of conception and gestation through investigation of sequence data.