63 Effect of sire conception rate on bovine early embryo development

Abstract

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 (<−1, n = 25), average (−1 to 1, n = 19), and high (> +1, n = 21) SCR were characterised for their ability to produce embryos using an in vitro embryo production (IVP) system. For each sire, 100 cumulus–oocyte complexes (COCs) were used. COCs were matured for 22 h, fertilized by co-incubation with sperm selected from density gradient centrifugation for 18 h, 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 < 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.