The evaluation of sperm viability on fertilization outcomes using a heterospermic insemination model in beef cattle

Abstract

Predicted fertility of sires used in artificial insemination programs is based on two semen quality parameters: motility and morphology. While these characteristics are critical for assessing the potential of spermatozoa to reach the egg and complete fertilization, additional factors may further differentiate sire fertility. The longevity of sperm viability within the female reproductive tract could influence the likelihood of fertilization, especially when insemination and ovulation are not ideally synchronized in many timed AI protocols. Therefore, the objective of this study was to indirectly evaluate differences in the duration of semen viability within the female tract using heterospermic insemination composed of two bulls. Semen collected from two bulls in each trial was evaluated for morphology and motility before combining into straws containing equal proportions of motile sperm. Single-sire straws for each bull were stored for in vitro comparisons. All females across four field trials were set up for breeding using a 7-day CO-Synch+CIDR protocol to synchronize estrus. Insemination timing for each trial was as follows: Trial 1, heifers and cows were inseminated at 36 and 48 hours post-PGF/CIDR removal, respectively; Trials 2 and 3, heifers and cows were inseminated at 48 and 54 hours, respectively; and Trial 4, cows were inseminated at 60 hours post-PGF/CIDR removal. All females in Trials 2, 3, and 4 received a GnRH injection at the time of insemination. Blood samples were collected from calves for parentage verification. In Trials 2, 3, and 4, z-test analysis of confirmed AI-sired calves indicated a significant difference in sire contribution between the Hereford and Angus bulls within each respective trial (p < 0.01). Within Trial 4, single-sire ejaculate analysis revealed significant between-bull differences in both total capacitation ability via zinc signature analysis (p = 0.01) and progressive motility following 1 hour of in vitro capacitation (p = 0.01). No other significant pairwise comparisons in zinc signature or progressive motility were observed across the remaining single-sire straws within trials. Another objective of this study was to evaluate fertilization ability and early embryonic development between two bulls in an in vitro heterospermic fertilization assay. The two bulls were chosen because they demonstrated a skewed distribution of calves when used in heterospermic field trial inseminations. An in vitro fertilization (IVF) protocol from ivf Bioscience, Ltd., was performed, with minor modifications. Oocytes were obtained via follicular aspiration from abattoir-derived ovaries and matured under incubation at 38.5°C and 5.5% CO₂ for 21–24 hours before introduction into fertilization medium. The Angus and Hereford bulls from Trial 4 were selected for IVF, with semen distributed across 12 wells: 3 single-sire Angus, 3 single-sire Hereford, and 6 heterospermic mixtures of the two bulls. All presumptive zygotes were subsequently cultured for 5 days and evaluated for cell-stage development. Single-sire Hereford and the heterospermic semen resulted in a greater number of zygotes developing past the 2-cell stage (p < 0.05) compared to single-sire Angus semen. Verification of progeny from heterospermic inseminations served as an indicator of relative fertility differences between bulls within this study. However, capacitation and progressive motility were not identified as primary contributors to these fertility differences. Collectively, these findings support heterospermic insemination as a viable tool for comparing relative sire fertility alongside other semen characteristics.