56 Paternal contributions to early embryonic stress affect development in the bovine

Currently, it is known that the ability of an embryo to develop to the blastocyst stage is influenced by sire. The aim of this study is to elucidate mechanisms that contribute to the variation observed between sires, focusing on those related to stress in early embryonic development. Previous studies from our group have shown an increase in autophagic activity, impacted by sire, at the 4- to 6-cell stage of development under controlled culture conditions (38.5°C with air containing 5% CO₂, 5% O₂, 90% N₂). It was hypothesised that embryos produced from low-performing sires have increased basal levels of stress compared with embryos produced from high-performing sires, and that embryos produced from high-performing sires have a more robust mechanism for mitigating stressful events. To test this hypothesis, embryos were produced in vitro, under controlled culture conditions, using semen from sires previously classified as high- (sire 1) or low- (sire 2) performing in their ability to produce blastocysts. A total of 20 to 40 embryos per stage were collected at four stages of development (2- to 6-cell, 8- to 16-cell, morula, and blastocyst) over the course of two replicates, and stained with the CellROX deep red to target reactive oxygen species (ROS), and Cyto-ID green as a marker of autophagic activity. The mean fluorescent intensity (MFI) of each image was measured using ImageJ software. Data were analysed using a generalised linear mixed model in SAS (SAS Institute Inc.), and are expressed as means ± s.e.m. At the 2- to 6-cell stage, ROS was increased (P < 0.0001) in embryos produced by sire 2 (21.201 ± 0.84 MFI) compared with sire 1 (12.602 ± 1.13 MFI). Autophagy was also increased (P = 0.0012) in embryos from sire 2 (12.561 ± 0.55 MFI) compared with sire 1 (9.541 ± 0.73 MFI). At the 8- to 16-cell stage, autophagic activity in embryos produced by sire 1 (14.399 ± 11.01) was increased (P = 0.0036) compared with that of sire 2 embryos (11.008 ± 0.82), while there was no difference in ROS at this stage. At the morula stage, ROS was higher (P < 0.0001) in embryos produced by sire 2 (23.210 ± 1.46 MFI) compared with sire 1(12.831 ± 1.26 MFI). Autophagy was also increased (P = 0.0044) in embryos from sire 2 (11.275 ± 0.95 MFI) compared with sire 1 (7.651 ± 0.82 MFI). There were no differences between embryos that reached the blastocyst stage. The cleavage rate was 87.50% and 61.57% for sire 1 and 2, respectively. Blastocyst rate was 32.40% and 15.73% for sire 1 and 2, respectively. Blastocyst rate was calculated as the number of blastocysts divided by the number of putative zygotes per sire. These results indicate that embryos produced by sire 2 began development under increased stress, which affected their ability to undergo cleavage and continue development. These data also suggest that their autophagic response alone is not robust enough to mitigate this stress by the morula stage, preventing a proportion from reaching the blastocyst stage. Further research will elucidate whether embryos from low-performing sires are more susceptible to external stressors that may contribute to higher embryonic mortality.

This research was supported by USDA-NIFA AFRI Competitive Grant No. 2019-67015-28998.