45 Paternal effects on early embryo development in bovine

To create a predictor of sire fertility, it is essential to identify how sires influence different processes throughout pregnancy. In a previous experiment, 65 sires with sire conception rates ranging from −14.2 to 5.3, were run through in vitro embryo production (IVP), and pictures of embryos were taken on days 3, 5, and 8. Sire performance was classified based on blastocyst/cleavage (BL/CL) rates into quartiles. Sires from quartile 1 were termed low performing (LP, n = 9) and had BL/CL rates <18%; sires from quartile 4 were termed high performing (HP, n = 8) and had BL/CL rates >40%. Analysis of pictures determined that the major arrest stage was the 4- to 6-cell stage, regardless of sire performance, and HP sires had fewer arrested embryos than LP sires (P < 0.05). To validate these results, putative zygotes from 3 HP and 3 LP sires were live imaged in pairs, across three IVP replicates, using a different straw of semen and a different pool of oocytes obtained from abattoir ovaries for each replicate. A total of 50 embryos per sire were imaged under culture conditions (38.5°C, humidified atmosphere of 5% CO₂ 5% O₂) every 3 h from 19 to 118 h post-insemination (hpi) using a Leica DMi8 scope. Video analysis was done in LASX (version 3.7.4.23463; Leica Inc.). The stage of each embryo was recorded as 1, 2, 3–4, 5–6, 7–8, 9–16+ cell stages, morula, or degenerated, at each timepoint. The percentage of embryos that reached each stage per timepoint was determined using a binomial logistic regression with an effect of performance across hpi using SAS software v 9.4 (SAS Institute Inc.). Difference in means was determined with the pdiff option of LSMEANS, and significance was defined as P ≤ 0.10. Embryos produced from LP sires were developmentally delayed and more likely to arrest at the 5–6 cell stage compared with embryos from HP sires. Lastly, 4-cell embryos from HP and LP sires were collected for RNA-sequencing to identify differentially expressed pathways and help elucidating the causes of sire-influenced embryonic arrest. Embryos (n = 100) were produced with each HP (n = 3) and LP (n = 3) sire. At 42 hpi, all 4-cell embryos were incubated in 0.1% pronase for 60 s, then pooled according to performance. This experiment was repeated three times. Samples were sequenced to an average of 50 million reads. Reads were aligned with Hisat2, mapped with feature counts, and differentially expressed genes were determined using the package Robust from R, with a false discovery rate < 0.05. Gene ontology analysis was performed using ToppGene (https://toppgene.cchmc.org/). Embryos from HP sires had increased expression in 1411 genes involved in processes such as sperm mitochondrial clearance, regulation of mRNA, and the cell cycle. Embryos from LP sires had increased expression in 687 genes related to DNA damage, apoptosis, and cellular stress. In conclusion, embryos from LP sires are delayed in development, and have increased arrest at the 5–6 cell stage, possibly due to a genetic or nongenetic contribution from the sperm. Future research is needed to determine the mechanisms influenced by sire that affect early embryo development.

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