Trimethylation of histone 3 at lysine 4 in cryopreserved bovine embryos produced in vivo with sexed semen

Abstract

The production rates of viable embryos using sexed semen through the conventional methodologies of multiple ovulation and embryo transfer are generally not satisfactory. However, the cryopreservation of these embryos is considered efficient. Knowledge of epigenetics can provide new tools or allow for adapting new protocols that could enhance the efficiency of reproductive biotechnologies. The aim of this study was to characterize the pattern of trimethylation of histone 3 at lysine 4 (H3K4me3) in bovine embryos produced in vivo with sexed semen that were submitted to cryopreservation. Bos taurus × Bos indicus cows (n = 5) were superovulated and inseminated with sexed (two sessions) or conventional (two sessions) semen. A portion of the embryos collected on Day 7 was immediately stored in paraformaldehyde (3%) and another portion was stored in paraformaldehyde after cryopreservation/thawing. All embryos from the four groups (fresh, conventional semen; fresh, sexed semen; cryopreserved, conventional semen; and cryopreserved, sexed semen; 15 embryos per group) were evaluated by immunofluorescence under confocal microscopy to identify and quantify the H3K4me3 status. In total, 190 embryos were recovered, 100 of which were produced with conventional semen and 90 with sexed semen. The use of conventional semen after superovulation yielded 72% (72 of 100) viable embryos, which were mostly (81%; 59 of 72) in advanced stages of development (blastocysts and expanded blastocysts). Embryos produced with sexed semen had a lower viability rate (36.7%; 33 of 90), and most of them were collected at earlier stages of development (morulae and early blastocysts; P < 0.05). The H3K4me3 signal was similar among groups; however, there was a difference between morulae and blastocysts. A high intensity of H3K4me3 was observed in bovine embryos produced in vivo, and this pattern did not vary using sexed semen and the slow cryopreservation process. The lower viability of bovine embryos produced with sexed semen could be not explained by differences in H3K4me. Cryopreservation did not alter the pattern of H3K4me3; in this sense, we suggest that it is a process that exerts minimal damage to the embryos.

Keywords: Cattle; Epigenetics; H3K4; Superovulation; semen.