Transgenerational Epigenetic Effect of Cryopreservation of F0 Rooster Sperm (Gallus gallus domesticus) on microRNA-Regulation and Histological Parameters of the Reproductive System of F1 Offspring

Sperm cryopreservation is an integral part of gene pool conservation programs for local poultry breeds. It is known that cryostress can cause significant changes in the expression profiles of microRNAs and their target genes—key players in spermatogenesis—in Gallus gallus domesticus. However, the transmission of these changes across generations remains poorly understood. The aim of this study was to evaluate the transgenerational epigenetic effects of rooster sperm cryopreservation on molecular genetics and histological parameters in the gonads of offspring (F1) during the embryonic (10 days) and postnatal (1 day) periods. The analysis included a comprehensive histomorphometric analysis of the gonads and a quantitative assessment of the expression of microRNAs (gga-miR-6701-3p, gga-miR-301a-5p) and their target genes (DMRT1, TGFB2), using qRT-PCR. Histological analysis of the gonads of 10-day-old embryos revealed early morphological abnormalities in the F1 (n = 10) offspring obtained from frozen–thawed semen (experimental group). It was found that day-old F1 chicks (n = 17) obtained from frozen semen had testes with a significantly reduced number of seminiferous tubules (−36%, p < 0.05) with an increased diameter (+22%, p < 0.05) and an increased number of undifferentiated gonocytes (+53%, p < 0.001) compared to chicks obtained from native semen (control group, n = 20). A decrease in the expression of DMRT1 and TGFB2 in the gonads of embryos (−48% and −29%, respectively, p < 0.05) and day-old chicks (−12% and −43%, p < 0.001 for TGFB2) was found, accompanied by an inversion of microRNA dynamics: miR-6701-3p was decreased and miR-301a-5p was increased. The obtained data provide important evidence of transgenerational effects in birds and contribute to the search for solutions to problems associated with maintaining sperm quality after cryopreservation, and indicate that cryopreservation does not simply reduce the level of molecular activity, but disrupts the ontogenetic regulatory program embedded in the genome.