This study was investigated to test whether the zygote recognized the topoisomerase II beta (TOP2B) mediated DNA fragmentation in epididymal spermatozoa or the nuclease degradation in vas deferens spermatozoa by testing for the presence of gammaH2AX (γH2AX). The γH2AX is phosphorylation of histone protein H2AX on serine 139 occurs at sites flanking DNA double-stranded breaks (DSBs). The presence of γH2AX in the pronuclei of mouse zygotes which were injected with DNA broke epididymal spermatozoa was tested by immunohistochemistry at 5 and 9 h post fertilization, respectively. Paternal pronuclei that arose from epididymal spermatozoa treated with divalent cations did not stain for γH2AX at 5 h. On the other hand, in embryos injected with vas deferences spermatozoa that had been treated with divalent cations, γH2AX was only present in paternal pronuclei, and not the maternal pronuclei at 5 h. Interestingly, both pronuclei stained positively for γH2AX for all treatments and controls at 9 h after sperm injection. In conclusion, the embryos recognize DNA that is damaged by nuclease, but not by TOP2B because H2AX in phosphorylated in paternal pronuclei resulting from spermatozoa treated with fragmented DNA from vas deferens spermatozoa treated with divalent cations, but not from epididymal spermatozoa treated the same way.

Genotyping-by-sequencing (GBS) is a cost-efficient method which can be useful for SNP marker discovery in a population of interest. GBS is genome reduction sequencing method using restriction enzyme. The quality of DNA is a key factor which could have an influence in downstream analysis. However, there have not been many studies which investigated the impact of DNA degradation and the quality of the data on marker discovery. In this study, GBS data of 6 Hanwoo samples (H1~6) showing differing level of DNA degradation were compared. Re-sequencing pipeline was followed to investigate the impact of DNA degradation on marker discovery. As a result, we found that the quantity and quality of SNPs were not affected in the sample H5 and H6 with moderately degraded DNA. On the other hand, marker discovery was greatly affected in samples with severe DNA degradation (H3 and H4). The findings in this study support that GBS is a robust genotyping method towards moderate DNA degradation.