Seminal Analysis and Testicular RNA Sequencing of a Ram with Low Fertility

One ram of the Rasa Aragonesa breed (M) exhibited low reproductive performance throughout his reproductive life (ewes that lambed/ewes inseminated: median: 0.57, interquartile range: 0.35; lambs born/ewes inseminated: 0.96 ± 0.513). In this exploratory case comparison, our objective was to determine the causes of M's reproductive problems. Another ram (O) was chosen as the reference animal due to its history of high fertility and fecundity (fertility: median: 0.73, interquartile range: 0.33; fecundity: 1.22 ± 0.523). Semen samples from both males were submitted to a complete quality study. M exhibited lower sperm concentration, reduced membrane integrity, altered ROS/live-dead sperm profiles, increased phosphatidylserine inversion, and reduced DNA integrity. RNA sequencing, mapping against the reference genome (Ovis aries, GCF_016772045.2), differential gene expression, and GO enrichment analysis were performed on testicular biopsies of M and O. Despite the limitations of this study, which is based solely on two rams with extreme reproductive performances, it was detected that the KRT2 gene was downregulated in M with respect to O, with possible deleterious effects on membranes. Since only two single individuals were considered, this transcriptomic finding should be interpreted as hypothesis-generating rather than confirmatory. Therefore, for KRT2 to be considered a likely candidate to explain the observed differences in sperm quality and fertility outcomes, a larger number of animals with either high or low fertility should be considered, and validation using qRT-PCR should be performed.