Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation—From Field Trials to Research Bench

Simple Summary The use of antioxidant compounds could be a successful tool to improve sperm cryopreservation protocols in ovine species. These molecules have been widely employed in different mammalian species with this purpose. It is important to consider the existence of a species-specific antioxidant effect discarding the extrapolations from other animal species. To corroborate the real effectiveness of these compounds is important to combine two approaches: in vitro sperm quality analyses and in vivo field trials based on fertility. In the first scenario, a multiparametric analyses and novel tests based on spermatozoa redox balance, as the main target of antioxidants, could improve the accuracy on antioxidant effectiveness on sperm quality. Moreover, an extensive field insemination study provides the definitive tool to select the best antioxidant treatment. All these aspects have been applied and extensively discussed throughout this manuscript. Novel approaches have been incorporated, such as RedoxSYS, to provide more accuracy in the integrative studies of Redox status in spermatozoa. The effectiveness of an antioxidant treatment, as trolox in our study, should be demonstrated in an integrative way, from in vivo (fertility trials) to in vitro analyses (sperm quality assays), especially when the final aim is to reach AI implementation. Abstract The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.