Frequency of Semen Collection Affects Ram Sperm Cryoresistance

SIMPLE SUMMARY: Sperm cryopreservation is an important step to optimize artificial insemination protocols for rams. Traditionally, the improvement of ram sperm cryopreservation protocols has been addressed through different methods: formulation of different cryoprotectants, supplementation with antioxidants or carrying out different freezing-thawing rates. However, the influence of the male sexual regime as a factor for the improvement of freezability has not been studied until now. The frequency of sperm collection in the work protocols of reproduction centers can be variable depending on the dose demand. To corroborate the variability of the quality of thawed semen according to the frequency of collection, in addition to traditional analyses, new tests based on the redox balance of spermatozoa were performed. All these aspects have been applied and discussed extensively throughout this manuscript. In addition, new approaches, such as antioxidant activity of some enzymes have been incorporated to provide more precision in integrative studies of the redox state in spermatozoa. The deleterious effect triggered by a high dose demand should be demonstrated as a consequence of a redox imbalance. ABSTRACT: The improvement of frozen-thawed sperm quality has been mostly approached from the view of cryopreservation protocol optimization in terms of cryoprotectant solutions, freezing-thawing rates and antioxidant supplementation, while the impact of sperm collection frequency remains unknown in rams. In this work, a multiparametric study was carried out in cooled and frozen-thawed semen to evaluate sperm quality after different semen collection frequencies during a month: zero sperm collection (0 CW), four sperm collections per week (4 CW), and ten sperm collections per week (10 CW). Traditional analyses have been applied, in combination with novel technologies related to redox balance. Frozen-thawed semen quality showed a significant decrease (p < 0.05) in 0 CW and 10 CW in comparison to 4 CW, concerning motility and kinetics parameters. However, apoptosis showed a significant increase (p < 0.05) in 10 CW in comparison to 0 CW and 4 CW. The employment methods related to redox balance provided us with the definitive probe to ensure the influence of collection frequency on balance redox after thawing. Specifically, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity showed a significant decrease (p < 0.05) in 10 CW compared to 0 CW and 4 CW. The characterization of alternative strategies to sperm cryopreservation based on consideration of male sexual regimes, could improve the quality of frozen-thawed sperm.