Effects of Three Semen Extenders, Breeding Season Month and Freezing–Thawing Cycle on Spermatozoa Preservation of Portuguese Merino Sheep

SIMPLE SUMMARY: Spermatozoa (SPZ) viability, morphology and the kinetics of fresh and thawing Portuguese Merino Semen were evaluated at different breeding season months using an owner egg yolk-based semen extender (S-EXT) and two commercial lecithin-based semen extenders. The main significant differences between S-EXT were observed for thawed semen. The viability (30% vs. 15% of alive SPZ; p < 0.001), total motility (74% vs. 34%; p < 0.001), total progressive motility (18% vs. 5%; p < 0.001), straight line velocity (37 vs. 22 μm/s; p < 0.001) and beat cross frequency (13 vs. 6 Hz; p < 0.05) were higher in the owner egg yolk than in egg yolk-free-based S-EXT. Significant interactions between S-EXT, semen processing and/or months of semen collection were observed on several of the 22 evaluated SPZ parameters and should be elucidated in further studies. The egg yolk-based S-EXT was more consistent across the months in cryopreserving SPZ than commercial egg yolk- free-based S-EXT and still seems, to the present day, the most appropriate S-EXT to be used in (Merino) sheep. ABSTRACT: This study aimed to evaluate and compare the effect of three semen extenders (S-EXT) on 22 spermatozoa (SPZ) parameters (subjective and computer-assisted sperm analysis evaluations), before and after semen cryopreservation throughout different months of the breeding season in the Portuguese Merino breed. According to the multivariable model, the SPZ viability (alive %), kinetics subjective individual motility, total motility, total progressive motility and its subpopulations, and beat cross frequency) were higher in the egg yolk-based S-EXT improved by Estação Zootécnica National (Portugal) than in Ovixcell(®) or Andromed(®) extenders. All the differences were only observed in thawed semen, except for total motility and total progressive motility, in which Ovixcell(®) also showed the poorest results on fresh semen. An interaction effect between S-EXT and semen processing was observed on 72.3% (17/22) of the evaluated parameters, evidencing a variable cryoprotective action between S-EXT. The SPZ viability was poorer in the onset of the breeding season (end of April/early May) than in the previous middle breeding season (November/early December), suggesting the influence of a short anoestrous season on ejaculate quality, even though the volume and SPZ concentration of the ejaculates remained stable throughout the experiment. Additionally, S-EXT x semen processing x month interaction effect on 59.1% (13/22) of the evaluated parameters evidenced the importance of SPZ time collection in a natural environment to cryopreserve ram’s semen. We concluded that, overall, the egg yolk-based S-EXT provided a greater value to the cryopreservation of Merino rams´ semen. Nevertheless, the causes of the interaction effect between S-EXT, semen processing and/or month on several SPZ parameters should be addressed, including SPZ molecular research in new studies, in order to improve egg yolk-based as well as in egg yolk-free-based S-EXT.