Optimization of the Equine-Sperm Freeze Test in Purebred Spanish Horses by Incorporating Colloidal Centrifugation

SIMPLE SUMMARY: The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations for equine-sperm cryopreservation. We investigated if the incorporation of single-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing. Using colloidal centrifugation, the percentage of ejaculates available to be frozen was increased from 35% to 71%, allowing us to obtain from poor-quality fresh ejaculates thawed sperm doses with similar sperm quality to that of good-quality fresh ejaculates. These results could potentially be of great interest in the equine reproductive industry when dealing with other individuals or breeds in which, initially, low sperm quality prevents or limits their inclusion in sperm-cryopreservation programs. ABSTRACT: The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations for equine-sperm cryopreservation. This means that artificial insemination with frozen semen from these stallions is less widespread than in other breeds. In this study, we investigated if the incorporation of single-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing, observing the influence of centrifugation and freezing extender protocol on post-thawed sperm motility. Using colloidal centrifugation, the percentage of ejaculates available to be frozen was increased from 35% (6/17) to 71% (12/17), doubling the number of samples that could have been subjected to cryopreservation. We only found significant differences in linearity (LIN) and lateral head displacement (ALH) after 5 min of incubation at 37 °C between colloidal and simple centrifugation processing techniques. No significant differences were found between the two different colloidal protocols in any of the variables considered. Colloidal centrifugation allowed us to obtain, from worse fresh-quality ejaculates, thawed sperm doses with similar quality to that of good-quality ejaculates. BotuCrio(®) produced, in general, higher motility parameters and its characteristics than the other extenders analyzed, with significant differences found in comparison to Inra-Freeze(®) and Lac-Edta in both total (MOT) and progressive motility (PMOT) when using colloidal centrifugation and only in PMOT when applying simple centrifugation. Colloidal centrifugation optimized the efficiency of cryopreservation, as it allowed us to increase the number of ejaculates of Purebred Spanish Horses suitable to be frozen. Including these semen processing techniques in the freeze test could help to optimize equine-sperm cryopreservation protocols, especially when dealing with individuals or breeds for which initially low sperm quality prevents or limits their inclusion in sperm cryopreservation programs.