Assessment of bovine spermatozoa viability using different cooling protocols prior to cryopreservation

The aim of our study was to evaluate the effect of different cooling rates on the post-thawing quality of bovine spermatozoa. Ejaculated semen from a 24-month-old Jersey bull was collected using an artificial vagina and diluted in a commercial extender to evaluate spermatozoan concentration and motility subjectively before cooling and freezing and after thawing. Straws were allocated to four cooling curves: rapid (RD), semi-rapid (SRD), semi-slow (SSLW) and slow (SLW). The temperature was decreased from 25°C to 4°C in 10, 50, 110 and 135 min, which represents a cooling rate of 2.06, 0.40, 0.18 and 0.15°C/min, respectively. Then straws were frozen and stored at -196°C. After thawing, one aliquot of each straw was used for evaluation. Spermatozoan integrity and mitochondrial function were evaluated using a combination of fluorescent probes containing 100 mg/mL FITC-PSA, 0.5 ^g/mL PI and 153 ^M JC-1. At the end of cooling, spermatozoan motility did not differ among RD (63.3%), SRD (66.7%), SSLW (66.7%) and SLW (80.0%). However, normal spermatozoan morphology was lower in SRD (84.8%) compared to RD (91.7%), SSLW (91.7%) and SLW (90.3%) (P<0.05). In thawed semen, spermatozoan motility and normal morphology did not differ among RD (40.0%; 88.8%), SRD (43.3%; 82.5%), SSLW (40.0%; 87.2%) and SLW (36.7%; 88.0%). The percentage of damaged spermatozoa, including plasma and acrosome membrane damage and low mitochondrial potential, was higher in RD compared to the others (P<0.05). In conclusion, a rapid cooling curve is detrimental to the spermatozoa and affects the post-thaw sperma-tozoan integrity of bovine frozen semen.