Optimization of a Protocol for Cryopreservation of Electroejaculated Beef Bull Semen Under Ambulatory Conditions

Abstract

The overall goal was to optimize and simplify a cryopreservation protocol under ambulatory conditions. It was hypothesized that removing seminal plasma by centrifugation would improve postthaw semen quality, and that increasing equilibration to 24 h and freezing in liquid nitrogen vapor within the neck of the tank would not affect post-thaw quality of electroejaculated bull semen. One ejaculate was collected from nine beef bulls using electroejaculation, and was divided into four aliquots: NC5: noncentrifuged, equilibrated for 5 h; NC24: non-centrifuged, equilibrated for 24 h; CE5: centrifuged, equilibrated for 5 h; CE24: centrifuged, equilibrated for 24 h. Straws were held horizontally on a rack 3cm above liquid nitrogen for 10min before plunging in liquid nitrogen. In addition, some straws equilibrated for 5h were held vertically 2cm over liquid nitrogen within the neck of the tank (NCneck, CEneck). Post-thaw sperm motility (CASA), membrane integrity (SYBR14/PI), acrosome integrity (FITC-PNA) and apoptosis (ANV) were evaluated and compared using ANOVA. Sperm motility, membrane integrity or apoptosis did not differ among treatments. However, centrifuged semen equilibrated for 24h had significantly lower DSL, VSL, LIN, STR and WOB than non-centrifuged semen equilibrated for 5h. Centrifugation increased the percentage of acrosome-damaged spermatozoa (NC5 5.8±3, CE5 42.2±10.7, NC24 4.8±2.5, CE24 32.5±8.9, NCneck 17.9±7.2, CEneck 42.3±12.5%; P=0.002). Extending the equilibration time had no effect on the parameters evaluated in non-centrifuged semen. Removal of seminal plasma by centrifugation is not recommended since it had a negative effect on acrosome integrity.