Effect of Cryopreservation Protocol on Post-Thaw Characteristics of Stallion Spermatozoa

Three ejaculates from each of eight stallions were initially centrifuged in INRA 96 extender and spermatozoal pellets were resuspended in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Extended semen was exposed to a fast pre-freeze cooling rate (FAST - semen immediately subjected to cryopreservation) or a slow pre-freeze cooling rate (SLOW - semen pre-cooled at a controlled rate for 80 minutes prior to cryopreservation). After thawing, semen was diluted in initial freezing medium (FM) or INRA 96 prior to analysis of 9 experimental endpoints: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; um/sec), average-path velocity (VAP; ?m/sec), straight-line velocity (VSL; ?m/sec), linearity (LIN; %), intact acrosomal and plasma membranes (AIVIAB; %), intact acrosomal membranes (AI; %), and intact plasma membranes (VIAB; %). Eight of nine experimental endpoints (MOT, PMOT, VAP, VSL, LIN AIVIAB, AI, and VIAB) were affected by extender type, with LE extender yielding higher values than MF extender for these variables (P<0.05). Exposure of extended semen to a slow pre-freeze cooling period resulted in increased values for seven of nine endpoints, as compared to a fast pre-freeze cooling period (P less than 0.05). Mean VAP and VSL were unaffected by prefreeze cooling rate (P>0.05). As a post-thaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, VAP, and VSL (P less than 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and VIAB (P less than 0.05). Extender x rate interactions (P less than 0.05) were detected for the variables MOT, AIVIAB, AI and VIAB. Mean values for these endpoints were higher following spermatozoal exposure to a slow pre-freeze cooling period, regardless of freezing extender type (P less than 0.05). The effects of pre-freeze cooling rate on MOT, AIVIAB, AI, and VIAB were more pronounced in spermatozoa cryopreserved in MF extender, as compared to LE extender. Within treatment groups SLOW and FAST, mean MOT, AIVIAB, AI, and VIAB were higher (P less than 0.05) for spermatozoa cryopreserved in LE extender, as compared to MF extender. Extender x diluent interactions (P less than 0.05) were detected for MOT, PMOT, VCL, VAP, VSL, and LIN. Within Group MF, mean MOT, PMOT, VCL, VAP, and VSL were higher in INRA diluent, as compared to FM diluent (P less than 0.05). Within Group LE, FM diluent yielded slightly higher values than INRA diluent for PMOT, VAP, VSL, and LIN (P less than 0.05). In conclusion, a slow pre-freeze cooling rate was superior to a fast pre-freeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory post-thaw diluent prior to semen analysis.