Canine spermatozoa-cryopreservation and evaluation of gamete interaction

Successful cryopreservation of spermatozoa for genome banking or transport for artificial insemination (AI) in domestic and nondomestic Canidae depends on understanding the species-specific conditions required. Experiment 1 was designed to evaluate the post-thaw effects of 5 cooling rates on percentages of motile cells, progressive motility, morphology and acrosomal integrity of domestic dog spermatozoa. Semen samples (n=18) extended in glycerol-Tris egg yolk buffer were cooled to 0 degrees C, drawn into 0.5-ml straws and subjected to A) cooling at 0.5 degrees C/min to-20 degrees C and at 2 degrees C/min from -20 degrees C to -70 degrees C, B) cooling at 12 degrees C/min, C) cooling at 28 degrees C/min, D) cooling at 99 degrees C/min, and E) cooling at 214 degrees C/min. Samples were thawed by placing straws into 70 degrees C water for 6 sec. The contents were poured into microcentrifuge tubes, kept at 0 degrees C, and evaluated at 0, 2, 4 and 6 h. Overall, the smallest decline in the proportion of motile spermatozoa and in the progressive motility score was with the 12 and 28 degrees C/min freezing rates, and the greatest with the 214 degrees C/min rate. There were no differences in morphology of spermatozoa among the protocols, but the proportions of viable spermatozoa and of undamaged acrosomes were highest for the 12 and 28 degrees C/min rates. In Experiment 2 the capacity of fresh spermatozoa to penetrate fresh, cooled or salt-stored canine oocytes in vitro was evaluated. Canine oocytes harvested from ovaries were either immediately placed into TALP (fresh ova), or into hypertonic salt solution (salt-stored ova): for cooled oocytes the ovaries were stored in PBS with BSA at 5 degrees C overnight before the ova were harvested. Oocytes and spermatozoa were co-incubated for 24 h, stained with Hoechst 33258 and examined under fluorescent light at x 400. Penetration and attachment of spermatozoa to the zona did not differ significantly between fresh and cooled oocytes (73 and 65%; 4.1 and 2.9 sperm/ovum, respectively), but cooled ova tended to show fewer attached spermatozoa. Salt-stored oocytes showed the lowest number of spermatozoa penetrating or attaching to the zona (0.7 sperm/ovum; P<0.001), and only 36.5% of the oocytes were penetrated (P<0.02). The results suggest that the optimum cooling rate for freezing canine spermatozoa is approximately 30 degrees C/min and that canine spermatozoal function can be evaluated by measuring their penetration of or attachment to homologous oocytes.