Canine spermatozoa — cryopreservation and evaluation of gamete interaction

Abstract

Successful cryopreservation of spermatozoa for genome banking or transport for articifial 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°C, drawn into 0.5-ml straws and subjected to A) cooling at 0.5 °C/min to − 20 °C and at 2 °C/min from −20°C to −70°C, B) cooling at 12 °C/min, C) cooling at 28°C/min, D) cooling at 99 °C/min, and E) cooling at 214 °C/min. Samples were thawed by placing straws into 70 °C water for 6 sec. The contents were poured into microcentrifuge tubes, kept at 0 °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 °C/min freezing rates, and the greatest with the 214 °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 °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 (saltstored ova): for cooled oocytes the ovaries were stored in PBS with BSA at 5 °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 ~30 °C/min and that canine spermatozoal function can be evaluated by measuring their penetration of or attachment to homologous oocytes.