Freezing sperm from cauda epididymis of castrated stallions

Abstract

The goal of this work was to compare cryoresistance of epididymal spermatozoa with that of sperm collected with an artificial vagina. For this study, semen was collected from ten stallions using an artificial vagina. After that the stallions were bilaterally castrated and both epididymes dissected. The epididymides were maintained at ambient temperature until sperm recovery from the epididymal tails as described by Granemann et al. (2006). Three groups were established: (1) semen collected with artificial vagina prior to castration, (2) semen collected from the left epididymis, and (3) semen collected from the right epididymis. Spermatozoa were diluted in Botu-Crio® extender to a concentration of 400× 106 sperm/ml and stored in 0.5-ml straws. After 20 min at 5 ◦C, the straws were placed in nitrogen vapour (approximately −120 ◦C) for 15 min, and then plunged into liquid nitrogen (−196 ◦C). The straws were thawed in water bath at 37 ◦C for 30 s. Total and progressive motility and acrosome alterations were evaluated before freezing and after thawing by light microscopy. For the evaluation of acrosomal integrity, the sperm smears were stained with Cerovsky stain (Cerovsky, 1976). Total and progressive motility before freezing were higher (p< 0.05) in group 1 (73 and 58%) than in group 2 (45.7 and 32%) or in group 3 (38.8 and 21.9%). Acrosomal defects did not differ among the groups: 7.6, 10.4, and 7.1% for groups 1, 2, and 3, respectively (p< 0.05). After thawing, total and progressive motility and acrosome defects were similar in all groups: 38.5, 27.7% and 21.7, 25.5% and 18, 12.2% for groups 1, 2 and 3, respectively. Progressive motility of fresh collected spermatozoa was higher in all groups, when compared with progressive motility of epididymal sperm after thawing (p< 0.05). It was possible to preserve only <20% of the epididymal sperm. Recovery techniques for epididymal stallion sperm need to be improved to increase the efficiency of cryopreservation of epididymal sperm, similarly to what has been demonstrated in the post-mortem collection of bovine epididymal sperm (Herrick et al., 2004).