Function and culture requirements of snow leopard (Panthera uncia) spermatozoa in vitro

Abstract

Electroejaculates from eight snow leopards were used to determine how the motility of spermatozoa was influenced by (i) type of media (Ham's F10, PBS, human tubal fluid or RPMI-1640); (ii) holding temperature (23°C versus 37°C); (iii) washing of spermatozoa and (iv) a sperm metabolic enhancer, pentoxifylline. The duration of sperm motility was assessed by evaluating samples in each treatment every hour for 6 h and a sperm motility index (a value combining percentage sperm motility and rate of forward progression) calculated. Spermatozoa from the Ham's F10, PBS and PBS plus pentoxifylline treatments were also co-incubated with zona-intact, domestic cat eggs that were fixed and evaluated for spermatozoa bound to the zona pellucida, penetrating the outer and inner layers of the zona pellucida and within the perivitelline space. During the 6 h co-incubation, the sperm motility index in PBS with pentoxifylline was greater (p < 0.05) than in PBS alone which, in turn, was greater (P < 0.05) than in the other three test media. Washing the spermatozoa enhanced (P < 0.05) motility in both PBS and PBS plus pentoxifylline relative to unwashed samples, but there was no effect (P > 0.05) of holding temperature. Pentoxifylline supplementation enhanced (P < 0.05) the proportion of cat eggs with bound, but not penetrated, snow leopard spermatozoa. Only one to three of the 120 total cat eggs/treatment group had snow leopard spermatozoa in the inner layer of the zona pellucida, and there were no spermatozoa in the perivitelline space. Failure of sperm penetration did not appear related to an inadequacy in the culture system because nine of 27 snow leopard eggs co-incubated in PBS with conspecific spermatozoa cleaved in vitro and nine of the 18 unfertilized eggs contained spermatozoa in the perivitelline space. The snow leopard appears unique among felid species in that (i) sperm viability is highly sensitive to type of culture medium, (ii) sperm motility and function in vitro appear enhanced by a simple rather than complex culture medium and (iii) there is a mechanism that prevents these spermatozoa from fully penetrating heterologous, salt-stored, domestic cat eggs.