Challenges in cryopreservation of clouded leopard (Neofelis nebulosa) spermatozoa.

The clouded leopard (Neofelis nebulosa) is an endangered species that is difficult to breed in captivity. Species management could benefit from the use of artificial insemination (AI) with frozen-thawed spermatozoa, but there have been no detailed studies of sperm cryosensitivity. The purposes of this study were to: (1) re-characterize seminal characteristics in the clouded leopard 20 years after the first descriptive studies Wildt et al., [Wildt DE, Howard JG, Hall LL, Bush M. Reproductive physiology of the clouded leopard. I. Electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. Biol Reprod 1986; 34: 937-947]; and (2) conduct a comparative cryopreservation study on the feasibility of sperm from this species surviving a freeze-thawing stress. Ejaculates were collected from five adult males and subjected to standard analysis, followed by a two-step straw freezing protocol that evaluated the impact of thawing, dilution, centrifugation and in vitro culture (through 4 h) on sperm motility and acrosomal integrity. Additionally, we assessed the impact of both a traditional permeating cryoprotectant (glycerol at a final dilution of 4%) and an unconventional nonpermeating trisaccharide; raffinose (R) at a final dilution of 4% or 8%, with or without 4% glycerol on sperm cryosurvival. The clouded leopard produced an extremely poor quality ejaculate; although approximately 70% of fresh sperm were motile, >80% were malformed. Phase contrast microscopy revealed that 40% of all sperm had abnormal acrosomes, but Coomassie blue staining indicated that acrosomal abnormalities existed in almost 70% of spermatozoa. Upon freeze-thawing, sperm motility declined markedly (P < 0.05) by an average of 40%, regardless of diluent used. Interestingly, raffinose was as effective as glycerol in protecting both sperm motility and acrosomal integrity. Although no acrosomal damage was seen immediately after thawing, < 6% morphologically normal intact acrosomes were present by the last measured time point. In conclusion, the clouded leopard is a rare felid that (at least in North American zoos) is producing extraordinarily poor quality ejaculates. There are so many sperm with unexplained deranged acrosomes that it will be particularly challenging to use traditional AI with thawed sperm as an adjunct management tool.

Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant.

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.