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.