First Evaluations and Cryopreservation of Semen Samples from Sunda Clouded Leopards (Neofelis diardi)

SIMPLE SUMMARY: The Sunda clouded leopard is a medium-sized cat from Borneo and Sumatra. The captive population has less than 20 individuals on both islands. Conservation breeding of this species has been unsuccessful due to the small numbers of individuals and the difficulty of pairing them. The present study aimed at evaluating for the first time the reproductive organs, semen characteristics, and sperm freezing for two wild-caught males (8 and 11 year old) living singly. The morphometric and ultrasound examination showed some degree of abnormalities in both leopards. Semen evaluation obtained by electro-ejaculation revealed a high proportion of abnormal sperm cells (similar to mainland clouded leopards). While only the younger male produced samples that could be frozen, post-thaw evaluations showed that the few surviving spermatozoa could potentially be used for in vitro fertilization or sperm injection. Studies on more individuals are needed to validate those encouraging but preliminary results. ABSTRACT: A better understanding of semen characteristics and resilience to freezing temperatures is necessary before developing assisted reproductive techniques and systematic biobanking for the Sunda clouded leopard. The objective of this study was to evaluate for the first time the semen and sperm quality (in fresh and frozen samples) of two captive Sunda clouded leopards in Malaysia. A total of 17 examinations of the reproductive tract (using ultrasonography) and electro-ejaculations were performed on the two leopards over a 2-year period. Samples obtained from Leopard 1 (8 years old) varied in terms of volume (402 ± 92 µL), pH (7.9 ± 0.9), sperm motility (54.5 ± 24.2%), sperm concentration (122.4 ± 84.7 × 10(6) sperm/mL), normal morphology (23.9 ± 12.3%), and viability (55.2 ± 18.2%). Midpiece defects represented the most common structural abnormality followed by abnormal tail and head defects. Samples from Leopard 2 (11 year old with abnormal testicular tissue) were of lesser quality. Two frozen semen samples from Leopard 1 were thawed and examined for acrosome integrity. Post-thawed samples contained <10% of motile spermatozoa but almost 50% of abnormal acrosomes. The present results emphasized the high incidence of structurally-abnormal spermatozoa, similar to the mainland clouded leopard. Post-thaw evaluations showed that the few surviving spermatozoa could potentially be used for in vitro fertilization or sperm injection. However, more individuals must be studied to validate those first findings that are exciting but still preliminary.