Characterization and cryopreservation of Amur leopard cats (Prionailurus bengalensis euptilurus) semen collected by urethral catheterization

The Amur leopard cat (Prionailurus bengalensis euptilurus) is a globally endangered species, and there is thus an urgent need to increase its population. The objectives of this study were to: (1) confirm the utility of urethral catheterization (UC) for semen collection from Amur leopard cats; (2) investigate proper dilution media for fresh semen; and (3) investigate the effectiveness of sperm cryopreservation, including examining the effect of glycerol concentration. Six adult males in captivity (mean weight 5.03 ± 0.44 kg, aged 2-6 years) were included. All study procedures were performed during the breeding season (February to April) over two consecutive years. Semen samples (n = 28) were collected four or five times from each animal (four times for two animals and five times for four animals) by UC under general anaesthesia, and their characteristics (including sperm motility) were evaluated. First, the sperm motility in semen diluted in Ham's F-10 or phosphate buffer saline (PBS) was compared. Next, semen diluted with TEST-yolk buffer containing 2%, 4%, or 6% glycerol was frozen in a liquid nitrogen tank, and sperm motility and acrosome integrity were evaluated after thawing. No difference in motility was observed between sperm diluted in Ham's F-10 and PBS. The percentages of sperm motility and kinetic values in semen frozen in 2% or 4% glycerol were higher than those in semen stored in 6% glycerol. In conclusion, the UC method for semen collection is recommendable for Amur leopard cats and should be useful for artificial insemination. Although sperm motility decreased after thawing, samples thus preserved may be usable for advanced reproductive techniques, such as intracytoplasmic sperm injection or in-vitro fertilization.