OVERCOMING SPERM ABNORMALITIES BY SPERM SELECTION IN CLOUDED LEOPARDS (Neofelis nebulosa)

Abstract

Clouded leopard (CL) is an endangered felid species of Southeast Asia. Wild populations are declining due to habitat loss and hunting in range countries. CL breeding success in captivity is limited due to male aggression, high incidence of abnormal sperm and uncertain ovarian activity of females. Therefore, the development of optimal assisted reproductive technologies for this species is recommended by CL specialist group. This study aimed to 1) investigate sperm characteristic of captive clouded leopards in Thailand; 2) determine the functional properties of sperm after selection with the single layer centrifugation (SLC) method; 3) identify the origin of coiled tail defects and mitigate them using a demembranation approach; 4) assess the reproductive organs and ovarian activity after hormonal treatment; and 5) develop laparoscopic oviductal artificial insemination technique for CL. Captive CL (n=17, 11 males and 6 females) have been studied during 2013 - 2015. In male study, twenty-two ejaculates were collected by electroejaculation and evaluated. The high proportion of morphologically abnormal sperm (63.9 ± 2.0%) was observed with tightly coiled tail as a major defect (13.5 ± 0.5 %). CL males that are paired with females showed significantly higher percentage of sperm with intact acrosome, and lower number of sperm with bent midpiece/cytoplasmisc droplet than the males housed singly (P <0.05). Two sperm preparation methods including 1) simple washing; or 2) SLC, was applied prior to cryopreservation (n=12). Sperm motility, % sperm with intact acrosome and % sperm with normal tail in chilled and frozen-thawed semen samples were increased after SLC treatment. The heterologous IVF in the SLC-processed group showed significantly higher fertilization success than the simple washing group (P <0.05). To investigate the potential origin of coiled tail, 11 ejaculates were evaluated and equally allocated to simple washing (control group; resulting in about 10% coiled tails after treatment) and SLC (treated sample; resulting in about 6 % coiled tails after treatment). Aliquots of semen were subjected to hypo-osmotic swelling (HOS) test and demembranation test using 20% Triton X-100 (TX). After TX treatment, most of the coiled tail in raw ejaculates could not be uncoiled or opened by TX indicating that the cause of coiled sperm tails may be caused by testicular origin. SLC demonstrated its ability to decrease the primary sperm defects of raw semen. SLC-selected spermatozoa were prone to be mitigated after demembranation. In females study, fecal estradiol (E2) and progesterone (P4) metabolites of adult female were quantified by enzyme immunoassay (EIA). Females were given exogenous gonadotropins; Group 1 (n=2) 300 IU eCG/1500 IU of pLH (82 h interval); and Group 2 (n=3) 200 IU eCG/1000 IU pLH (82 h interval). Ovarian assessment was performed at 29 h, 44 h and 96 h post pLH administration using laparoscopy. Estrus signs have been observed in all females. Oocytes were collected from two females of group 1 by OPU technique and were subjected to homologous IVF with post-thawed SLC-selected spermatozoa. A two-cell stage embryo was transferred to recipient (n=1) with no conception. For AI, the ovulated female (n=1) were inseminate with frozen-thawed selected sperm at 44 h after pLH administration. However, no pregnancy occured after 90 days post insemination. The optimized stimulating protocol for AI (eCG 200 i.u. and pLH 1000 i.u. with 82 hr interval) was applied in four CL females. Ovarian assessment was performed at 44 hr after pLH administration. One nulliparous female ovulated from both ovaries. Semen of two adult males with total motile sperm of 8 x 106 (M1) and 2.7 x 106 (M2) were used for AI by laparoscopic oviductal technique. Increasing of fecal progesterone concentration after AI presented the peak at day 25 post pLH injection and maintained at 128.4 µg/g dry feces during 65 day post AI indicating that the female had conceived. Delivery of two healthy CL cubs occurred on day 90 of gestation. In conclusion, sperm selection for good quality spermatozoa with optimal assisted techniques can be promising tools to assist in conservation breeding and sustaining genetic variation of this endangered species in the future.