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Zoo ResearchDeveloping in vitro Fertilization in the Clouded leopard (Neofelis nebulosa) for Assessment of Oocyte and Embryo Quality

JoGayle Howard, Theriogenologist, Co- Investigator, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC.

Rebecca Hobbs, Post-doctoral Fellow, Co-investigator, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC.

The North American clouded leopard population managed by the Species Survival Plan is not a genetically healthy, self-sustaining population. The major problem affecting captive breeding of clouded leopards is the high incidence of male aggression. This has severely limited the number of naturally breeding pairs, and consequently, most animals are housed individually. It is integral that all animals within the SSP contribute to the population ensuring greater genetic diversity.

The Smithsonian Conservation Biology Institute at the National Zoo has studied the reproductive biology of clouded leopards for over 20 years. We have used this information to develop ovarian control and assisted reproductive techniques to enhance reproduction in this species. Unfortunately, attempts at artificial insemination (AI; placing sperm directly into the uterus) have not been successful in clouded leopards as they have in other cat species.

AI failure may be due to multiple factors, including: changes in oocyte (egg) quality, reduced sperm function, poor fertilization rates, compromised embryo development, corpora lutea (ovulation site) insufficiency, or uterine abnormalities. To analyze these problems, we are developing in vitro fertilization (IVF) for clouded leopards. IVF is a tool that allows direct evaluation of oocyte and embryo quality through observational assessment of fertilization, embryo development and molecular analysis of mRNA (the message read from DNA and translated into protein) in follicle cells. IVF involves hormonal stimulation to induce ovarian activity and oocyte maturation, collection of oocytes by laparoscopy and placement of these oocytes in culture medium with sperm for fertilization in vitro. Not only can IVF be used to diagnose problems with AI, there also is potential to cryopreserve and store the IVF embryos for future use.

The primary objectives are to:
a) characterize clouded leopard oocyte fertilization in vitro;
b) assess the quality of oocytes following the AI hormone stimulation protocol; and
c) assess the quality of embryos generated from young (under 8 years) versus aged (over 9 years old) clouded leopards.
The overall goal of this project is to develop an IVF protocol for clouded leopards to identify any potential problems with oocyte quality leading to AI failure and ultimately to improve reproductive and genetic health in the North American Clouded Leopard SSP program.