Reproductive Biotechnology and International Training Programs for the Conservation of Brazilian Felids
Brazil possesses the broadest diversity of felids of any country in Latin America. Within its borders exist eight endemic felid species, consisting of two large-sized (>35 kg body weight) and six small-sized (<20 kg BW) cats. For each of these species, habitat loss and/or poaching are imperiling wild populations.6 For the past 7 yr, conservation of Brazilian felids has been the primary focus of a collaborative reproductive research and training program implemented both in Brazil and the U.S. Scientists from the Cincinnati Zoo’s Center for Conservation and Research of Endangered Wildlife (CREW) and the Smithsonian Institution’s Conservation and Research Center (CRC) have been working extensively with Brazilian colleagues based at five institutions in Brazil (Universidade Federal do Paraná, Zoológico do Curitiba, Itaipu Binacional, Associaçao Mata Ciliar, Universidade de São Paulo). The objectives of this partnership have been to: 1) provide intensive training in reproductive research techniques to Brazilian scientists, 2) conduct collaborative studies investigating basic and applied reproduction in endangered Brazilian felids, and 3) establish a trained cohort of Brazilian scientists to conduct independent conservation-based research in Brazil.
Initial training in reproductive research techniques was provided to several Brazilian veterinarians in 1995 as part of a genetics/reproductive survey of felids in Latin American zoos.7 Results of the reproductive survey established species’ norms for reproductive traits in eight Latin American felid species and provided insight into several management factors negatively impacting on reproductive success in Latin American zoos. This informal training was followed by three formal training courses (1995, 1996, 1998) conducted at the Zoológico do Curitiba and the Universidade Federal do Paraná in Curitiba and at Itaipu Binacional in Foz do Iguaçu. These courses focused on reproductive research in endangered felids, providing both didactic lectures and hands-on training in research techniques, including semen collection, laparoscopy, and laparoscopic artificial insemination (AI), in vitro fertilization (IVF) and sperm/embryo cryopreservation. During these courses, a total of ∼30 Brazilian veterinarians received training, including several participants in subsequent collaborative studies. Some of the latter scientists also traveled to the U.S to receive instruction in conducting fecal hormone metabolite analysis at the CRC. Three of these scientists have recently completed doctoral dissertations derived from their reproductive research with Brazilian felid species.
In collaborative studies, Brazilian colleagues have used these new skills to characterize basal reproductive traits in captive ocelots (Leopardus pardalis), margays (Leopardus weidi) and tigrinas (Leopardus tigrinus). Scientists conducted detailed reproductive evaluations (including testes measurement and semen collection/analysis) of males of each species, gathering valuable data on basal sperm production and quality.3,4 Fecal hormone metabolites (testosterone, estradiol, progesterone, cortisol) also were monitored in male and female felids to investigate reproductive cyclicity and the influence of seasonality and stress levels on reproductive parameters.2-5 These studies established that males of each species can produce high-quality ejaculates throughout the year, with minimal fluctuations due to season, when maintained off-exhibit on nutritionally complete diets. Findings also indicated that female ocelots and margays (and possibly tigrinas) exhibit ovarian cyclicity year-round, and that ocelots and tigrinas are induced ovulators whereas margays may ovulate spontaneously.
In applied studies in U.S. zoos, the feasibility of laparoscopic artificial insemination (AI) in ocelots was investigated as a means to improve gene flow between isolated populations. This research demonstrated that, compared to other felid species, ocelots were relatively insensitive to the exogenous gonadotropins used to stimulate ovarian activity. This study culminated in the production of viable offspring at the Dallas Zoo after laparoscopic AI with frozen-thawed sperm.8 In subsequent collaborative studies in Brazilian zoos, Brazilian colleagues used laparoscopic AI with freshly collected sperm to produce offspring in two cat species, the ocelot and tigrina.1 Fecal hormone monitoring allowed comparison of estrogen and progestin profiles in pregnant and nonpregnant females. Ongoing research is exploring the application of laparoscopic AI for propagation of margays and jaguarundis (Herpailurus yaguarondi). Findings to date suggest that the relative insensitivity to exogenous gonadotropins is a conserved trait among species in the ocelot genetic lineage but that offspring may be produced with AI after determination of appropriate hormone dosages.
In the most recent collaborative efforts, the potential of in vitro fertilization (IVF), embryo cryopreservation and embryo transfer are being investigated for genetic management of ocelots and tigrinas. To date, over 75 Brazilian ocelot and 50 tigrina embryos have been created, cryopreserved, and stored in liquid nitrogen tanks in Brazil.10 Notably, the frozen ocelot embryos represent ∼15 potential founders for the development of a North American population of Brazilian ocelots (a primary goal of the Ocelot Species Survival Plan and a major focus of the recently initiated Brazilian Ocelot Consortium). The validity of this approach was recently demonstrated by the birth of a generic ocelot kitten after transfer of frozen-thawed embryos to a female ocelot at the Cincinnati Zoo.9 In early 2002, the first transfers of frozen-thawed Brazilian ocelot and tigrina embryos were conducted at the Associaçao Mata Ciliar in Brazil (pregnancy results still pending). With the initiation of the Brazilian Ocelot Consortium, importation of frozen Brazilian ocelot embryos and additional transfers into generic female ocelots in the United States are anticipated to occur in late 2002.
These collaborative research projects are providing invaluable information that is directly benefiting the conservation of Brazilian felid species. Findings derived from these studies are proving useful for improving husbandry, population management, and breeding of Brazilian felids in captivity as insurance against extinction. In particular, the importance of providing adequate nutrition and minimizing stress to optimize reproductive success among small felids has received strong emphasis. In addition, continued advances in assisted reproduction eventually may provide an alternative route for exchanging genetic material among populations within Brazil as well as between Brazil and other countries, an important consideration in developing global management programs (such as the Brazilian Ocelot Consortium). Most importantly, this collaborative program has been essential for capacity building within Brazil, in establishing a core group of highly trained reproductive biologists that will continue applying their new knowledge and skills to the conservation of Brazilian felids.
The research and conservation efforts described in this paper involved the dedicated participation of numerous individuals in Brazil and the United States. In Brazil, the authors thank Drs. Rosana Morais, Nei Moreira, Wanderlei Moraes, Cristina Adania, Regina Paz, Ronaldo Morato, Marcelo Guimaraes, and Maria Lucia Gomes and, in the U.S., Drs. JoGayle Howard, Laura Graham, David Wildt, Mark Campbell, Ken Cameron, and Terri Roth, in addition to Helen Bateman, Jennifer Bond, Molly McRae and Lynn Patton. The assistance of animal keeper staff at all collaborating institutions is greatly appreciated. Funding for these projects was provided by the National Institutes of Health, Friends of the National Zoo, British Airways, the Philip Reed Foundation, American Zoo and Aquarium Association, Fundação de Amparo á Pesquisa do Estado de São Paulo, and the Consejo Nacional de Ciencia y Tecnologia.
1. Moraes W, RN Morais, N Moreira, O Lacerda, MLF Gomes, RG Mucciolo, WF Swanson. 1997. Successful artificial insemination after exogenous gonadotropin treatment in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrina). Proc. Amer. Assoc. Zoo Vet. Pp. 334–335.
2. Morais RN, N Moreira, W Moraes, RG Mucciolo, O Lacerda, MLF Gomes, WF Swanson, LH Graham, JL Brown. 1996. Testicular and ovarian function in South American small felids assessed by fecal steroids. Proc. Amer. Assoc. Zoo Vet. Pp. 351–356.
3. Morais RN, RG Mucciolo, MLF Gomes, O Lacerda, W Moraes, N Moreira, WF Swanson, JL Brown. 1997. Adrenal activity assessed by fecal corticoids and male reproductive traits in three South American felid species. Proc. Amer. Assoc. Zoo Vet. Pp. 220–223.
4. Morais RN, RG Mucciolo, MLF Gomes, O Lacerda, W Moraes, N Moreira, LH Graham, WF Swanson, JL Brown. Seasonal analysis of seminal characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus). Theriogenology. In press.
5. Moreira N, ELA Monteiro-Filho, W Moraes, WF Swanson, LH Graham, O. Pasquali, MLF Gomes, RN Morais, DE Wildt, JL Brown. 2001. Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus. Zoo Biol. 20:103–116.
6. Nowell K, P Jackson. 1996. Wild Cats: Status Survey and Conservation Action Plan. IUCN/SSC Cat Specialist Group. Gland, Switzerland. 382 pp.
7. Swanson WF, DE Wildt, RC Cambre, SB Citino, KB Quigley, D Brousset, RN Morais, N Moreira, SJ O’Brien, WE Johnson. 1995. Reproductive survey of endemic felid species in Latin American zoos: male reproductive status and implications for conservation. Proc. Amer. Assoc. Zoo Vet. Pp. 374–380.
8. Swanson WF, JG Howard, TL Roth, JL Brown, T Alvarado, M Burton, D Starnes, DE Wildt. 1996. Responsiveness of ovaries to exogenous gonadotrophins and laparoscopic artificial insemination with frozen-thawed spermatozoa in ocelots (Felis pardalis). J. Reprod. Fertil. 106:87–94.
9. Swanson WF. 2001. Reproductive biotechnology and conservation of the forgotten felids - the small cats. Proceedings of the 1st International Symposium on Assisted Reproductive Technology for the Conservation & Genetic Management of Wildlife. Omaha, NE, Pp. 100–120.
10. Swanson WF, RCR Paz, RN Morais, MLF Gomes, W Moraes, CH Adania. 2002. Influence of species and diet on efficiency of in vitro fertilization in two endangered Brazilian felids - the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus). Theriogenology. 57:593.