International training programs in reproductive ... - Semantic Scholar

Animal Reproduction Science 82–83 (2004) 21–34

International training programs in reproductive sciences for conservation of Latin American felids W.F. Swanson a,∗ , J.L. Brown b a

b

Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, 3400 Vine Street, Cincinnati OH 45220, USA Conservation and Research Center, Smithsonian Institution, 1500 Remount Road, Front Royal VA 22630, USA

Abstract Survival of the ten non-domestic felid species endemic to Latin America is imperiled by habitat loss, poaching and poor captive management. Over the past 10 years, conservation of these felids has been the primary focus of a reproductive research and training program conducted in Brazil, Mexico, and the USA. The objectives of this program were to: (1) provide intensive training in reproductive sciences to Latin American scientists, (2) conduct collaborative studies investigating basic and applied reproduction in endangered felids, and (3) establish a highly-trained scientific cohort to conduct independent conservation-based research. Four formal training courses, consisting of didactic lectures and hands-on instruction in research techniques, including semen collection, sperm cryopreservation and laparoscopic artificial insemination (AI), were taught in Brazil and Mexico between 1995 and 1998. Several of these scientists received further training in conducting fecal hormone analysis in the USA, and a number of research studies, many in collaboration with American scientists, were initiated in Latin American felids. Research findings have characterized basal reproductive traits in several felid species, including ocelots, margay, tigrinas and jaguars, and established that Latin American felids exhibit only minimal seasonal variation in most reproductive traits. Other studies have explored the impact of acute and chronic stressors on adrenocortical activity and demonstrated the importance of environmental enrichment in captivity, especially in small felids. Additional research has examined ovarian and immunological responsiveness of Latin American felids to exogenous gonadotropins and assessed the impact of nutrition on sperm production and oocyte quality. Applied reproductive studies have investigated sperm cryopreservation in both captive and wild felid populations and demonstrated the production of viable offspring in ocelots and tigrinas following laparoscopic AI. Ongoing studies are investigating the potential of in vitro fertilization (IVF), embryo cryopreservation and embryo transfer for genetic management of ocelots and tigrinas. To date, over 75 Brazilian ocelot and 50 tigrina IVF embryos have been cryopreserved and two pregnancies have been established in ocelots following transfer of frozen-thawed embryos. Findings from these studies are helping to improve husbandry, population management, ∗ Corresponding author. Tel.: +1 513 961 2739; fax: +1 513 569 8213. E-mail address: [email protected] (W.F. Swanson).

0378-4320/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2004.05.008

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W.F. Swanson, J.L. Brown / Animal Reproduction Science 82–83 (2004) 21–34

and breeding of Latin American felids in captivity. Continued advances in assisted reproduction eventually may provide an alternative route for exchanging genetic material among Latin American felid populations. Most importantly, this collaborative program has been essential for building scientific capacity, within Brazil and Mexico, in establishing a core group of highly-trained reproductive biologists that will continue applying their new knowledge and skills to the conservation of Latin American felids. © 2004 Elsevier B.V. All rights reserved. Keywords: Cats; Fecal hormones; Laparoscopy; Electroejaculation; Cryopreservation; Research training

1. Introduction Latin America is comprised of 21 countries within North, Central and South America, a vast geographic area totaling nearly 20,000,000 km2 . Within this broad region, there exist ten non-domestic felid species—jaguar (Panthera onca), puma (Puma concolor), ocelot (Leopardus pardalis), margay (Leopardus wiedii), tigrina (Leopardus tigrinus), Geoffroy’s cat (Oncifelis geoffroyi), pampas cat (Oncifelis colocolo), jaguarundi (Herpailurus yaguarondi), Andean mountain cat (Orealilurus jacobitus), and kodkod (Oncifelis guigna) (Nowell and Jackson, 1996). Most Latin American felids are threatened with extinction in all or part of their natural range, with habitat loss and/or poaching threatening wild populations (Nowell and Jackson, 1996). Of the worlds’ 24 biodiversity hotspots, six are located in Latin America, containing 16% (43,700 species) of global plant diversity (Mittermeier et al., 1998) and all ten endemic felid species. Conserving these six hotspots could protect representative populations of most Latin American felids. Conservation of felids in Latin America, however, is challenging in the face of human population growth, increasing resource consumption and a host of other societal pressures on habitats and wildlife populations. Concerted conservation efforts, involving a host of complementary strategies and disciplines, are needed to ensure species survival. An improved understanding of reproduction in Latin American felids likely would benefit the conservation of both captive and wild populations. Moreover, capacity building in all aspects of wildlife conservation, including reproductive sciences, may be essential for effecting meaningful progress in conserving biologically-threatened populations in range countries. Previous conservation training programs in Latin America and other regions have addressed the need for skilled zoo biologists (Waugh, 1988; Wemmer et al., 1990) and wildlife ecologists (Bennett and Rabinowitz, 2001; Rudran and Wemmer, 2001), providing impetus for incorporating other scientific disciplines that serve to optimize these conservation efforts. Mexico and Brazil possess the broadest diversity of felids in Latin America, due to their relatively large geographic size (i.e. 52% of total area) and diversity of habitats found within their borders. In these two countries, there exist eight of the ten endemic felid species (all except the kodkod and Andean mountain cat). In Mexico and Brazil, there also is a growing conservation ethic among the general populace, a well-established, scientific infrastructure and nascent financial resources available to support conservation-based, reproductive research programs. Accordingly, these two countries were the logical choices as focal points for initiating a research training program in reproductive sciences in Latin America.


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Over the past 10 years, conservation of Latin American felids has been the main focus of a collaborative reproductive research and training program, based primarily in Mexico and Brazil. During this time period, 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 worked extensively with Mexican and Brazilian colleagues from six institutions in Mexico and Brazil (Universidad Nacional Autonoma de Mexico, Universidade Federal do Paraná, Zoológico do Curitiba, Itaipu Binacional, Associaçao Mata Ciliar, Universidade de São Paulo). The objectives of this partnership were to: (1) provide intensive training in reproductive sciences to Brazilian and Mexican colleagues; (2) conduct collaborative studies investigating basic and applied reproduction in endangered Latin American felids; and (3) establish a highly-trained cohort of Latin American scientists to conduct independent conservation-based research within their native countries.

2. Application of reproductive sciences to conservation Reproductive sciences encompass a diversity of inter-related fields, including gamete biology, embryology, endocrinology, and cryobiology. In the past 20 years, the application of reproductive sciences has matured as a conservation discipline. From the initial excitement about the potential use of assisted reproduction (i.e. artificial insemination, AI; in vitro fertilization, IVF; embryo transfer, ET) for propagation came the eventual recognition of the inherent difficulties and inefficiency of that approach, given limitations in species-specific reproductive knowledge (Wildt and Wemmer, 1999; Wildt et al., 2001). Research emphasis subsequently shifted to using reproductive sciences as a means to systematically defined basal reproductive traits and assess the reproductive status of individuals, populations and species. Development of a comprehensive physiological database for each species is considered a necessary pre-requisite to applying reproductive sciences for animal propagation, whether through improved management for natural breeding or assisted reproduction. Substantial progress has been made in expanding our basic understanding of felid reproduction, primarily through comparative studies in domestic cats as models for endangered felids but also from targeted research using endocrine analysis and periodic semen collection or laparoscopy in selected non-domestic species. In particular, the use of fecal hormone metabolite analysis as a non-invasive method to assess reproductive patterns and adrenocortical activity has revolutionized this aspect of endangered species research (Graham and Brown, 1996; Brown et al., 1996; Brown and Wildt, 1997). Information gained from these systematic characterization studies forms the basis for modifying management strategies and husbandry to improve animal health and reproduction and for increasing the effectiveness of assisted reproductive technology. Assisted reproduction still retains tremendous potential for conserving endangered populations. An adequate level of basal reproductive knowledge may now exist in several felid species to more effectively apply assisted reproduction as a conservation management tool. A summary of references documenting the application of reproductive sciences in Latin American felids is provided in Table 1.

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Table 1 Application of reproductive sciences to Latin American felids Felid species

Endocrine analysis

Semen collection

Sperm Artificial cryopreservation insemination

In vitro Embryo Embryo fertilization cryopreservation transfer

Jaguar Puma Ocelot Margay Tigrina Geoffroy’s cat Pampas cat Andean mountain cat Kodkod Jaguarundi

+a , b , c ,d ,e +j , c ,d ,k +o , p , q , r , s , c ,t ,u , d +o , p , q , r , s , w , d +n ,p ,c , d +x , d +d –

+f ,g ,a ,b ,h , i ,d +j , l , f , m ,n , d ,k +f ,p ,q , r ,t ,v , d +f ,p ,q , r ,d +f ,p ,q , r ,d +f ,d +f ,d –

+h , i , d +d +t , d +d +d +d +d –

– +l , n +p , t +p +p +y – –

+g +m +u , v – +v +y – –

– – +u , v – +v +y – –

– – +u – +y – – –

– +o , c ,d

– +f ,d

– +d

– +y

– +z

– –

– –

a

Morato et al., 2001. Morato et al., 2004. c Nogueira and Silva, 1997. d Swanson et al., 2003. e Wildt et al., 1979. f Howard, 1993. g Morato et al., 2000. h Paz et al., 2002. i Swanson et al., 1996a. j Barone et al., 1994a. k Wildt et al., 1988. l Barone et al., 1994b. m Miller et al., 1990. n Moore et al., 1981. o Brousset, 2003. p Moraes et al., 1997. q Morais et al., 1997. r Morais et al., 2002. s Moreira et al., 2001. t Swanson et al., 1996b. u Swanson, 2001. v Swanson et al., 2002. w Moreira et al., 2002. x Carlstead et al., 1992. y Swanson, unpublished. z Pope et al., 1998. b

3. International training programs From 1993 to 1995, a broad-based genetics and reproductive survey was conducted investigating endemic felid species found in zoos throughout Latin America (Johnson et al., 1999; Swanson et al., 2003). During the reproductive survey, a total of 44 zoos in 12 Latin American countries were visited by the USA research team. In Mexico and Brazil, several veterinarians, either affiliated with a collaborating national university or employed by the host zoological park, received their initial exposure to the use of reproductive research techniques with non-domestic felids and were able to contribute to completing the reproductive survey. Although the primary objective of the survey was to assess the reproductive status of Latin American felid species, an added, unanticipated benefit was the fostering of these initial interactions and communication between USA and Latin American scientists. This


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dialogue eventually developed into working relationships and the initiation of a formal reproductive research training program for Latin American scientists in Mexico, Brazil and the United States. In Brazil, three formal training courses were conducted in 1995, 1996 and 1998 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, in vitro fertilization and sperm/embryo cryopreservation. During these three courses, a total of ∼30 Brazilian veterinarians received training, including several participants in subsequent collaborative studies. In Mexico, a continuing education course for veterinarians and veterinary students was taught in 1997 at the Universidad Nacional Autonoma de Mexico in Mexico City, involving ∼100 students and including didactic lectures and demonstration of semen collection, cryopreservation and laparoscopic procedures. In some instances, course instructors and participants collaborated on designing research studies and writing grant proposals to fund these projects. Following in-country training, several Mexican and Brazilian scientists traveled to the US to receive further instruction in semen collection and analysis, laparoscopy and fecal hormone metabolite analysis. To date, five of these scientists have completed doctoral dissertations derived from their research with Latin American felids. Findings from their studies, some completed in collaboration with American colleagues and some conducted independently, have greatly broadened our understanding of basal reproductive physiology in several Latin American felids, including ocelots, margays, tigrinas and jaguars. Their research results also have provided the scientific basis for improving captive management by addressing issues of animal well-being, exhibit quality, nutrition and genetic heterozygosity. Finally, these studies have helped to increase the efficiency of assisted reproductive technology as a potential genetic management tool. A brief review of some of these research findings and their implications for conservation are provided in the following sections. 4. Characterization of basal reproductive parameters 4.1. Seminal and endocrine analysis Until recently, normative physiological data from Latin American felids were nearly nonexistent. In the mid-1990s, the Latin American reproductive survey was completed, assessing the reproductive status of 185 male cats (>95% wild-born) representing eight endemic Latin American felid species (Swanson et al., 2003). This comprehensive survey established species-specific norms for seminal and endocrine traits in all eight cat species and identified several management factors that were affecting reproductive success. Among other findings, this study documented that proven breeders had superior values for many reproductive traits compared to nonbreeders, providing criteria for assessing fertility and/or selecting breeding stock. In small cats, it was shown that males housed alone or paired had improved reproductive traits compared to males in group exhibits, possibly reflecting varying stress levels under different exhibit conditions. Of concern, the survey also found

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that most (>80%) of the study population had never reproduced in captivity, more than half had very low sperm counts and several species (puma, margay, tigrina, jaguarundi) had high percentages (>50%) of morphologically-abnormal spermatozoa. Collectively, the results suggested that modification of both exhibits and diets were warranted in Latin American zoos to improve reproductive values and breeding success. Beginning in 1995, a series of collaborative studies were conducted in Brazil to further characterize basal reproductive traits in captive ocelots (Leopardus pardalis), margays (Leopardus weidi) and tigrinas (Leopardus tigrinus) (Morais et al., 1997; Moreira et al., 2001; Morais et al., 2002; Moreira et al., 2002). In this study, males were maintained under more optimal conditions (i.e. individually housed in relatively large, off-exhibit cages with daily vitamin/mineral-supplemented diets) than found at most institutions in the reproductive survey. Each month, detailed reproductive evaluations (including testes measurement and semen collection/analysis) were conducted to gather data on basal sperm production and quality (Morais et al., 2002). Fecal testosterone metabolites also were monitored to investigate the relationship to seminal traits and influence of seasonality on reproductive traits. This study established that all species produce high-quality ejaculates throughout the year with minimal fluctuations due to season. Among the few seasonal differences noted were apparent seasonal peaks in sperm recovery during the summer months. These findings suggested that males of each species were capable of reproducing year-round at the southern extent of their natural range. Of importance, males in this study also produced three to seven times more spermatozoa per ejaculate and showed a 20–30% improvement in percentage of normal sperm forms compared to mean values reported in the reproductive survey (Swanson et al., 2003). These pronounced differences were attributed primarily to improved nutrient content of the diets and a decrease in potential stressors associated with housing singleton cats in off-exhibit caging. An ongoing collaborative project with ocelots in Mexico is investigating the reproductive effect of supplementing all-meat diets with a commercially-available, human multi-vitamin (Ugaz and Swanson, unpublished data). The goal is to provide Latin American zoos with an easily accessible, inexpensive means to improve the nutrient composition of felid diets. Results to date suggest that vitamin supplementation produces a dramatic increase in steroid production in male ocelots but the impact on other reproductive traits remains to be determined. Other studies conducted in Brazil have assessed seminal and endocrine traits in jaguars and investigated the effect of seasonality on male reproductive patterns (Morato et al., 2001; Morato et al., 2004). In one study (Morato et al., 2001), seminal traits and serum testosterone levels were compared between wild and captive jaguars. Although captive jaguars had a larger seminal volume, wild jaguars tended to produce more spermatozoa per ejaculate and a higher percentage of morphologically-normal spermatozoa, suggesting that the captive environment may be compromising their reproductive status. In a subsequent study (Morato et al., 2004), semen collection and fecal androgen analysis were used to assess potential reproductive seasonality in captive jaguars. Fecal testosterone and seminal traits did not differ among males based on the four traditional seasons of the year. However, androgen levels were found to be higher during the wet season compared to the dry season, indicating that other seasonal cues, besides day length, may influence reproductive seasonality in neotropical felids.


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Fecal hormone analysis also has been used to assess ovarian activity and seasonality of female cats in southern Brazil (Moreira et al., 2001). Findings suggest that ocelots and margays (and possibly tigrinas) exhibit ovarian cyclicity year-round at the southern extent of their range. All three species were found to be polyestrus with a similar estrous cycle duration of approximately 16–19 days. Fecal progesterone analysis and periodic laparoscopic exams also established that ocelots and tigrinas were induced ovulators, whereas margays ovulated spontaneously on a fairly frequent basis. Interestingly, the male margay is the only Latin American felid completely lacking penile spines (Swanson et al., 2003), keratinized structures that help to induce ovulation in other cat species via a neuroendocrine response. 4.2. Assessment of adrenocortical activity Additional studies in Brazil have addressed basal adrenocortical activity in ocelots, margays and tigrinas and the effect of acute stressors, such as anesthesia and electroejaculation, and chronic stressors, such as inappropriate exhibits (Morais et al., 1997; Moreira et al., 2002). Measurement of fecal corticoid metabolites in males housed off-exhibit as singletons indicated that all species showed increased cortisol secretion in response to acute stressors (Morais et al., 1997). However, ocelots had lower basal and peak fecal cortisol levels than margays and tigrinas, a relationship also observed with serum cortisol levels (Swanson et al., 2003), and suggestive of an inverse correlation between body size and adrenocortical activity among small felids. Small felids (margays and tigrinas) also showed a differential stress response to exhibit modification (Moreira et al., 2002). In this study, singleton cats were housed successively in large environmentally-enriched enclosures, small barren enclosures and small enriched enclosures. Stereotypic pacing activity increased in both species after transfer to small barren cages, corresponding to increased fecal corticoid levels. However, corticoid levels decreased in margays, but not tigrinas, after cage enrichment, suggesting species-specific differences in susceptibility and adaptation to environmental stressors. In Mexico, the effect of environmental enrichment on behavior and adrenocortical activity was assessed in three small cat species (ocelot, margay, jaguarundi) housed in four Mexican zoos (Brousset et al., 2000; Brousset, 2003). Assessment of adrenocortical activity found that jaguarundis had much higher basal corticoid levels than either ocelots or margays. Husbandry changes, such as alterations in the timing, frequency and method of food presentation, increased the amount of exploratory behavior and decreased stereotypic pacing, but without affecting basal adrenocortical activity. Collectively, results of these studies in Mexico and Brazil suggest that relatively low-cost, environmental enrichment programs can significantly improve the psychological and physiological well-being of captive felids, especially important with the more ‘stress-prone’ smaller-sized cats. 5. Assisted reproductive technology 5.1. Sperm cryopreservation Genome resource banking, and the systematic cryopreservation and storage of gametes and embryos, can facilitate maintenance of genetic heterozygosity, while minimizing move-

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ment of living animals between institutions or countries (Wildt et al., 1997). As an example of this conservation potential, a total of 59 high-quality ejaculates were collected from eight felid species during the Latin America reproductive survey and processed for sperm cryopreservation (Swanson et al., 2003). Based on pre-freeze sperm concentrations and motility, these samples potentially would be adequate for ∼100 AI or ∼26,000 IVF procedures. Because almost all cats were wild-born, non-proven breeders, this frozen spermatozoa represents nearly 60 founders for the captive populations. The ultimate usefulness of this resource, however, is dependent of the efficiency of cryopreservation protocols. With current freezing techniques, post-thaw sperm viability and acrosome status frequently are compromised, indicating the necessity for more systematic cryobiology research with felid sperm (Swanson et al., 1996a; Pukazhenthi et al., 1999). There also is a need to develop ‘field-friendly’ cryopreservation techniques to permit freezing of semen collected from free-ranging cats under more primitive conditions. In the USA and Latin America, a number of studies have been initiated addressing sperm cryopreservation in Latin American felids. Our earlier research with jaguars in the USA and Brazil compared sperm freezing in straws at a controlled rate over liquid nitrogen versus pelleting samples on dry ice (Swanson et al., 1996b). Straw freezing provided similar post-thaw acrosome and motility values as pelleting, eliminating the need to use relatively labile dry ice in the field or other remote locations. Furthermore, jaguar spermatozoa frozen in straws appeared to be functional in vitro, based on a low of rate of penetration of salt-stored domestic cat oocytes (Swanson et al., 1996b) and zona-free hamster oocytes (Paz et al., 2002). Subsequent research with ocelots has confirmed that straw freezing provides comparable post-thaw characteristics as pelleting, and that frozen-thawed ocelot spermatozoa are fully functional in vitro as assessed by heterologous IVF with viable domestic cat oocytes and by production of offspring after AI (Swanson et al., 1996a; Stoops and Swanson, unpublished). However, pelleting and straw freezing are known to induce substantial acrosome damage (>50%) in both jaguar and ocelot spermatozoa. A recent study in Brazil has demonstrated that even slow cooling of ocelot spermatozoa from room temperature to 9 ◦ C is sufficient to induce significant acrosome loss (Queiroz et al., 2002). Application of these sperm freezing techniques with wild felid populations is currently being investigated in both Mexico and Brazil. In Mexico, semen has been collected successfully from two wild ocelots captured in the field for radiotelemetry studies and preserved by straw freezing over liquid nitrogen vapor (Ugaz, Caso and Swanson, unpublished data). In Brazil, efforts are now underway to collect and freeze spermatozoa from wild jaguars and pumas (Morato, personnal communication). 5.2. Exogenous gonadotropin treatment and laparoscopic artificial insemination For AI, domestic and non-domestic felids typically are treated with exogenous gonadotropins, such as equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), to induce ovarian follicular development and ovulation, followed by laparoscopic insemination directly into the uterine horns (Howard et al., 1992; Howard et al., 1997). In an earlier study in American zoos, the feasibility of laparoscopic AI in ocelots was investigated as a potential means to improve gene flow between isolated populations (Swanson et al., 1996a). Our findings indicated that ocelots were relatively insensitive to


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Table 2 Comparative ovarian sensitivity of Latin American felids to exogenous gonadotropins Felid species

Body weight (kg)

eCG/hCG Dosagea

eCG dose/BW (IU/kg)

No. of cats/dosage

Ovarian responseb

Litter size

Domestic cat Cheetah Clouded leopard Ocelot Margay Tigrina Geoffroy’s cat Pampas cat

3.5 35 15 9 3 1.7 3.8 2.9

100/75 200/100 100/75 400/200 200/150 200/150 200/150 100/75

29 6 7 44 67 118 53 35

30 18 5 28 4 11 14 2

9.4 10.7 10.8 9.7 8.7 10.3 15.2 14.0

4 4 3 1 1 2 2 2

References: Howard et al., 1992; Howard et al., 1997; Moraes et al., 1997; Sunquist and Sunquist, 2002; Swanson et al., 1996b; Swanson et al., 2002; Swanson, unpublished data. a Dosage(IU) of equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG). b Average number of ovarian structures (follicles and CL) per female treated at listed dosage.

the exogenous gonadotropins used to stimulate ovarian activity compared to other felids (Table 2). This study culminated in the production of one pregnancy following insemination with frozen-thawed spermatozoa and the birth of a single viable kitten (Swanson et al., 1996a). In subsequent collaborative studies in Brazilian zoos, laparoscopic AI with freshlycollected spermatozoa was used to produce two pregnancies and multiple viable offspring in two cat species, the ocelot and tigrina (Moraes et al., 1997). Fecal hormone monitoring also was used during this latter study to allow comparison of estrogen and progestin profiles in pregnant and non-pregnant females. More recent collaborative research in Brazil has explored the preliminary application of laparoscopic AI for propagation of margays, jaguarundis, and pampas cats. In addition, AI has been attempted with Geoffroy’s cats in the USA, using frozen-thawed spermatozoa originally collected from wild-born males in Latin America (unpublished). All seven small, spotted cats found in Latin America (ocelot, margay, tigrina, Geoffroy’s cat, pampas cats, kodkod, Andean mountain cat) are classified in the ocelot lineage, based on taxonomy and molecular genetic analysis (Johnson et al., 1999). Our findings in five of these species suggest that the relative insensitivity to exogenous gonadotropins is a conserved trait among cats in the ocelot lineage (Table 2). These species require gonadotropin dosages that are 50–400% higher on a per body weight basis than that used in domestic cats or other felids. This genetic bias against induced ovarian hyperstimulation also may be reflected in average litter sizes (i.e. 1–2 kittens) typically observed following natural breeding (Table 2). Because exogenous gonadotropins are large, immunologically-complex foreign proteins, development of anti-gonadotropin immunoglobulins is an expected sequelae in cats after repeated gonadotropin treatment, especially with short treatment intervals (Swanson et al., 1995). In studies conducted in the USA, gonadotropin treatment intervals of 6–12 months were adequate to maintain ovarian responsiveness and prevent formation of persistent antibody titers in several felid species, including ocelots and Geoffroy’s cats (Swanson et al., 1999). In recent research with ocelots and tigrinas in Brazil, alternating gonadotropin

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regimens (eCG/hCG and pFSH/pLH) were used at 4-month intervals for up to six total treatments (Paz et al., 2004). Although females tended to develop antibodies with successive treatments, ovarian responsiveness did not decrease, suggesting minimal attenuation of gonadotropin activity by humoral immune responses in these two species. 5.3. Laparoscopic oocyte recovery and in vitro fertilization Exogenous gonadotropin treatment and laparoscopy also form the basis for ovarian stimulation and oocyte recovery for IVF procedures (Howard, 1999). Although efficiency varies, IVF techniques developed in the domestic cat have proven to be readily adaptable across most cat species (Pope, 2000). To date, IVF has been used successfully to generate embryos in several Latin American felids, including the jaguar (Morato et al., 2000), puma (Miller et al., 1990), ocelot (Swanson et al., 2002), tigrina (Swanson et al., 2002), Geoffroy’s cat (unpublished) and jaguarundi (Pope et al., 1998). Recent research conducted in Brazil has focused primarily on using IVF in the jaguar, ocelot and tigrina. In jaguars, ovarian stimulation with pFSH/pLH produced ∼25 follicles per female with most (>80%) recovered oocytes being classified as high quality. Several embryos were produced after IVF, although fertilization percentages were fairly low (70%) (Pope, 2000). In general, protocols developed initially for freezing embryos of other species, such as livestock, appear to be adequate for cryopreservation of cat embryos. Demonstrating the in vivo viability of non-frozen or frozen-thawed IVF cat embryos has been more problematic, primarily due to difficulties in synchronizing recipients to provide an appropriate environment for embryo and fetal survival. There have been no systematic studies investigating the cryobiology of embryos from any non-domestic cat species and only a few transfer trials have been attempted. To date, offspring have been produced in only three non-domestic felid species (ocelot, wild cat, caracal) following transfer of frozen-thawed embryos (Pope et al., 2000; Swanson, 2001;


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Pope, personal communication). In ocelots, embryo transfer studies have been conducted in both the USA and Brazil, using laparoscopic transfer protocols extrapolated from our domestic cat studies (Swanson, 2001, 2002). For these procedures, an estrone enzyme immunoassay (EIA) was used with fecal samples to identify natural estrus phases for subsequent ovulation induction with gonadotropin-releasing hormone (GnRH). Two embryo transfer procedures have been attempted, with both resulting in pregnancies and natural delivery of term offspring. In the USA, three frozen-thawed ocelot embryos were transferred laparoscopically into the oviduct of a recipient female, resulting in the birth of one viable ocelot kitten (Swanson, 2001). Subsequently, a second pregnancy was produced in Brazil from transfer of three frozen ocelot embryos, resulting in the birth of one stillborn Brazilian ocelot kitten after a prolonged labor (Swanson, 2002). These ocelot studies, using a host of reproductive science techniques, epitomize the value of systematic reproductive research in domestic and non-domestic felid species. Importantly, this proven capacity to produce, freeze and successfully transfer ocelot embryos has immediate application for achieving conservation goals with this endangered species. These studies represent one component of a comprehensive, multi-institutional conservation program, termed the Brazilian Ocelot Consortium, focused on conserving the Brazilian ocelot subspecies (L.p.mitis) both in the wild and in captivity (Swanson, 2002). The Consortium is striving to establish a genetically-viable population of Brazilian ocelots in American zoos, through importation of both captive-born offspring and frozen embryos from Brazil. Cooperative management programs and periodic use of assisted reproduction will allow the two separate captive populations in the USA and Brazil to be managed as a larger metapopulation, greatly benefiting overall genetic heterozygosity.

6. Conclusions Over the past decade, this training program has succeeded in teaching Brazilian and Mexican scientists to use reproductive sciences effectively in investigating the reproductive physiology of endangered Latin American felids. Results from their research studies are providing invaluable information that is directly benefiting the conservation of these species. Most importantly, this training program has been essential for building scientific capacity within Mexico and Brazil. There now exists a core group of highly-trained reproductive biologists in these two countries that will continue applying their new knowledge and skills to studying and conserving Latin American felids. Their strong connections with a network of North American colleagues will ensure future opportunities for knowledge sharing and collaboration. The success of their efforts ultimately will depend on building additional research and conservation capacity in Latin America. Latin American zoos must continue growing into true conservation organizations by implementing genetic management programs and developing in situ connections for their captive populations. In addition, this new cohort of reproductive biologists must assume the responsibility for training the next generation of scientists in their native countries and expanding the application of reproductive sciences to other Latin American species and regions. Lastly, it is imperative that sufficient resources be made available in Latin America to support these young scientists and their conservation-focused research.

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Acknowledgements We are indebted to our colleagues and friends in Brazil and Mexico whose extraordinary energy, willingness to learn and eagerness to conserve their native wildlife have made this research training program so successful. In Brazil, the authors thank Drs. Rosana Morais, Nei Moreira, Wanderlei Moraes, Cristina Adania, Regina Paz, Ronaldo Morato, Marcelo Guimaraes, Marcelo Gomes, Eduardo Dias, Claudia Yumi, Vinicius Queiroz, Renato Barnabe, and Maria Lucia Gomes and, in Mexico, Drs. Cristian Ugaz, Dulce Brousset, Marta Romano, Claudia Lewy, Carlos Esquivel, Francisco Galindo and Arturo Caso. In the USA, the authors thank Drs. JoGayle Howard, Laura Graham, David Wildt, Warren Johnson, Mark Campbell, Ken Cameron, Monica Stoops and Terri Roth, in addition to Helen Bateman, Jennifer Bond, Molly McRae, Lynn Patton, Len Davidson and Russ Kelly. Funding for these projects was provided by the National Institutes of Health, USA Fish and Wildlife Service, 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. References Barone, M.A., Roelke, M.E., Howard, J.G., Brown, J.L., Anderson, A.E., Wildt, D.E., 1994a. Reproductive characteristics of male Florida panthers comparative studies from Florida, Texas, Colorado, Latin America, and North American zoos. J. Mammal. 75, 150–162. Barone, M.A., Wildt, D.E., Byers, A.P., Roelke, M.E., Glass, C.M., Howard, J.G., 1994b. Gonadotropin dose and timing of anaesthesia for laparoscopic artificial insemination in the puma (Felis concolor). J. Reprod. Fertil. 101, 103–108. Bennett, E.L., Rabinowitz, A.R., 2001. It has to be done on the ground: in-country training courses in wildlife management and research. In: Conway, W.G., Hutchins, M., Souza, M., Kapetanakos, Y., Paul, E. (Eds.), The AZA field conservation resource guide. Zoo Atlanta, Atlanta, pp. 232–235. Brousset, D.M., Galindo, F., Valdez, R., Romano, M., Aluja, A.S., 2000. Behavior and fecal cortisol as noninvasive indicators for the assessment of welfare in captive small Mexican felines. In: Proceedings of the 34th International Congress of the International Society of Applied Ethology. Florianopolis, Brazil, p. 118 (abstr.). Brousset, D.M., 2003. Efecto del enriquecimiento ambiental sobre el bienestar de tres especies de felinos Mexicanos en peligro de extincion (ocelote, margay y jaguarundi) mantenidos en cautiverio. Tesis Doctora en Ciencias Veterinarias, Universidad Nacional Autonoma de Mexico, Mexico, DF. Brown, J.L., Terio, K.A., Graham, L.H., 1996. Fecal androgen metabolite analysis for non-invasive monitoring of testicular steroidogenic activity in felids. Zoo. Biol. 15, 425–434. Brown, J.L., Wildt, D.E., 1997. Assessing reproductive status in wild felids by non-invasive faecal steroid monitoring. Int. Zoo. Yrbk. 35, 173–191. Carlstead, K., Brown, J.L., Monfort, S.L., Killens, R., Wildt, D.E., 1992. Urinary monitoring of adrenal responses to psychological stressors in domestic and nondomestic felids. Zoo. Biol. 11, 165–176. Graham, L.H., Brown, J.L., 1996. Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids. Zoo. Biol. 15, 71–82. Howard, J.G., Barone, M.A., Donoghue, A.M., Wildt, D.E., 1992. The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. J. Reprod. Fertil. 96, 175–186. Howard, J.G., 1993. Semen collection and analysis in carnivores. In: Fowler, M.E. (Ed.), Zoo and Wild Animal Medicine: Current Therapy, third ed. Saunders, Philadelphia, pp. 390–399. Howard, J.G., Roth, T.L., Byers, A.P., Swanson, W.F., Wildt, D.E., 1997. Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard. Biol. Reprod. 56, 1059–1068.


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