Sperm cryopreservation in endangered felids: developing linkage of in situ-ex situ populations, WF Swanson, MA Stoops, GM Magarey

Tags: Swanson, Wildt, spermatozoa, Theriogenology, captive populations, cat species, sperm motility, captive population, fishing cats, cryopreservation, conservation, Journal of Zoo and Wildlife Medicine, population, cat, sperm morphology, zoo populations, Sperm cryopreservation, frozen cat, comparative studies, Cincinnati Zoo, W.F. Swanson, International Zoo, cats, endangered species, Semen collection, North American zoos, Howard, sperm concentration, Philadelphia, PA USA, Fort Worth Zoo, Minnesota Zoological Garden, Carnivore Preservation Trust, semen analysis, field studies, mobile laboratory, wild cats, Marbled cat Oncilla Pallas, Pallas, IUCN Red List, J. Howard, fishing cat, International, laboratory microscope, Zoo and Wild Animal Medicine, semen volume, Nashville Zoo, IVF procedures, Henry Doorly Zoo, International Pallas, IVF Heterologous IVF Gonadotropin, domestic cats, domestic cat, financial support, total population, Pallas cat, Meredith Brown, Feline Conservation Center, W.B. Saunders Company, North Carolina State University, IVM IVM, the Cincinnati Zoo, American Zoo and Aquarium Association, Oklahoma City Zoological Park, Beryl Wilson, feline immunodeficiency virus, Corne Anderson, airport security screening, Nadine Lamberski, Reproduction, Fertility and Development
Content: Sperm cryopreservation in endangered felids
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Sperm cryopreservation in endangered felids: developing linkage of in situ-ex situ populations
W.F. Swanson, M.A. Stoops, G.M. Magarey and J.R. Herrick Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo & Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA
Many of the world's cat species face growing threats to their continued survival in nature. For some species, managed captive populations may provide a reservoir for future reintroduction or genetic augmentation. Because most zoo populations are derived from small founder sizes and are subject to loss of genetic variation over time, periodic infusion of founder alleles is necessary to avoid the dire consequences of inbreeding. Collection and freezing of semen from free-living nondomestic felids offers a viable option for introducing founder genes into captive populations without removal of animals from the wild. The effective application of this strategy requires established protocols for safely capturing and anaesthetizing wild cats coupled with suitable methods of semen recovery, processing and cryopreservation under field conditions. In small-sized non-domestic felids, the general feasibility of this approach is being explored in two studies of black-footed cats and Pallas' cats. Two factors ­ relatively low sperm numbers per ejaculate and compromised status of frozen-thawed cat spermatozoa ­ suggest that in vitro fertilisation (IVF) and embryo transfer present the most efficient use of this limiting resource in small-sized cats. Our studies with captive felids have explored alternative methods of sperm cryopreservation that are adaptable to field situations and shown that frozen-thawed spermatozoa from Pallas' cats, ocelots, and fishing cats exhibit adequate function to fertilise heterologous and/or homologous oocytes in vitro. Most recently, we investigated the fertilising capacity of frozen-thawed spermatozoa obtained from wild Pallas' cats in Mongolia. Combined with improved methods for embryo culture and transfer in small cat species, sperm banking in situ will facilitate introduction of new founders into captive populations without causing further depletion of their wild counterparts. As one component of holistic conservation programs, including ongoing support of field ecology studies in range countries, this reproductive strategy serves to further strengthen linkages among imperiled ex situ and in situ cat populations.
Corresponding author E-mail: [email protected]
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Conservation of the forgotten felids ­ the small cats Among carnivores, the 36 extant species of non-domestic felid have been fairly resilient over their recent evolutionary history. Although several cat populations, most notably three subspecies of tigers, have been extirpated by man in the past century, there have been no species level extinctions of wild cats in at least several thousand years. However, persistent habitat loss, combined with poaching and the escalating threats associated with global climate change, have created an altered environment in which several cat species are now in imminent danger of disappearance (Nowell and Jackson, 1996). The International Union for Conservation of Nature (IUCN, 2003) includes 24 species of the world's wild cats on its Red List under some degree of risk for extinction (i.e., critically endangered, endangered, vulnerable, near threatened; Table 1).
Table 1. In situ and ex situ conservation status of the 7 large-sized and 17 small-sized nondomestic felid species classified under some degree of risk on the IUCN Red List.
Common name
Cat species Latin name
In situ statusa
Ex situ populationb
Captive managementc
Cheetah Clouded leopard Lion Jaguar Puma Snow leopard Tiger African golden cat Andean mountain cat Asian golden cat Black-footed cat Bornean bay cat Chinese desert cat Fishing cat Flat-headed cat Geoffroy's cat Iberian lynx Kodkod Marbled cat Oncilla Pallas' cat Pampas cat Rusty-spotted cat Sand cat
Acinonyx jubatus
Vu
Neofelis nebulosa
Vu
Panthera leo
Vu
Panthera onca
NT
Puma concolor
NT
Uncia uncia
En
Panthera tigris
En
Profelis aurata
Vu
Oreailurus jacobita
En
Catopuma temmickii
Vu
Felis nigripes
Vu
Catopuma badia
En
Felis bieti
Vu
Prionailurus viverrinus
Vu
Prionailurus planiceps
Vu
Oncifelis geoffroyi
NT
Lynx pardinus
Cr
Oncifelis guigna
Vu
Pardofelis marmorata
Vu
Leopardus tigrinus
NT
Otocolobus manul
NT
Oncifelis colocolo
NT
Prionailurus rubiginosus
Vu
Felis margarita
NT
Yes
SSP/EEP
Yes
SSP/EEP
Yes
SSP/EEP
Yes
SSP
Yes
-
Yes
SSP/EEP
Yes
SSP/EEP
No
-
No
-
Yes
EEP
Yes
SSP/EEP
No
-
No
-
Yes
SSP/EEP
Yes
-
Yes
EEP
Yes
-
No
-
Yes
-
Yes
-
Yes
SSP/EEP
Yes
-
Yes
-
Yes
SSP/EEP
aIUCN classification categories (Cr, critically endangered; En, endangered; Vu, vulnerable; NT, near threatened) (IUCN, 2003) bPresence or absence of the species in captivity, based on the most recent ISIS data (ISIS, 2006) cSSP, Species Survival Plan of the Association of Zoos and Aquariums (AZA); EEP, European endangered species Program of the European Association of Zoos and Aquaria (EAZA)
Most of the world's cat species are small in size, with individuals of 28 species averaging less than 20 kg in body mass (Sunquist and Sunquist, 2002). Compared to the eight large (> 20 kg body mass) cat species, small-sized cats have been relatively neglected in both conservation and science circles. In particular, comprehensive in situ studies have been virtually non-exis- tent in the majority of small cat species, creating a dearth of reliable information on the popu-
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lation status and ecology of these forgotten felids (Nowell and Jackson, 1996; Sunquist and Sunquist, 2002). Based on limited in situ data, the IUCN (2003) has listed 17 of the 28 small cat species as facing varying degrees of risk for extinction in the wild (Table 1). Most small cats have been only sporadically maintained in zoos and few species are currently managed effec- tively in captive breeding programs. Of the 24 total cat species included on the IUCN Red List, the International Species Inventory System (ISIS, 2006) indicates that the 7 large cat species and 12 of the 17 small cat species currently are housed in the world's zoos. Although all large cats species, with the exception of the puma (Puma concolor), are intensively managed within cooperative breeding programs in North American and/or European zoos, only a handful of small cat species are provided with similar oversight by these regional zoo associations. In North American zoos accredited by the Association of Zoos and Aquariums (AZA), just five small cat species ­ the Brazilian ocelot (Leopardus pardalis mitis), fishing cat (Prionailurus viverrinus), Pallas' cat (Otocolobus manul), Arabian sand cat (Felis margarita harrisoni) and blackfooted cat (Felis nigripes) - are managed by Species Survival Plans (SSPs) (Table 1). European zoos that are members of the European Association of Zoos and Aquaria (EAZA) have compa- rable management programs (European Endangered Species Programs, EEPs) in place for six small cat species, including four of the SSP species (Table 1). SSPs and EEPs represent the most intensive management programs available within the world's zoos, with program coordinators using demographic and genetic analyses of studbook data to make breeding recommendations, limit inbreeding and maximise genetic diversity within participating institutions. Achieving these management goals are a difficult proposition in small cats, given the limited founder numbers, small population sizes, short generation intervals and reproductive life spans, and breeding challenges that are typical of most of these species (Swanson, 2006).
The need for in situ linkage Captive felid populations managed by SSPs or EEPs are potentially important as genetic reservoirs for possible reintroduction of living animals into nature, provided that suitable habitat remains in situ and the tremendous difficulties of integrating captive-born individuals into a hostile wild environment can be overcome (Beck, Rapaport, Stanley Price and Wilson, 1994). The number of species, however, that can be effectively managed in zoos within conventional breeding programs is limited. In a seminal paper on the carrying capacity of zoos, Conway (1986) calculated that all of the worlds' zoos, working in concert, could only maintain a total of 330 mammalian species in captivity, given space restrictions in zoos and the need for minimal population sizes for genetic viability. Included in his calculations was the yearly cost of caring for individual animals of various species, illustrating the financial obligations incurred with large zoo populations. These costs have only increased in the past twenty years, with zoos investing more money in building naturalistic exhibits while continually striving to improve the husbandry of their charges by providing imaginative forms of enrichment, higher quality diets, and more sophisticated veterinary care. It might be illustrative to revisit the genetic constraints and maintenance costs for Zoo animals in the twenty-first century, specifically using the Pallas' cat as an example for the small cats that are the primary focus of this paper. In North American zoos, SSP population managers use computer software programs to collate studbook data (ISIS, 2003) and conduct pedigree analysis and population modeling (Ballou and Foose, 1996; Pollack, Lacy and Ballou, 2001). From these analyses, we find that the Pallas' cat SSP population currently consists of 65 animals derived from 24 founders (i.e., primarily wild-born cats, presumably unrelated) imported into North America in the 1990's (Caron, 2006). Taking into consideration the total cage spaces available for all small cats in North American
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zoos, this species has been allocated 100 exhibit spaces. Over the past ten years, this population has grown at an average rate of 5.7% per year with an effective breeding population size (Ne) equal to ~24% of the total population (N) and a generation interval of 4.7 years. At present, the SSP population retains 93.6% of the species' existing genetic diversity (GD) with the goal being to maintain 90% GD over the next 50 years. However, population modeling indicates that 90% GD is sustainable for only 6 more years and, after 50 years, this value will have declined to 74%. With a moderate improvement in breeding success (i.e., Ne/N = 50%; maximal growth rate = 20%), 90% GD for 50 years can be achieved but the maximal population size must be allowed to grow to 392 cats (Fig. 1A). If the maximal population size is held at 100 cats, our genetic goal is sustainable for just 16 years and GD declines to 84% by 50 years (Fig. 1B).
400
100
% Gene diversity
Population size
300
95
90 200 85 100 80
0
75
0 10 20 30 40 50
Year
100
100
% Gene diversity
Population size
80
95
60
90
40
85
20
80
0
75
0 10 20 30 40 50
Year
100
100
% Gene diversity
Population size
80
95
60
90
40
85
20
80
0 0
10 20 30 40 5075
Year
Figure 1. Number of captive Pallas' cats required to maintain 90% genetic diversity over a 50 year period under three scenarios: (A) unlimited total population size with improved management to increase the effective population size (Ne) and maximal population growth rate; (B) improved management with a total population size limited to 100 cats; and (C) improved management of 100 total cats combined with introduction of 2 new founders from the wild every 5 years.
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Even if ~400 cage spaces somehow could be reserved for Pallas' cats in zoos, the financial implications of managing 392 cats versus 100 cats over 50 years are profound. At the Cincinnati Zoo, we estimate that housing, feeding and caring for each of our Pallas' cats costs $2,300 per year (Table 2). Accordingly, to maintain 392 cats over 50 years, North American zoos would need to provide a total of $45,080,000 in financial support. In contrast, housing 100 cats over 50 years would require $11,500,000 in total funding for a projected cost savings of almost $33,500,000 or ~$700,000 per year. One means to realise this cost savings while still meeting the SSP's genetic goals is through the periodic introduction of new founders into the captive population. By adding two new founders every 5 years for the next 50 years, the SSP population can be sustained at 100 total cats with 90% GD (Fig. 1C). Traditionally, zoos have acquired founders through the capture and importation of free-living wild-born animals, as with the establishment of the Pallas' SSP population. In most cases, the removal of a handful of animals from a vibrant wild population is unlikely to have any long-lasting detrimental impact on species' survival. For example, over the past 10 years, 105 wild-born Pallas' cats have been brought into captivity internationally (Caron, 2006). Although ecological studies have just begun to assess wild Pallas' cats (Munkhtsog and Ross, in press), it is unlikely that this limited offtake from multiple geographic sites has had any significant adverse effects.
Table 2. Financial costs (USD, $) for maintaining a captive population of Pallas' cats over the next 50 years based on a projected total population size of 392 cats versus 100 cats.
Parameter Labor Food Vet care Facility overhead Total cost
Cost/cat/yr $1,750 $310 $100 $140 $2,300
Cost/cat/50 yr $87,500 $15,500 $5,000 $7,000 $115,000
Cost/392 cats/50 yr $34,300,000 $6,076,000 $1,960,000 $2,744,000 $45,080,000
Cost/100 cats/50 yr $8,750,000 $1,550,000 $500,000 $700,000 $11,500,000
For many species, the periodic capture of wild founders remains as one option for zoos interested in genetic augmentation of captive populations. In essence, this practice represents one variation on the concept of ecosystem services in which intact functional environments provide essential biological services to humankind - in this case, providing genetic resources to zoos (Costanza, d'Arge, de Groot, Farber, Grasso, Hannon, Limburg, Naem, O'Neill, Paruelo, Raskin, Sutton and van den Belt, 1997; Pimental, Wilson, McCullum, Huang, Dwen, Flack, Tran, Saltman and Cliff, 1997). As zoos have evolved to focus more on conservation as their primary mission, the need for zoos to support habitat preservation has received greater emphasis (Hutchins, 2003; Miller, Conway, Reading, Wemmer, Wildt, Kleiman, Monfort, Rabinowitz, Armstrong and Hutchins, 2004). One direct incentive for supporting in situ conservation is the notion of zoo reserves, in which zoos provide financial support for wildlife reserve management in exchange for occasional removal of animals for zoo collections (Conway, 1999). In the Pallas' cat example, the substantial financial savings gained by managing a smaller captive population could be invested in support of Pallas' cat reserves, providing major funding for habitat preservation in the range countries of Central Asia. However, if zoos are sincere in their intent to become true conservation organisations and be perceived as such by other conservationists and the public, then they should continue to distance themselves from the traditional "bring `em back alive" method of founder acquisition whenever possible. Even if sustainable from an ecological perspective, capturing any animal in the wild for placement in zoos harkens back to an earlier era when zoos were major extractive
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and destructive forces on wild animal populations. Although nearly 90% of zoo animals today are born in captivity (ISIS, 2006), this perception of zoos as consumers of wildlife persists and, with each transfer of a formerly free-living animal into captivity, is reinforced. Many zoo professionals, including the authors, struggle with this apparent contradiction of exhorting society to conserve endangered species in situ while simultaneously advocating the removal of these same species from the wild. Times change, science advances, and alternatives do exist.
In situ genetic resource banks Beginning in the 1980's, Conway (1986) and others (Seager, 1983; Benirschke, 1984; Wildt, Schiewe, Schmidt, Goodrowe, Howard, Phillips, O'Brien and Musch, 1986; Holt and Moore, 1988) noted that the carrying capacity of zoos could be substantially enhanced by the application of assisted reproductive technologies, including the use of sperm cryopreservation, in combination with artificial insemination (AI) or in vitro fertilisation (IVF) and embryo transfer (ET). Twenty years later, the true potential of assisted reproduction for population management has yet to be realised but we are progressing incrementally closer to being able to apply these technologies as genetic management tools (Swanson,2006). In theory, assisted reproductive technologies would allow gene flow to be established between disjunct locations, including linking of in situ and ex situ populations (Lasley, Loskutoff and Anderson, 1994; Johnston and Lacy, 1995; Wildt, Rall, Critser, Monfort and Seal, 1997; Wildt and Roth, 1997). In the near future, unidirectional movement of genetic material from the wild into captivity presents the most viable option for creating this initial linkage. In fact, the general feasibility of this approach already has been demonstrated in cheetahs (Acinonyx jubatus), using frozen-thawed spermatozoa collected from wild males in Namibia for AI, culminating in the production of three litters of offspring in North American zoos (Wildt et al., 1997; Howard, Marker, Pukazhenthi, Roth, Swanson, Grisham and Wildt, 2002). In our laboratory at the Cincinnati Zoo's Center for Conservation and Research of Endangered Wildlife (CREW), one of the goals is to develop and apply similar reproductive strategies of genetic linkage for small cat SSP species. In effect, small cat populations in the wild would serve as living in situ genetic resource banks with the periodic withdrawal of founder genes in the form of frozen spermatozoa to augment the genetic diversity of captive populations. In return, a portion of the substantial cost savings accrued by zoos from reducing captive population sizes, as seen with the Pallas' cat example, would be redirected to support in situ research and conservation efforts with wild populations. Substantial progress already has been made in developing the basic and applied scientific knowledge and establishing the in situ collaborative partnerships that will be necessary for implementing this in situ-ex situ linkage strategy with small cats.
Semen collection and cryopreservation in situ For in situ-ex situ genetic linkage to be a realistic option, it is imperative that the wild cats can be physically located in their natural habitat within a defined time period and then safely captured, anaesthetised and subjected to electroejaculation before being released back into the wild. Recovered semen must be evaluated, processed and cryopreserved under field conditions in a manner that allows recovery of adequate post-thaw sperm function for IVF or AI procedures. Lastly, frozen semen must be transported internationally with proper government permits and awareness of airport security screening hazards. Each of these steps present logistical
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challenges that require some flexibility in experimental design and methodology. Comparative studies with domestic cats and captive nondomestic felids are essential for establishing effective protocols for semen collection, analysis and cryopreservation before venturing into the field to study cats in the wild.
Capture and restraint Earlier studies of semen collection in wild felids focused on two African plains species, the lion (Panthera leo) and the cheetah (Wildt, O'Brien, Howard, Caro, Roelke, Brown and Bush, 1987a; Wildt, Bush, Goodrowe, Packer, Pusey, Brown, Joslin and O'Brien, 1987b). Because these populations already were acclimated to the presence of field researchers, capture prior to anesthesia was unnecessary and the cats could be easily approached and darted with anesthetic agents delivered remotely from a nearby vehicle. Subsequent studies with wild Florida panthers and Brazilian jaguars (Panthera onca) proved more challenging, requiring the use of trained dogs to track the cats and force the animals to climb trees for subsequent darting (Barone, Roelke, Howard, Brown, Anderson and Wildt, 1994; Morato, Conforti, Azevedo, Jacomo, Silveira, Sana, Nunes, Guimaraes and Barnabe, 2001). With small-sized cats, animals typically have been captured either in baited live traps (Shindle and Tewes, 2000; Grassman, Austin, Tewes and Silvy, 2004; Sliwa, 2004) or found by visual sighting and then physically restrained after a short chase and netting or cornering in an underground burrow (Sliwa, 2004; Brown, Lappin, Brown, Munkhtsog and Swanson, 2005; Munkhtsog and Ross, in press). In an optimal situation, the wild cats will have been captured initially at an earlier date and fitted with radiocollars as part of ongoing field ecology studies. This approach in Mongolia allowed us to relocate and recapture four radiocollared Pallas' cats over a two day period, greatly shortening the time commitment needed for biological sampling and reducing field expedition expenses.
Anaesthesia Among wildlife species, felids are among the safest to anesthetise using injectible anesthetic agents alone or injectible anesthetics combined with inhalant gas anesthesia (Swanson, 2004). Traditionally, many biologists working with wild cats in the field have conducted anesthesia without veterinary support, using ketamine, sometimes combined with xylazine, or tiletaminezolezapam to achieve adequate anesthetic depth for blood sampling, physical measurements and/or placement of radiocollars (Franklin, Johnson, Sarno and Iriate, 1999; Shindle and Tewes, 2000; Grassman et al., 2004). Ideally, an experienced veterinarian should be present to administer the anesthestic agents and monitor the animal's vital signs throughout the sampling period, especially when a greater depth of anesthesia is required for stimulatory procedures such as electroejaculation. To the authors' knowledge, veterinarians have been responsible for conducting anesthesia in all previous and ongoing studies involving semen collection of cats in the wild (Wildt et al., 1987ab; Barone et al., 1994; Morato et al., 2001; Herrick, Lamberski, Wilson, Anderson, Swanson and Sliwa, 2005; unpublished). Ketamine alone has been used for semen collection procedures in some species, including the cheetah and Pallas' cat (Wildt et al., 1987a; Swanson et al., 1996), but combination regimens are now preferred for providing a greater level of sedation and analgesia during electroejaculation. Common injectible agents used for electroejaculation in field situations include tiletamine-zolazepam (Wildt et al., 1987a; Morato et al., 2001) and ketamine-medetomidine (Herrick et al., 2005), but each combination has drawbacks, such as prolonged recovery times with the former anesthetic drug and blood pressure fluctuations with the latter. In some species, inhalant anesthesia has proven feasible in
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a field situation, such as in our ongoing study of wild Pallas' cats in Mongolia. One advantage is that the dosage of injectible anesthestic may be reduced, allowing for quicker recovery times after cessation of gas anesthesia. The main disadvantage is the need to acquire oxygen tanks and compatible flow regulators in the study region and transport this bulky equipment into the field. A recent study, however, has shown that a non-rebreathing system attached to a flowover anesthestic vaporizer may be used with ambient air alone, eliminating the need for compressed oxygen gas cylinders (Lewis, 2004). With continual monitoring of oxygen saturation using pulsoximetry, this approach provides an adequate margin of safety for effective field anesthesia in a variety of species, including felids.
Electroejaculation and semen analysis Semen collection from wild felids requires the electroejaculation of anesthetised individuals. Most felids are easily collected in this manner (Howard, Bush and Wildt, 1986; Howard, 1993), although some species, such as ocelots, fishing cats and jaguarundis, are prone to urination during electrostimulation. In field situations, a mobile laboratory approach is required (Swanson, 2003), including transport of electroejaculation equipment to the study site and provision of a suitable power source. In some of the earlier field studies, a standard alternating current (AC) powered electrostimulator (PTE Electronics, Boring OR USA) was used with electricity provided from a portable gas generator capable of generating a minimum of 300-500 watts (Wildt et al., 1987ab; Barone et al., 1994). In more remote study locations where the transport of heavy equipment must be minimised, battery-powered direct current (DC) electrostimulators (Lane Manufacturing, Denver CO USA; Eletrovet electronic equipment, Sao Paulo Brazil) have proven to be adequate for semen collection (Barone et al., 1994; Morato et al., 2001; unpublished). Semen analysis in the field entails evaluation of most of the same parameters that are assessed in a normal laboratory setting, specifically semen volume, sperm concentration, sperm progressive motility and rate of progressive movement, sperm morphology and acrosomal integrity (Howard et al., 1986). If a gas-powered electrical generator is available and equipment transport is not an issue, then a standard-sized phase contrast laboratory microscope may be used to assess sperm concentration, motility and morphology (Wildt et al., 1987ab; Barone et al.; 1994). Alternatively, sperm motility parameters may be evaluated using a small, lightweight phase-contrast field microscope (Swift Instruments, San Jose CA USA) with a small flashlight as the source of illumination (Morato et al., 2001; Swanson, 2003). Sperm concentration may be determined by fitting the field microscope eyepiece with a calibrated optical grid and counting the total number of spermatozoa within the grid area on a thin counting chamber slide (Microcell, Conception Technologies, San Diego CA USA). Since sperm morphology assessments require higher magnification (>400x), aliquots of raw semen usually are fixed in glutaraldehyde for later evaluation with a standard laboratory microscope. Lastly, sperm acrosome evaluation may be conducted in the field using stable colorimetric stains, such as a rose bengalfast green stain (Pope, Zhang and Dresser, 1991) or a Coomassie blue stain (Thiangtum, Swanson, Howard, Tunwattana, Tongthainan, Wichasilpa, Patumrattanathan and Pinyopoommintr, 2006; Crosier, Pikazhenthi, Henghali, Howard, Dickman, marker and Wildt, 2006). With both stains, spermatozoa are fixed in either alcohol or paraformaldehyde so acrosomal degradation prior to microscopic evaluation is of minimal concern. Most knowledge of basal seminal parameters in felids is based on studies of captive populations (Howard, 1993; Wildt, Brown, Bush, Barone, Cooper, Grisham and Howard, 1993; Morais, Mucciolo, Gomes, Lacerda, Moraes, Moreira, Graham, Swanson and Brown, 2002; Swanson,
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Johnson, Cambre, Citino, Quigley, Brousset, Morais, Moreira, O'Brien and Wildt, 2003; van Dorrser and Strick, 2005; Thiangtum et al., 2006). Although these data are valuable for assessing the reproductive potential of our breeding populations, their validity as true species norms, especially in relation to wild individuals, is unknown for most cats. Multiple factors associated with the captive environment, including levels of genetic heterozygosity and inbreeding, nutrition, seasonal and latitudinal temperature and photoperiod fluctuations, and physiological or psychological stressors, may affect measured reproductive characteristics of felids in zoos. Comparative seminal data from wild felid populations presently exist for only a few large cat species (cheetah, Wildt et al., 1987a; lion, Wildt et al., 1987b; puma, Barone et al., 1994; jaguar, Morato et al., 2001). Our ongoing studies of black-footed cats in South Africa and Pallas' cats in Mongolia are providing the first comparative information about the reproductive status of small cat species in the wild. In black-footed cats and Pallas' cats, data collection has been limited thus far to analysis of semen from two adult males of each species but reproductive values for the wild cats appear similar to those of captive individuals (Swanson et al., 1996; Herrick et al., 2005; Herrick et al., in press; Table 3). Notably, wild Pallas' cats evaluated in October produced minimal sperm numbers, similar to that seen in this seasonally breeding species in captivity during the nonbreeding season (Swanson et al., 1996), but sperm quality was still adequate for cryopreservation for future use with IVF procedures.
Table 3. Mean (+ SEM) values for seminal and testicular traits of wild South African black-footed cats and Mongolian Pallas' cats versus captive black-footed cats and Pallas' cats in North American zoos.
Parameter
Black-footed cats
Captivea
Wild
Pallas' cats
Captiveb
Wild
No. cats No. ejaculates Semen volume (ml) Total sperm/ejaculate (x 106) Progressively motile sperm (%) Normal sperm morphology (%) Testicular volume (cm3)
5 12 0.25 + 0.02 28.4 + 6.3 80.4 + 2.5 48.8 + 3.3 1.64 + 0.08
2 2 0.13 + 0.03 26.6 + 4.6 82.5 + 7.5 46.3 + 4.3 2.19 + 0.46
1 5 0.19 + 0.01 0.7 + 0.3 78.0 + 2.0 34.6 + 4.9 1.6 + 0.1
2 2 0.18 + 0.06 1.7 + 0.2 67.5 + 2.5 46.5 + 4.5 2.6 + 0.3
aHerrick et al., in press; bSwanson et al., 1996 (for the non-breeding season only)
Sperm cryopreservation Effective techniques for cryopreservation of domestic cat spermatozoa are still under development (Pukazhenthi, Pelican, Wildt and Howard, 1999; Pukazhenthi, Spinder, Wildt, Bush and Howard, 2002) and methods used at present are far from ideal for retaining maximal post-thaw sperm motility and intact acrosomal membranes. Variables such as cooling rate, glycerol concentration and timing of addition, packaging method, and freezing and thawing rate all can affect post-thaw semen parameters (Watson, 2000). Extrapolating these cryopreservation methods to nondomestic cats introduces the further complication of interspecies variability in sperm cryobiological properties (Holt, 2000). For example, in fishing cats, cooling the semen sample prior to glycerol addition benefits post-thaw motility and function whereas the temperature of glycerol addition has little impact on cheetah spermatozoa (Crosier et al., 2006; Thiangtum et al., 2006). For in situ banking of spermatozoa from nondomestic cats, cryopreservation techniques that are adaptable to a field environment also are a necessity. In some situations, anesthetised males, such as black-footed cats, have been transported to an indoor mobile lab facility for semen collection (Herrick et al., 2005) or semen recovered at a remote location has
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been transferred to a more conventional laboratory for further processing and freezing, as in cheetahs (Crosier et al., 2006). At other study sites, semen collection, processing and freezing all have been conducted under more primitive conditions in the field, such as with Pallas' cats in Mongolia. Based on current knowledge, the most `field-friendly' cryopreservation method involves adding glycerol at room temperature, packaging the semen in sealed straws, cooling slowly in an ice chest or refrigerator and then freezing over liquid nitrogen vapor or directly in a dry shipper. In a recent study, we have shown that ocelot spermatozoa can be processed using similar methods and frozen either on dry ice, over liquid nitrogen vapor or in a dry shipper, resulting in minimal differences among treatments in post-thaw sperm longevity and in vitro function (unpublished data). All of these factors in combination create major challenges for being able to freeze cat spermatozoa in the field. In small cats, an additional consideration is the relatively low number of spermatozoa typically recovered in each ejaculate (Table 4). The amount of frozen spermatozoa banked from small cats in the wild represents the major variable dictating the use of this limiting resource. For assisted reproduction, AI is relatively uncomplicated and easy to perform (Howard, 1999; Wildt, 2003), but IVF represents a more economical choice for assisted reproduction when sperm numbers are at a premium and post-thaw sperm parameters are compromised. Insemination of each IVF drop requires only ~50,000 motile sperm as compared to a minimum of 10,000,000 motile sperm for each AI procedure. With small cats, cryopreservation of each ejaculate would permit 1 - 7 AI attempts; in contrast, 300 - 1500 IVF drops could be inseminated with the same number of motile spermatozoa (Table 4). Although IVF adds the additional challenge of transferring either freshly-produced or frozen-thawed embryos to synchronised recipients, recent research progress has led to improved ET success with some cat species (Pope, 2000; Swanson and Brown, 2004; Pope, Gomez and Dresser, 2006; Swanson, 2006).
Table 4. Relative merit of using frozen-thawed spermatozoa for artificial insemination versus in vitro fertilisation for assisted reproduction in small cat species.
Cat species
No. cats (no. ejaculates)
Total sperm/ ejaculate (X 106)*
Pre-freeze motility (%)*
AI doses/ IVF doses/ ejaculate** ejaculate***
Fishing cat
8 (8)a
56.4 + 17.7
73.0 + 4.0
3
600
Ocelot
10 (10)b
129.4 + 39.4
76.9 + 5.6
7
1470
Black-footed cat
5 (12)c
28.4 + 6.3
81.3 + 2.3
1
345
Sand cat
7 (10)d
43.5 + 11.0
77.0 + 2.3
2
495
Pallas' cat
1 (5)e
25.2 + 3.2
78.0 + 2.0
1
290
*Mean (+ SEM). **Based on 10 x 106 motile sperm per AI with an estimated 20% decline in post-thaw motility. ***Based on 5 x 104 motile sperm per IVF drop with an estimated 20% decline in post-thaw motility. aThiangtum et al. (2006); bStoops, unpublished; cHerrick et al., in press; dHerrick et al. (2006); eSwanson et al. (1996) (breeding season only).
Given the potential pitfalls associated with effective banking of semen from wild felids in the field, assessing the functionality of frozen-thawed cat spermatozoa before applied usage is a necessity. Since routine collection of oocytes from non-domestic cats for fertility evaluations is not feasible, heterologous IVF of viable domestic cat oocytes has been developed as an alternative. Freshly-collected spermatozoa from at least nine nondomestic cat species, including three of five small cat SSP species, have proven capable of fertilising viable domestic cat oocytes, allowing evaluation of all sequential aspects of sperm function from capacitation to syngamy (Swanson, McRae, Davidson, Levens and Campbell, 1998; unpublished). With fro-
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zen-thawed spermatozoa from Pallas' cats (Swanson, Maggs, Clarke, Newell, Bond, Bateman and Kennedy-Stoskopf, in press), ocelots (unpublished) and fishing cats (Thiangtum et al., 2006), heterologous IVF percentages have ranged from 33% to 46%, including 36% fertilisation using frozen-thawed spermatozoa from two wild Pallas' cats in Mongolia (Table 5; Fig. 2). Because in vitro matured (IVM) oocytes frequently have reduced nuclear maturation compared to in vivo matured oocytes, fertilisation percentages may be adjusted based on mature oocytes only. For frozen-thawed fishing cat and ocelot spermatozoa, assessing fertilisation of mature oocytes only increases heterologous IVF percentages to 59 - 62% (Table 5). Direct comparison to values from homologous IVF with frozen-thawed spermatozoa is difficult, given limited data using mobile laboratory techniques for sperm cryopreservation and/or both homologous and heterologous IVF (Magarey, Herrick, Thiangtum, Tunwattana and Swanson, 2006; Swanson et al., in press). In a conventional laboratory setting, homologous IVF with frozen-thawed fishing cat and caracal spermatozoa has resulted in high (65-71%) fertilisation success (Pope et al., in press).
Table 5. Homologous in vitro fertilisation (IVF) of conspecific oocytes and heterologous IVF of domestic cat oocytes with frozen-thawed spermatozoa from small-sized nondomestic felids.
Homologous IVF
Heterologous IVF
Gonadotropin Total oocytes Cat Species treatment** inseminated
Fertilisation percentage
Oocyte source**
Total oocytes Fertilisation inseminated*** percentage
Pallas' cat
eCG/hCG
30
Ocelot
Fishing cat eCG/hCG
29
FSH/LH
313
Caracal
FSH/LH
392
66.7%*a 37.9%*e 70.6%f 64.8%f
eCG/hCG eCG/hCG IVM IVM
45 201 298 (157) 110 (58)
35.6%*b 46.1%*c 36.0%*d (59.1%) 32.7%*g (62.1%)
*Sperm cryopreservation and/or IVF procedures conducted using a mobile laboratory approach. **Oocytes for homologous IVF were recovered from eCG/hCG or FSH/LH treated conspecific females whereas domestic cat oocytes for heterologous IVF were either recovered from eCG/hCG treated females or after in vitro maturation (IVM) of oocytes recovered after spaying. ***For IVM oocytes, fertilisation percentage also was calculated based on the total number of oocytes (in parentheses) that had completed nuclear maturation. aSwanson and Kennedy-Stoskopf, unpublished; bSwanson, unpublished (semen from wild Pallas' cats); cSwanson et al., in press; dStoops, unpublished; eMagarey et al. (2006); fPope et al. (2006); gThiangtum et al. (2006).
Disease and transportation concerns Two other considerations with in situ sperm banking of wild cats are the potential for disease transmission in frozen cat semen and the impact of airport security screening on sperm viability. The scientific literature contains little to no information on these two concerns in cats but either could have a significant impact on the applied usage of this genetic resource. In other mammalian species, infectious viruses have received the most attention as pathogens in semen (van Oirschot, 1995; Guerin and Pozzi, 2005). Of the common viruses found in cats (feline immunodeficiency virus, FIV; herpes virus, FHV; leukemia virus, FLV; corona virus, FCV; panleukopenia virus, FPV), only FIV has been thoroughly investigated as a potential contaminant of cat semen. In domestic cats, FIV is found in semen of infected males and may be
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Figure 2. Pallas cat - domestic cat hybrid embryos (48 h post-insemination; 5-8 cell stage) produced from heterologous IVF of domestic cat oocytes using frozen-thawed spermatozoa from wild Mongolian Pallas' cats. transmitted via laparoscopic AI (Jordan, Howard, Barr, Kennedy-Stoskopf, Levy and Tompkins, 1998), but no comparative studies have been conducted in non-domestic felids. To our knowledge, FHV is the only other virus that has been assessed for presence in cat semen. In FHVinfected Pallas' cats, viral DNA was not detectable by PCR in either seminal fluid or frozen sperm cells, although conjunctival biopsies showed recurrent viral shedding (Swanson et al., in press). Given the limited data available, the safest strategy for banking semen from wild felids would be to screen blood samples from all sperm donors for viral exposure and freeze samples in sealed cryostraws for added biosecurity. For ex situ usage, frozen cat semen typically must be transported internationally in liquid nitrogen dewars (or dry shippers) using commercial airlines as the means of conveyance. With recent terrorist activity, airline security has become more restrictive and now requires that all checked luggage undergo x-ray screening. A recent study has shown that a single screening procedure causes a significant decline in post-thaw cat sperm motility, possibly due to mitochondrial injury, and that multiple screenings induce double-stranded DNA breakage (Gloor et al., in press). For the near term, transporting dry shippers via overnight carrier services (such as Fedex or DHL) on cargo aircraft is one safe option; however, in some countries, these carriers either are not available or ship their cargo on passenger aircraft, necessitating x-ray exposure. These companies currently are developing high-intensity x-ray screening methods that can be applied to large cargo containers which, when implemented, will effectively eliminate overnight carriers as a transport option. Holistic conservation programs Linkage of in situ and ex situ populations is one essential step in the development of holistic conservation programs that incorporate captive breeding, habitat preservation and environmental education. This process involves identifying native collaborators in range countries, forming cooperative partnerships with local conservation organizations and engaging the local human communities in each region in these conservation efforts. An integral component of these partnerships has been scientific capacity building, in disciplines such as field ecology and reproductive biology, so that resident scientists acquire the skills to independently study indig-
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enous wildlife and apply their new knowledge to species management and preservation (Rudran and Wemmer, 2001; Swanson and Brown, 2004). Zoos have played an integral role in these research training programs and increasingly are providing funding to support field ecology studies and habitat preservation (Hutchins, 2003; Miller et al., 2004). Among small cats, field ecology projects are ongoing in all five SSP species, including camera trap surveys of fishing cats in Thailand, radiotelemetry studies of Pallas' cats in Mongolia and sand cats in Saudi Arabia, distribution and biomedical surveys of black-footed cats in South Africa and habitat restoration for ocelots in Brazil. The Cincinnati Zoo provides financial support for each of these projects with other North American zoos contributing funding as well. In situ sperm banking already has been incorporated into field ecology studies in Pallas' cats and black-footed cats and likely will become a component of these other field projects within the next few years. We anticipate producing offspring in several of these small cat species using frozen-thawed spermatozoa from wild males in the near future, laying the foundation for more effective genetic management of captive cat populations. Provided that this linkage strategy shows adequate efficiency, the intriguing but even more challenging prospect of reversing gene flow (i.e., from ex situ to in situ) then may be explored for future management of geneti- cally impoverished, geographically isolated cat populations in the wild.
Acknowledgements The authors gratefully acknowledge the following individuals for assistance with in situ studies: Dr. Meredith Brown, Bariushaa Munkhtsog, Steve Ross and Dr. Amanda Fine (Mongolia) and Dr. Alex Sliwa, Beryl Wilson, Morgan Hauptfleisch, Dr. Corne Anderson and Dr. Nadine Lamberski (South Africa). Our appreciation also is extended to the multiple zoos and universities that collaborated on research in ocelots (African Wildlife Safari, Carnivore Preservation Trust, Dallas Zoo, Fort Worth Zoo, Nashville Zoo, Oglebay's Good Zoo), fishing cats (Feline Conservation Center, Kasetsart University, Khao Kheow Open Zoo, Oklahoma City Zoological Park, San Francisco Zoological Gardens), black-footed cats (Central Florida Zoological Park, Oklahoma City Zoological Park, Omaha's Henry Doorly Zoo, Riverbanks Zoo & Garden, San Antonio Zoological Gardens), sand cats (Erie Zoo, Feline Conservation Center, Living Desert Zoo, Saint Louis Zoo) and Pallas' cats (North Carolina State University). At the Cincinnati Zoo, the authors also thank Dr. Mark Campbell, Dr. Greg Levens, Jennifer Bond, Helen Bateman and Jackie Newsom for help with animal procedures and John Dinon and Barbara Lintzenich for estimates of Pallas' cat maintenance costs. Funding for these studies was provided, in part, by grants from the National Institutes of Health (RR001588) and the Morris Animal Foundation (D04ZO-72).
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