Abstract
Reproductive biotechnologies are still considered critical tools for saving and maintaining endangered species. Some successes have been reported with the use and integration of artificial insemination (with fresh or frozen-thawed samples) in conservation programs. However, not a single species is currently managed through oocyte freezing or embryo-based technologies.
The limited contribution of fertility preservation techniques to species conservation principally stems from the lack of knowledge of species biology, as well as inadequate facilities, space, expertise, and funding needed for their successful application.
More fundamental studies on animal reproductive biology as well as more fertility preservation options are needed with all parties involved (reproductive technologists, zoo biologists, and conservationists) adopting parallel efforts to sustain wild populations and habitats.
Introduction
Extinction currently is occurring at a much higher rate than speciation because of detrimental human activities, such as habitat destruction, over-hunting/fishing, and poaching. Animal conservation aims at understanding and sustaining biodiversity because the disappearance of even a single species can compromise the functioning of entire ecosystems (Comizzoli et al., 2009). The International Union for Conservation of Nature (IUCN) estimates that 25% of mammals, 12% of birds, 20% of reptiles, 30% of amphibians, 20% of fishes, 30% of invertebrates, and 55% of plant species are threatened with extinction (IUCN, 2014). Many of these wild species populations are small and fragmented in their habitat with little or no genetic exchange, which increases homozygosity and inbreeding that, in turn, leads to a bad adaptive capacity to environmental changes and fertility problems (Wildt et al., 2010). In addition to protecting species in their natural habitat (in situ conservation), it is critical to maintain viable populations in captivity (ex situ) for eventual reintroductions. However, reproduction fitness may be impaired in captivity by small space, health and husbandry problems, non-adapted diets, modified sexual behavior, or infertility (Lasley et al., 1994; Wildt et al., 2010). Therefore, conservation breeding can be optimized with assisted reproductive techniques (ART) to overcome the issues listed above. These approaches have been widely promoted over the past decades for enhancing breeding management and sustaining small populations of rare species (Holt and Lloyd, 2009). Besides the techniques of artificial insemination (AI), embryo transfer (ET), and in vitro fertilization (IVF), a wide range of methods and tools have been developed (Comizzoli et al., 2000; Wildt et al., 2010). These include non-invasive hormonal assessments for accumulating fundamental knowledge in diverse species (e.g., ovulatory mechanisms, seasonality, pregnancy, and infertility) and manipulating the reproductive activities (e.g., superovulation and estrous synchronization). Among these critical tools, germplasm cryobiology also has played a key role in establishing biorepositories for capturing extant genomic diversity (Comizzoli et al., 2012). However, critical knowledge of reproductive traits is first needed before developing ART. Unfortunately, we know very little about species biology (reproduction in only 250 species has been properly described) with our efforts still remaining mainly concentrated on mammals and birds (Holt et al., 2014).
Even though this is not the aim of this article, it is also important to keep in mind that one of the biggest challenges facing zoos or some natural habitats is overly abundant populations. The genetic management programs of zoos dictate breeding of the most valuable individuals but also help to ensure that animals of unknown origin or those already well represented are not reproducing. Separating individuals into same-sexed groups is fraught with problems, especially severe aggression among adults housed together, thereby requiring significant additional space to place “excess” individuals. As a result, there is an urgent need to develop safe and effective reproduction control measures relevant to the management of wildlife. This requirement extends beyond the zoo world to wild habitats where native landscapes are being destroyed by overabundant or invasive species (Garside et al., 2014).
The objective of the present article is to review 1) existing reproductive biotechnologies to preserve the fertility of wild species populations and 2) emerging technologies associated with the need to change the paradigm that also are critical to solve conservation issues.
Development and Use of Reproductive Biotechnologies for Wild Species Conservation
For the past 20 yr, major progresses in wildlife reproductive science have been made with the help of non-invasive endocrine monitoring (measuring fecal or urine steroid metabolites) to either 1) study reproductive traits such as ovarian cyclicity or seasonality of testicular activity, 2) monitor pregnancies, 3) assess the stress though cortisol level, or 4) design the best protocols to enhance fertility or induce ovulation (Schwarzenberger et al., 1996; Brown et al., 2001; Monfort, 2003). Before AI or ET in herbivores, for instance, ovulation may be induced artificially by PGF2 injection or by removal of progesterone-releasing implants (Morrow et al., 2009). In felids, the induction of ovulation is possible with injections of eCG followed by hCG or luteinizing hormone (LH) administration (Howard and Wildt, 2009). Unfortunately, as in domestic species, ovarian response is highly variable, and the oocyte quality may be impaired by the exogenous hormones.
Giant panda cub born after artificial insemination at the National Zoological Park,Washington DC.
Artificial insemination with fresh or frozen-thawed semen
Artificial insemination is currently the most extensively applied ART. Initial successes were achieved in bovids because of the significant development of ART in the cattle production. Artificial insemination has been successfully applied to produce live offspring in 14 species of non-domestic bovids and seven cervid species (Roldan et al., 2006; Morrow et al., 2009). However, AI is not integrated in the routine management of endangered ungulates yet. Too few zoos actually possess the facilities or expertise to permit animals to be safely handled for administration of exogenous hormones to control ovarian activity. In wild carnivore conservation, the progress in AI is best illustrated by the basic research on ferret reproduction seasonality, semen cryopreservation methods, and laparoscopic AI (Howard and Wildt, 2009). To date, more than 150 kits (60% success with fresh sperm) have been produced by AI, including multiple litters of kits that have been produced from frozen founder sperm stored for as long as 20 yr. Many of the individuals produced by AI have subsequently reproduced, and some of their offspring have been reintroduced into the wild, representing a direct example of how ART have tangibly contributed to a successful species recovery program. Since 1987, more than 8,000 black-footed ferrets have been produced and more than 3,000 of these have been released into prairie dog colonies across North America. The genetic management of the giant panda population also is using AI as a really powerful tool with 25 to 50% success after AI and the significant production of panda cubs derived from fresh or frozen-thawed semen (Huang et al., 2012). However, AI in other carnivore species (e.g., felids and canids) is far from being routinely used and still has a really poor success rate (Crosier et al., 2006; Jewgenow and Songsasen, 2014).
Embryo transfer
The earliest report of ART in a wild species was from Kraemer et al. (1976) who actually succeeded in the first embryo transfer in baboons. It was followed by successful interspecies embryo transfers from eland (Dresser et al., 1982) and gaur (Stover and Evans, 1984) to domestic cow and bongo to eland (Dresser et al., 1985). This early emphasis on embryo technologies in the 1980s as well as in the 1990s (Loskutoff et al., 1995) was then followed by a strong interest in somatic cloning (Holt et al., 2004) and, more recently, in genomic approaches for “rescuing” or even resurrecting extinct species (Zimmer, 2013); however, major technical and ecological challenges remain for their application in conservation (Monfort, 2014). Thirty years after the first successful interspecies embryo transfer in a wildlife species, there is not a single example of genetic management based on that technique (Monfort, 2014). Successes related to the transfer of embryos produced by IVF also remain limited even though this technique (from the oocyte recovery through the IVF with fresh or frozen-thawed semen to the in vitro culture of embryos) is, in theory, the fastest and most efficient way to propagate small populations. For instance, significant but still anecdotal successes in IVF and ET have been reported in ocelots (Swanson, 2012) and deer species (Locatelli et al., 2012; Thongphakdee et al., 2012). But the technical complexity associated with the high procedural costs is limiting the development and implementation in conservation programs. In addition, the scarce knowledge on the kinetics of embryo development and foeto-maternal recognition also leads to many losses of pregnancies.
ART in birds, amphibians, and fish
Based on the progress made in the development and application of AI in the domestic poultry industry for more than a half-century (USDA National Agricultural Statistics Service, 2014), AI has been used to produce chicks in numerous species of raptors, cranes, waterfowl, psittacines, and passerines (Gee, 1995). The technology also has played a key role in successful species recovery programs for the peregrine falcon (Hoffman, 1998), houbara bustard (Saint Jalme et al., 1994), and whooping crane (Ellis et al., 1996). The success of these excellent programs was underpinned by systematic research in diverse disciplines, including behavior, genetics, animal husbandry, veterinary medicine, and chick rearing (Ellis et al., 1996). For all bird species, successful application of AI still requires pre-emptive research in semen collection and processing (much more complex than in mammals because of the fragile sperm cells), access to sufficient numbers of birds for basic and applied research, baseline knowledge of species biology, and appropriate facilities and expertise (Blanco et al., 2009).
Amphibian unique reproductive patterns and mechanisms, key to species propagation, have only been explored in a limited number of laboratory models. The development of applied reproductive technologies for amphibians has been useful for a few threatened species only. These include non-invasive fecal and urinary hormone assays, hormone treatments for induced breeding or gamete collection, and artificial fertilization (Kouba et al., 2013; Clulow et al., 2014). The hormonal control of reproduction in amphibians has hardly been studied in comparison with fish and mammals. The use of injectable gonadotropin preparations, especially when coupled with egg and sperm collection for artificial fertilization is effective. Currently administered to a wide variety of anuran species are hCG and eCG, sometimes given in combination to maximally stimulate gonadal function. Regardless of the gonadotropin preparation chosen, much work remains to determine optimal species-specific injection protocols, given the large variations in responses to gonadotropins (Clulow et al., 2014). Optimization of egg and sperm collection methods for fertilization also is highly species-specific (good progress in anurans vs. lack of results in urodeles and caecilians).
Zoos and aquariums are solicited to help conserve endangered fish species using both ex situ and in situ approaches (Reid et al., 2013). However, it appears that simply producing and releasing large numbers of hatchery-reared fish is not sufficient to sustain and/or recover endangered fish populations. Conservation aquaculture is in its infancy, and it is clear that more research is required to understand the impacts of diverse factors such as genetics (inbreeding, outbreeding), broodstock sourcing, maturation and development, growth rate modulation, environmental enrichment, anti-predator conditioning, as well as an improved understanding of anthropogenic impacts on aquatic environments, such as habitat loss/fragmentation, pollution, and climate change (Flagg and Nash, 1999; Reid et al., 2013).
Germplasm cryopreservation and genome resource banking efforts
Genome resource banking (GRB) refers to the collection, processing, storage, and use of germplasms (sperm, eggs, embryos, ovarian, and testicular tissues) and other biomaterials (blood products and DNA samples) that can be used for understanding and sustaining biodiversity. If used properly in association with ART, GRB have the potential to decelerate the loss of gene diversity in captive populations by reintroducing original genetic material (without removing genetically valuable individuals from the wild) and decrease the interval between generations (Wildt et al., 1997; Comizzoli and Holt, 2014). The number of GRB has grown over the past decades, but the integration and significant roles into the management of captive populations are still limited. Fertility preservation strategies using cryopreservation have enormous potential for helping sustain and protect rare and endangered species. However, wide-scale applications currently are difficult because of the significant physiological variations among species and a sheer lack of fundamental knowledge in germplasm cryobiology. The best examples (mentioned above) can be found within the conservation programs of the giant pandas and black-footed ferrets (Comizzoli and Holt, 2014). Cryo-studies have been conducted in more species (mainly vertebrates) in the recent years, but a vast majority still remain unstudied.
Semen freezing.
Semen cryopreservation represents the most extensive effort, with live births reported after AI. Physico-chemical properties related to cryo-resistance differ between species as shown, for instance, by tolerances to glycerol concentrations in the extender: 5% in cattle, no more than 4% in deer, 3% in pigs, 1.75% in mice, 6% in chinchillas, and large differences observed in marsupials (Comizzoli et al., 2000, 2012). Recent progress in vertebrates have been reviewed (Comizzoli and Holt, 2014) and include pioneering studies on endangered gazelles and Iberian lynx (Garde et al., 2003; Gañán et al., 2009). Sperm processing challenges also are illustrated from amphibian to fish studies. Generally, these types of spermatozoa remain immotile in seminal plasma until released into the environment. In the case of frogs, spermatozoa are actually excreted in the urine (pH = 7.5; 85 mOsmol/L) that, in turn, naturally activates motility due to a lower osmolarity than is present in testicular tissue (Kouba et al., 2013). A similar phenomenon occurs in fish spermatozoa (mostly from salmonids, sturgeons, carp, turbot, halibut, and cod) that are initially immotile in seminal plasma but then are activated by fresh or saltwater (Cabrita et al., 2008). Spermatozoa from some fish species maintain motility for < 1 min whereas others retain this function for several days. As a result of these characteristics, amphibian and fish protocols generally focus on processing and storing inactivated cells by collecting samples into a buffered saline solution that imitates the original seminal plasma environment. In endangered amphibians, excellent progress has been made in developing sperm cryopreservation methods for anuran species with embryos or more advanced offspring generated from frozen sperm in several species, including sperm successfully cryopreserved after non-invasive collection by hormonal induction (Kouba et al., 2013). Recently, there has been some success with cryopreserving sperm cells from a variety of coral species (in vitro production of larvae). Based on that, GRB have been established to help offset these threats to the Great Barrier Reef and other areas (Hagedorn and Spindler, 2014).
Eld's deer born after in vitro fertilization and embryo transfer at the Zoological Park Organization, Thailand.
Semen collection on a Pananian golden frog at the National Zoological Park, Washington DC.
Oocyte cryopreservation.
Oocyte freezing remains challenging and unsuccessful in wild species and will require more research before becoming a standard procedure (Comizzoli and Holt, 2014). Despite extensive efforts conducted in different wild mammals, not a single individual has been produced from a frozen–thawed egg. In amphibian and fish, the potential for cryopreservation of the female is also challenging, with no offspring reported to date from cryopreserved oocytes. Egg size and structure and yolk composition appear to create technical barriers to cryopreservation.
Gonadal tissue preservation.
As an alternative to fully grown gametes, gonadal tissue preservation has become a promising option in vertebrates (Comizzoli et al., 2012). Ovarian and testicular tissues are systematically banked, but the production of mature gametes (through xenografting or long-term in vitro culture) has not happened yet in wild species. In amphibians, the direct cryopreservation of immature ovarian follicles holds promise but would need to be combined with procedures such as xeno-transplantation to generate mature, ovulated oocytes. Cryopreservation of primordial germ cells also holds promise but would likely need to be combined with the generation of chimeras to obtain adults that can produce viable gametes (Clulow et al., 2014). This approach also seems to be the future for birds and fish ART (Comizzoli and Holt, 2014).
Even though the results are not satisfactory using classical ART, more fertility preservation options are necessary to save species. It also is worthwhile thinking beyond systematic characterizations and considering the application of cutting-edge approaches to universally preserve the fertility of a vast array of species (Comizzoli and Wildt, 2013). Regardless of the specific technology to be explored, new tools will require the significant use of “models” (usually domestic animals) for comparable wildlife species. This need has been recognized and adhered to for three decades (Wildt et al., 2010). It is essential to consider the practicality of initial testing and application, which will likely require exploration first in a taxonomically related “model.” Even then, if a certain technique works efficiently in the model, it may require further modifications to be used effectively in the species of interest. Traditionally, close relatives have been selected: for example, the domestic cat (for wild felids), domestic dog (for wild canids), red- or white-tailed deer (for wild cervids), brushtail possum (for endangered marsupials, or common frogs or toads (for rare amphibians). Finally, there are species that are so specialized that models may be unavailable. Examples of these include the elephant, rhinoceros, and giant panda (among hundreds of others), all of which will most likely require direct studies, although based on best available knowledge or predictions from work performed in other species (Wildt et al., 2010).
Genomic tools (next-generation sequencing of the DNA) enable scientists to better understand the origins and patterns of biological functions. Biodiversity genomics will soon be yet another tool to add to the ART toolbox. However, without a parallel effort in reproductive science and ART development, biogenomic applications will only continue to attract attention disproportionate to their potential for sustainably managing reproduction in endangered species. Whether it is the successful application of AI or the use of cloning to sustain an endangered living species or even “resurrect” an extinct one, success will always depend on knowledge of a species biology, ecology, social structure, reproductive cycle, seasonality, embryo implantation, placentation, gestation, parturition, maternal behavior, neonatal care, nutrition, disease susceptibilities, and causes of endangerment. These important physiological traits are still poorly understood and monitored (like pregnancy for instance).
Filling the Gap between Technology and Animal Conservation
As already emphasized, success in producing new individuals with the help of ART requires, as a first step, a greater knowledge in the basic aspects of reproductive biology and cryobiology. The complexity and diversity of reproduction in the animal kingdom was vastly underestimated, and we have certainly overestimated our ability to develop and apply ART that can be used to aid reproductive management and contribute to biodiversity conservation (Holt and Lloyd, 2009; Holt et al., 2014). As seen above, the barrier to the successful application of ART is not a shortage of new techniques, but rather a fundamental lack of “conservation capital”—trained scientists, sufficient numbers of research subjects, funding, and appropriate facilities designed specifically to study and manage nondomestic species. The zoo community has been too slow to recognize that current management paradigms are insufficient for sustaining hundreds of species across diverse taxa (Monfort, 2014). Likewise, conservationists have often minimized the role of zoos and resisted biotechnology when their own efforts to stem the loss of biodiversity and wild places have fallen short. Reproductive technologists, zoo professionals, and conservation biologists all have the same goal—to save species and the ecosystems they require for survival. Success will require collective efforts to identify extant limitations and fundamental gaps in knowledge, both intellectual and practical, and joint efforts to secure long-overdue improvements.
Aligning technological capability with good animal management and sound conservation principles will make it increasingly possible to apply ART to increase reproductive efficiency; to readily transport gametes (sperm, eggs, and embryos), raw DNA, or genomes to overcome increasingly onerous international animal importation restrictions; to facilitate zoo-to-zoo animal exchanges (e.g., elephant AI already serves this purpose); and eventually to permit the routine exchange of genetic material between zoo and wild populations (Holt and Lloyd, 2009). As Monfort (2014) clearly highlights: “The justification for a return to building basic knowledge boils down to this: what is the ultimate value of using ART to produce endangered animals, or even resurrect extinct species, if we lack the capacity to manage and sustain these species in the first place? If we cannot now sustainably manage an oryx, Eld's deer, or cheetah with or without ART, then what chance do we have of sustaining resurrected woolly mammoth, quagga, or dodo in the future? Our strategy and focus must change or the true potential of ART for managing endangered species will never be fully realized.”
Conclusion
The application of reproductive biotechnologies for the preservation of endangered mammalian species is limited by several factors. Obtaining a healthy and genetically valuable offspring after AI or IVF/ET depends on the existing knowledge of the reproductive physiology of each particular species, and little is known about the physiology of most wild animals. Captivity and poorly available biological material (often in disparate locations) also increase obstacles for research progress. The role and relevance of ART for contributing to species conservation is inextricably linked to whether or not zoos and conservation centers invest in developing an improved understanding of the overall biology, and reproduction in particular, of species. Reproductive biotechnologies, combined with sound husbandry and management, appropriate facilities, and parallel efforts to sustain wild populations and places, offers the best chance for conservation success. Zoos and conservation centers must adopt such holistic conservation strategies, or they risk becoming living museums exhibiting relic species that no longer exist in nature.
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