Author's address (for correspondence): P Comizzoli, Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC 20008, USA. E-mail: firstname.lastname@example.org
Innovations are emerging from the growing field of fertility preservation for humans and laboratory animals that are relevant to protecting and propagating valuable domestic and wild carnivores. These extend beyond the ‘classical’ approaches associated with sperm, oocyte and embryo freezing to include gonadal tissue preservation combined with in vitro culture or xenografting, all of which have potential for rescuing vast amounts of unused and wasted germplasm. Here, we review approaches under development and predicted to have applied value within the next decade, including the following: (i) direct use of early-stage gametes for in vitro fertilization; (ii) generation of more mature gametes from gonadal tissue or stem cells; (iii) simplification, enhanced safety and efficacy of cryopreservation methods; and (iv) biostabilization of living cells and tissues at ambient temperatures. We believe that all of these fertility preservation strategies will offer knowledge and tools to better manage carnivores that serve as human companions, valuable biomedical models or require assistance to reverse endangerment.
We are fascinated by carnivores for reproductive studies for two reasons. First, these taxa present many challenging mechanisms associated with propagation, for example, from year round activity vs extremely short breeding seasons, to spontaneous vs induced ovulation, and to resistance to treatments for stimulating or suppressing gonadal activity (Wildt et al. 2010). We have suggested that there are as many mechanistic differences in carnivore reproduction (especially within the Felidae and Canidae families) as there are species (Wildt et al. 2010). Thus, the highest priority is to understand these species-specificities, including the normative, baseline data that still lag far behind the vast information already known about humans, farm and laboratory animals (Comizzoli et al. 2009, 2010). The second reason our laboratory has devoted more than 35 years to carnivore reproduction is that, beyond the scholarly benefits of such work, findings can be applied to enhance management of individuals, populations and entire species. In some cases, this has simply involved more confidence in how to improve natural breeding. For example, knowing that more vertical enclosure space reduces adrenal activity (i.e. stress) in the rare clouded leopard (Neofelis nebulosa) has been used to increase cub production (Wildt et al. 2010). Specific physiological information also is essential for advancing assisted reproduction, especially artificial insemination (AI). Our interest has included ensuring the ability to propagate domestic cat and dog models (used to address human metabolic disorders) and especially wild carnivore species (Comizzoli et al. 2009, 2010). There now are examples of how intensive science directed at understanding fundamental reproduction has allowed AI to help turn around the dire status of endangered carnivores. One example is the black-footed ferret (Mustela nigripes), once reduced to only 18 individuals and now reintroduced to the plains of the American West (Comizzoli et al. 2009, 2010). Another is the giant panda (Ailuropoda melanoleuca) where an intensively studied captive population has almost tripled in the last 15 years (Wildt et al. 2010). We predict far more demonstrations on the practical value of assisted reproduction in ‘conservation breeding’, especially with trends now moving towards not only creating genetically viable, but also sustainable ex situ populations as insurance for wild counterparts.
Although the value of AI has been proven for the domestic dog, an few felids and the occasional ursid and mustelid, there is a need to advance in vitro fertilization (IVF) and embryo technologies (so important to humans, farm and laboratory animals) to both domestic and wild carnivores. This is clearly a formidable challenge, and although IVF and embryo technologies have been used for producing a few ‘milestone’ births in domestic and wild carnivores (Wildt et al. 2010), these approaches are not used for routine reproductive management. This largely is because of insufficient knowledge about embryogenesis, complications in recovering germplasm (especially oocytes from canids, ursids and mustelids) and identifying an appropriate surrogate (recipient) for any laboratory-produced embryos (Wildt et al. 2010). Furthermore, the reproductive physiology of certain carnivores is highly sophisticated and complex (e.g. the dog in particular), and certain taxa or species express unique reproductive traits (e.g. teratospermia or gamete hypersensitivity to cryopreservation; Comizzoli et al. 2012).
Over the last decade, fertility preservation has evolved rapidly in human reproductive medicine, offering new approaches to patients at risk for compromised reproduction because of cancer treatments or other therapies (Comizzoli et al. 2012). Research has primarily centred on the salvage, storage and use of gonadal tissue and gametes. These approaches have intriguing applications for domestic and wild carnivores, especially for individuals that have not yet produced adequate numbers of descendants to ensure passing on of their genome. This issue is critical when managing the breeding of rare animals where maintaining gene diversity is essential to sustain species integrity and avoid the menacing effects of inbreeding. Here, we briefly review concepts that have the potential of (i) improving now, or in the future, the propagation of individuals that are living, but failing to reproduce by conventional means, (ii) rescuing the genome after death and (iii) protecting fertility in young, pre-pubertal animals or extending reproductive potential in older individuals.
Within the Gonad as a Source of Gametes or Parental Genomes
The ovary and testis have a wealth of untapped, arrested or developing gametes, most of which never participate in fertilization. An ability to artificially mature early-stage oocytes or spermatozoa in culture could provide unlimited germplasm to generate embryos, including from animals that are pre-pubertal, nearing the end of their reproductive lifespan or that die unexpectedly. Based on successes in mouse and non-human primate models (Comizzoli et al. 2012), we are conducting studies to culture ovarian tissues or isolated follicles from the domestic cat and dog. Encouraging progress is being made towards producing viable antral follicles, especially for the cat, a topic addressed in detail by Songsasen et al. (2012).
Less work has been directed at in vitro culture of testicular tissues to produce fully formed carnivore spermatozoa that have the capacity to fertilize. We recently initiated investigations that have revealed the feasibility of using collagenase and hyaluronidase to isolate living cat and dog seminiferous tubules for preservation and culture (Fig. 1a). More than 50% of germ line and somatic cells remain alive and continue to differentiate in vitro for at least 4 weeks (Fig. 1b). Such investigations should be pursued, especially given recent encouraging data from Sato et al. (2011) who demonstrated that mature mouse sperm cells can be produced in vitro. Certainly, a next high priority for carnivores is to determine the mechanisms related to acquisition of motility and centrosomal maturation in testicular spermatozoa grown in vitro, phenomena not well understood for any species. Testis tissue from the common ferret has been xenografted into the body of immunodeficient mice that then produce mature spermatozoa from the original donor (Gourdon and Travis 2011). While having theoretical relevance to other carnivores, the challenge can be the normally abbreviated lifespan of the rodent host (much shorter than for more carnivores) and the protracted (>35 weeks) duration required for gamete maturation from the tissue grafts.
Another innovative approach is to focus simply on the oocyte's germinal vesicle (GV) – the source of the maternal genome – rather than the whole, more complex female gamete. We have advanced this concept in the cat by demonstrating the feasibility of transferring GVs between oocytes (Comizzoli et al. 2011). These organelles retain both structure and function, including the capacity to resume meiosis and participate in fertilization and embryo formation. Particularly intriguing has been the ability to recover and transfer GVs from oocytes from the early antral follicular stage into the ooplasm from large antral follicles to allow producing viable IVF embryos (Comizzoli et al. 2011). Thus, it may be possible to use this GV rescue approach to salvage the maternal genome from individuals who die early or late in life before reproducing, or who are experiencing cytoplasmic deficits in the oocyte or follicular anomalies. It also has been determined that cat GV chromatin withstands artificial compaction for subsequent injection to reconstruct a viable oocyte (Fig. 1d,e), all without encountering the need for the usual complex membrane electrofusion (Graves-Herring et al. 2011).
Stem cell technologies also are promising for producing gametes, in this case from oogonial or spermatogonial progenitors or from differentiated cells. Characterization, isolation and transfer of spermatogonial stem cells have been attempted in the cat and dog with mixed results (Travis et al. 2009). In brief, this has involved isolating the spermatogonial stem cells followed by transfer into a germ-cell-depleted (via radiation) host. On occasion, it has been possible to recover approximately 20% of mature sperm cells derived from the donor (Travis et al. 2009). Others have transplanted germ cells from a wild felid (ocelot; Leopardus pardalis) into the domestic cat to produce spermatozoa successfully from the donor (Silva et al. 2012). Eventually, it probably will be more efficient to differentiate embryonic stem cells or induced pluripotent stem cells in vitro for this purpose, the latter being accomplished recently for the snow leopard (Panthera uncia) (Verma et al. 2012). The cat GV chromatin potential of these strategies also has been strikingly demonstrated in the mouse where in vitro–differentiated embryonic stem cells have given rise to sperm-like cells (Nayernia et al. 2006) and oocyte-like cells derived from newborn mouse skin (Dyce et al. 2011).
Ultra-Rapid Freezing Approaches for Gametes and Gonadal Tissues
Vitrification still is regarded by many as ‘novel’, despite Rall and Fahy's pioneering report more than 25 years ago on its usefulness for preserving mouse embryos (Comizzoli et al. 2012). We continue to be enthusiastic about vitrification because of its relative simplicity, low cost and ‘field-friendliness’ (i.e. the ability to vitrify biomaterials even in harsh, remote environments using only a liquid nitrogen dry shipper). Compared to studies of embryos, there continues to be few reports on the efficacy of ultra-rapid freezing, or vitrification, of spermatozoa. However, encouraging results recently have been reported for human (Isachenko et al. 2011) and dog (Kim et al. 2012) spermatozoa. In the latter case, the gametes were exposed to 5% glycerol and freezing vapours for <1 min before plunging into liquid nitrogen; >50% of sperm were motile after thawing. There also is a trend towards freeze-preservation in lower cryoprotectant concentrations, which appears especially important for dog spermatozoa and cat oocytes that are susceptible to cryoprotectant toxicity (Comizzoli et al. 2012). Another alternative explored in our laboratory is the effectiveness of vitrification solutions that rely on non-permeating (non-toxic) cryoprotectants, including natural sugars such as sucrose and trehalose. In the case of cat oocytes, we have observed >80% survival after vitrification in saturated trehalose solutions.
For preserving gonadal tissues, priorities are trending towards formulating ‘closed’ systems that seal the vitrified biosamples from direct contact with liquid nitrogen, thereby avoiding pathogen transmission during storage (Comizzoli et al. 2012). In one study on cat and dog ovarian tissues (Comizzoli et al. 2012), we determined that 75% of follicles were viable after warming, a value comparable to the 85% measured in a conventional open system. More recently, we have observed as high as 60% survival in cat seminiferous tubules that were cultured for 7 days after vitrification in sealed straws.
Novel Preservation Strategies
Although isolated cells and tissues can be successfully vitrified and warmed without detrimental formation of ice crystals, the challenge remains that low temperature storage can trigger injury to DNA, membranes and cell junctions (Yavin and Arav 2007). For this reason, our laboratory is exploring alternative opportunities for preserving carnivore spermatozoa via desiccation or storage at supra-zero temperature (Comizzoli and Wildt 2012). The advantages of both include much simpler sample processing and transport of genetic material, and most impressively, no need for liquid nitrogen. In theory, the latter could markedly reduce the costs and complexity of biomaterials storage. However, results also have revealed a significant loss in sperm motility and the potential of compromised centrosomal function post-rehydration. For example, we observed poor sperm aster formation after injecting dehydrated (at ambient temperature in trehalose) cat spermatozoa into conspecific oocytes (Comizzoli and Wildt 2012). Centrosomal dysfunction post–freeze drying has been less apparent for non-human primate and bull spermatozoa, although rhesus monkey sperm desiccated in trehalose are known to lose fertilizing capacity (Comizzoli and Wildt 2012). Results from preliminary studies of freeze-drying canine spermatozoa have revealed pronuclear formation after injection into mouse oocytes (Watanabe et al. 2009). Also encouraging are the recent findings of Ringleb et al. (2011) who found early (albeit limited) embryo development after injecting freeze-dried cat spermatozoa into conspecific oocytes. Finally, it is worth noting that desiccation likely has excellent potential for preserving the maternal genome. For example, our laboratory has determined that GVs from cat oocytes that are artificially compacted (with histone deacetylation enhancers), air-dried and then rehydrated can resume meiosis after injection into a fresh (enucleated) cytoplast (Fig. 1c–e; Graves-Herring et al. 2011). This approach, never reported for other species, may well evolve into a simple, inexpensive and biologically viable means of storing the female genome (without the cytoplasm) of carnivores as well as other taxa.
Finally, there are recent approaches in laboratory or conceptual development, one being preservation of single cells in liquid or moist environments at supra-zero or ambient temperatures. For example, we have effectively stored cooled (4°C), viable cat spermatozoa for up to 2 weeks in a trehalose solution while retaining DNA integrity and centrosomal structure (i.e. the presence of centrin) and function (i.e. normal sperm aster formation; Comizzoli and Wildt 2012). For multi-cellular structures (e.g. gonadal tissue), there is exciting potential in the process of biostabilization at room temperatures, that is, using combinations of cell membrane stabilizers, enzyme inhibitors and toxic chelators. In theory, this approach could preserve the structural and functional integrity of tissue at ambient temperature while eliminating the complexities and costs from using liquid nitrogen. As yet, there are few data supportive of this concept, although recently our laboratory demonstrated the ability to sustain cat ovarian tissue structure and some functions after maintenance in a biostabilization environment for 2 weeks.
There is no doubt that the usual methods of preserving carnivore germplasm will continue, with studies involving the conventional cooling, freezing and storage approaches that rely on liquid nitrogen. But as demonstrated with encouraging data presented here, we assert that it is time to break away from customary practices and to explore novel and likely more cost-effective strategies. We are especially excited about mining the germplasm within the gonads, that is, the premature stage spermatozoa and oocytes that represent an enormous reserve of genetic material normally never used for actual reproduction. In this arena, we believe the priorities should include exploring the developmental potential of early gamete stages, developing in vitro culture systems to secure more mature stages and determining how stem cells can be converted into gametes that can produce viable embryos. In terms of preserving fertility, we believe there is great promise in the simplified storage of genomes without the intricacies and expense associated with liquid nitrogen. Therefore, it seems prudent to invest more research into the areas of desiccation and biostabilization at ambient temperatures. It also always is wise to monitor the literature for new information on yet-to-be-discovered storage and reanimation phenomena that normally are found in nature. For example, how can we take the knowledge that bat and bee spermatozoa remain viable in the female reproductive tract for months (Wildt et al. 2010) and transform it into laboratory techniques to preserve carnivore germplasm? Lastly, it is essential that our ability to preserve viable carnivore germplasm, embryos and the entire genome short and long term does not surpass our capacity to use it to produce viable young. Therefore, a continued priority for the carnivore science community is to advance assisted reproductive technologies, including developing more consistent artificial insemination, ovulation induction, ovarian cycle synchronization and embryo transfer protocols. Simultaneous progress with all of these tools will allow the improved production and management of genetically valuable companion animals, models to understand human diseases and rare wild carnivore species.
A portion of the described studies was funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH), and is currently supported by the Office of Research Infrastructure Programs/Office of the Director (R01 OD 010948).
Conflict of interest
None of the authors have any conflict of interest to declare.