Special collection of papers in honor of Dr. John K. CritserComparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species
Introduction
Most of our contemporary knowledge on reproductive mechanisms is derived from a few laboratory and livestock species, as well as substantial research on humans. What we actually know about animal reproduction is limited (and prejudiced), especially when considering how “unconventionally studied” species (i.e., wildlife) self-perpetuate [1]. Species-specificities in reproductive form and function have long been recognized, but have been vastly undervalued. Although of fascinating scholarly interest, such data also have practical use in helping better manage endangered species and rare genotypes, in some cases to avoid extinction. Certain detailed effects of species and genotypic variations in reproductive mechanisms have been well documented. The most compelling evidence has arisen by conducting comparative studies of related species in the same taxonomic family (e.g., carnivores [1]) or by examining populations that undergo inbreeding and losses in heterozygosity (e.g., Florida panther; Puma concolor coryi) [2]. Specifically, comparative assessments across various subspecies of Puma concolor led to the discovery of the extraordinarily poor sperm quality in the rare Florida panther [3]. That finding, in turn, helped to drive relocation and genetic restoration protocols to save the subspecies [2]. Another example is the comparative study of the reproductive biology of three species of ferrets (Mustela sp.), one of which (the critically endangered black-footed ferret; Mustela nigripes), was reduced to the last 18 individuals [4]. Those investigations eventually led to applying artificial insemination (AI) with fresh or frozen-thawed spermatozoa to help recover and return black-footed ferrets to the wild in the American West [4]. It is well established that the successful application of AI, in vitro fertilization (IVF), or embryo transfer (ET) for enhancing propagation is directly related to the amount of basic reproductive information available from each species. This is why, of course, assisted reproductive technologies have become so well incorporated into certain domestic animal breeding and human infertility programs—because so much is known about the whole organism, cellular and molecular biology of livestock and people [5]. We and others have advocated for more studies in wildlife 1) to identify the wondrous ways of how diverse animals naturally reproduce and 2) how such fundamental information can be applied to enhance population and genetic management, including by assisted reproduction [1], [6].
This philosophy extends to more comparative cryopreservation studies of gametes, embryos and gonadal tissues. The practical benefits of the freezing, storing and thawing of such biomaterials is well-established for improving breeding efficiency in livestock, sustaining specific laboratory animal genotypes, and for addressing certain subpar fertility conditions in humans [7]. Less well known is that these same cryo-strategies have enormous implications for developing sustainable populations of rare species and genotypes. The benefits for wildlife include preserving genetic vigor, distributing valuable genes without the stress/expense of moving sensitive, fractious animals and “insuring” all existing genetic diversity that protects fitness and species integrity [8], [9]. Yet, most of the specific details that will optimize cryo-storage of biomaterials from people, livestock and laboratory animals often have marginal relevance to wildlife species [9], [10]. This largely is due to remarkable variations in germ plasm structure and function across species, especially elements that regulate tolerance to osmotic and toxic effects of cryoprotectants, as well as resistance to chilling injuries [11], [12], [13]. Although most of the focus has been on spermatozoa and oocytes (to a lesser extent), activities are increasingly oriented to freeze-storage of ovarian tissues and somatic cells that may be useful for rescuing early stage ovarian follicles [14] or generating induced pluripotent stem cells [15]. Most importantly, the concept of biodiverse frozen and living repositories is no longer futuristic, but rather a contemporary collection and use strategy to better address conservation challenges. Our home organization, the Smithsonian, has been developing a Pan-Institutional Cryo-Initiative involving hundreds of thousands of samples of cryopreserved germ plasm, embryo, blood products, tissues, DNA, feces, and urine. These biomaterials are archived and proactively managed in ways that increase collection and diffusion of knowledge, as well as help sustain genetically diverse, sustainable populations of rare species and genotypes. In contrast to a DNA Bank, or a traditional museum collection, researchers interested in topics from evolution to infectious diseases can access the entire complement of cellular machinery (with any accompanying pathogens), for studies of proteins, RNA, mitochondrial DNA, and even epigenetics. This is not only a conservation issue, but rather a unique and invaluable resource for scholarly investigations.
This paper is presented for two reasons, the first as a reminder of the magnitude of species differences (and, more rarely, similarities) in cryosensitivity of gametes and gonadal tissues. The point is to emphasize that the ability to consistently and successfully preserve viable samples after low temperature storage is only possible after securing basic cryobiological information on each target species. Most of the evidence supporting this assertion will be related to gametes and gonadal tissues, as a recent review has highlighted comparative differences in embryo freezing success in wild species [16]. Our second purpose is to celebrate and honor the philosophies and strategies of our friend and colleague, Dr John Critser. John understood the challenges and opportunities afforded by conducting comparative studies across widely divergent species. He long argued that studying wildlife species could generate information useful for addressing issues in human reproductive health. John was a strong advocate for generating fundamental knowledge where the interested audience might be small, but that informational tidbits would gradually build to answer major intellectual and practical questions. Because of our common interests and John's collegial personality, our laboratories collaborated for more than two decades, studying species ranging from mice to elephants (the latter as a grand adventure in Kruger National Park in the Republic of South Africa). John was a world authority on cryopreservation (with >150 published articles and book chapters) and an activist for just doing more and more basic research until we learned enough to affect real-life change. He loved to brainstorm what might be possible in the future, even rather outrageous ideas. Therefore, we end this manuscript with a brief, but hopefully useful perspective on short-term priorities and an outlook for the future. Regardless, we know that our ideas here will not be as thoughtful (or radical) as if John Critser would have been here to dream with and co-author with us.
Section snippets
Seminal volumes and concentrations
Appropriate handling of semen samples is a critical step to retain the viability and functionality of spermatozoa before any exposures to cryoprotectant solutions and low temperatures. Species-inherent seminal and sperm traits eventually influence cellular survival prefreezing and post-thawing; yet these data are unknown for the vast majority of vertebrates and invertebrates. Seminal characteristics are almost as unique as the species themselves, starting with differences in ejaculate volume,
Oocyte cryopreservation
As Critser, and colleagues [12] have emphasized, oocytes are remarkably different from sperm cells or embryos in cryosensitivity properties and requirements. Because the volume of a round mammalian oocyte (generally ∼120 μm in diameter) is exponentially larger than a spermatozoon, there is a smaller surface-to-volume ratio and a correspondingly higher sensitivity to chilling and intracellular ice formation [67]. The naturally fragile cytoskeleton of eggs also lessens the resistance to
Gonadal tissue cryopreservation
The ability to successfully cryopreserve testicular and ovarian tissue has potential importance in genetic management of rare species and genotypes. Having a viable bank of tissues that could be used in combination with germ cell in vitro culture, transfer or xenografting and IVF would be useful in cases of unexpected death of under-represented adults or from prepubertal individuals. There also could be circumstances when gametes are needed year-round for breeding, but are unavailable because
Emerging preservation approaches and priorities for the future
Vitrification still is regarded by many as “novel”, despite Rall and Fahy's pioneering report (>25 yrs ago) on its usefulness in preserving mouse embryos [92]. We continue to be enthusiastic about applying vitrification because of its relative simplicity, low cost and “field-friendliness” (e.g., the ability to obtain vitrification in a dry shipper even in harsh, remote environments). Its utility across a wide range of taxa also is attractive, as recently demonstrated by the survival of coral
Conclusions
Most contemporary research in this field has been concentrated on cryopreservation of spermatozoa in diverse wild species. There are few studies on oocytes and gonadal tissues (similar to the paucity of research in this area for domesticated species). The overall goal needs to be creating the ability to preserve any germ plasm from a valuable animal of any age or reproductive state using reasonably simple, cost-effective techniques. This includes “occasions of opportunity”, e.g., when an
Acknowledgments
The authors thank Elizabeth Critser and the many staff and students of John Crister's laboratories for the years of productive collaboration, hospitality, and information interchange.
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Cited by (67)
How can mating systems inform future biobanking strategies? An illustration using two Indonesian bovids, banteng (Bos javanicus) and lowland anoa (Bubalus depressicornis)
2022, Animal Reproduction ScienceCitation Excerpt :The storage of genes in the form of cryopreserved spermatozoa permits the utilization of assisted reproductive technologies (ART) to integrate genes back into future populations (Watson, 2001). Basic understanding of ejaculate characteristics is critical to proper handling and successful cryopreservation of sperm (Comizzoli et al., 2012). It is well understood that differences in socio-sexual behavior and sperm competition are evolutionary forces influencing ejaculate characteristics such as volume, concentration, total sperm output, sperm motility and morphology.
Toxicity of cryoprotective agents to semen from two closely related snake species: The endangered Louisiana pinesnake (Pituophis ruthveni) and bullsnake (Pituophis cantenifer)
2021, CryobiologyCitation Excerpt :Cryopreservation of sperm was initially developed for use in the production of livestock, but cryotechnologies have successfully been transferred to some species of non-domesticated animals [5,3,8]. However, only a small number of wildlife species have been studied and huge gaps in taxonomic coverage remain, particularly for non-avian reptiles [9]. One in five species of reptile are currently facing extinction [10], and yet development of assisted reproductive technologies and specifically cryopreservation and banking of genetic material for reptiles has received little attention with limited success [5,3,11,12].
Reproductive technologies for the conservation of wildlife and endangered species
2020, Reproductive Technologies in Animals