Between 1970 and 2012, vertebrate abundance has declined by 58% with an average annual decline of 2%, calling for serious action to prevent a mass extinction and an irreversible loss of biodiversity. Cryobanks and cryopreservation have the potential to assist and improve ex situ and in situ conservation strategies by storing valuable genetic material. A great deal of studies concerning cryopreservation have been performed within the class Mammalia, although no systematic overview has previously been presented. The objective of this study is therefore to evaluate the status, pattern and future of cryopreservation within Mammalia. A strong disproportional distribution of studies in examined orders is displayed. For the majority of examined orders less than 10% of species has been examined. However, the cryopreservation of germplasm has in several cases been successful and resulted in successful applications of assisted reproductive techniques (ARTs). Various obstacles are associated with the development of cryopreservation protocols, and among them the most prominent is interspecific differences in cryotolerance. Extrapolation of protocols in closely related species is considered the most applicable procedure, and a future supplement to overcome this problem is the examination and comparison of cryobiological traits. Successful protocols have been developed for the vast majority of domesticated mammals, which gives incentive for the further extrapolation of protocols in threatened species.
INTRODUCTION
Biodiversity on earth is rapidly declining. The current rate of species extinction is unprecedented in human history and is already consistent with a mass extinction episode unmatched in the last 65 million years (Ceballos et al., 2015). Between 1970 and 2012, vertebrate abundance has declined by 58% with an average annual decline of 2% (WWF, 2016). The most common threat to declining populations is habitat loss and degradation (Rondinini et al., 2011; Heinrichs et al., 2016). This is evident for mammals living in terrestrial and freshwater habitats. However, the most common threat to marine mammals is overexploitation (WWF, 2016). Declines in population size reduce genetic diversity and increase the probability of inbreeding, leading to higher risk of extinction due to loss of adaptability and inbreeding depression (Wright et al., 2008; Hedrick and Garcia-Dorado, 2016). The aforementioned threats are consequences of anthropogenic activity and we are therefore already finding ourselves in the middle of the Anthropocene epoch (Waters et al., 2016).
Due to the rapid loss of mammalian species, there is a desperate need for conservation strategies. The ideal solution is provided by in situ conservation, e.g., habitat preservation, however predictions of future exploitation of land make this strategy seemingly impossible (WWF, 2016). A less favorable approach is ex situ conservation, e.g., captive breeding programs. However, ex situ conservation should primarily be used as a complement to in situ conservation (Kasso and Balakrishnan, 2013). As an interface between these strategies, cryopreservation of biological material offers the opportunity to preserve endangered animals (Holt and Pickard, 1999). By storing cryopreserved gametes, embryos, or somatic cells, genetic diversity from existing wild or captive populations can be preserved (Johnston and Lacy, 1995; Leon-Quinto et al., 2009). To accommodate this, genome resource bank initiatives such as the Frozen Ark Consortium ( https://www.frozenark.org) and Frozen Zoo ( http://institute.sandiegozoo.org/resources/frozen-zoo) Contribute to the preservation of genetic material. Recovery of genetic material requires different extraction methods, depending on which material is to be preserved. These include, but are not limited to, electroejaculation (EE), manual stimulation (MS) or use of an artificial vagina (AV) for sperm, and post-mortem recovery of reproductive organs, e.g., epididymis or ovaries, for sperm or oocyte collection. Furthermore, optimal freezing methods and cryomedia are necessary. The most commonly applied freezing method is storage of the material in liquid nitrogen at -196°C (Prieto et al., 2014). Chilling and freezing procedures often face problems with cold-shock stress, inflicting injuries and low quality rates in cryopreservation. To avoid this, the procedure often involves diluting the material in different media before chilling or freezing. The so-called extenders, such as egg yolk and antibiotics, are added to enrich and increase quality of the material. Cryoprotectants, such as glycerol, non-ionic sucrose, or lipoproteins, are added to prevent osmotic stress and intracellular ice formation (Fickel et al., 2007).
The susceptibility of biological material to injury during cryopreservation shows inter-specific differences, and optimal methods differ even between species that belong to the same phylogenetic group, e.g., order (Thurston et al., 2002). This requires examination of cryopreservation in virtually all (particularly endangered and unique) species to ensure the development of successful assisted reproductive techniques (ART). Aspects concerning cryopreservation of biological material have previously been outlined (Goodrowe et al., 2000; Mocé and Vicente, 2009; Rodger et al., 2009; Silva et al., 2016). However, a broad overview of Mammalia as a whole is lacking. The objective of this study is therefore to evaluate the status, pattern, and future of cryopreservation within the class Mammalia.
THE STATUS OF CRYOPRESERVATION WITHIN MAMMALIA
In this review, the state of the art and the application of cryopreservation techniques in Mammalian species is presented. Motility has been emphasized for characterization of sperm quality and the development rate for characterization of embryo quality due to the prevalence of these parameters. Furthermore, every attempted use of ARTs has been included, regardless of success. Emphasis has been put on the attempt to present the progress of cryopreservation within each order by including all examined species. However, to represent the current progress of cryotechniques, few well-examined species have been thoroughly described. This review reserves its position on the inclusion of every cryopreservation study conducted to date.
EMERGING PATTERNS OF CRYOPRESERVATION WITHIN MAMMALIA
A great deal of studies has been performed on species within Mammalia and has in several cases been successful and resulted in the successful application of ARTs (Table 1). Considering the vast number of species within this class, at least 2.7% of species has been examined, however further research is critically needed. For the majority of examined orders less than 10% of species has been examined, where some species have been subject to intense study and others have been subject to few. Further development of cryopreservation techniques could benefit from increased sharing of knowledge between researchers. This review serves as an overview of the class and as a preliminary foundation for the development of increased sharing of knowledge.
The most intensively examined species primarily consist of domesticated and captive wild animals. These protocols can be extrapolated to field conditions for wild animals to increase the genetic diversity of the current reserves of cryopreserved material within each species. This has already been accomplished in African elephant (Loxodonta Africana) (Hildebrandt et al., 2012) and Japanese black bear (Ursus thibetanus japanicus) (Okano et al., 2006). Furthermore, a large proportion of examined species consists of non-threatened animals. Extrapolation of protocols from non-threatened to threatened species is another promising procedure, which have already been observed from common squirrel monkey (Saimiri sciureus) to black-headed squirrel monkey (Saimiri vanzolinii) (Oliveira et al., 2016) and generic grey wolf (Canis lupus) to Mexican grey wolf (Canis lupus baileyi) (Zindl et al., 2006).
Extraction methods
Electroejaculation is the most prevalent extraction method of mammalian sperm, although this method has been observed to yield a lower sperm quality compared to other extraction methods. This has been observed in several species including domestic stallions (Equus caballus) (Cary et al., 2004) and grey wolf (Christensen et al., 2011). MS and AV in wild species require intensive animal training and conditioning, which has been successfully performed on captive whales (Robeck et al., 2010; Montano et al., 2012), monkeys (Takasu et al., 2016), and zebra (Crump and Crump, 1994). However, MS leads to other complications as seen in Asian elephant (Elephas maximus) where the mix of seminal plasma components can vary with each ejaculate and the risk of urine contamination is increased (Imrat et al., 2012). Due to poor results with the application of AV, MS, and EE in rhinos, a post-coital extraction method was applied as it includes the natural ejaculation of sperm. The small fluid volumes emitted by MS or EE may not consist of the appropriate mixture of seminal fluids needed to maintain sperm longevity and, ultimately, fertility (O'Brien and Roth, 2000b).
Another less invasive extraction method than EE is urethral catheterization which yielded superior motility figures for fresh sperm compared to EE in African lion (Panthera leo) (Lueders et al., 2012; Fernandez-Gonzales et al., 2015). The ejaculate volume was low, yet sperm motility was higher than sperm collected by EE and from cauda epididymes (Lueders et al., 2012). Therefore, urethral catheterization and post-coital extraction should be considered as alternative extraction methods in the future. In some species, female germplasm is extracted following euthanasia reducing the effective population size (Asada et al., 2000; Fujihira et al., 2006). However, oocytes can be collected surgically from live animals by follicular aspiration as seen in cynomolgus macaque (Macaca fascicularis) (Curnow et al., 2002) and vervet monkey (Clorocebus aethiops) (Sparman et al., 2007).
When extracting germplasm from both males and females, reproduction seasonality should be taken into account. Understanding the reproductive physiology of animals can contribute to optimizing extraction protocols (Santos et al., 2015). For example, tufted deer (Elaphodus cephalophus) sperm traits were observed to peak during autumn (Panyaboriban et al., 2016) and North American bison (Bison bison) sperm motility peaked during late summer and autumn (Krishnakumar et al., 2011). A similar tendency was indicated in Grant's zebra (Equus quagga burchelli), but was absent in the related Grevy's zebra (Equus grevyi) (Crump and Crump, 1994). Knowledge of the reproductive biology of each individual species is needed to enable optimal extraction.
Freezing methods
The most frequently applied freezing method of the examined species is the conventional slow-freezing method, although other freezing methods have shown promising results. An alternative freezing method is vitrification, which has shown superior results in cryopreservation of testicular tissue from house mouse (Yokonishi et al., 2014) and blastocysts from house mouse (Yeoman et al., 2001). Vitrification offers the advantages of low cost, ease of operation, and the avoidance of extracellular ice formation (Rall and Fahy, 1985; Yeoman et al., 2001; Liu et al., 2009; Comizzoli et al., 2012). Several improvements to the vitrification method have been developed, to more efficiently vitrify biological material. These consist of ultra-rapid vitrification methods using smaller volumes and higher freezing rates, such as the cryotop method (Kuwayama, 2007) used for Canis lupus baileyi (Czarny et al., 2009) and Sus scrofa domesticus (Sakagami et al., 2010) oocytes. Freeze-drying is another alternative freezing method. Freezing of sperm by both slow-freezing and freeze-drying showed no significant difference in fertilization rates in rhesus macaque (Sánchez-Partida et al., 2008) and golden hamster (Muneto and Horiuchi, 2011). However, the freeze-drying method is convenient due as it does not require storage in liquid nitrogen, which makes it less expensive and well suited for long-term preservation combined with easier shipping at ambient temperature (Ward et al., 2003; Sánchez-Partida et al., 2008). Furthermore, the estimation of blastocyst development was calculated to have no significant decrease after fertilization with freeze-dried sperm kept at -80°C for 100 years (Kawase et al., 2005). However, a downside of freeze-drying sperm is the immotility after rehydration, which excludes most ARTs, except intra cytoplasmic sperm injection (ICSI) (Sánchez-Partida et al., 2008; Muneto and Horiuchi, 2011).
An alternative to the conventional protocols for germplasm is the freezing of whole bodies. In Ogonuki et al. (2006), the successful fertilization of oocytes using ICSI was conducted with 15 year old sperm extracted from frozen whole bodies of house mouse kept at -20°C. This investigation provides an incentive for further experiments using frozen whole bodies, which could simplify future cryopreservation methods. This may also enable de-extinction, as ARTs could be performed using animals preserved in permafrost (Ogonuki et al., 2006).
Interspecific and intraspecific differences
The development of universal cryopreservation protocols is problematic as cryotolerance appears variate between species (Holt, 2000; Thurston et al., 2002). Interspecific variation was observed in closely related species after the application of identical cryopreservation protocols in rhinos (Portas et al., 2009) and squirrel monkeys (Oliveira et al., 2016). Moreover, sperm quality and cryotolerance have been observed to vary among individuals of the same species, which might relate to the genotype of the individual (Thurston et al., 2002; Gagliardi et al., 2008; Portas et al., 2009). This hypothesis is supported by the observation that cryotolerance did not differ within individual ejaculates from the same rhesus macaque (Macaca mulatta) (Gagliardi et al., 2008). Intraspecific sperm quality and cryotolerance have been found in Asian elephant. (Thongtip et al., 2004; Imrat et al., 2012) and white rhino (Ceratotherium simum) (Portas et al., 2009). Intraspecific differences are especially problematic, because not only must cryopreservation protocols be developed for the specific species, but it must also be tailored to suit the individual. If this is not taken into consideration, there is a possibility that cryopreservation protocols favor a specific genotype within each species. This is an unfavorable direction as it conflicts with the overall aim of cryobanking, which is to preserve as much genetic diversity as possible (Imrat et al., 2012).
Transport and disease transmission risks
Cryobanking has demonstrated useful applications in ex situ conservation programs. The transport of frozen material is a less comprehensive procedure compared to the transport of live animals (Hermes et al., 2013; Saragusty et al., 2015). The application of frozen material in ex situ conservation programs was investigated in African elephant (Hildebrandt et al., 2012; Hermes et al., 2013). Cryopreserved sperm from wild African elephants were shipped from South Africa to Europe, where artificial insemination was performed on a captive female with the purpose of introducing new genes to the captive population. One pregnancy was successfully established (Hermes et al., 2013) and a later study reports the birth of two calves and one more pregnancy (Saragusty et al., 2015). These results are of great importance, as transport-induced stress in elephants increases the risk of mortality (Clubb et al., 2008). Furthermore, frozen epididymis and testis from house mouse (Mus musculus) were successfully shipped from the United Kingdom to Japan (Ogonuki et al., 2006). These successful endeavors are unique, because health legislation restricts the transport of genetic material across borders (Hermes et al., 2013; Saragusty et al., 2015). For the purpose of transportation, donors have to be investigated for a variety of pathogens, which excludes a lot of already cryopreserved material. In the successful transport of African elephant sperm, it was therefore important that a thorough clinical examination was performed on each donor, and blood samples were collected for disease screening at the time of collection (Hermes et al., 2013). Despite these efforts, cryopreservation protocols of male and female germplasm are not performed under sterile conditions (Bielanski et al., 2003). Furthermore, liquid nitrogen is not sterile as pathogenic organisms can be conserved on immersion. During transportation, these pathogenic organisms may be released back into the environment as nitrogen vapor cools dry shippers (Grout and Morris, 2009). These precautions should be considered not only during transportation, but also at storage sites. Nitrogen vapor cools programmable freezers, which can release dormant pathogens to the surroundings (Grout and Morris, 2009) and contaminate samples, which are being prepared for cryopreservation or thawing. A problem arises when contaminated material is used in ARTs and thereby transferred to a live animal. However, it has been concluded that no direct evidence of disease transmission by transferred cryopreserved animal embryos have been seen in over 25 years (Bielanski, 2012).
Implementation of cryopreservation
Cryobanking can work as a supporting tool for ex situ and in situ conservation programs (Leon-Quinto et al., 2009). However, which species should be prioritized is a matter for continuing discussion. It can reasonably be argued that focus should be on Critically Endangered (CR) listed species, as they might be on the brink of extinction. Cryopreservation of these species could work as a supplement to in situ conservation with the purpose of reversing the loss of heterozygosity in susceptible populations by introducing more genetically diverse material into the gene pool (Wildt, 2000).
The cryopreservation of threatened species could nevertheless face some obstacles. Firstly, inaccessibility of biological material and expenses related to the collection of this could prove to be an obstacle due to the small population size. Secondly, an increase in the genetic diversity of small populations could be insufficient as the selective pressure could be overwhelmed by the effects of genetic drift, resulting in no adaptive reaction to selective pressure (Pertoldi et al., 2007).
Further implementation of cryopreservation in ex situ and in situ conservation strategies could be prioritized, as transportation of cryopreserved material is more favorable than the transportation of live animals (Hildebrandt et al., 2009; Hermes et al., 2013). Cryopreserved germplasm could play a key role in continuous gene flow between captive and wild populations of the same species, effectively increasing the genetic diversity of ex situ populations and preserving the genetic diversity of the species as a whole. Cryopreserved germplasm and captive bred individuals conceived using cryopreserved germplasm, could then be reintroduced into the wild, increasing the population size sufficiently and reducing the effects of genetic drift (Holt and Pickard, 1999; Hermes et al., 2013). Alternatively, the future priority of cryopreservation could lie in the selection of species, which have a sufficient population size.
The future of cryopreservation
In the future, efforts should be concentrated on the rather large gaps, particularly within the species-rich orders Rodentia and Chiroptera. This is especially relevant to Chiroptera spp., as to our knowledge no successful cryopreservation has been conducted within this order. Furthermore, focus is needed on the remaining Mammalia orders, which have not been examined at all.
The extraction of sperm post-coital or by urethral catheterization offers alternative extraction methods to the species, where prevalent extraction methods have been unsuccessful. These methods need further investigation in other species to acknowledge their encouraging successes.
Promising and alternative freezing methods include vitrification and freeze-drying. However, these methods have not been implemented to the same extent as conventional freezing methods and further studies are needed to determine their application to different species. Also, little information is available of the long-term storage of freeze-dried sperm from other species than laboratory house mouse. Further research is needed on the possibility of storing freeze-dried sperm at a higher temperature than -80°C for long periods of time (Kawase et al., 2005; Muneto and Horiuchi, 2011). Furthermore, estimations from Kawase et al. (2005) can be extrapolated to other cryopreservation protocols and thereby estimate the future success rates of freezing procedures.
Future experiments with the aim of simplifying freezing methods might also be an option. Both frozen and cooled sperm without cryoprotectants have shown successful results, which could give incentives to further protocols without cryoprotectants. Furthermore, the success of ICSI using sperm from a frozen whole mouse (Ogonuki et al., 2006) may encourage zoological gardens worldwide to store deceased animals in an ordinary deep freezer, when equipment for standard cryopreservation methods is unavailable.
Protocols developed for laboratory conditions serve as important groundwork for the development of protocols for field conditions. Protocol adjustments have to be made when extracting and handling biological material from wild populations, as field conditions rarely provide sufficient equipment for proper cryopreservation.
Examination of cryobiological traits prior to cryopreservation could be performed, as optimal protocols depend on these traits. Application of methods previously deemed successful for a particular set of traits could prove to be the optimal foundation when working with non-examined species. This has the potential of overcoming the difficulties associated with interspecific differences in cryotolerance (Comizzoli et al., 2012).
In the future, it will be necessary to exercise precaution against the risk of contamination; sterilization of liquid nitrogen by UV irradiation (Parmegiani et al., 2010) and disinfection of storage units (Bielanski, 2012) should therefore be implemented in cryopreservation protocols. Recommended methods and procedures to diminish the risk of disease transmission from post-thaw embryos and sperm to live animals is summarized in Bielanski (2012). Additionally, a thorough health examination of the donor animals could be considered to increase the chances of a later approval of transport across borders.
It has recently been suggested that the microbiome of animals may have implications for the successful reintroduction of animal species into the wild (Bahrndorff et al., 2016). Consequently, it could be argued that characterization of the microbiome and development of protocols for cryopreservation of symbiotic microorganisms should be considered, when developing cryopreservation protocol for a species of conservation interest.
To augment the overview of cryopreservation in Mammalia beyond the accomplishments of this review, the development of a peer-reviewed online database could be considered as it offers an easy and accessible overview, which provides incentives for scientists to continuously submit their work.
Table 1.
Species and subspecies included in this study in which the effect of cryopreservation on biological material has been examined. ‘Cryopreservation’ refers to the commonly used slow freezing method. Deviations around the mean are presented as standard error of mean, unless followed by * in which case it presents the standard deviation. ** Denotes range of values.
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CONCLUSION
The accelerating decline in biodiversity calls for the implementation of cryobanks and cryopreservation in conservation strategies, which have the potential to assist and improve ex situ and in situ conservation. Cryopreservation of germplasm from wild populations has been successfully implemented in ex situ breeding programs. In the class Mammalia, at least 2.7% of species has been subject to examination in which the extent of successful cryopreservation and ARTs vary. The species examined belong to less than half of all orders, and a strongly disproportionate distribution of studies across orders has been observed. The application of cryopreservation should be considered in the species-rich or non-examined orders. The cryopreservation of germplasm has in several cases been successful and resulted in successful applications of ARTs. Domesticated species and species relevant for general research have been extensively examined. Protocols for threatened species have successfully been extrapolated from these examinations, which gives incentives for future conservation of genetic diversity in threatened species. Interspecific and intraspecific differences complicate the extrapolation of protocols from non-threatened to threatened species. One approach to be considered as a supplement to the extrapolation of protocols in closely related species is the examination and comparison of cryobiological traits. For the implementation of new genes from wild populations in ex situ breeding programs, the contamination and disease transmission risks are to be taken seriously, before routine transportation of cryopreserved material can be utilized. For the future development of cryopreservation, the alternative techniques mentioned should be considered. The development of a peer-reviewed online database should be considered, as it would offer an easy and accessible overview.
