Application of sperm sorting and associated reproductive technology for wildlife management and conservation

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.     

Short-term storage and cryopreservation of black grouse Tetrao tetrix and capercaillie T. urogallus semen

Assisted reproductive technologies can be an important part of programs directed for maintenance and protection of genetic variability. The objective of this study was to develop methods for liquid storage and cryopreservation of semen capercaillie and black grouse semen. Our results provide for the first time evidence for successful short-term storage and cryopreservation of capercaillie and black grouse semen using criteria of sperm motility characteristics as quality indices. Sperm motility could be protected up to 48 h liquid storage; however, 24-h storage should be preferable. Cryopreservation secured 40–60% post-thaw motility (as compared with control), both for freshly collected and 24-h-stored semen. In conclusion, a procedure for short-term and cryopreservation of capercaillie and black grouse semen are now available. These assisted reproductive technologies can be implemented into captive breeding programs for these species.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Artificial insemination technology for the emu--improving sperm survival

For the emu, where monogamous mating is normal, artificial insemination (AI) promises much faster genetic improvement and a considerable reduction in production costs by reducing the number of male birds needed for mating. Semen collection is now a routine procedure so the next step is to develop successful protocols for sperm storage. In this paper, we briefly overview our recent progress on the development of protocols for liquid storage and cryopreservation of emu spermatozoa. We have shown that emu semen can be stored at 10 °C for up to 48 h with a minimal loss of viability, and that cryopreservation with dimethylacetamide (DMA) as a cryoprotectant is feasible because we have observed no adverse effects of this cryoprotectant on the emu sperm membrane integrity, morphology and motility. We now need to establish the predictability of the various tests in vivo, but the proportions of live normal and motile sperm with good egg membrane penetration potential suggest that acceptable numbers of competent sperm are preserved and that this will be sufficient for AI.     

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.     

Reproduction and larval rearing of amphibians

Reproduction technologies for amphibians are increasingly used for the in vitro treatment of ovulation, spermiation, oocytes, eggs, sperm, and larvae. Recent advances in these reproduction technologies have been driven by (1) difficulties with achieving reliable reproduction of threatened species in captive breeding programs, (2) the need for the efficient reproduction of laboratory model species, and (3) the cost of maintaining increasing numbers of amphibian gene lines for both research and conservation. Many amphibians are particularly well suited to the use of reproduction technologies due to external fertilization and development. However, due to limitations in our knowledge of reproductive mechanisms, it is still necessary to reproduce many species in captivity by the simulation of natural reproductive cues. Recent advances in reproduction technologies for amphibians include improved hormonal induction of oocytes and sperm, storage of sperm and oocytes, artificial fertilization, and high-density rearing of larvae to metamorphosis. The storage of sperm in particular can both increase the security and reduce the cost of maintaining genetic diversity. It is possible to cryopreserve sperm for millennia, or store it unfrozen for weeks in refrigerators. The storage of sperm can enable multiple parentages of individual females' clutches of eggs and reduce the need to transport animals. Cryopreserved sperm can maintain the gene pool indefinitely, reduce the optimum number of males in captive breeding programs, and usher in new generations of Xenopus spp. germ lines for research. Improved in vitro fertilization using genetic diversity from stored sperm means that investigators need the oocytes from only a few females to produce genetically diverse progeny. In both research and captive breeding programs, it is necessary to provide suitable conditions for the rearing of large numbers of a diverse range of species. Compared with traditional systems, the raising of larvae at high densities has the potential to produce these large numbers of larvae in smaller spaces and to reduce costs.     

Methods for Cryopreservation of Guinea Fowl Sperm

Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet) and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide). The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining). Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols (based on in vitro assessment of post-thaw semen quality) were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively). During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.     

The importance and potential of artificial insemination in CANDES (companion animals, non-domestic, endangered species)

Artificial insemination (AI) is the least invasive assisted reproductive technology, and is therefore of great interest to breeders of companion animals, non-domestic, and endangered species (CANDES). This most fundamental artificial breeding technique circumvents physical or behavioral impediments to natural mating and provides the means for genetic exchange between populations without transfer of live animals. In addition, because oocytes grow, mature and are fertilized in vivo and embryos are not subjected to in vitro culture conditions, AI eliminates the epigenetic effects on the female gamete that are inherent in more invasive assisted reproductive technologies. Although the management of CANDES differs significantly from current livestock husbandry practices, the cattle industry is a powerful example of the potential for AI to enhance the genetic health and sustainability of animal populations. Ultimately, successful AI requires sperm of adequate quality and quantity, oocytes that have attained nuclear maturation and cytoplasmic competence, operational gamete transport systems, accurate timing, and proper placement of sperm in the female reproductive tract. Increased understanding of semen collection, evaluation and preservation techniques, estrus synchronization and superovulation, estrus and ovulation detection, and insemination instrumentation is needed for each CANDES before AI success rates will approach those of the livestock industry. Concentrated, collaborative research in these areas must be encouraged among private breeders, universities and zoological institutions to realize the full potential of AI in the management of CANDES.     

Application of Testis Germ Cell Transplantation in Breeding Systems of Food Producing Species: A Review

A major benefit of advanced reproduction technologies (ART) in animal breeding is the ability to produce more progeny per individual parent. This is particularly useful with animals of high genetic merit. Testis germ cell transplantation (TGCT) is emerging as a novel reproductive technology with application in animal breeding systems, including the potential for use as an alternative to artificial insemination (AI), an alternative to transgenesis, part of an approach to reducing generation intervals, or an approach toward development of interspecies hybrids. There is one major difference in TGCT between rodents and some other species associated with immunotolerance in heterologous transplantation. In particular, livestock and aquatic species do not require an immunesuppression procedure to allow donor cell survival in recipient testis. Testicular stem cells from a genetically elite individual transplanted into others can develop and produce a surrogate male—an animal that produces the functional sperm of the original individual.

Spermatozoa produced from testis stem cells are the only cells in the body of males that can transmit genetic information to the offspring. The isolation and genetic manipulation of testis stem cells prior to transplantation has been shown to create transgenic animals. However, the current success rate of the transplantation procedure in livestock and aquatic species is low, with a corresponding small proportion of donor spermatozoa in the recipient's semen. The propagation of donor cells in culture and preparation of recipient animals are the two main factors that limit the commercial application of this technique. The current paper reviews and compares recent progress and examines the difficulties of TGCT in both livestock and aquatic species, thereby providing new insights into the application of TGCT in food producing animals.     

Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 10⁶ cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females'' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 10⁶ motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.     

Assisted reproductive technology in canid species

Assisted reproductive technologies in dogs began as early as the 18th century. The first scientifically recorded artificial insemination (AI) was performed in Italy by Spallanzani and lead to the birth of three pups. Progress in the area was slow, and subsequent development included AI equipment and methods for short-term preservation of fresh, and later, for frozen semen which led to the world's first litter produced from frozen semen in 1969. Improvement of freezing methods and AI equipment from 1970 onwards has rendered AI useful as a breeding technique for dogs. In parallel, AI in foxes was developed in Scandinavia in the early 1980's; this resulted in the economically valuable crossbreeding of silver and blue foxes for the production of bluefrost pelts. Unfortunately, due to the particular physiology of the canine female, progress in other artificial breeding techniques has lagged behind. Only in the last few years have these techniques been successfully applied in basic research to study oocyte maturation, in vitro fertilization, embryo cryopreservation and embryo transfer in canids.     

Cryopreservation of poultry sperm: the enigma of glycerol.

This review summarizes recent data for cryopreservation of poultry sperm and data establishing the contraceptive effect of glycerol. Successful cryopreservation protocols for bovine sperm are compared to the requirements for rooster sperm, with emphasis on glycerol-induced alterations in avian reproductive systems. It has been shown that molar concentrations of glycerol can affect (a) physical features of the cytoplasm (cytoplasmic organization and viscosity), (b) permeability and stability of the membrane bilayer(s), and (c) noncovalent attachment of proteins to the sperm surface. Perturbing effects of glycerol on sperm metabolism and the essentiality of maintaining bioenergetic balance during the temperature changes associated with any cryopreservation protocol are discussed. Emphasis is placed on the processes in avian reproduction that may be altered by interactions with glycerol. Finally, we discuss the potential value of using available genetic models (lines of roosters differing in the capacity of their sperm to survive a freeze-thaw cycle) to clarify and overcome damage to poultry sperm induced by cryopreservation.     

Birth of offspring following artificial insemination in the common marmoset,Callithrix jacchus

The objective of the study was to develop a method for artificial insemination (AI) in the common marmoset, a New World primate species. For AI to be successful, sperm must be deposited at an appropriate site and time in the female reproductive tract, details of which are currently not available for Callitrichid species. Epididymal sperm were deposited in the cervix of 18 marmoset monkeys (Callithrix jacchus) around the time of expected ovulation using either 3, 2, or 1 inseminations. Six out of 18 females conceived, resulting in the first reported births following AI in this species. These pregnancies show that the presence of coagulum in the vagina and the stimulus of the female reproductive tract by natural mating are not essential for effective sperm transport in this species. Although 3 different timing regimes for sperm deposition relative to ovulation were employed, no protocol was demonstrably better than the others in terms of number of conceptions. The proportions of motile, live, and morphologically normal sperm in the suspensions used for AI were comparable with published values for ejaculates from fertile male macaques. These preliminary results indicate that births are possible following AI in marmosets: the technique could be used to aid effective genetic management of the species and possibly to facilitate captive breeding of endangered Callitrichids. Am J Primatol 41:37–43, 1997. © 1997 Wiley-Liss, Inc.     

Factors affecting commercial application of embryo technologies in New Zealand: a modelling approach

New reproductive technologies include sexed sperm and embryo-based technologies. The technology of sperm sexing, for various reasons, is not available in New Zealand and its use has not been modelled. Embryo technologies are however already in use on a limited scale and various scenarios for their use in both the dairy and beef industries in New Zealand have been modelled. This review briefly discusses the various technologies available and some of their potential strengths and weaknesses. In the dairy industry, modelling has been used to simulate the production of breeding bulls for large breeding companies and the production of replacement heifers in dairy herds. For the beef industry, similar modelling has been carried out to determine the opportunities for more efficient beef production.All the models confirmed that at current levels of performance, embryo-based reproductive technologies are usually not profitable in New Zealand except in niche market situations where the returns from the resulting offspring are significantly greater than can be obtained from natural mating or artificial insemination (AI) reproduction systems. This is confirmed by the low uptake of these technologies in this country to date. Even if performance lifts to levels similar to AI, profitability is likely to occur only if the costs of pregnancies to embryo-based reproductive technologies can occur at prices less than two to four times greater than AI or natural mating. This break-even requirement depends on the returns that can be achieved and the advantages that can be captured by the technology over and above those available from AI or natural mating. Two new uses for reproductive technologies in dairy cattle could be the proliferation of novel or rare genotypes from gene discovery programs and improving the female reproductive rate for optimal marker assisted selection. In both these uses the technology is not at present competing with AI or natural mating. The challenge exists therefore for the biological scientists to satisfy these requirements, coupled with the ethical and human factors involved in the introduction of any new technology.Potential end users of the technologies have been surveyed. They are quite positive about the technologies provided they can use them profitably and are keen to obtain more information about them.     

Advances in the cryopreservation of salmonid semen and suitability for a production-scale artificial fertilization program

During the past decade, semen cryopreservation techniques in salmonid fish have progressed to where fertilization rates obtained with preserved sperm approach those produced with fresh sperm. Recent advances in the collection, dilution and storage of salmonid spermatozoa are reviewed. The problems encountered during the development and examination of artificial insemination programs in warm-blooded animals are contrasted with those encountered in fish. This paper discusses the advantages of artificial fertilization, and the manner in which advances in cryopreservation techniques could aid in the development and expansion of concentrated, production-scale artificial fertilization programs in salmonid fish.     

Genome-wide association studies for sperm traits in Assaf sheep breed

Sperm quality traits routinely collected by artificial insemination (AI) center for rams progeny test are related with the capacity to produce sperm doses for AI and, in more or less grade, with males' fertility. Low-quality ejaculates are unuseful to perform AI sperm doses, which suppose high economic loses for the AI center. Moreover, sperm quality traits have low heritability values which make traditional genetic selection little efficient to its improvement. In this work, a genome-wide association study (GWAS) was conducted by using sperm quality traits data and 50 K Affymetrix custom chip genotypes of 429 rams of Assaf breed from OVIGEN AI centre. Furthermore, 47 of these rams were also genotyped with the Illumina HD Ovine BeadChip, and therefore HD genotypes were imputed for all rams with phenotype data. Previous to the GWAS, a linear regression model was fitted including sperm traits as dependent variables; the flock of origin, date of sperm collection, and jump number as fixed effects; rams age at collection in months as covariate; and ram permanent effect as random. Pseudo-phenotypes obtained from this model were used as input for GWAS. Associations at the chromosome-wise level (FDR 10%) of 76 single-nucleotide polymorphisms (SNPs) in 4 chromosomes for ejaculate concentration (CON), 20 SNPs in 3 chromosomes for ejaculate volume (VOL), 32 SNPs in 1 chromosome for ejaculate number of spermatozoa (SPZ), and 23 SNPs for spermatozoa mass motility (MOT) in 17 chromosomes were found. Only SNPs associated with MOT overcame the genome-wide significance level. Some candidate genes for sperm traits variability were SLC9C1 (OAR1), TSN (OAR2), and FUT10 (OAR26) for MOT;. DOCK2, CPLANE1, SPEF2, and RAI14 (OAR16) for CON; SCAPER and PSMA4 (OAR18) for VOL; and PARM1 and LOC101110593 (OAR6) for SPZ. SNPs associated with sperm traits were not found to be correlated with milk production genetic variation; however, the high frequencies of some SNPs with negative effect over sperm traits found in animals at the top milk yield estimated breeding values (EBVs) ranking would allow to exert some selective presure to improve rams sperm performances. Effects and frequencies of some of the SNPs detected over sperm quality traits make these variants good candidates to be used in marker-assisted selection to improve sperm characteristics of Assaf rams and AI center efficiency to produce sperm doses.     

Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.     

Effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds

The interest to develop assisted reproductive technologies and cryobanking for farm animal genetic resource conservation has recently increased. However, cryopreservation for ex-situ management of genetic diversity sometimes is not routinely feasible, owing to the lack of facilities (AI centres, laboratories) and expertise near the local breed farming area. In these cases, epididymal sperm obtained from slaughtered or castrated animals, associated with the possibility of managing rather long periods between animal death, sperm recovery and freezing, would increase the opportunities to create semen storages. This investigation addresses the pre-freeze/post-thaw quality of goat epididymal sperm as a function of testicle storage temperature (environment or +5°C) and time elapsed between animal’s death and sperm recovery (0, 24, 48, 72 h) to establish the optimal protocols for the recovery and cryopreservation of epididymal sperm in this species. Testicles of 50 mature bucks collected at the abattoir were divided in two groups: half of the testicles (n=50) were transported to the laboratory at environment temperature (E), whereas the remaining half (n=50) at a refrigeration temperature (R) of +5°C. In the two groups (E) and (R), one testicle from each pair was processed after slaughter forming the time 0 groups (0E and 0R). The contralateral testicle was processed after 24, 48 or 72 h of storage, at the corresponding temperature. Sperm motility and kinetic parameters, viability and morphology were assessed in pre-freeze and post-thaw samples. Until 48 h postmortem, both E and R temperatures are able to maintain good pre-freeze epididymal sperm quality. After 48 h postmortem, R temperature is fundamental to reduce epididymal sperm quality decay in pre-freeze samples. Moreover, testicle refrigeration also has a positive impact on post-thaw samples, allowing a lower decline through time considering total motility, kinetics parameters, sperm viability and sperm abnormalities. Therefore, when sperm cryopreservation is not immediately practicable, goat testicles should be transported and stored at 5°C up to a maximum of 48 h postmortem to ensure an acceptable sperm quality.     

Potential contribution of cryopreserved germ plasm to the preservation of genetic diversity and conservation of endangered species in captivity.

Demographic and genetic objectives of captive propagation programs for endangered species focus on establishing demographically secure populations that maintain adequate levels of genetic diversity. Long-term storage and utilization of cryopreserved germ plasm could extend the population's generation length and allow higher levels of genetic variation to be maintained in smaller populations. Since fewer breeding animals would be needed, more species would be "rescued" from extinction using the cage facilities currently available at existing institutions. Doubling generation lengths for callitrichid primates through use of cryopreservation could almost triple the number of species that could be rescued in world zoos. Additionally, long-term cryopreservation would allow for a third population, that of the frozen zoo. Three-way exchange of germ from germ plasm banks to captive and wild populations would increase genetic diversity at reduced risk and expense. Advances in reproductive technology and better understanding of the reproductive physiology of these animal populations are necessary to permit routine application of artificial insemination and embryo transfer using frozen-stored germ plasm.     

Advancements in cryopreservation of domestic animal embryos.

The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced (IVP) or micromanipulated embryos is less than that of in vivo-derived embryos. This review outlines recent efforts in livestock embryo cryopreservation. In the near future, use of preserved embryos could be a routine breeding alternative for all livestock producers providing 1) preservation methods for maternal germplasm, 2) global genetic transport, 3) increased selection pressure within herds, 4) breeding line regeneration or proliferation, and 5) methodology for genetic rescue.