A review on reproductive biotechnologies for conservation of endangered mammalian species

This review describes the use of modern reproductive biotechnologies or assisted reproductive techniques (ART) including artificial insemination, embryo transfer/sexing, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, genome resource banking, and somatic cell nuclear transfer (cloning) in conservation programs for endangered mammalian species. Such biotechnologies allow more offspring to be obtained from selected parents to ensure genetic diversity and may reduce the interval between generations. However, the application of reproductive biotechnologies for endangered free-living mammals is rarer than for endangered domestic breeds. Progress in ART for non-domestic species will continue at a slow pace due to limited resources, but also because the management and conservation of endangered species is biologically quite complex. In practice, current reproductive biotechnologies are species-specific or inefficient for many endangered animals because of insufficient knowledge on basic reproduction like estrous cycle, seasonality, structural anatomy, gamete physiology and site for semen deposition or embryo transfer of non-domestic species.     

Reproductive physiology studies in zoological species: Concerns and strategies

A sophisticated understanding of reproductive physiology of domestic animals has evolved over only the past several decades. Related technical advances in this field have permitted propagation through artificial insemination and/or embryo transfer using fresh or frozen-thawed gametic or embryonic material. More recent studies in genetic engineering and embryo micromanipulation have generated additional potential for improved reproductive efficiency in the farm-animal industry. Although these procedures provide incentive for solving management problems and improving the endangered status of many zoological species, achieving these objectives will be neither simple nor immediate. Major obstacles to confront include the poor image of research, unavailability of research stocks, lack of financial support, scarcity of trained personnel, and the idea that new reproductive techniques offer a “quick fix” to the problem of inadequate reproduction in an endangered species. Zoological researchers should now concentrate on establishing basic reproductive relationships in species of interest. Comparative studies in domesticated animals will accelerate progress. The immediate goal should be the production of sound scientific data of publishable quality. Only then can artificial propagation be considered as a viable alternative to preserving wildlife species.     

Sperm cryopreservation in wild animals

Extinction of a species represents the loss of a resource evolved through eons of mutations and natural selection. Reproductive technologies, including artificial insemination, embryo transfer, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, and genome resource banking (GRB) have all been developed with the aim of solving existing problems and preserving genetic material for conservation purposes. Although protocols from domestic or non-threatened related species have been extrapolated to nondomestic and endangered species, usually these reproductive technologies are species-specific and inefficient in many nondomestic species because of insufficient knowledge on their basic reproduction biology and the need for species-specific customization. Since spermatozoa are usually more accessible and come in large numbers compared to oocytes and embryos, they are considered the primary cell type preserved in most emerging GRBs. For this purpose, semen from endangered species is currently cryopreserved to avail long-term storage. Due to the intractability of most exotic species, semen collection without chemical restraint is limited to only a handful of species and individuals. Viable epididymal spermatozoa can be obtained from dead or castrated animals, but this resource is limited. Electroejaculation, artificial vagina, abdominal massage, and/or transrectal, ultrasound-guided, massage of the accessory sex glands of living animals are viable alternative methods of semen collection. The ultimate goal is to adapt and optimize collection and cryopreservation protocols for each species, making it feasible, among other things, to collect gametes in the wild and introduce them into captive or isolated populations to increase genetic diversity. Recent advances in these fields have allowed the establishment of GRBs for many threatened species.     

Artificial insemination in marsupials

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.     

Sex and wildlife: the role of reproductive science in conservation

This essay explains the role of reproductive science, including what are termed reproductive technologies (i.e. artificial insemination, in vitro fertilization, embryo transfer, cloning), in conservation biology. Reproductive techniques (high- and low-tech) find their greatest application in understanding species uniqueness, adaptations and physiological mechanisms, not in the large-scale assisted breeding and the production of offspring. Models of how to use these tools to study reproductive fitness are emerging to help insure gene diversity and even propagate endangered species, but only after fundamental databases have been developed. Examples are provided of how non-invasive hormone metabolite monitoring, artificial insemination and genome resource banking are being used ex situ and in situ to understand wildlife biology. We predict that as the fundamental, multi-species database grows, so will the applied benefits for: (1) developing genome banks for insuring extant genetic diversity; (2) assessing the relationship of physiology, behaviour and environmental perturbations; (3) managing small populations; and (4) dealing with dilemmas ranging from contraception to skewed sex ratios to animal welfare. Most progress will be made in using these tools in systematic studies to solve the mystery of how thousands of unstudied species reproduce. Carried out appropriately, financial costs will be consistent with any approach for generating sound scientific knowledge.     

The breeding of captive wild species by artificial methods

The growing interest in many zoos, wildlife parks and game ranches in the captive breeding of wild species has brought about much interest and research in developing and testing artificial methods of breeding. Much of this work has been concentrated in endangered species. The author outlines the relevance and utilization of such techniques and reviews successful attempts in this area and elaborates on future potential ones. A brief review is given of successful artificial methods of breeding domestic species and how this may be applied to non-domestic ones. Areas that are covered are semen collection, semen freezing, induction of estrus, artificial insemination, embryo transfer including interspecies transfer and embryo surgery. It is acknowledged that it is highly unlikely that an artificial program could save a species from extinction but such methods could greatly assist a comprehensive natural breeding program.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.     

Artificial insemination in South American camelids and wild equids

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the "stallion effect", extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.     

Artificial fertilization for amphibian conservation: Current knowledge and future considerations

Amphibian populations in the wild are experiencing massive die-offs that have led to the extinction of an estimated 168 species in the last several decades. To address these declines, zoological institutions are playing an important role in establishing captive assurance colonies to protect species in imminent danger of extinction. Many of the threatened species recently placed into captivity are failing to reproduce before they expire, and maintaining founder populations is becoming a formidable challenge. Assisted reproductive technologies, such as hormone synchronization, gamete storage and artificial fertilization, are valuable tools for addressing reproductive failure of amphibians in captive facilities. Artificial fertilization has been commonly employed for over 60 years in several keystone laboratory species for basic studies in developmental biology and embryology. However, there are few instances of applied studies for the conservation of threatened or endangered amphibian species. In this review, we summarize valuable technological achievements in amphibian artificial fertilization, identify specific processes that need to be considered when developing artificial fertilization techniques for species conservation, and address future concerns that should be priorities for the next decade.     

Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm

Assisted reproductive techniques are needed urgently to facilitate the captive breeding of many New World primate species which are endangered in the wild and to assist the effective genetic management of small colonies. A protocol was devised for artificial insemination in the common marmoset, Callithrix jacchus, using ejaculated sperm obtained by vaginal washing after copulation. A double insemination protocol was employed, with the first insemination taking place the day before ovulation was expected to occur and the second 48 h later. All six females inseminated with fresh ejaculated sperm became pregnant, delivering a total of 16 offspring at term. The gestation lengths and litter sizes were not statistically different from those observed in pregnancies following natural mating. The insemination protocol was adapted for use with cryopreserved ejaculated sperm by including an additional insemination on the day of expected ovulation, to take into account differences in the capacitation time of frozen–thawed sperm compared to fresh sperm. Three out of six females inseminated according to this triple insemination schedule, conceived, although one female subsequently resorbed twin foetuses approximately 100 days later. The remaining two pregnant females delivered four babies at term, one singleton and one set of triplets. In the final group, six females were inseminated with low doses of cryopreserved epididymal sperm using the same triple insemination protocol used for frozen–thawed ejaculated sperm. One female conceived, delivering triplets.     

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.     

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.     

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.     

The application of artificial reproduction techniques to the propagation of selected endangered species

Artificial reproduction techniques were applied to individual animals not capable of natural reproduction. All individuals represented endangered species. An aged and debilitated Arabian oryx was treated with prostaglandin F2 α (PG) and pregnant mare serum gonadotropin (PMSG) prior to euthanasia. She responded to the treatment with mild superovulation. Four follicular oocytes were recovered, all of which subsequently matured in vitro and were frozen. A group of three Soemmering's gazelles, including a crippled male and a three-legged female, was evaluated for reproductive potential. The male was electro-ejaculated and determined to be producing normal sperm; semen was frozen. Estrus was induced and synchronized in the two females with PMSG and PG prior to the introduction of the male. Copulation attempts were unsuccessful. A second modified estrus induction attempt did not result in behavioral estrus in either female. The handicapped female was subsequently superovulated with PMSG before euthanasia. Four oocytes were recovered but failed to mature in vitro. Two Przewalski's horse mares are not able to mate naturally due to chronic laminitis. Preliminary studies have been undertaken to determine, through ultrasound examination, the estrous cycles and ovarian dynamics of the two horses, in preparation for artificial insemination and embryo transfer. Cycle length and variability, ovarian response to PG and rate of follicle growth have been determined to be similar to the domestic horse.     

Ultrasound examination and behavior scoring of captive broadnose sevengill sharks,Notorynchus cepedianus (Peron, 1807)

Serial ultrasound examination of four mature female sevengill sharks (Notorynchus cepedianus) was carried out over 18 months. Monitoring the reproductive cycle and development of follicles and fetuses in sharks in a noninvasive manner using this technique has not been reported previously. Sharks were caught out of the “Oceanarium” tank by divers using a specially made catch‐out bag, and brought to a holding area for examination. A behavior scoring system was used to monitor the impact of regular handling on the well‐being of the animals. Ultrasound showed the growth and regression of follicles in sevengill ovaries, and allowed an approximation of the reproductive stage of these sharks. Monitoring behavior at five time points during the procedure showed that regular handling of sharks for clinical studies could be done with minimal impact on animal welfare. The ability to follow reproductive events in elasmobranches using ultrasonography is an important step in the application of assisted reproductive technology in these species. Assisted reproductive technology, such as monitoring female reproductive cycles and artificial insemination, could potentially be used to maintain genetic diversity and compliment aquaria‐based breeding programs for endangered species such as the gray nurse shark (Carcharias taurus). Zoo Biol 26:383–395, 2007. © 2007 Wiley‐Liss, Inc.     

Successful embryo transfer in the silver fox (Vulpes vulpes)

Surgical embryo transfer in the silver fox was investigated as part of a larger project concerning the conservation of endangered canine species using modern artificial reproduction techniques with the farmed fox as a model. The animals were chosen on the basis of synchrony in natural oestrus. The timing of ovulation and artificial insemination was determined by measuring electrical resistance in the vagina. Twenty-nine embryos were flushed from eight humanely killed donor females and transferred surgically into the uteri of eight recipients. One recipient female gave birth to two male pups 47 days after the transfer of four expanded blastocysts and one embryo at the 16-cell stage derived from a donor female flushed 10 days after artificial insemination.     

Effect of dietary arginine and N-carbamoylglutamate supplementation on reproduction and gene expression of eNOS, VEGFA and PlGF1 in placenta in late pregnancy of sows

Artificial insemination has changed the small ruminant industry and has allowed increased genetic improvement, better control of reproduction and sexually transmitted diseases, dissemination of valuable genetics and preservation of the genetics of endangered breeds. Recent developments in this technology have focused on preserving the vitality/fertilizing capability of fresh and frozen spermatozoa by improving the composition of extenders, and by changing cooling/freezing protocols. The other main issue is the development of minimal invasive techniques for proper deposition of fresh or frozen semen. The paper discusses state of the art in methodology and technology currently used in small ruminant artificial insemination, as well as future perspectives after their wide application in these animal species.