Embryo Transfer

Embryo transfer is a technique by which fertilized ova and early embryos are recovered from the reproductive tract of a donor female, the genetic mother, and are transferred into the tract of a recipient female, the foster mother, in whom the embryos develop into full-term fetuses and live young. The first successful transfer of mammalian embryos was performed in the rabbit by Heape in 1891. His observations stimulated relatively little further research until about 1950. Since then, there has been an explosion of research in this area. Numerous published reviews and textbooks describe both the methods and the fundamental principles on which the technique of embryo transfer rests [see reviews by Betteridge (1977), Mapletoft (1984), Seidel (1981), and Sreenan (1983), and texts by Adams (1982), Cole and Cupps (1977), and Daniel (1978)]. Although the specifics of the methods depend upon the species used, the general principles are the same whether performed in laboratory animals such as mice and rabbits, in large domestic animals such as horses and cattle, or in wild species such as baboons and antelope.

Embryo transfer in two species, mice and cattle, has become absolutely routine. It is not an exaggeration to state that tens of thousands of living mice and cattle have been produced by embryo transfer. In general terms, the transfer of embryos of other species has been modeled on techniques devised for these two species. Although there are fewer live young, probably thousands of live rabbits, pigs, and horses have been produced by embryo transfer. The application of embryo transfer to wild species is a relatively recent event. Its history can be highlighted as follows:

Synchronization of the donor and recipient animals in embryo transfer can be accomplished through precisely timed injections of prostaglandins, such as Lutalyse and Estrumate. These hormone analogs serve to stimulate the ovaries to begin a new cycle. Superovulation of the donor is accomplished through the injection of fertility hormones such as follicle-stimulating hormone (FSH). Superovulation has been fairly successful with the ungulates, but optimal drugs and dosages have yet to be refined for most other species. As many as 31 embryos have been collected from one FSH-stimulated eland cow (Dresser, 1983). On the other hand, fertility drugs seem to have little or no effect on the equids (such as zebras and Przewalski's horse) (Hearn and Summers, 1986). Clearly, the hormone regimen that produces the optimal superovulation response within a given species seems to be fairly individualized and much work is needed in this particular area.

In felines, the superovulation of donors and the synchronization of donors and recipients is complicated by the fact that most cats are induced or reflex ovulators, meaning that they ordinarily do not ovulate without the stimulation of copulation. Human chorionic gonadotropin (HCG) has been administered to domestic cats to cause ovulation to occur (sometimes in conjunction with stimulation by a vasectomized male) (Bowen, 1977). Researchers in several institutions have been working on embryo transfer in domestic cats with limited success (Dresser et al., 1987; Goodrowe et al., 1986; Kraemer et al., 1979), but little work has been done thus far on wild cats (Bowen et al., 1982; Reed et al., 1981). It is hoped that domestic cats may be able to serve as surrogates for incubating embryos from small endangered wild cats such as the black-footed cat (Felis nigripes). A great deal more work needs to be done in this area to determine the best regimens for stimulating cat's ovaries to produce more than the usual number of follicles and to cause the ovulation of these follicles and, thus, to obtain embryos.

Figure

Gaur calf born to Holstein surrogate on 5/25/87 at Kings Island Wild Animal Habitat. This was the first nonsurgical interspecies embryo transfer between an endangered species and a domestic species. Photo by Dr. Betsy L. Dresser.

The development of embryo transfer techniques is essential if genetic diversity within captive populations is to be maximized. The ability to introduce new bloodlines into a captive population through the transfer of nonlocal embryos into surrogates would be far preferable to the transport of adult animals for breeding purposes, or to the depletion of wild herds to add new breeding stock to captive populations. In addition, it is a goal of many zoo researchers to develop interspecies embryo transfers to the point at which embryos can be collected from endangered species and transferred to surrogates of a more common species, thereby greatly increasing the reproductive potential of the donor species.

Other important benefits result from embryo transfer. For example, it has been found that disease transmission between different populations can be dramatically reduced by embryo transfer (see Hare, 1985, for review). This happens because the intact embryo collected from a diseased mother is almost always free of the microbial or viral disease agent and does not transmit the disease to the foster mother. Alternatively, the surrogate mother may confer passive immunity to her offspring that develop from transferred embryos. This may occur either through the placental blood supply or via the colostrum, the first milk. That is, it has been found that cattle of a given breed may quickly succumb to local diseases when imported into a new location. However, live calves produced by transfer of embryos from a foreign breed into another (native or endemic) domestic breed will usually be as disease-resistant as this domestic breed. This accident of biology should have important consequences for the transplant of both domestic and wild species from one location to another.

Interspecies embryo transfer has enjoyed limited success in wild animals, but much more research needs to be done in this area. Intergeneric, e.g., eland to cow (Dresser et al., 1982a) or water buffalo to cow (Drost, 1983), as opposed to interspecies, e.g., tiger to lion (Reed et al., 1981), embryo transfers have never been successful. It seems that most embryos can develop to the early blastocyst stage in the oviducts of unrelated species (Daniel, 1981), but further development requires a much closer relationship between donor and recipient and similarity in time and type of implantation, placenta formation, rate of ovum transport, length of gestation, birth weight, and both neonatal and maternal postpartum behavior. Several techniques have been tried to help overcome the surrogate mother's immune response and prevent the rejection of the foreign embryo in an interspecies embryo transfer. Recently, a domestic horse mare gave birth to a donkey foal after she had been injected with donkey white blood cells (Antczak, 1985). Prior to this treatment, all other donkey embryo transfers into domestic horses had failed. It is not yet clear why the procedure was successful.

Although various attempts have been made to collect embryos from nonhuman primates at various zoos and primate centers over the years, very few of these procedures have been reported in the scientific literature. For a review of the existing literature on superovulation and ova collection attempts in the more common nonhuman primates, see Bavister et al. (1985), Clayton and Kuehl (1984), Hodgen (1983), and Kraemer et al. (1979).

There has been dramatic progress in embryo micromanipulation in little over 6 years, especially as it applies to domestic animals. The research of Willadsen (1979, 1980) and Willadsen et al. (1981) is most notable in this regard.

In the most important application of micromanipulation to exotic animals, an embryo is microsurgically bisected into two or more pieces, thereby producing genetically identical twins or triplets. This technique, although not yet successful with exotic species, could help to quickly increase the numbers of endangered or rare animals.

Another result of micromanipulation has been the production of chimeras, which are embryos that are a product of combining two embryos at a relatively early stage of development. It is possible to prevent the surrogate uterus from recognizing the foreign embryo by combining different blastomeres. The younger cells tend to form the trophoblast that gives rise to the placenta, and the older cells tend to form the inner cell mass that will form the fetus. Chimeric embryos can be constructed so that the cells that constitute the trophoblast belong to the surrogate species and the cells of the inner cell mass belong to the donor species. A reupholstered embryo such as this can effectively trick the surrogate's uterus into thinking it is carrying a native embryo (Vietmeyer, 1984).

Interspecies embryo transfer and inner cell mass transfer should not be confused with hybridization of species. Offspring contain only genetic material of the original species. Many people now recognize that the method of chimera production might be utilized to rescue endangered species. It might be possible to construct chimeras consisting of an embryo from an endangered species plus an embryo of a common, but related species. The common species might then carry the fetus of the endangered species to term.

In summary, then, embryo transfer has become a widely used technique to produce live young animals, especially of domestic species. Although it is as yet still a novel procedure in wild species, continued research will inevitably make transfer of wild animal embryos as successful as the transfer of domestic animal embryos.

Cryopreservation of Embryos

The first successful freezing of mammalian embryos was reported in 1972 (Whittingham et al., 1972; Wilmut, 1972). Since those reports, more than 300 articles and 100 abstracts have been published on this one subject. There have also been three full meetings (Ciba Foundation, 1977; Muhlbock, 1976; Zeilmaker, 1981) and numerous symposia devoted exclusively to the freezing of mammalian embryos. Numerous reviews have also been published (Lehn-Jensen, 1981; Leibo, 1977, 1981; Maurer, 1978; Rall et al., 1982; Renard, 1982).

Freezing of mouse, rabbit, and bovine embryos has now become a routine procedure. Altogether, embryos of 11 mammalian species have now been successfully frozen. Again, success means that live young have been born from frozen embryos. To date, the species that have been successfully preserved include the mouse (Whittingham et al., 1972), cow (Wilmut and Rowson, 1973), rabbit (Bank and Maurer, 1974), sheep (Whittingham et al., 1972), rat (Whittingham, 1975), goat (Bilton and Moore, 1976), horse (Yamamoto et al., 1982), human (Trounson and Mohr, 1983), baboon (Pope et al., 1984), antelope (Dresser et al., 1984a), and cat (Dresser et al., 1987).

The freezing of mouse embryos has become so routine that banks of tens of hundreds of thousands of mouse embryos have been frozen to preserve valuable genetic stocks for extended times. For some species, most notably the mouse, rabbit, and cow, preservation by freezing has reached such a high level of sophistication and reliability that approximately 80 to 90% of frozen embryos will develop in vitro when thawed and cultured. Moreover, the procedures to freeze embryos have become increasingly simplified. Regardless of the methods used, it can be reasonably estimated that thousands, if not tens of thousands, of live young of domestic species have been produced from frozen-thawed embryos.

Again, because of extremely limited experimental material, only a few attempts have been made to transfer previously frozen wild animal embryos into recipients.

The procedure has yielded some limited success (Cherfas, 1984; Dresser et al., 1984a; Hearn and Summers, 1986; Kramer et al., 1983).

Cryopreservation will undoubtedly prove to be an important adjunct to reproductive research in nondomestic animals. Geneticists Thomas Foote and Ulysses Seal have determined that a population of 250 properly managed animals of a particular species can theoretically preserve 95% of the original genetic diversity of the group after 50 generations (400 years) (Myers, 1984). The world's zoos, however, have limited facilities and often cannot accommodate large numbers of animals of each species they maintain. Success in the area of cryopreservation will allow zoo professionals to overcome the limited space in zoos and wild animal preserves by maintaining the bulk of the desired genetic diversity in liquid nitrogen freezers. Embryos containing new bloodlines could be recovered in the wild and brought back to zoos in the frozen state to improve the bloodlines of captive populations without depleting the wild herds. U.S. government restrictions currently prohibit the importation of embryos from other countries, but scientists have been actively lobbying for a change in these restrictions. It appears that a change may be possible in the future.

Artificial Insemination

Artificial insemination is the introduction of semen into the vagina or cervix by artificial means. This procedure was supposedly used by the Arabs in ancient times, but the first documented success in the modern world occurred in 1784 with the artificial insemination of a dog (Betteridge, 1981). In the 1930s, artificial insemination of livestock was used extensively in Russia. Arthur Walton demonstrated its potential as an effective method to transport genes in the 1920s and 1930s by shipping fresh rabbit, sheep, and bull semen from England to other European countries (Betteridge, 1981). The ability to successfully freeze semen resulted from the discovery of the cryoprotective action of glycerol by Polge, Smith, and Parkes in 1949 (Betteridge, 1981). Artificial insemination is very common in the agricultural industry today. Foote (1981) estimated that close to 90 million head of cattle were produced worldwide in 1977 by artificial insemination with previously frozen semen samples. Artificial insemination has had limited success in wild animals thus far, especially with certain species of mammals and birds. Success has been attained for the following species:

Much more semen has been collected from wild species than has actually been evaluated and used. The first successful artificial insemination of a wild species with previously frozen semen occurred in 1973 with the wolf (Seager, 1981). This was followed by the successful insemination of a gorilla (Douglass and Gould, 1981). There has been a great deal of time and effort spent trying to artificially inseminate wild-caught felidae (cats); most attempts have resulted in failure (Dresser et al., 1982b). The London Zoo finally produced a puma in 1980 through surgical artificial insemination with a fresh semen sample (Moore et al., 1981). This was followed in 1981 by a successful nonsurgical artificial insemination of a Persian leopard with fresh semen at the Cincinnati Zoo (Dresser et al., 1982b).

There are three methods of semen collection: manual stimulation of the male reproductive tract, use of an artificial vagina, and electroejaculation (Cherfas, 1984). Electroejaculation was invented by two French workers, Jonet and Cassou, and is by far the most common collection mode for wild animals. Electroejaculation works by inserting a lubricated probe into the rectum of an anesthetized animal. This conveys mild pulsating electrical stimuli to the nerves of the reproductive tract, resulting in ejaculation. There is some question about the fertility of sperm collected through electroejaculation, but there is also a question about how viable a semen sample must be to be effective. For example, the successful gorilla insemination at the Memphis Zoo in 1981 was accomplished with a previously frozen sample whose motility was 10% and judged to be poor at the time of insemination (Douglass and Gould, 1981).

A great many problems are associated with artificial insemination as it applies to wild animals. First of all, it necessitates the use of anesthesia, which is always a risk, for both semen collection from the male and insemination of the female. In addition, as mentioned above for electroejaculation, the fertility of semen obtained from artificial collection techniques is sometimes questionable when compared to that produced in a natural ejaculation. Sperm usually begin to die as soon as they are collected. Even if a fresh sample is used for insemination, it is likely to have undergone a certain amount of sperm loss.

Semen Cryopreservation

The freezing process is somewhat detrimental to sperm, and it is very unlikely for sperm to come out of a thaw as motile as they were going into the freeze (Cochran et al., 1985). Much work needs to be done in the area of semen cryopreservation for exotic animals. To date, sperm from at least 200 different species has been frozen, but very little of it has actually been thawed and tested (Seager, 1981). The ultimate test is production of offspring. From semen that has been tested, cryobiologists have found that sperm from each species needs to be extended and frozen under slightly different conditions to produce the optimal results.

Extenders used to preserve the collected semen basically consist of a buffered solution that contains a cryoprotectant (e.g., glycerol), antibiotics, and either egg yolk or milk. Many variations have been tried in the basic recipe for semen extender. The agricultural industry has found that the optimal extender for a given species seems to be very species-specific and that is turning out to be true with wild animals also. Perhaps some clues for semen preservation can be found in the natural world from studying certain female reptiles who have the potential for keeping sperm viable within their bodies for up to 6 years after mating (Cherfas, 1984).

Other problems with artificial insemination include difficulty in predicting the optimal time for inseminating the female and the fact that artificial insemination cannot occur as frequently as a female would have been inseminated naturally for the duration of her estrous cycle.

On the positive side, artificial insemination can be a great boon toward improving the genetic diversity of a captive population of animals. As with embryo transfer, the risk and expense of transporting semen is far less than that of transporting a male animal for breeding purposes. Artificial insemination could also be used to overcome quarantine restrictions and the risks of disease. Often, the strict agricultural legislation has made transport of zoo animals more difficult and costly than is perhaps necessary.

Future Prospects

Since preservation of genetic material from a species is one of the keys to ensuring diversity, the development of reproductive technology for exotic species, such as cryopreservation of gametes, embryo transfer, and artificial insemination, should be emphasized and supported. Ex situ animal conservation programs that are dependent upon the long-term preservation of genetic variation should apply this technology as it becomes available because of the increasing realization that captive breeding programs are essential to prevent many species from becoming extinct. Loss of genetic diversity could also limit the potential of a population to adapt to new environments when reintroduced to the wild.

A large amount of basic research is urgently needed before application of new technology will be routine for maintaining captive populations. It is hoped that the urgency will be recognized by many more scientists worldwide than at present, and that ex situ conservation programs will become the nuclei of genetic material for dwindling populations of wild animals. Extinction for some may be softened by the frozen zoo concept, which may turn out to be the single most important reproductive technology developed for exotic animals during this decade. Its effects will reach centuries into the future for many species.

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