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Chapter 2
Applications of embryo transfer

INCREASE REPRODUCTION OF FEMALES

The main use of embryo transfer in cattle has been to amplify reproductive rates of valuable females. Because of low reproductive rates and long generation intervals, embryo transfer is especially useful in this species. Cattle may be valuable for many reasons, including scarcity, proven genetic value, or having unique characteristics such as disease resistance. Ideally, embryo transfer is used to satisfy both genetic and financial objectives simultaneously, i.e. milk or meat production increase or greater efficiency, and the investment returns financial benefits as well. It is possible to increase reproductive rates of valuable cows by an average of tenfold or more in a given year and fivefold or more per lifetime with current embryo transfer techniques. This amplification will increase substantially as new technologies, such as maturing oocytes in vitro, are perfected.

Increased reproductive rates of donors with routine embryo transfer procedures are nearly always at the expense of reduced reproductive rates of recipients. This means that fewer calves will be produced from donors and recipients combined that if both reproduced conventionally. This is because potential recipients must be on hand to await embryos, which means recipients do not become pregnant as soon as they would conventionally. This waiting can be minimized with good management, and is often justified by the increased reproductive rates of donors. An exception to decreased reproduction with embryo transfer can occur when a substantial proportion of recipients receive two embryos (or two demi-embryos). Such twinning programmes, however, are currently a very minor use of embryo transfer.

The degree of amplification of reproduction of donors by embryo transfer will be considered in detail and in the context of other factors in Chapter 12. A caveat is that people tend to advertise their spectacular successes and minimize their failures. There is even a bias in scientific reports because experiments with poor results tend not to be published. Thus one must be careful to analyse the complete picture. Success rates can be especially poor in new programmes and in hostile environments; with good management, they can also be excellent.

CIRCUMVENT INFERTILITY

It is possible to obtain offspring from genetically valuable cows that have become infertile due to injury, disease, or age by means of superovulation and embryo transfer (Bowen et al., 1978; Elsden et al., 1979), although success rates are only about one-third of those achieved with healthy, fertile donors. Infertile heifers and cows with genetically caused subfertility should not be propagated. Although success rates are low, it is possible to recover oocytes from genetically valuable, moribund cows, fertilize them in vitro, transfer them, and obtain offspring (Shea, 1978); techniques require only systematic optimization before they are applied in the field.

Choice of superovulatory and recovery procedures varies with the type of infertility (Table 1). For example, cows with cystic ovaries might be most effectively superovulated according to a regimen based on the insertion and removal of a progestin implant or intravaginal device rather than on injection of prostaglandin F2 alpha.

IMPORT/EXPORT

The desire to improve herds of cattle, to increase variation in the gene pool, and to introduce new breeds, has motivated the importation/exportation of breeding stock. In the past, trade has been primarily either in young animals with outstanding pedigrees or semen. Animals have the advantage of being 100 percent of the desired new genotype and are usually of breeding age so that impact on the herd is immediate. The disadvantages are that costs, especially for transportation, are very high, and that there is a high morbidity rate if the new environment is markedly different in management, climate, or endemic pathogens. Moreover, if cows are imported, the genetic influence on the general population is limited until their bull calves reach breeding age. While the genetic influence of imported semen can be distributed over a larger portion of the herd, offspring have only 50 percent of the new genes and will not become producing members of the herd for two to three years. With imported embryos, the resulting offspring have 100 percent of the desired genes, but as with artificial insemination, it will be several years until the resulting animals become producers. The relative advantages and disadvantages of importing animals, semen and embryos are summarized in Table 2.

TABLE 1
Therapy for various types of infertility based on embryo transfer procedures

Cause of infertilityProcedure
Uterine infectionIn cases of persistent pyometra in which volumes of fluid and debris build up in the uterus, it is often efficacious to flush the uterus with 0.9 percent NaCl until the recovered fluid is clear, and then to administer prostaglandin F2 alpha. Penicillin or oxytetracycline may be infused for three to four days but, if not done carefully, can do more harm than good. Normal embryo transfer procedures can be followed after the next oestrus.
In cases of subclinical or recurrent endometritis, repeated superovulation and embryo transfer may result in the “rescue” of viable embryos from the toxic environment to develop in the healthy uterus of the recipient.
Repeat breederHeifers in this category should not be propagated. With normally cycling, parous cows one should try at least three times to recover a single ovum to diagnose if there is ovulation, and if the ovum has been fertilized. It may be helpful to use raw semen. If a morphologically normal embryo at the expected stage of development is recovered, consider progesterone therapy during gestation.
Aged repeat breederProblem likely due to “worn-out” uterus; normal superovulatory treatment and transfer of embryos to uterus of younger recipient is often effective.
Chronic abortionRecovery of the embryo before the abortion-causing condition is active can often circumvent the resulting infertility.
Cystic ovarian diseaseOften superovulatory treatments based on the insertion and removal of a progestin implant rather than on prostaglandin F2 alpha injection are effective (see Mapletoft et al., 1980, for details). If the cystic ovarian condition appears to be hereditary, no propagation should be attempted.
Adhesions of ovaries or blocked oviductsSuperovulation increases the chances that at least a few ova will be picked up if there are adhesions. A combination of superovulation with surgical recovery or laparoscopy helps to diagnose the cause of the infertility and can result in recovery of viable embryos for transfer. Some conditions can be corrected surgically (e.g., flushing plugs of debris from the oviduct), although relieving adhesions is rarely of lasting benefit. In the future, it may be efficacious to recover oocytes from the ovaries of such cows laparoscopically for fertilization in vitro.

Valid and serious sanitary and economic concerns have resulted in strict regulation of trade in breeding stock and semen (International Zoosanitary Code, 1986). For example, in order to prevent the introduction of infectious diseases, calves and adult cattle must often undergo a lengthy quarantine and testing programme before they may be imported; the collection and processing of semen is similarly regulated. Conditions of importation vary widely and frequently require months to years to carry out, many of them in advance of a proposed sales agreement. Thus, logistics are quite complicated and costly.

TABLE 2
Comparison of importing germplasm as postparturient animals, as semen or as embryos

AdvantagesDisadvantages
Postparturient animals
Animals productive quicklyExpensive
 Animals often succumb to disease
 Chance of introducing exotic disease
 Complex transportation logistics
 Limited immediate genetic influence if females are imported
Semen
InexpensiveNeed to grade up to get pure-bred animals*
Low risk of disease transmissionNeed for Al technology
Hybrid vigour, F1 and F2*Long wait until animals productive
Simple transportation logistics 
Passive immunity from native dam 
Embryos
Very low risk of disease transmissionNeed for ET technology
Costs may be lower than animalsLong wait until animals productive
Simple transportation logistics 
Passive immunity from native dam 

* If changing from one breed to another

Few infectious organisms are spread by routine embryo transfer procedures (Hare, 1986), and such procedures do not result in rates of abortion or incidence of abnormalities among offspring that differ from those of the normal population of cattle (King et al., 1985). Such characteristics of embryos as protection by the zona pellucida, minute size, exposure only to a very circumscribed environment, and lack of body systems to host pathogens (e.g. respiratory, digestive, circulatory systems) result in significant barriers to infection. In addition, it is possible to wash, treat, and physically examine the individual embryo, which provides additional, very effective safeguards. Thus, importation of genetic material in the form of embryos is innately safer than importation of post-natal animals or semen (Stringfellow, 1985; Hare and Seidel, 1987). Regulatory officials recognize this fact and are drafting realistic conditions for importation that are less time-consuming than those required for post-natal animals. Health regulations pertaining to the collection and processing of the semen used to produce embryos intended for export, however, may still apply.

The decreased risk of compromising the health of national herds in itself makes embryo transfer the method of choice for importing breeding stock in many cases. Other advantages are that the offspring will be 100 percent of the desired genotype and will adapt more readily to the new environment because of passive immunity acquired from the recipient. There is still a potential for problems of unthriftiness and disappointing production if the type of cow is inappropriate for the new environment, as for example, a high-producing North American dairy cow would be for an extensive management system based on range foraging.

Costs of importing embryos are often lower than importing post-natal animals, and it is possible to change the breed of a herd within a single generation. Nevertheless, costs are still a great deal higher than importing semen, and conventional embryo transfer remains a less potent tool for genetic progress than artificial insemination programmes based on intensive selection.

MOET PROGRAMMES

The term MOET, multiple ovulation and embryo transfer was coined by Nicholas and Smith (1983) to consider embryo transfer and related technology in the context of optimizing genetic improvement of cattle. Most MOET schemes require one or a few large nucleus herds. The resulting genetic improvement would be disseminated to the general population by embryo transfer, artificial insemination, or more practically by young bulls to be used in natural breeding. MOET procedures rely on advanced technology, which at first seems inappropriate for less developed countries. However, nearly all of the advanced technical procedures would be carried out at one or a few central sites, which may be especially appropriate for some applications in many less developed countries. There are both practical and theoretical advantages to MOET, which will be discussed after a description of MOET procedures.

To appreciate why MOET procedures are effective, it is necessary to consider briefly conventional animal breeding procedures. Improved animals result from the following practices.

Identify genetically valuable animals accurately so that the best can be used as parents of the next generation. This can be done by performance testing, progeny testing and pedigree analysis. Performance testing measures the animal itself, e.g. rate and efficiency of growth, milk production or the degree of calving difficulty (as a trait of the calf or the mother). Because there is some genetic component to such performance, a partial measure of genetic value is obtained. Advantages of performance testing include low cost, rapid availability of data and ability to test many or all of the animals in the population. Disadvantages are low accuracy (in many cases one measurement per animal), confusion by environmental factors (in some cases deliberate manipulation in order to make certain animals look good) and sex limitations, e.g. one cannot performance test a bull for milk production.

Progeny testing measures traits in offspring of animals and in many respects is the converse of performance testing. It is not sex limited and can be done over a variety of environments in ways that are not likely to be misleading. However, it is expensive, data are not available until the next generation and only limited numbers of animals can be progeny tested. Accurate progeny testing is difficult with cows because of limited numbers of offspring. In many cases a performance test is used to pre-select animals for progeny testing.

Pedigree analysis simply uses information available on relatives, for example, the genetic value of parents or siblings.

Use high selection intensity so that only the best animals genetically are selected as parents. Genetically superior cattle are propagated selectively by artificial insemination and embryo transfer, by keeping offspring from only the best cows and by using only the few best bulls in natural breeding systems. Because of low reproductive rates of cows, most genetic progress is made by selecting bulls and obtaining many progeny per bull.

Minimize the generation interval. If selection steps can be made every three years, genetic progress will be nearly twice what it would be with selection every six years. Progeny testing lengthens the generation interval because data are not available until the next generation, which often dissipates the advantages of the increased accuracy.

The main objective of MOET is to select on the basis of performance tests and pedigree analysis in order to reduce the generation interval, in comparison to progeny testing procedures used currently. Selection intensity is increased on the female side with superovulation and embryo transfer. In MOET schemes, genetic progress increases slightly if embryos are split so that more accurate assessments of genetic value are obtained. This occurs because two phenotypic measurements are made on the same genotype. Furthermore, reliability of measurements is increased because all animals are kept in one or a few herds under controlled conditions, and thus can be compared to each other accurately without bias.

MOET procedures should be especially useful for improving milk production (Nicholas and Smith, 1983) therefore this discussion is based on dairy cattle. However, MOET can also be used for beef cattle. Populations of the order of 1 000 animals (donors, calves, recipients) are required to make MOET procedures work optimally without increasing inbreeding more than 0.5 percent per generation.

To begin such a herd, the best females available are gathered, superovulated and bred to the best bulls available. The embryos are then collected and bisected to maximize production (or, in the future, cloned by nuclear transplantation so that many identical females result per embryo). Sexing of sperm or embryos would further improve this system. The progeny are then compared for such traits as milk production and milk composition, and the best sets are used to become parents of the next generation. Embryos may also be collected from heifers and then frozen, so that embryos are available as soon as selection has occurred in the fourth or fifth month of the first lactation. These procedures are repeated continually with each generation.

After several generations, the average genetic value of selected animals in these nucleus herds will exceed the average genetic value of selected animals from outside the herd, even most progeny-tested bulls. Thus, bulls that are siblings of the best females in the nucleus herd are selected as sires for the next MOET generation and for the general population because they are genetically superior (on the average) to bulls available elsewhere, even though they are not progeny tested.

A variation on MOET that may be an option in the future is to use cloning in another context. Some embryos of a clone would be frozen so that if a particular clone proved to be valuable, many cloned embryos would be made by serial nuclear transplantation. These embryos could then be disseminated to the population, thus greatly increasing the genetic value of the population. For genetic progress to continue, females of the best clones would be mated to the best bulls to obtain even better clones in the long run. It is likely to be some years before such a scheme becomes feasible, even in developed countries. However, MOET schemes are potentially very useful without cloning at all, and may be especially valuable in the absence of contemporaneous genetic improvement schemes, which require sophisticated data gathering systems. Such systems for cattle populations are frequently unavailable in less-developed countries.

TWINNING

Most of the world's cattle are of beef breeds, and most of the calves born to dairy cows are used primarily for meat. Beef production is inherently inefficient biologically. Since about 70 percent of nutrients consumed by dams are for body maintenance and the other 30 percent go to producing the foetus and milk to feed the calf (Seidel, 1981b), it should theoretically be possible to produce twice as many calves with only 30 percent more nutrients if cattle had twins. Probably a 60 percent increase in feed costs is more realistic due to higher morbidity and mortality and slower growth rates with twins. In practice, one would probably decrease cow numbers and increase calf numbers (due to twins) so that the amount of nutrients used per farm would remain constant. There is a great advantage to twinning if nutrients are limited and management capabilities are high.

There are dozens of studies (for example, Anderson, 1978) demonstrating that twinning can be successful, both in terms of calf survival and high fertility with acceptable intervals between parturition and conception while cows are suckling twins. However, most of these studies were conducted by highly motivated researchers with considerable resources. In routine cattle management programmes, farmers universally show an aversion to twinning, because the calves often die or do poorly, and there is a higher incidence among cows of death, retained placenta, decreased milk production and lower fertility after parturition. The majority of these problems are attributable to the fact that the farmers were not expecting twins to be born, and therefore, did not adjust management procedures accordingly.

Many studies have involved the production of twins by embryo transfer (Anderson, 1978). To date, other methods, for example administering low doses of gonadotrophins to cause twin ovulations, have not been efficacious, while twinning by embryo transfer has proved too complex and expensive to be profitable. The schemes that have worked at all for farmers have been heavily subsidized. However, in some situations, it may be profitable to obtain oocytes from slaughterhouse ovaries, mature and fertilize them in vitro, culture them to the early blastocyst stage and freeze them for twinning purposes (Lu et al., 1987). Such an enterprise would have to rely on huge volumes of embryo production, and delivery of embryo transfer services very inexpensively, for example by systems similar to artificial insemination programmes.

At the time of writing, a huge effort is under way in several countries in the European Economic Community to exploit twinning by embryo transfer because of a marked shortage of calves to grow for beef purposes. The shortage and consequent high value of calves was caused by surplus numbers of dairy cows being used for milk production, whereas traditionally they produced most of the calves for beef. This shortage of calves is likely to moderate as more farmers switch to production of beef calves, but mean while considerable progress in technology of oocyte maturation and in vitro fertilization for commercial purposes is likely. Undoubtedly, there will be other situations in various countries in which twinning cattle will be profitable. However, due to the complex management requirements, such programmes will be appropriate only in very special situations, at least for the rest of this century.

EMBRYO TRANSFER AS PART OF OTHER BIOTECHNOLOGY

Many other potential applications of embryo transfer could be cited, but only three will be considered.

Detection of carriers of undesirable Mendelian recessive traits via embryo transfer is very effective for both cows and bulls. For certain traits like syndactyly and dwarfism, there is a shortage of homozygous, fertile females to use as mates for suspected carrier bulls. Embryo transfer is an obvious means of amplifying gamete (and embryo) production of such females so that bulls can be tested for carrier status. Embryo transfer also provides a method of testing daughters of carrier bulls to determine which half does not have the deleterious allele. Since at least seven defect-free calves are required to be 99 percent certain that a given animal is not a carrier, it would normally take longer than the average reproductive lifespan of a cow to test this; furthermore, all the calves produced during the test would be carriers because of using semen from a double recessive bull. With superovulation and embryo transfer, one or two courses of superovulation will provide enough embryos to test most cows; moreover, recipients can be twinned and the foetuses examined at about two months of gestation to diagnose many of these defects. Thus, with embryo transfer a quick answer is possible to a problem that is otherwise intractable.

Exploitation of other technologies that require manipulating the oocyte or embryo in vitro depends on good embryo transfer techniques for success. Such technologies include in vitro fertilization, sexing, production of transgenic animals, bisection of embryos and cloning by nuclear transplantation.

From the standpoint of research, embryo transfer is a powerful tool for separating foetal and maternal effects. For example, is declining reproductive efficiency with age due to an aged ovum or an aged reproductive tract? Applications in research are considered in detail by Kuzan and Seidel (1986). As is described in Chapter 10, production of identical twin animals by transfer of bisected embryos for use as experimental animals greatly reduces research costs since much smaller treatment groups are needed to obtain statistically significant results.


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