It took more than 80 years from the demonstration that embryo transfer was possible in laboratory animals, until it started to be used commercially. However, the interval from demonstration to application was less than a decade for cryopreservation of embryos. While, in selected cases, the pace is becoming even more rapid for the transition from basic laboratory science to application, some other seemingly practical technologies never find a use, or only a limited use. Artificial insemination is such an example: it is used widely in dairy cattle and some species of poultry, but only to a very limited extent in most countries for beef cattle, pigs, sheep, goats and horses. For that matter, embryo transfer currently has only very limited use in all species, although some of these uses are very important. This chapter deals with emerging technologies related to embryo transfer, but not in routine commercial use. Because it is impossible to predict which of these technologies will be useful in commercial agriculture in the near future, methodology will not be presented. Nevertheless, it was felt that a brief introduction to each would be appropriate.
Theoretically, the ideal method for sex control in cattle is separation of X-and Y-bearing sperm. Unfortunately, to date, there has been no clear example of a method that accomplishes this in mammals without damaging the sperm (Seidel, 1988a), although several methods have been used to sex embryos successfully and convincingly (Seidel, 1988a). As all of these methods have limitations, commercial application has been limited. Nevertheless, sexing embryos will be used commercially in the near future.
There are, however, several intrinsic constraints to sexing embryos. If the embryos of the undesired sex are to be discarded, the process is innately inefficient because of the high cost of obtaining embryos. Moreover, in many instances, embryos of either sex are valuable from the particular donors that justify the cost of embryo transfer, which makes sexing irrelevant.
Methods of sexing embryos are likely to remain imperfect. With current techniques, many embryos are not sexable at all, and some are sexed incorrectly. Procedures are clumsy, time consuming, slightly damaging to embryos and costly. Many of these problems will be solved, but it will probably be some years before most embryos collected will be sexed. The three most common procedures for sexing embryos are described briefly below. Other procedures are discussed elsewhere (Seidel, 1988a).
With this procedure, a biopsy of the embryo is obtained and cultured with colchicine or a related drug that causes cells to stop dividing at the metaphase stage of mitosis. After some hours, cells are lysed osmotically, and the preparation is fixed and stained so that chromosomes can be examined microscopically. The main advantage of the method is that it is quite accurate for the embryos that produce at least one readable metaphase set of chromosomes (about half for day-7 embryos). Another advantage is that gross chromosomal abnormalities can be detected. A third advantage is that, except for the biopsy procedure, the equipment needed, primarily a good microscope, is already part of many laboratories. Also, reagents are inexpensive and easy to obtain.
There are three main disadvantages to karyotyping: readable sets of chromosomes are frequently not obtained, particularly from embryos recovered before day 10; the embryo must be biopsied; and the procedure requires considerable training and can take as long as 12 or more hours. Because of these problems, most people feel that this approach is unsuitable for everyday use.
Antibodies to male-specific antigen
This procedure requires antibodies to cell-surface molecules specific to male tissues (sometimes referred to — probably incorrectly — as the anti-H-Y antigen method). Embryos are incubated for 30–60 minutes with antibodies, and then for an additional 30–60 minutes with an antibody to the first antibody containing a fluorescent dye. Embryos are then briefly examined with a fluorescence microscope. Male embryos fluoresce. The advantages of this approach are its speed and lack of need to biopsy embryos. The disadvantages are need for a fluorescence microscope, commercial unavailability of reagents, the subjective nature of determining what is and what is not specific fluorescence and limited accuracy (80 percent). Despite these problems, many people feel that this approach may be developed into an acceptable procedure for routine use.
Y-chromosome-specific DNA probes
This approach is based on a molecular biological technique. Pieces of DNA (probes) can be made that bind to DNA on the Y-chromosome, but not other chromosomes. Embryos are biopsied, the DNA is extracted from the cells, and an enzymatically or radioactively labelled DNA probe is incubated with the extracted embryonal DNA. If Y-chromosomes are present, the probe binds. This procedure suffers from many of the same problems as karyotyping: the need to biopsy and the long time and complex skills required. The time required can probably be shortened to several hours with further research. The main advantages are that it is highly accurate, and a higher percentage of embryos can be sexed than with karyotyping since cells need not be in metaphase. This method is offered commercially by one company for frozen embryos (which presents problems for exportation to some countries since the zona pellucida is damaged during biopsy). A slight variation on this procedure is to use the polymerase chain reaction so that a segment of DNA can be observed directly.
This procedure usually comprises four separate steps in vitro: oocyte maturation, capacitation of sperm, fertilization, and culture of embryos until they can be frozen or transferred to the uterus. The actual in vitro fertilization step is the easiest of the four, but success requires that the other steps work well. Oocyte maturation, capacitation, and culture of embryos can all be done in vivo, but as the number of in vivo steps increases, the practicality decreases greatly. Recently, there have been significant advances in the in vitro fertilization process with cattle (Lu et al., 1987; Goto et al., 1988). A major advance has been co-culture of oocytes and embryos with cumulus cells or oviduct epithelial cells (see also Gandolfi and Moor, 1987). The references just cited provide information on successful methodology which, however, still leads to fairly variable results.
Potential applications of in vitro fertilization include supplying embryos from slaughterhouse oocytes for twinning programmes to increase calf crops without increasing the number of cows. Another obvious application is to circumvent certain kinds of infertility, rather as it is used for humans. A third possible application is as an alternative to harvesting gametes from valuable cows by superovulation: by removing an entire ovary, recovering thousands of oocytes and allowing them to mature and be fertilized in vitro. However, current methodology with cattle is limited to maturing a few dozen oocytes per ovary at most. Oocyte maturation could also provide material for cloning by nuclear transplantation and for making transgenic animals. Despite its promise, in vitro fertilization has resulted in fewer than 150 calves to date, and has not been used commercially at all in cattle. This is likely to change soon, as has been described in Chapter 2, although in vitro fertilization techniques may not be commercially profitable for some time.
Chimeras are animals with cells of two or more different genotypes in their bodies. They are usually made either by mixing cells of two or more embryos just before compaction or by injecting cells of one embryo into the inside of another, generally at the blastocyst stage. With cattle, chimeras can also be produced by transferring embryos so that fraternal twins occur. Due to placental anastomoses of blood vessels, haemopoetic tissue of such twins contains both genotypes. Chimeric cattle have been made by several variations on the techniques just described. Some of these have been quite valuable from the standpoint of basic science. We are not aware of any agricultural applications of such technology to date.
Amphibia have been cloned by nuclear transplantation since the early 1950s. Results of studies with mice, which began in the early 1980s, have not been clear-cut until recently. It is now generally accepted that nuclei from cells of mouse embryos greater than the four-cell stage are unsuitable for nuclear transplantation into one-cell ova. However, there are recent reports of cloning sheep and cattle by nuclear transplantation from more advanced embryos (Willadsen, 1986; Robl et al., 1987). It appears that nuclei from 32-cell embryos and even the inner cell mass of blastocysts of these species can be used successfully. It is not yet clear what percentage of nuclei from 32-cell bovine embryos make suitable donors for this purpose, but it could well be more than 50 percent.
There are a number of ways of effecting the actual transplantation of nuclei, and the recipient ovum can be either an unfertilized oocyte, a one-cell embryo, or one cell of a two-cell embryo. The genetic material of the recipient cell must be removed or inactivated so that the resulting animal will have the genotype of the donor nucleus and, more important, so that it will not have excess chromosomes. Details of procedures can be found in papers by Willadsen (1986) and Robl et al., (1987).
Currently, success rates with nuclear transplantation appear to be quite low with cattle, although very little has been published. There is no doubt that success rates will eventually improve so that the procedure can be used commercially. Key technologies to make it affordable include in vitro oocyte maturation and in vitro culture of embryos to the late morula or early blastocyst stage.
Perhaps the biggest problem with this technology at present is that it is not possible to clone animals, only embryos. To circumvent this problem, one strategy is to clone 16- to 32-cell stage embryos by transplanting nuclei to one-cell ova, allow the resulting embryos to develop to the 16- to 32-cell stage, and reclone them repeatedly until sufficient viable embryos accumulate. If only three successes occurred per round on the average, the number would increase fairly rapidly after several rounds: 3, 9, 27, 81, 243, etc. Note that all embryos will be of the same sex and that diagnosing the sex of the embryo prior to cloning would be extremely important. It is not yet known how well repeated recloning of embryos will work. Most of the cloned embryos would be transferred and allowed to mature in order to measure the productivity of the resulting animals, but some would be kept frozen. If the animals proved outstanding, the frozen embryos would be thawed, and their nuclei transplanted to produce as many clones of that given individual as would be profitable. In a sense, one is cloning adults with this strategy. However, it is necessary to freeze embryos prior to the time that the resulting adults exist. This technology can clearly work, even though it is somewhat inexpedient. Genetic progress will be much slower than it would be if adults could be cloned directly.
In mice, it is possible to remove the inner cell mass from the blastocyst and culture these cells in vitro so that they continue to divide without further differentiation, resulting with time in millions of these embryonic stemcells. It is possible to inject such cells back into a blastocyst to form a chimeric foetus. In up to 30 percent of cases, the germ-cells of such foetuses are partly or exclusively derived from the embryonic stem-cells, which are therefore the parents of the next generation. Embryonic stem-cells can be frozen, have genes added to them, or be manipulated in various other ways, thus forming ideal in vitro parents.
One of the most exciting technologies is to make transgenic animals. Genes are added to, replaced, or deleted from embryos, one gene at a time, to make interesting animals for research or more productive animals for agriculture. There are several methods of doing this (Renard and Babinet, 1987; Murray et al., 1988). As with many other new technologies, costs are high, success rates are low and results are highly variable. No two transgenic animals are alike, although once one is made, it transmits the new gene to half its offspring. The genes to be added can originate from many sources, including other species or from computer-controlled gene machines. There are huge logistical problems in making homozygous transgenic lines of farm animals. Perhaps the biggest problem is that we know so little about genetic control of development, growth, lactation, reproduction and disease resistance that few genes have been identified that could reasonably lead to improved animals. Nevertheless, this technology has potential importance in production agriculture and already has many research applications.