There are two broad criteria for selecting donors for most embryo transfer programmes: (1) genetic superiority, that is animals that contribute to the genetic objectives of the programme, and (2) likelihood of producing large numbers of usable embryos. In the majority of embryo transfer programmes, in both developed and less-developed countries, superiority is determined in practice by market forces. For example, it makes good sense to select donors whose offspring can be sold at a profit above embryo transfer expenses. Obviously, it is inappropriate to produce animals that will not be accepted by farmers. Educational programmes and demonstration projects may be required before new types of cattle can be introduced into an area.
In some cases, the sole criterion for selection is scarcity, and embryo transfer is used to increase numbers of animals available. This may be required to determine whether a new type of animal fits the environment or to get enough animals to develop appropriate management systems. If the objective is to conserve germplasm of indigenous breeds by cryopreservation of embryos, one may wish to select a random sample of donors (and sires) or insure that a range of phenotypes within the breed is used.
In many cases, objectives measures of genetic superiority can be used, for example milk production, milk composition, growth rates, calving ease and disease resistance. Because phenotypic superiority may not indicate genetic superiority, it is usually desirable to consult someone trained in animal breeding so that the best donors are selected to meet objectives.
Selection of donors for embryo production is frequently overlooked; indeed, in using embryo transfer to circumvent infertility one often selects against this trait. Although embryo production should be secondary to genetic superiority, it should be considered seriously. Healthy, cycling cattle with a history of high fertility make the most successful donors. When there is a choice, animals without calving problems, such as retained placenta, should be used. Donors at least two months post-partum produce more embryos than those closer to calving. Young cows seem to yield slightly more usable embryos than heifers under some conditions (Hasler et al., 1987). Lactation in either beef or dairy cows does not decrease response to superovulation provided that cows are cycling and not losing weight. Extremely fat cows make poor donors, both because they do not respond well to superovulation and because their reproductive tracts are more difficult to manipulate. Sick animals usually do not produce many good embryos.
Frequently, the factors described above are beyond the control of personnel working in an embryo transfer programme, but the following steps can sometimes be taken. First, when there is a choice, use animals for donors that are intrinsically fertile, are at least two months post-partum and otherwise in good reproductive health. Second, encourage management practices that minimize or circumvent potential problems such as having animals gaining weight at the time of embryo transfer. Third, develop strategies to deal with problems caused by the embryo transfer programme itself. For example, repeated superovulation of the same donor means that it will not be going through an annual reproductive cycle; cows tend to get fat under such circumstances.
Since half of the genes come from the male, it is extremely important to use genetically superior bulls. In fact, selecting the male is usually more important than selecting the donor female because males will normally be bred to many females and can be selected more accurately than females. Likewise, it is necessary to select fertile bulls and fertile semen. Sperm transport is inhibited in superovulated cows (Hawk, 1988), which makes it especially important to use high quality semen.
Donors are located either on the farm under production conditions or at an embryo transfer centre, frequently under intensive management. Both situations have advantages and disadvantages. Keeping donors on the farm is usually the less expensive alternative. Also, less input for labour and management is required on the part of embryo transfer personnel, especially if donors are lactating. However, it is extremely important to have good communication with the personnel who manage donors on the farm. Simple, written protocols are essential. Planning is very complex, since a series of steps occurs over a period of weeks. In most cases, personnel will visit the farm to collect the embryos. However, donors can be trucked to the embryo transfer centre without lowering success rates if they are not stressed unduly. Three to four hours of travel in trucks or trailers does not seem to be a problem.
It is important that suitable facilities are available for on-the-farm programmes. This includes equipment for handling cattle, such as chutes and head catches, a refrigerator for keeping drugs, and an appropriate site to work with the embryos. Personnel at the farm must have certain skills and, above all, be extremely conscientious. For best results, palpation skills are required to determine if a corpus luteum is present when donors are superovulated; in most cases artificial insemination and semen handling skills are also needed.
If it is necessary for embryo transfer personnel to go to the farm frequently to perform most of the steps, the financial advantage of having donors on the farm will be lost. On the average, success rates with donors on the farm are lower than if donors are at an embryo transfer facility, primarily because they are monitored more closely at embryo transfer centres. Nevertheless, success rates on some farms are as good as or better than those at embryo transfer centres. Obviously, success is highly correlated with the management skills of the farmer. In most cases of embryo collection on the farm, the facilities and management capabilities are also needed for recipients (see below).
If reasonable numbers of donors (e.g. 25–100) are assembled at a central facility, embryo collection, processing and transfer can be done efficiently. Clearly, under these circumstances, facilities for large numbers of donors (and recipients) are required. Concentrating cattle in this way invites disease. This is exacerbated by assembling animals from various sources which may introduce diseases from each source. The net result is that great attention must be directed to herd health management for successful programmes, particularly when valuable cattle are involved. This includes quarantine of incoming animals, and vaccination and testing programmes. If done systematically and conscientiously, herd health programmes tailored to local conditions are usually very effective.
Another problem encountered when concentrating cattle is their feed. In many climates pasture is not an option for a central embryo transfer facility because cattle would be too scattered to manage efficiently. Local conditions, such as availability of different kinds of feed, amount of rainfall, etc., dictate how this problem will be dealt with. Proper nutrition is extremely important, but beyond the scope of this manual. Donors definitely should not be losing weight at the time of superovulation.
Other aspects of donor management, such as oestrus detection, are covered below. Conscientious and gentle handling of donors is a very important component of successful donor management.
As with donors, management of recipients is fundamentally different if they are located at an embryo transfer centre rather than on the farm; therefore, these situations will be considered separately. Frequently, using the farmer's own recipients simply is not feasible because of insufficient suitable animals. Another consideration is management capabilities of the farmer. In North America, one simple criterion has been useful to assess management capabilities; if successful artificial insemination programmes have been carried out in previous years, there is a reasonable chance that an embryo transfer programme using the farmer's recipients will be successful. If artificial insemination programmes have failed, embryo transfer is also likely to fail.
A common question is whether to use cows or heifers as recipients. The big advantage in using cows is there is less difficulty with calving (King et al., 1985). Conversely, heifers are easier to manage than cows because they are not lactating, which requires milking or calf management. One must be cautious if candidates for recipients are not lactating (and not pregnant), because this frequently means that they were culled for reasons that will make them subfertile. Heifers generally have higher fertility than cows, especially dairy cows. On the other hand, it is more difficult to transfer embryos non-surgically in heifers than in cows. Clearly, cows and heifers both have disadvantages and advantages, and the choice depends on careful analysis of the factors just described for each particular situation.
On-the-farm recipient programmes must be tailored to the resources available. The following are essential:
On-farm recipient programmes can often only be efficient during a two-to three-week period of the year two to three months after the peak calving season. At other times, there are often too few animals available as suitable recipients without keeping cows non-pregnant for long periods. Also, with such short programmes, potential recipients which are not used or are not becoming pregnant to embryo transfer, can become pregnant at the next oestrous cycle and thus do not need to be culled because of not fitting into the annual breeding/calving programme. Sometimes such considerations are minor; for example, dairy heifers may be more available for extended times than beef heifers. Very large herds without a pronounced breeding season have more flexibility.
Management of recipient herds at embryo transfer centres can be a huge logistical undertaking. Effective herds usually number at least several hundred head if pregnant recipients are included. These herds are the most expensive aspect of embryo transfer programmes, primarily because normal, healthy, fertile females are deliberately kept non-pregnant (essentially out of production) waiting for embryos. The waiting time can be minimized by using frozen embryos as a buffer and having a smaller recipient herd. Excess embryos are frozen when insufficient recipients are available; conversely, when excess recipients are available, frozen embryos can be thawed and transferred. Despite the advantages of frozen embryos, recipients often remain in the recipient herd a fairly long time. Many fail to get pregnant, and usually a definitive pregnancy diagnosis can be made only four to six weeks after embryo transfer (five to seven weeks after oestrus), at which time non-pregnant animals can be recycled. Blood or milk can be tested for progesterone 22–24 days after oestrus or returns to oestrus can be monitored to identify some of the non-pregnant recipients sooner so that they can be reused.
How many opportunities should a recipient have to become pregnant before culling her? The consensus is that in nearly all circumstances a recipient should be given a second chance and, in most circumstances, a third chance. However, after three failures following transfer of an embryo of good to excellent quality with no evidence of technical problems, the recipient should be culled as a poor risk. The exception is if pregnancy rates are rather poor on the average, e.g. below 45 percent per transfer, in which case recipients should possibly be given a fourth chance.
The next logistical question is to what extent oestrus synchronization should be used (see Chapter 8 for details on methods). About 5 percent of recipients should be in oestrus spontaneously on any given day. If embryo transfer work is done on an essentially daily basis, with an average of two or more donors per day, most of the recipients coming into oestrus will be required as recipients, and oestrus synchronization will not be advantageous on most days. On the other hand, if embryo transfer is scheduled less than three or four times per week, oestrus synchronization will be very useful. There is some evidence that oestrus synchronization with prostaglandins may result in higher pregnancy rates than natural oestrus (Hasler et al., 1987).
The following summarizes essential components of successful recipient management at an embryo transfer centre:
The specific details of facilities, health tests, types of animals used, nutrition and so on vary so much from country to country that they are not presented here. However, the general principles discussed apply universally; failure to consider these factors will result in a propensity to failure of the entire programme.
Nearly all steps in embryo recovery and transfer are timed in relation to the onset of behavioural oestrus; clearly, accurate oestrus detection is essential. Physiological characteristics of the reproductive tract change greatly throughout the stages of the oestrous cycle. On day 1 after oestrus, for example, the oviduct provides an ideal milieu for the recently fertilized ovum, but the uterine environment on this same day is lethal.
Oestrus detection must be done carefully and conscientiously; accuracy is as important for recipients as it is for donors since embryo transfer success depends on the oestrous synchrony of both. Since considerable behavioural oestrus occurs at night, oestrus detected in the morning may have begun up to half a day earlier. Thus, recipients observed to be in oestrus one-half day out of synchrony with the donor may, in fact, be a full day off.
Oestrus detection is both art and science. The method that generally works best is to move around in each pen for 10–15 minutes or more while gently moving the cattle around and chasing up animals that are lying down. Detection cannot be done properly while sitting on the fence, although this may be an initial step. Some cows are more active in mounting other cows or stand to be mounted for a longer period than is normal (up to 30 hours); others show very few signs of oestrus and may not be observed to stand to be mounted (“silent” oestrus). It may help in the case of such cows to place them with a different group of cows to check oestrus. Display of behavioural oestrus among a group may be modified by treatment with synchronizing drugs such as progestagen or prostaglandin F2 alpha because so many are in oestrus at once. Removing cows already found in oestrus in this situation often improves the chances of detecting others.
Every donor and recipient should be checked visually for oestrus at least twice each day—early in the morning and late in the afternoon and, ideally, more often, especially in the case of donors. Each animal will be in one of three categories each time oestrus is checked: (1) not in oestrus, (2) suspicious, or (3) in standing oestrus. The latter two categories should be recorded, together with the date, time and the animal's identification number. Cows in oestrus stand when mounted by others. Suspicious signs include ruffled rump hair, restlessness, bawling, walking the fence, nudging, mounting, sniffing, tail raising, discharge of clear mucus from the vulva, and swelling and inflammation of the vulva. Not every cow showing one of these characteristics should be recorded as suspicious, but a cow should be watched closely for standing oestrus and recorded as suspicious if it displays most of these characteristics.
Metoestrous bleeding—blood from the vulva, which is also seen frequently on the tail or hind-quarters of the animal—often occurs one to three days after oestrus. This bleeding is a good sign that a cow is cycling normally and should always be recorded for donor and recipient cows, especially if standing oestrus was not detected one to three days earlier.
As a further aid to accurate oestrus detection, a calendar should be kept for donors and, in some cases, for recipients too. When oestrus is detected, the donor's identification should be recorded on the calendar 18 days later. Thus, donors that were in oestrus 18–24 days earlier can be observed closely for oestrus. As soon as practical after recording oestrous behaviour on the form that the technician carries with him while observing the cows (see Chapter 16, example 4), the information should be transferred to a notebook in which daily tabulations are kept, and to individual data cards for each animal. Another option is to use a microcomputer system. Whenever data are transcribed, they should be checked for accuracy by a second person.
Aids to oestrus detection, such as chalk on the tailhead, are useful as long as they do not become a crutch for visual detection. Such aids are recommended for donors for the oestrus resulting from superovulation. Foote (1975) reviews aids and management schemes for oestrus detection. For an embryo transfer programme, the use of detector bulls, vasectomized or with a blocked or deviated penis, is not advisable because bulls may spread venereal disease. Moreover, these procedures may not ensure total sterility. If a teaser animal is used, it should be an androgenized female.
A detailed discussion of pregnancy diagnosis is beyond the scope of this manual. Nevertheless, some comments with regard to pregnancy diagnosis within embryo transfer programmes seem desirable. The first good indicator of pregnancy is failure of the recipients to show oestrus 18–24 days after the pre-transfer oestrus; obviously, the converse, showing oestrus, indicates non-pregnancy, although a small percentage of pregnant animals are in behavioural oestrus about three weeks after the previous oestrus. Progesterone assay of milk or blood samples 22–24 days after the pre-transfer oestrus is = 95 percent accurate in diagnosing non-pregnancy and about 80 percent accurate for pregnancy. However, with good oestrus detection, one gets about the same information as with progesterone tests. In other words, if the accuracy of oestrus detection is so poor that progesterone tests provide much additional information, the embryo transfer programme is likely to fail because of poor oestrus detection. Another point is that neither oestrus detection nor progesterone assay gives sufficiently accurate information for definitive pregnancy diagnosis on individual recipients. The information from returns to oestrus is very useful on a population basis, however, because it gives early information concerning success or problems.
At about day 26 of pregnancy in heifers and day 28 in cows, pregnancy can be diagnosed accurately under field conditions by ultrasonography or even earlier in very skilled hands (Kastelic et al., 1988). In most embryo transfer programmes, ultrasonography equipment is not justified, although it is very useful for a variety of purposes. When costs of ultrasonography equipment decline to half current prices, such equipment will probably be considered indispensable and will come into general use. A serious problem with early pregnancy diagnoses is that about 10 percent of 28-day pregnancies will not go to term. It has been found that 95 percent of two-month and 97 percent of three-month embryo transfer pregnancies go to term (King et al., 1985).
Pregnancy diagnosis can usually be diagnosed definitively by palpation per rectum after day 35 of pregnancy. Of course, ultrasound can also be used at these later stages. We do not recommend palpation prior to day 45, both because the conceptus is more fragile at early stages and because the information is not definitive anyway due to occurrence of spontaneous abortion even in the absence of palpation. Thus, our recommendation is to palpate per rectum at 45–60 days of gestation and confirm this with another palpation one month later.
Cows that show oestrus may be checked earlier than 45 days by palpation or ultrasonography. If they are, in fact, non-pregnant they can be recycled for use as recipients.
It is often useful to distinguish between an embryo transfer pregnancy and one from artificial insemination or natural breeding during the next cycle. The following steps will make it possible for most non-pregnant recipients to be bred or reused as soon as possible. First, it is imperative not to breed recipients that show oestrus earlier than normal, that is artificial insemination or exposure to bulls should be delayed 17–18 days from pre-embryo transfer oestrus. This makes the 90–95 percent of non-pregnant recipients without short cycles eligible for breeding. Pregnancy diagnosis is then done in a window of time that permits unequivocal distinction between the embryo transfer pregnancy and a possible subsequent one, which will be at least 17 days younger. With rectal palpation, this window may extend to about 65 days after pre-transfer oestrus for skilled palpators, and perhaps to 60 days for less skilled ones. Note that a good portion of this window is needed if embryo transfer was done over a seven-to ten-day period, if all recipients are to be examined at once, and recipients are not palpated prior to a 45-day pregnancy.
One other point is that it is common to diagnose pregnancy per rectum by “slipping membranes”. This method should not be used, at least not prior to day 50, because it leads to abortion in a significant, though small, percentage of cases (Abbitt et al., 1978). Palpation should be done on the basis of fluids, tone and size of the uterine horn. The small percentage of false positives due to undiagnosed uterine pathology is lower than the abortion rate caused by slipping membranes.
Since pregnant recipients are carrying valuable calves, they should receive better than average care. Nutrition is clearly important as well as prevention of abortion. The most critical time is at parturition. It is easy to lose 10 percent of calves at and within a few days of birth (King et al., 1985); most of these losses are due to poor management. It is especially costly to lose the calf after the huge investment up to that point. Of course, recipients often calve on individual farms not under direct supervision of embryo transfer personnel. Even so, it is wise to provide information on management at calving (e.g. ensuring that calves receive colostrum) to the personnel responsible so that embryo transfer programmes do not get a bad reputation.