Chapter 8
Transfer of embryos

In cattle, embryos are routinely transferred to the uterine horn. This is because nearly all embryos are recovered non-surgically from the uterus, and therefore should be returned to this site. Furthermore, it is much easier to transfer embryos to the uterus than to the oviduct.

Both surgical and non-surgical methods of embryo transfer can be made to work well. Under most circumstances, non-surgical transfer is greatly preferred, although surgical transfer can be done quite rapidly, even in rather primitive circumstances.

SURGICAL TRANSFER

Although thousands of embryos have been transferred via mid-line abdominal incision to cows under general anaesthesia, in most circumstances a flank incision is far more practical. Recipients are placed in squeeze chutes that give access to either flank. The CL is located by rectal palpation and the flank ipsilateral to the CL is clipped, washed with soap and water, and sterilized with iodine and alcohol. About 60 ml of 2 percent procaine is given along the line of the planned incision. In everyday practice this seems more reliable than using a paravertebral block. Having scrubbed, the surgeon makes a skin incision about 15 cm long, high on the flank, just anterior to the hip. Muscle layers are separated, and the peritoneum is cut. The surgeon inserts a hand and forearm into the incision, locates the ovary, usually about 25 cm posterior to the incision, and visualizes or palpates the CL. The uterine horn is exteriorized by grasping and stretching with the thumb and forefinger the broad ligament of the uterus, which is located medial to the uterine horn. The uterine horn itself is very fragile. A puncture wound is made with a blunted needle through the wall of the cranial one-third of the exposed uterine horn. Using about 0.1 ml of medium in a small glass pipette (<1.5 mm outside diameter), an assistant draws up the embryo from the storage container. The pipette is then inserted into the lumen of the uterus, and the embryo is expelled. It takes some experience to be confident that the embryo has been deposited in the lumen. The incision is then closed, using two layers of sutures. With practice, the surgery takes about 15 minutes.

NON-SURGICAL TRANSFER

The big problem with non-surgical transfer is the difficulty in becoming proficient in this technique. First, it is necessary to be able to palpate ovaries accurately in order to select the side of ovulation. Pregnancy rates are markedly lowered if embryos are transferred to the uterine horn contralateral to the corpus luteum (Seidel, 1981a). Also, recipients should be rejected if no corpus luteum is present or pathology of the reproductive tract is noted. Even very experienced palpators make some errors in palpating corpora lutea.

The next step is to pass the embryo transfer device through the cervix. This is more challenging during the luteal phase, which is when embryos are transferred, than during oestrus, when artificial insemination is done and the cervix is more open. Heifers present a special challenge because of the small cervix; some breeds of cattle are more difficult than others, e.g. certain Bos indicus breeds require greater skill. The best training prior to undertaking non-surgical embryo transfer is experience in artificial insemination. Ideally, the trainee will have inseminated hundreds of cattle artificially, including a large number of heifers.

The third step with non-surgical transfer is to be able to insert the tip of the instrument into the desired uterine horn quickly, smoothly and atraumatically. Some people never master this technique, and others require hundreds of transfers to become proficient. This is not surprising since pregnancy rates from artificial insemination are usually markedly lower for the first 50–100 cows bred by a newly trained inseminator than after he or she has become proficient. Well-trained inseminators generally require 100–200 non-surgical transfers until their pregnancy rates plateau; others usually require more. Most technicians who are successful with non-surgical transfer had low pregnancy rates for their first 100 non-surgical transfers.

One approach for people starting an embryo transfer programme is to begin with surgical procedures until acceptable pregnancy rates are achieved. If one begins with non-surgical transfer and pregnancy rates are low, it is difficult to distinguish among problems such as identifying usable embryos, problems with media, problems in storing embryos from collection to transfer, poor non-surgical embryo transfer technique, recipient problems, etc. Pregnancy rates are frequently low with surgical embryo transfer also, but one of the problems just mentioned is usually the cause, not the surgical transfer. Once the entire sequence of superovulation, embryo recovery, surgical embryo transfer, etc., is working well, it is advisable to switch to non-surgical transfer until proficiency is achieved. A frequent error is to have a number of embryo transfer teams work in a given province or country, none of which become proficient because of insufficient opportunities to gain experience. The result is that proficiency is attained slowly or not at all, and the programme is abandoned because of poor results.

Some people believe that there is a 5–10 percent advantage in pregnancy rates with surgical transfer, even when very proficient technicians are doing the non-surgical transfer. Even if this is true, in most circumstances nonsurgical transfer is still preferred because it is less expensive, it is quicker and does not involve surgical procedures. This may also obviate the need for veterinary supervision, which is required for surgery in many countries.

LOADING STRAWS

We recommend loading straws for embryo transfer as illustrated in Figure 6. The first step is to take a sterile 0.25-cc straw, shortened by 1 cm before sterilization, label it and rinse it twice with medium to removed any toxic contaminants, taking care not to wet the cotton plug and to discard the rinses. A plastic 1-cc tuberculin syringe fits snugly over the straw for aspirating and expelling fluid. The straw is filled nearly one-third full of fluid, then with a 5-mm column of air, then another column of fluid containing the embryo, one-third the length of the straw, then another short column of air, and finally more fluid to fill the straw and wet the cotton plug. Care must be taken not to compromise the sterility of the tip of the straw or the internal surfaces.

NON-SURGICAL TRANSFER EQUIPMENT

The most commonly used instrument for non-surgical transfer is the standard Cassou inseminating gun for French straws (Figure 27). There are dozens of other basically similar devices. Many have been tried in the Embryo Transfer Laboratory at Colorado State University. None of these more expensive devices gave improved pregnancy rates with Bos taurus cattle, and many were difficult to load without compromising sterility. Thus, our recommendation remains to use the standard French straw gun with a 0.25-cc French straw because it is inexpensive and easy to use correctly. We advise cutting about 1 cm off the standard 0.25-cc French straw to give better control of the tip after covering the straw and gun with the sterile sheath. We also recommend placing a sterile plastic bag over the instrument as it is placed in the vagina, and piercing the plastic bag as the instrument enters the cervix.

FIGURE 26
Steps in loading a 0.25-cc plastic straw in preparation for transfer (or freezing) an embryo: labelling (A), aspirating embryo in second column of fluid (B), and the loaded straws (C). Note the air bubbles (arrows) to compartmentalize the straw. The top straw is loaded for freezing and the bottom straw is loaded for transfer, with a third column of medium as an added measure of safety

There are three situations in which an instrument other than the standard Cassou insemination gun may be advisable. Most instruments designed specifically for non-surgical transfer are longer, thinner and have a smoother tip than the sheath on the French straw gun for artificial insemination. This makes them somewhat easier to pass through the cervix. This can be especially helpful for beginners, although it is usually of little value to experienced technicians except for some heifers and certain breeds with difficult cervixes. This constitutes the second situation for the special instruments, i.e. when mostly heifers are used as recipients or difficult cervixes are experienced. The third situation in which a different instrument can be helpful is in large breeds of cows with long uteri, such as older Holstein-Friesian cows. Many of the instruments for non-surgical transfer are longer than the standard French straw gun, and these are somewhat easier to use for the long uterus. Perhaps the most used of these special instruments is the miniaturized embryo transfer syringe made by Cassou, which uses blue sheaths (see Chapter 17). Straws should be shortened by 0.5 cm for this instrument.

FIGURE 27
Non-surgical transfer equipment illustrating a 0.25-cc plastic straw attached to a syringe (A), a Cassou inseminating gun (B), and the sheath (C)

We reiterate, however, that while it may be desirable to invest in several of the expensive non-surgical transfer devices (which also use expensive sheaths), under most circumstances large numbers of these devices cannot be justified, and the standard Cassou gun should be used.

ANAESTHESIA

Epidural anaesthesia is recommended for routine non-surgical transfer (see Figure 2). This relaxes rectal musculature, making it easier to manipulate the reproductive tract gently as is required for high pregnancy rates. Very experienced technicians sometimes do not use epidural anaesthesia. However, under most conditions this is probably unwise because of the occasional difficult animal. Epidural anaesthesia clearly costs some minutes in time, and occasionally effective anaesthesia is not attained. The problem of waiting several minutes until the rectal muscles relax can be circumvented by having an assistant give the epidural injection about five minutes before embryo transfer while the technician is transferring the embryo to the previous recipient. The procedure for epidural anaesthesia is the same as for non-surgical embryo recovery.

TRANSFER PROCEDURE

The actual embryo transfer process is similar to the method used for artificial insemination, except that the transfer gun is passed well up the uterine horn ipsilateral to the corpus luteum. A good site to aim at is the palpable bifurcation of the uterine horns. Some technicians go a bit further by straightening the uterine horn progressively just before the gun is passed. The key is to pass the gun without damaging the endometrium. Therefore, it is better to insert the instrument less deeply and not cause damage. Speed is quite important once the cervix is passed but, at the same time, techniques must be gentle. Because cattle tend to move around when confined in a chute, there will be less chance of damaging tissues if the procedure is done quickly. As with artificial insemination, the plunger should be depressed firmly, but not too rapidly.

SYNCHRONY OF REPRODUCTIVE CYCLES

Perhaps the most venerable principle of embryo transfer is that the stage of the reproductive cycle of the recipient must correspond to that of the donor or physiological stage of development of the embryo. This is definitely true for cattle (see Seidel, 1981a). Two questions arise: to what extent is asynchrony of reproductive cycles tolerated, and what methods can be used successfully for synchronizing reproductive cycles pharmacologically?

Many studies indicate that pregnancy rates decline with asynchrony of donor and recipient (Seidel, 1981a, Hasler et al., 1987). In most studies with morphologically normal, unfrozen embryos, pregnancy rates were similar with perfect synchrony and asynchrony of 1 day. Several studies with large numbers per group are summarized in the following table. There is a hint that optimum pregnancy rates result when recipients are in oestrus slightly before donors, although there may be a statistical artefact in these data due to different oestrus detection practices between recipients and donors.

In any case, it appears that asynchrony of up to one and a half days does not result in marked reduction in pregnancy rates with unfrozen embryos collected six to eight days after oestrus. Pregnancy rates do not decline markedly even with + 2 days asynchrony. With embryos of lower quality asynchrony is less well tolerated, and slight negative asynchrony seems preferable to positive asynchrony for such embryos (Lindner and Wright, 1983; Hasler et al., 1987). Embryos collected three to four days after oestrus seem to be less tolerant of asynchrony than older embryos. Furthermore, several studies indicate that there is less tolerance of asynchrony with frozen embryos; more definitive data are required to substantiate this point.

METHODS OF SYNCHRONIZING OESTRUS

There are many methods of synchronizing reproductive cycles of recipients to match those of donors. In some circumstances, natural synchrony is feasible, but in most cases some recipients will need to be synchronized to augment those whose oestrous cycles match the donor's naturally. The most widely accepted procedure for synchronizing recipients is administration of a luteolytic dose of prostaglandin F₂ alpha or a suitable analogue during the luteal phase. This is probably superior to using natural oestrous cycles (Hasler et al., 1987). Injecting potential recipients with two doses of prostaglandin at 11-day intervals when stages of the reproductive cycle are unknown also works well if cattle are cycling.

TABLE 7
Percentage pregnant (No.) with varying degrees of donor-recipient oestrous cycle asynchrony

* + means recipient in oestrus before donor.

Various progestin withdrawal procedures also have been used successfully. Under some circumstances, one of these, Syncromate B (norgestomet) has resulted in lower pregnancy rates than prostaglandin controls (King et al., 1986). However, others have used the Syncromate B method successfully for recipients, therefore more data are required to analyse these results.

Refractoriness of cattle to repeated synchronization appears to be a nonproblem. Despite anecdotal reports of problems in the field, each of a number of experiments to study this problem systematically has indicated that no such refractoriness occurs with the prostaglandins.

EXAMPLE OF PROGRAMMING A HERD FOR EMBRYO TRANSFER

To illustrate combining the information in previous chapters in order to set up an embryo transfer schedule, key steps are listed in Table 8. In this example, it is assumed that regular oestrus detection is not being done prior to the start of an on-farm programme, therefore the system of two injections of prostaglandin F₂ alpha, or an analogue, given at 11-day intervals is used for both donors and recipients. This also synchronizes all donors and recipients, even if the stages of their oestrous cycles are known. If a large number of donors is used at one location, it is wise to stagger them by one or two days along with recipients so that embryo collection and transfer will be done on two or three consecutive days.

TABLE 8
Example of programming donors and recipients

On day 0, or preferably earlier, it is wise to palpate most of the donors and a sample of recipients to determine if the majority have a corpus luteum. If fewer than half have a corpus luteum, it may be wise to postpone the programme for several weeks because prostaglandin is effective only for cows in the luteal phase. Most of Table 8 is self-explanatory, but we call attention to the following. On day 27, potential recipients are given prostaglandin one day before donors since gonadotrophin-treated cows show oestrus one-half to one day earlier after prostaglandin injection than untreated cattle. On day 30, most donors will be in oestrus, although a few may already have been in oestrus the previous evening and some may be up to a day late (these cows usually have a poor response). Actually, most will first show oestrus 40–46 hours after prostaglandin. On day 37, after embryo collection, donors are given another prostaglandin injection to prevent multiple pregnancy from occasional uncollected embryos. The interval from this injection to oestrus is unpredictable; it may occur weeks later.

Obviously, this is only one of many possible programming schemes. However, it does illustrate the principles involved.