Embryo Transfer in Farm Animals: Introduction

In farm mammals, early embryos can be removed from the uterus of their dam (the donor) and transferred to the uterus of other females (recipients) for development to term. The main use of embryo transfer is increased productivity of selected females; others are identification of potential artificial insemination (AI) bulls through contract matings, disease control, importation and exportation of livestock, rapid screening of AI sires for genetically recessive characteristics (eg, syndactyly), and treatment or circumvention of certain types of infertility. Embryo transfer also is a useful research tool for evaluating fetal and maternal interactions.

Cattle:

In cattle, the day of the estrous cycle in donors and recipients is estimated by estrus detection (first day of estrus = day 0). Donors are induced to superovulate by treatment with equine chorionic gonadotropin (eCG; currently not available) or with follicle-stimulating hormone (FSH; activity varies, use according to label directions), typically given bid starting on day 9-14 of the cycle. Prostaglandin (PG) F_2α, 25-35 mg, IM, is administered on the third or fourth day of gonadotropin treatment, and estrus should be seen 36-48 hr later. Although there appears to be no difference in pregnancy rates among recipients ovulating from 1 day before to 1 day after the donor, synchrony is considered to be optimal when the recipient ovulates on the same day as the donor. Recipients with a single growing follicle take longer to come into estrus after prostaglandin administration than do donors that have multiple follicles. For this reason, PGF_2α is often given to recipient cows in mid-diestrus the evening before the donor cow is treated with prostaglandin. Donors are typically inseminated with high-quality semen at 12 and 24 hr after the onset of estrus, or at 72, 84, and 96 hr after treatment with prostaglandin. Estrus may also be synchronized by use of progestagen and estrogen regimens or gonadotropin and prostaglandin regimens (see hormonal control of estrus, Hormonal Control of Estrus: Introduction). While up to 90% of cows have been reported to respond to superovulatory treatments, 20-30% of cows flushed produce no transferable embryos. An average of 5 or 6 transferable embryos can be expected from each donor cow, but variability in response to superovulatory treatments is high. Induction of superovulation is particularly difficult in old or high-yielding, lactating dairy cows and may lead to loss of milk. Some practitioners use repeated single embryo collections during successive untreated cycles, while others superovulate and collect embryos from donor cows on the cycle before the cow is bred for pregnancy establishment (to initiate the next lactation) to maintain close to a 365-day calving interval. Cows can be successfully superovulated every 35-40 days.

To perform embryo collection, the rectum is evacuated (at this time the number of corpora lutea on the ovary may be estimated), and the cow is given an epidural anesthetic. The perineum is washed, and a 12- to 24-French Foley-type or 3-way catheter, stiffened by a stylet, is passed into the vagina. While palpating per rectum, the operator guides the catheter through the cervix into the uterus, about halfway up one uterine horn; the stylet is then withdrawn, and the cuff near the end of the catheter is inflated. In this way, the tip of each uterine horn is flushed separately. This may be done because cattle embryos do not migrate after descending into the horn.

About 20-35 mL of fluid (typically Dulbecco’s phosphate buffered saline) with added antibiotics (eg, 60 IU penicillin and 60 µg streptomycin/mL) and either 1% fetal calf serum or bovine serum albumin (4 mg/mL) is placed into the horn during each flush. Complete flush medium is also available. Medium containing polyvinyl alcohol in place of protein may foam less when used for flushing and can be stored at room temperature. When the horn becomes mildly distended, as detected by transrectal palpation, the fluid is drained from the horn. The flushes are repeated ~10-15 times if using a fluid-in/fluid-out technique, or for a total of 800 mL if using a continuous flow technique. Alternatively, the entire uterus may be flushed at one time; the uterus may take up to 1 L of medium per flush. An embryo filter is placed in the outflow, and embryos are washed from the filter for examination. Ova or embryos are located using a dissecting microscope at ~10× magnification.

Once embryos are located, they are transferred to a smaller volume of transfer medium (Dulbecco’s phosphate buffered saline with up to 20% serum or 0.4% bovine serum albumin; complete transfer medium is also available commercially) and examined morphologically at 50-100× magnification to evaluate their quality. Those selected as being viable and of transferable quality are held in the medium at room temperature until they are transferred to recipients or prepared for freezing, bisection (“splitting”), or more specialized treatment, such as sex determination. Alternatively, embryos can be refrigerated in transfer medium for up to 24 hr with no loss of viability. Splitting embryos into identical halves by microsurgery is practiced by a few commercial embryo transfer teams. The number of embryos to be transferred can be doubled with only a minor reduction in pregnancy rates. However, techniques of freezing manipulated embryos still need to be improved.

Almost exclusively, embryos are transferred nonsurgically. The embryo is loaded into a small pipette (embryo straw) in a specialized (Cassou-type) insemination “gun.” This is placed into the vagina and, with the aid of manual palpation per rectum, is threaded through the cervix into the horn ipsilateral to the corpus luteum. Although it is desirable to place the embryo into the cranial uterine horn, it is better to place it quickly and atraumatically into the caudal horn than to manipulate the tract for a prolonged period of time. Surgical transfer is performed rarely; paravertebral or local anesthesia is used, and the flank ipsilateral to the recipient’s corpus luteum is opened by a straight or grid incision. The uterus is grasped and a small puncture made, then a pipette holding the embryo is placed through the puncture, and the embryo is deposited into the horn with a minimal volume of medium. The flank incision is closed routinely.

After direct transfer of single fresh embryos, 60-70% of recipients become pregnant; frozen embryos result in pregnancy rates of 50-60%. When 2 embryos are transferred to each recipient, the pregnancy rate is 60-90%, with 40-60% embryo survival, but twin transfers are rarely performed commercially because of the risks of causing calving difficulties and of producing freemartins when the twins are not of the same sex. Splitting of embryos would overcome the latter problem.

Sheep and Goats:

Techniques and results in sheep and goats are basically similar to those in cattle, except that surgical or laparoscopic methods are almost always used for collection and transfer. Ewe and doe recipients are synchronized so that ovulation occurs ~12 hr before donor ovulation. Transcervical collection of embryos in the doe has been attempted with variable success. Per rectum manipulation of the tract is not possible, so flushes generally involve the whole uterus rather than individual horns as in the cow. Although this method is possible in goats, it is much less repeatable in sheep because the more convoluted cervix is difficult to cannulate.

Pigs:

Embryo collection and transfer in pigs is usually done surgically. Nonsurgical and endoscopic techniques for transfer have been reported but tend to result in lower pregnancy rates. Although sows can be superovulated, donors that are to be used repeatedly are typically allowed to cycle normally because the ovulation rate in sows is high and superovulation response can be variable. There is evidence of reduced embryo viability if superovulation response is too high. Survival of embryos from first-estrus gilts is ~20% lower than from older gilts. Embryos are collected via midventral laparotomy, under general anesthesia 4-6 days after the onset of estrus, by normograde flushing of the oviduct and collection of the flush fluid from the tip of the uterine horn. On day 4, embryos are in the 4- to 8-cell stage and are easily differentiated from unfertilized ova, whereas later stages are not. For surgical transfer, recipients should come into estrus 1 day before to 2 days after the donor; preference is given to those that come into estrus the same day or 1 day later than the donor. Methods of synchronization are more complicated than in cattle because the corpus luteum of swine is not responsive to prostaglandins until late in diestrus (see hormonal control of estrus, Hormonal Control of Estrus: Introduction). Embryos >2 cells are placed in the tip of the uterine horn of the recipient under general anesthesia. Bleeding and adhesion formation may be minimized by puncturing the oviduct near the uterotubal junction and threading a narrow-gauge catheter (eg, tomcat catheter) through this opening into the uterus; embryos are expelled into the uterus through the catheter. Embryos will migrate throughout the uterus after transfer. Nonsurgical transfer involves threading a spiral pipette into the cervix of the recipient, expelling the embryos into the uterus, and flushing the catheter with 10-12 mL of medium. There is some evidence that using nonsedated recipients results in higher pregnancy rates with this method. With either method, ³14 embryos should be transferred to help ensure that luteolysis is prevented.

Farrowing rate after surgical embryo transfer is ~70%. When transfers are done within 6 days of the onset of estrus, embryo survival rates in farrowing recipients are reported to be similar to those occurring naturally; about 30% of embryos fail to survive. Nonsurgical transfer resulted in a lower farrowing rate (~20%) and smaller litter size. Significant developments have been made with respect to the freezing of porcine embryos. These techniques, although still uncommonly used, include delipidating embryos prior to freezing and vitrification techniques.

Horses:

In mares, embryo transfer is used to increase productivity of a given mare or to obtain a foal from a mare that is not able to carry a foal to term. Superovulation is of variable efficacy in mares. Recent work with bid administration of equine FSH resulted in an increased ovulation rate (3-4 ovulations/mare) and increased embryo recovery (>1.5 embryos/treated mare, compared with 0.5 embryos/control mare). Equine FSH is now commercially available in the USA. For fertile, naturally ovulating donors, an embryo recovery rate of 60-70% and a pregnancy rate after transfer of 70% can be expected; these rates are 30% and 50% for subfertile donors. Breeding of the donor mare is done as for a conventional pregnancy; however, ultrasonography is essential to determine the exact day of ovulation, on which the day of embryo recovery is based.

The recipient mare should ovulate from 1 day before to 3 days after the donor mare ovulates. If a large recipient mare herd is available, mares that have ovulated at the appropriate time may be selected from the herd. If small numbers of recipients are used, the time of ovulation of the donor and recipient mares must be synchronized. This may be done using a progesterone and estrogen regimen (see hormonal control of estrus, Hormonal Control of Estrus: Introduction). The recipient mares should be started on the regimen 2 days after the donor to help ensure that they do not ovulate before the donor. Mares should be examined by ultrasonography daily to detect ovulation. To aid synchronization, ovulation may be speeded in the donor or recipient by administration of human chorionic gonadotropin (hCG) or deslorelin when a mature follicle is present. Two recipients should be synchronized for each donor to enable the recipient with the best synchronization to be chosen at the time of transfer; in addition, both recipients may be needed if the mare ovulates 2 follicles. Alternatively, ovariectomized or follicle-suppressed progesterone-treated mares may be used as recipients, eliminating the need for donor synchronization and for examination of recipient mares to detect ovulation. Recipients should be ovariectomized no more than 6 mo before transfer or should be treated with estrogen and then progesterone for a period (followed by at least 1 wk of no treatment) before being used as a recipient. Follicular suppression may be induced by administration of 2 deslorelin implants (2.2 mg, IM) to induce ovulation of a mature follicle, followed by prostaglandin administration when the resulting corpus luteum is 5-7 days old. This may result in up to 30 days of follicular suppression. The ovariectomized or suppressed recipient is untreated until donor ovulation is detected (day 0), or may be given estradiol (eg, 3.3 mg estradiol 17b, IM, sid) during this time. Starting on day 2, the recipient is given progesterone in oil, 300 mg, IM, sid. After embryo transfer, the recipient is continued on a progestagen daily until day 100 of gestation, after which placental progesterone is sufficient to maintain pregnancy. Recipients with intact ovaries may be able to discontinue progesterone sooner if secondary ovulations are seen after day 40 of pregnancy. Ovariectomized recipient mares have normal pregnancy rates after transfer, as well as normal parturition, lactation, and maternal behavior; they may also be used successfully again as embryo recipients after foaling. Fewer data are currently available on follicle-suppressed, hormone-treated mares, but no abnormalities in pregnancy or maternal function have been noted.

Embryos are usually recovered on day 7 or 8 after donor mare ovulation. Modified Dulbecco’s phosphate buffered saline with 1% fetal calf serum, or a commercially available equine embryo flush solution, is used for embryo recovery. Three 1-L flushes are performed. The mare is restrained in stocks with the tail wrapped and tied to the side, and the perineum is scrubbed and dried. A Foley-type catheter is passed manually through the vagina and cervix and into the uterine body. The cuff is then inflated with 30-40 mL of air and is pulled caudally to rest against the internal cervical os. The flush medium (1 L) is infused by gravity flow into the uterus. The fluid is then drained from the uterus by gravity flow. A wait of 3 min before draining may increase embryo recovery rates; alternatively, the fluid may be massaged throughout the uterus by palpation per rectum before it is drained. The fluid may be collected into a 1-L container and then poured through an embryo filter, or the filter may be placed directly in the outflow line. The latter may limit the speed of the outflow, potentially reducing embryo recovery. After the 3 uterine flushes have been performed, the donor mare is given PGF_2α, 10 mg, IM, to shorten the cycle and to reduce the chance of endometritis due to organisms introduced into the uterus during flushing.

The embryo is then located within the filter contents. Early embryos are similar to those of cattle, being still enclosed by the zona pellucida, appearing somewhat like a parasite egg ~200 µm in diameter. Later embryos have a hollow center and cellular rim or, if completely expanded, appear as a gray, semitranslucent sphere of up to 0.5 mm diameter on day 7 and 1 mm diameter on day 8. The embryo is transferred into a small (35 mm) Petri dish containing fresh medium with 10-20% neonatal calf serum or commercial handling medium, and is washed by transferring it at least 2 more times to droplets of fresh medium. As soon as the embryo is washed, it may be transferred, which should be done within 3 hr of collection.

For transcervical transfer, the recipient should be restrained and the perineum scrubbed as described above for the donor. Tranquilization of the recipient with xylazine or acepromazine, if necessary, does not appear to lower pregnancy rates. Tranquilization may relax the cervix and aid in a smooth transfer. The embryo is loaded into an insemination pipette or an embryo straw in the following sequence: medium, air, medium, air, medium containing the embryo, air, medium, air. If a straw is used, it is then loaded into an insemination gun. The pipette or insemination gun is passed manually into the vagina. The external os of the cervix is located but not cannulated with the finger. By stabilizing the cervix with the fingers, the pipette is passed into the external os, and the hand is withdrawn from the vagina. The pipette is manipulated through the cervix by palpation per rectum, and the embryo is expelled into the uterus. Surgical transfer may be done via flank incision as described for cattle (see above) but has no benefit over transcervical transfer.

Embryo freezing is still not widely used in horses because larger embryos (>6.5 days old) have poor viability after thawing. However, embryos in commercial handling media may be successfully cooled and stored for up to 24 hr in semen transport containers, allowing time for shipment to an embryo transfer facility, with no decrease in pregnancy rates after transfer.