Embryo Transfer in Swines (Female Pigs)
The major reason for performing commercial embryo transfer (embryo transfer technology) in swines (female pigs) is to prevent and/or control disease. Although swine producers invariably exploit superior females, this is seldom the primary reason given for doing embryo transfer in swine, because even the most valuable donors and embryos are inexpensive compared with costs incurred when some of the common diseases of swine are introduced into a susceptible herd.
Export of swine embryos is likely to become another important reason for doing embryo transfer once long-term storage of swine embryos becomes possible.
Synchronization of Estrus
There are two methods commonly used to synchronize estrus for embryo transfer purposes. The first method involves weaning a group of sows on the same day, with estrus occurring 4 to 10 days later. However, if the sows are injected subcutaneously with 500 to 750 IU of pregnant mare serum gonadotropin (PMSG) at weaning, a high proportion of sows will come into estrus 4 to 5 days later.
Another method frequently used to synchronize sows is breeding and then aborting sows when they are between days 16 and 45 of pregnancy. Sows are aborted with one injection followed 12 hours later by a second injection of prostaglandin F2α (PGF2α) or one of its analogues.
A high proportion of sows come into estrus 4 to 7 days after treatment, and conception rates are high. Better synchrony can be achieved by injecting 500 to 750 IU of PMSG 12 hours after the second injection of PGF2α. Often, a group of sows will be synchronized by using both the weaning and the abortion methods.
Two other methods are sometimes used to synchronize estrus in swine. Pseudopregnancy may be induced with daily injections of estrogen preparation on days 11 through 15 of the estrous cycle. The corpora lutea of pseudopregnancy, which can be maintained for as long as 90 to 120 days, can be induced to regress with PGF2α.
Most sows return to estrus 4 to 7 days later. Another synchronization method is to inject or to feed progestogens for about 14 to 16 days. However, most of these progestogens induce ovarian cysts and are seldom used.
Superovulation
Sows are usually superovulated with one injection of 1200 to 1500 IU of PMSG at weaning or 24 hours after the first injection of PGF2α in sows that were synchronized by first being made pregnant or pesudopregnant.
For gilts and sows in which embryos are collected on more than two consecutive estrous cycles, the time of estrus is not controlled, and the animals are not usually superovulated. If these sows are superovulated, PMSG is given on alternate estrous cycles 4 to 5 days before the expected onset of estrus.
As with other species, the superovulatory response is quite variable within and among breeds. However, the average response for small groups of sows to 1200 to 1500 IU of PMSG ranges from 30 to 45 ovulations.
Ovulation rates of 45 or more are not desirable because of the increase in the proportion of abnormal embryos and the proportion of unfertilized eggs.
Human chorionic gonadotropin (hCG), which can be used to control the time of ovulation, is seldom used by embryo transfer specialists. If hCG is used, 500 IU is given 3 to 4 days after administration of PMSG. Ovulation occurs about 40 to 42 hours after hCG injection.
Insemination
For optimum conception and fertilization rate, donors should be mated or inseminated every 12 hours throughout estrus. If hCG is used to control the time of ovulation, the most important inseminations of fresh and frozen semen are those done at 24 and 36 hours, respectively, after hCG injection.
The volume of the inseminate should be 50 to 100 ml and contain at least 4 to 5 billion live spermatozoa.
Embryo Collection
Timing
Swine embryos are usually collected 4 to 6 days after the onset of estrus.
Four days after the onset of estrus most embryos are at the four-to-eight –cell stage, whereas on the sixth day after the onset of estrus, most are in the expanded unhatched blastocyst stage.
Most collections of swine embryos are done 4 days after the onset of estrus because four-to-eight cell embryo are easily identified and evaluated.
In contrast, morulae and the early blastocysts, which are most frequently collected on day 5, are more difficult to identify and to distinguish from unfertilized eggs.
Collection and transfer are seldom done before day 4, not only because embryos which are usually located in the oviduct of the donor, must be transferred to the oviduct of the recipient, but also because it is more difficult to deposit embryos in the oviduct than in the uterus.
The collection of embryos on day 7 or later is not usually done because sows that receive hatched blastocysts may be less likely to farrow than sows that receive unhatched embryos.
Surgical Collection
Embryos are collected surgically in a clinic or laboratory. Anesthesia is induced by injecting a barbiturate into the marginal ear vein and is maintained with halothane using a closed-circuit anesthesia machine.
A small mid ventral incision is made to expose one ovary along with the adjacent oviduct and about 30 cm of the uterine horn. A small incision is made for insertion of a glass cannula on the antimesometrial side of the uterine horn at about 20 to 25 cm from the uterotubal junction.
To avoid contaminating the cannula with blood when it is introduced into the uterine horn, it is important (1) to squeeze the blood vessels on the mesometrial side with thumb and forefinger while forcing the blunt end of a scalpel handle through the wall of the uterus on the opposite side and (2) to insert the cannula into the uterine lumen as soon as pressure on the blood vessels is removed.
The glass cannula should be about 12 to 15 cm long and 9 to 11 cm in diameter. The end that goes into the uterine horn should be cut at a 45°angle and flared. The opposite end of the cannula should have a bend of about 45° located 1 to 2 cm from the end. The glass cannula is inserted about 2 to 3 cm into the uterine horn and is held in place with a towel clamp.
To collect the embryos about 40 to 50 ml of the medium warmed to 37° C is placed in a syringe fitted with a blunt 12 to 14-gauge needle. The needle is inserted into the oviduct, and the entire medium is flushed into the oviduct, through the uterine horn, out the cannula and into a Petri dish. After removing the entire flushing medium from the uterine horn, the incision is closed before repeating the entire procedure in the second uterine horn.
Following the surgical collection of embryos, swines are especially prone to form adhesions of the reproductive organs. Therefore, to reduce the possibility of adhesion forming, it is essential to (1) maintain asepsis throughout the procedure, (2) handle the reproductive organs gently and only when necessary and (3) keep the exposed reproductive organs moist at all times with saline or another physiological solution.
Handling, Storage and Evaluation of Embryos
Searching for and Handling Embryos
The flushings, are examined for embryo with a stereomicroscope. Searches are done at 10 to 20x magnification and the evaluation of embryos at 50x or 70x. Good optics and high magnification are particularly important for distinguishing morulae and early blastocysts from unfertilized eggs.
As embryos are located, they are transferred to culture plates or other dishes that contain fresh medium warmed to 37ºC. After several rinses in fresh medium, the embryos are stored until transferred to the recipient.
Tuberculin syringes fitted with a tom cat catheter or a glass pipette are frequently used to handle embryos.
Short-term Storage
The medium for the flushing procedure can also be used to store embryos in vitro for several hours. Some of the media used for flushing and storing embryos include Brinster‘s solution, Ham‘s F-10 and TCM-199 with bicarbonate.
Embryos should be stored in fresh medium at 37°C. Although not recommended, it is possible to obtain acceptable conception rates with embryos stored at room temperature for 2 hours.
Swine embryos have been cultured for 24 hours without a decrease in embryonic survival rates after transfer to recipients.
Evaluation of Embryos
Evaluation of the quality of embryos is done by examining the general morphologic appearance of the embryo at the time of collection. In general, the cleavage rate of embryos collected from a donor is quite uniform.
On days 4, 5 or 6 the stage of development for most normally developing embryos ranges from four to eight cells, eight cells to morulae and blastocysts, respectively.
Once embryos pass the eight –cell stage, cells fuse, making it difficult to identify and to count individual cells. Therefore, considerable experience is required to distinguish 8- to 16-cell embryos, morulae and early blastocysts from degenerating unfertilized eggs. Because four- to eight-cell embryos are easily identified and evaluated, most embryo transfer specialists prefer to collect embryos from donors when the embryos are expected to be at the four-to-eight-cell stage. However, in a recent study it was shown that sows that received morulae were more likely to farrow than sows that received four-to-eight-cell embryos.
Embryo Transfer
Practical methods for the nonsurgical transfer of swine embryos have not been developed. Surgical transfers are usually done on the farm rather than in a clinic or a laboratory to reduce the risk of introducing disease. Anesthesia is induced and maintained by injecting a barbiturate into the marginal ear vein. The reproductive tract is reached through a mid ventral incision. Corpora should be examined for appropriate stage of development before embryos are transferred to the recipient. The uterine horns should also be examined for abnormalities, especially if gilts are used as recipients.
Embryo are transferred to the recipient by one of two methods. In one method a fine catheter or pipette that contains the embryos is passed through a small puncture wound into the lumen of the uterus. The embryos are deposited wound does not require sutures. Inexperienced individuals should be especially careful not to deposit the embryos into the endometrium or the myometrium. Depositing embryos into the wall of the uterus is more easily done in swine than in bovine or in ovine. However, this complication and hemorrhaging of the puncture wound, which sometimes occur, can be avoided. This is accomplished by introducing a tom cat catheter or a piece of rubber tubing, which contains the embryos, into the oviduct. The distal end of the tubing is held firmly in place while the embryos are flushed into the uterus with a syringe that is attached to the other end of the tubing.
