A200
Embryo Transfer

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superovulation | embryo recovery | recipient synchronization | transfer of bovine embryos



Introduction: The practice of bovine embryo transfer (ET) is an ever changing science that involves three major events. It begins with selection, superovulation, and artificial insemination (A.I.) of the donor animal. Next, the embryos are recovered from the donor through either surgical or nonsurgical means, evaluated, and then frozen or transferred fresh. Lastly, the recipient animals are synchronized to be in the same stage of the estrous cycle as the donor when the embryo was recovered and receive the embryos through surgical or nonsurgical techniques. This article will briefly discuss the history and benefits of embryo transfer. Then the remainder of the article will focus on the details of ET and discuss the various pros and cons of the surgical and nonsurgical approaches.

History: The first successful embryo transfer was performed in 1890 using rabbit embryos. The first bovine embryo was recovered by Hartman, Lewis, Miller and Swett in 1930 at the Carnegie Laboratory of Embryology in Baltimore. In the 1950s, embryo transfer technology in cattle expanded with the first successful transfer performed by Umbaugh and the first calf born through a joint effort by the USDA and the University of Wisconsin. Until the 1970's progress was slow, with many ideas ending in failure. As nonsurgical methods advanced through the efforts of Elsden, Hasler, Seidel and others, the commercial use of embryo transfer exploded. In 2002, over 25,000 ET calves were registered in the United States.

Why Perform Embryo Transfer: There are many reasons a producer might select embryo transfer for his/her particular operation. The first reason would probably be the potential for genetic improvement in the herd. Through artificial insemination, superior male genetics can be spread across a herd; with embryo transfer, superior female genetics can now be spread across a specific herd or even many herds. Superovulation and embryo transfer allows one particular female to produce many offspring in a given year and many more over her reproductive lifetime. Each of these offspring would potentially carry the superior traits of the mother, such as increased weight gain, improved carcass merit, or even increased milk production. Embryo transfer may also eliminate the stress of parturition on a desirable animal, thereby increasing her reproductive life span. Disease control, salvage of reproductive function, and potential twinning are a few of the other benefits of embryo transfer. Finally, the impact embryo transfer has had and will have on the research environment cannot be overlooked. Techniques such as gene insertion, embryo splitting, and pronuclear DNA injections would not be as feasible without embryo transfer technology.

Superovulation and Artificial Insemination (A.I.)

Selection of a proper donor is essential to the success of the embryo transfer program. A potential donor can be selected based on the following criteria:

  1. Possesses superior traits desired in the herd.
  2. Must be at least 15 months of age.
  3. Has regular, normal estrous cycles.
  4. Has not had greater than two services per conception.
  5. Has not had previous conformational, parturition, or reproductive problems.
  6. Must be disease free.

Superovulation of the donor animal traditionally involved 1500 to 3000 I.U. of pregnant mares serum gonadotropin (PMSG). However, due to the ease of administration, superovulation treatments often utilize 5-6 mg of follicle-stimulating hormone (FSH) given twice daily for 4 days. This dose is gradually decreased each day (for example 6,6,4,4,2,2,2, and then 2 mg of FSH) and then is joined by a luteolytic dose of prostaglandin F2a (PGF2a) on day 3-4. This results in ovulation approximately 48 hours after the prostaglandin administration. This particular treatment regime requires the donor has a well formed corpus luteum (CL) and is between day 8-13 of her cycle. In superovulated animals, the ova are released over a 6-12 hour period. Therefore, the cow should be inseminated 2-3 times, at 12 hour intervals, beginning 12 hours after the onset of standing heat. Each cow will vary tremendously on the dose of FSH required and the response she has to the FSH. Most cows will produce 5-7 viable embryos, while others may produce very low numbers of embryos.

Great attention should be placed on heat detection, semen selection, and proper semen handling. Proper heat detection involves three areas for consideration and attention:

  1. Watch for indications of approaching estrus.
    • donor stands and allows another cow to mount her (#1 sign)
    • restlessness
    • decreased milk production
    • off feed
    • clear, mucoid vaginal discharge
  2. Donor animal(s) should not be left alone (a group of approximately 10 other animals is sufficient).
  3. Pens used should not be slippery or too crowded.

Because semen is so sensitive to temperature changes, proper semen handling is essential for good results. The semen used should be evaluated for proper morphology and intact acrosomes prior to use. A high quality semen tank should be used with liquid nitrogen levels checked regularly. Straws should be carefully removed and thawed in a 95 F (35 C) water bath for 40 seconds. The straws are then wiped dry and placed in a prewarmed insemination rod. The insemination rod is then placed under clothing to prevent damage from direct sunlight and/or cold shock. The donor animal must then be inseminated immediately. The semen is placed in the body of the uterus about 2 cm in front of (cranial to) the internal cervical os. Failure to follow any one of the above procedures will result in a decreased number of fertile ova. See pages A716 and A718 for additional details about the insemination process and semen handling.

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Embryo Recovery: The nonsurgical method for recovery of bovine embryos begins on day 7 after the onset of estrus. Embryos before day 6 are usually found in the oviduct and therefore would require surgical removal. There are many different techniques and devices for the nonsurgical removal of embryos. Only one of these procedures will be discussed here. On day 7 the donor is placed in a squeeze chute and the rectum is evacuated. An epidural is performed and the perineal region is washed thoroughly. A 40 cm long, 3.6-6 mm diameter 3 or 2-way Foley catheter, with stylet for rigidity, is inserted into the uterine horn with rectal guidance. A sterile vaginal speculum is also used to prevent contamination as the catheter is placed. The balloon is inflated with 10-20 mLs of air and the stylet is removed. The inlet valve of the catheter is connected by siliconized, sterile tubing to 500 mL of flushing solution. The outlet is connected to a conical, prewarmed glass covered with tinfoil. The flushing medium flows with pressure or gravity into the uterus and is collected in the glass receptacle. The tract can be manipulated per rectum to help facilitate the embryo recovery. This procedure is repeated on the opposite horn using a separate sterile device. The entire recovery process takes approximately 30 minutes. Benefits of this procedure when compared to surgical techniques include fewer adhesions, ability to perform under field conditions, inexpensive, and fewer anesthetic risks. The embryos collected can then either be frozen or directly transferred fresh.

Freezing and thawing protocols involve a series of specific steps for successful transfer. There are basically two methods of freezing and thawing embryos: conventional and direct transfer. In the conventional method, the embryo is frozen with glycerol. Because this substance can kill the embryo, it must be removed by "washing" the embryos multiple times during the thawing process. This can be a time consuming procedure. In recent years a process called direct transfer has been developed. With this method, the embryos are frozen in ethylene glycol and transferred directly to the recipient cow after thawing, without the time consuming washing procedure. Another process called vitrification has also been developed by researchers at Colorado State University.

Surgical approaches to embryo collection are limited by the skill of the surgeon, expense, possible adhesions, and inability to perform on the farm. However, if properly done, successful collection of oviductal stage embryos can be accomplished in almost every donor.

Recipient Synchronization: There are many options for recipient synchronization in bovine embryo transfer. No matter what option is chosen the outcome must be that the embryos are transplanted on day 6, 7 or 8 of the recipient's cycle. One common method used is a luteolytic dose of prostaglandin (PGF2a) given to animals with a significant corpus luteum (CL) present. Other methods of estrus synchronization may include gonadotropin-releasing hormone (GnRH) in conjunction with PGF2a. Probably the most critical step in recipient embryo transfer is proper heat detection. Page A714 has additional details about estrus synchronization and heat detection.

Transfer of Bovine Embryos: The nonsurgical approach of embryo transfer has multiple variations in methods and equipment. One common technique uses a Cassou A.I. gun and either a 0.5 or 0.25 mL French straw. With this procedure each embryo is loaded into a straw, the cow is cleaned and the embryo is slowly deposited in the uterine horn on the same side as the corpus luteum (CL). It may be helpful to deposit the embryo in front of (cranial to) the external uterine bifurcation, while trying to avoid a prolonged, traumatic placement.

The surgical placement of bovine embryos is often times more successful than the nonsurgical technique. However, these procedures are filled with the same problems that we find in the surgical collection of embryos. Most surgical transfers of embryos involve a flank approach with a prior palpation of a CL on the same side as the incision. The flank is prepared and local anesthesia is administered. The proper uterine horn is brought to the flank and a small puncture is made. A pipette is inserted and the embryo is deposited in roughly the same location from which it was collected in the donor cow.

Costs: The costs associated with ET can vary greatly from one farm or situation to another. It is important to understand the possible cost and determine if the benefits of ET outweigh the added expenses. For example, an average cost per pregnancy using ET can range anywhere from $125-$250. The cost will vary greatly depending on the number of eggs flushed per donor, the cost of the semen, and any collection, freezing and transfer fees. Increased labor expenses (more heat detection, more insemination time, more times each animal must go through the chute, etc.) and costs for synchronization programs should also be considered. Do not forget that the most significant expense associated with ET will be the cost of owning and maintaining the necessary recipient cows. This can range anywhere from $400-$650 per year and would be in addition to the above costs for simply getting an ET pregnancy.

Summary: The techniques involved with embryo transfer are not extremely difficult or confusing. With sufficient training and experience embryo transfer can be a very viable method for genetic improvement or manipulation within any situation. As the science behind bovine embryo transfer progresses, applications for its use will continue to become apparent in the laboratory, university, large commercial enterprise, and even the individual operation.

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