Although collection of a single embryo is occasionally done in cattle, most embryo transfers in cattle are done after a hormonal treatment to superovulate donor cows and to maximize recovery of embryos during the collection procedure. In cattle, there are two generally accepted methods of superovulation. One method consists of administering a single injection (2,000–2,500 U, IM) of equine chorionic gonadotropin (eCG), typically on day 10 of the estrous cycle (in which day 0 is defined as the day cows are observed in estrus), followed 2–3 days later by two injections of prostaglandin F2alpha (dinoprost or cloprostenol) 12–24 hours apart.
Currently, the method of choice in several countries is to induce superovulation by serial administrations of follicle-stimulating hormone (FSH). The superovulatory response induced by eCG treatment is often greater than that induced by FSH; however, more embryos of transferable (good) quality are produced on average after FSH treatment. For this reason, FSH has become the preferred method of superovulating cows for commercial use.
The two most commonly used commercial FSH preparations are made from porcine pituitary extracts and contain some contamination with luteinizing hormone (LH). Whereas high amounts of LH in FSH preparations may interfere with optimal superovulatory response, it is believed that a low level of LH contamination does not interfere and may even be needed for superovulation. Studies using recombinant FSH have found no improvement on the number of transferable embryos.
Although some good results have been reported with a single administration, FSH is typically administered IM over 4–5 consecutive days, twice daily in decreasing doses. Both commercial FSH preparations can be diluted into 20 mL of saline (0.9% NaCl) solution and administered over 4–5 days. A typical 4-day FSH treatment protocol is as follows: day 1, 4 mL every 12 hours; day 2, 3 mL every 12 hours; day 3, 2 mL every 12 hours; day 4, 1 mL every 12 hours (total volume = 20 mL). Treatments typically begin on day 10 after estrus. On the third or fourth day of FSH treatment, a luteolytic injection of prostaglandin F2alpha is administered and repeated 12 hours later. Estrus can be expected 36–48 hours later, and the artificial insemination can be scheduled based on the observed heat (12 and 24 hours after onset of estrus) or by appointment (72, 84, and 96 hours after administration of PGF2alpha) that is accompanied by administration of an agent to trigger ovulation (eg, human chorionic gonadotropin or gonadotropin releasing hormone [GnRH]).
Several variations of methods to increase the efficiency of superovulation programs in cattle exist. For example, the induction of a new follicle wave before gonadotropin treatments is desirable to increase the ovary response to superovulatory treatments. Response to FSH treatments is improved if treatments are initiated at the time of emergence of a new follicle wave. Examples of approaches to induce a new follicle wave include estradiol injection, GnRH injection, and transvaginal aspiration (follicle ablation) of the dominant follicle (known as dominant follicle removal [DFR]).
A new follicle wave emerges 4, 2, or 1–2 days after treatments with estradiol, GnRH, or DFR, respectively. Responses to gonadotropin treatments to induce superovulation are best when treatments are started when the follicle wave emerges. These treatments aim to promote a more uniform follicle recruitment and growth that ultimately may result in more viable embryos per procedure. In the US and many other countries, estrogen preparations are not approved for use in reproduction.
Management of recipients can be done after observation of natural estrus or based on synchronization protocols. In either scenario, a luteolytic dose of PGF2alpha is administered ~12 hours before the first treatment of PGF2alpha of donor cows. Recipient cows ovulating on the same day as donor cows are preferred for embryo transfer; however, asynchrony of ±1 day has produced pregnancy rates comparable to those of perfectly synchronized recipients. It is expected that 75%–90% will respond to the superovulation treatment; however, 20%–30% of flushed cows do not produce embryos of transferable quality. For cows that successfully respond to the FSH treatment, there is still a chance for notable variability in producing good-quality embryos (from 0 to >20 per flush). A production of 5–7 embryos of good quality (transferable) per embryo collection is considered an adequate commercial outcome.
Embryo collection is done on day 7 of the cycle when uterine stage embryos (morula and blastocysts) are expected to be recovered. Before the embryo collection procedure, the donor cow is palpated and the ovarian response to the superovulation treatment is assessed by palpation per rectum and ultrasonography to determine the number of corpora lutea in the ovaries. At this time, early detection of presumptive follicular cysts may prove helpful in developing hormonal treatments after the embryo collection procedure to resolve the cystic condition.
The procedure to collect the embryos from the uterus (flush) involves the following steps:
An epidural with 5–7 mL of lidocaine is administered.
The perineum is carefully washed with hand soap or an antiseptic scrub (chlorhexidine or iodine); depending on the animal and cervical size, a 12F to 24F Foley-type catheter placed over a metal rod (stylet) is introduced in the vagina as the palpating arm is placed in the rectum to manipulate it through the cervix. The size of the catheter varies according to the body size. It is important to wet the stylet with sterile water or saline (0.9% NaCl) solution before introducing it into the catheter to ease its removal from the catheter after its placement into the uterus.
Once inside the uterus, the catheter is advanced beyond the uterine body until it is located at the first third of one of the uterine horns; the stylet inside the catheter is then withdrawn and the cuff is inflated with 15–20 mL of air, depending on the size of the uterine horn (which varies with breed, age, parity, etc).
Each uterine horn is then flushed with commercially available, complete, and ready-to-use flushing media; these embryo flushing solutions also contain antimicrobials and bovine serum albumin as a source of protein. Some commercial preparations of complete flush media also contain surfactants to minimize the formation of foam and bubbles in the embryo search dish. In the past, the flush media had to be prepared for each collection and consisted primarily of Dulbecco phosphate-buffered saline (PBS) solution with added antimicrobials and 1% fetal bovine serum (FBS) or bovine calf serum (or alternatively, 0.1% bovine serum albumin); around the world, many flushes are still prepared in this manner.
The uterine horn may be lavaged by repeated small volume (25–50 mL) infusions of flush media that are allowed to drain into an embryo filter. Alternatively, the uterine horn may be flushed continuously; typically, 1–2 L of flush media is used to flush a cow uterus.
After the flush is completed, the cuff is deflated and the contents of the catheter are carefully allowed to flow into the embryo filter located at the end of the outflow tubing.
The embryo filter is then taken to the laboratory, and its contents are dispensed into a search dish with grid and examined under a stereoscope (dissecting microscope) at 10× magnification.
All embryos are transferred to a clean dish (with wells) containing holding media similar in composition to the flush media except for a higher concentration of FBS or bovine calf serum (10%–20%) or bovine serum albumin (0.4%). As with flush media, complete holding media are also available commercially. Embryos are then examined at a higher magnification (40–60×) and classified according to their morphology, stage of development (unfertilized oocytes, early morula, tight morula, blastocyst, expanded blastocyst, hatched blastocyst, etc), and quality (excellent, good, fair, poor, or degenerate). The quality score is based on morphological assessment of the physical integrity of embryos and morphological characteristics according to the stage of embryonic development, compaction status, color of cytoplasm, areas of cellular degeneration, number of extruded blastomeres, size of perivitelline space, and the size and sphericity of the embryo.
Only embryos classified as fair, good, or excellent should be transferred. Embryos are kept in holding media after being washed at least three times. Embryo washing is performed by transferring embryos into different, clean wells containing holding media. This procedure, recommended by the International Embryo Transfer Society, aids in removing cellular debris and potential pathogens adhered to the zona pellucida. Embryos are kept in holding media at room temperature until they are transferred to recipients or prepared for freezing, bisection (splitting), or embryo sexing. Alternatively, embryos can be refrigerated in transfer medium for up to 24 hours with no appreciable 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 decrease in pregnancy rates. However, techniques to freeze manipulated embryos require improvement.
Since 1978, the current preferred and most common method to transfer cattle embryos into synchronized recipients is via nonsurgical techniques. The embryo is loaded into a small plastic straw (0.25 mL) fitted into a specialized (Cassou-type) pipette (ie, artificial insemination gun). The embryo transfer gun is then placed into the vagina protected by a disposable sterile cover sheath to prevent contamination of the tip of the gun with potential perineal and vaginal contaminants. The gun is passed through the cervix, aided by palpation per rectum, and advanced into the uterine horn ipsilateral to the corpus luteum. The contents of the straw are then deposited as cranial as possible into the uterine horn with care to avoid prolonged and unnecessary manipulation.
After direct transfer of single fresh embryos, 60%–70% of recipients are expected to become pregnant; transfer of two embryos may result in pregnancy rates as high as 90%. A notable proportion of bovine embryo transfers are made with frozen-thawed embryos (~46%) and, with good management, pregnancy rates routinely are >50%. Under the same management conditions and well-trained staff, the pregnancy rate of frozen-thawed embryos is expected to be 10% lower than that derived from transfers of fresh bovine embryos.
Although increased pregnancy rates may be obtained with transfer of multiple embryos, the risk of dystocia and retention of fetal membranes associated with twin pregnancies and the increased probability of producing freemartin calves preclude widespread use of this approach. The use of sexed semen in embryo transfer programs is considered economically viable, especially if attention is given to inseminating donor cows with sperm numbers comparable to those used in artificial inseminations with conventional nonsorted semen.