With the average herd bull siring 20-30 calves each year and the average cow producing one calf per year, making genetic progress using natural breeding is a gradual process. However, the advent of artificial insemination (AI) hastened the rate of genetic progress by widely disseminating the genetics of superior bulls. Although great genetic strides were made possible by AI, half of the calves’ genetic potential remained largely unimproved until conventional embryo transfer (ET) became commercially available nearly 40 years later. Conventional ET allows maternal genetics to be exploited in much the same way that artificial insemination propagates paternal genetics. A valuable cow who would once only raise one calf per year can now potentially produce 50 or more calves each year with ET technologies.
Understanding the Conventional ET Process
The overall goal of conventional ET is to generate numerous viable embryos from genetically superior donor cows and sires for transfer into recipient (or surrogate) cows. This is accomplished by superovulating and inseminating the selected donor, flushing the donor to harvest the resulting embryos, and then transferring the embryos into recipient cows or freezing the embryos for transfer at another time.
During a normal estrous cycle, a cow typically ovulates only one oocyte that, if fertilized, will develop into a single embryo. In order to generate a number of embryos for ET, we must first recruit a number of oocytes. This is accomplished by superovulating the donor. The donor is set up on a four-day regimen of twice-daily injections of follicle-stimulating hormone (FSH) to stimulate follicular growth. On the third day of FSH treatments, the donor is also given an injection of prostaglandin F2α (PGF2α). This brings the donor into estrus (or heat) within the next 24 to 48 hours. The donor is then bred by artificial insemination either 14 and 20 hours or 12, 18 and 24 hours post-estrus.
The fertilized oocytes, now referred to as embryos, are allowed to develop in the uterus of the donor for seven days. The embryos are then harvested, or flushed, from the uterus via non-surgical collection. The technician first administers epidural anesthesia to the donor, relaxing the sacral nerves and preventing the cow from straining during the procedure. The technician then rectally palpates the reproductive tract and guides a silicone catheter into the uterus. The catheter cuff is inflated to seal the opening of the cervix to prevent embryos from flowing past the catheter and out of the uterus. A Y-connector with both inflow and outflow tubes is connected to the exterior end of the catheter. A specially-designed flush media is passed through the inflow tube into the uterus. The fluid is then drained from the uterus by gravity through the outflow tube. The outflow tube is connected to a filter that collects the embryos as the fluid passes through. The flush fluid is added to and removed from the uterus multiple times to flush the embryos from the uterus and into the filter.
Once the flush is complete, the embryos are rinsed from the filter and evaluated under a microscope for their quality and stage of development. The embryos are then prepared either for fresh transfer into recipient cows (whose estrous cycles have been synchronized to match the donor’s) or for freezing for transfer at a later time.
Fresh embryo transfer
Using the microscope, each embryo is individually loaded with holding media into a ¼ cc straw. Each straw is then loaded in an ET gun to facilitate the transfer. The technician rectally palpates the ovaries of the recipient to identify a structure on the ovary known as the corpus luteum (CL). The CL indicates which ovary has ovulated. The technician guides the loaded ET gun into the uterine horn on the same side as the ovary with the active CL. The embryo is gently expelled from the gun into the uterine horn, taking great care not to damage the lining of the uterus which could significantly diminish the likelihood of a successful pregnancy being established. If no CL (or a weak CL) is detected, the recipient is passed and will not receive an embryo as her uterine environment is not suitable for establishing and maintaining a pregnancy.
If the embryos are not going to be transferred immediately, they can be frozen for later use. While various methods can be utilized for freezing embryos, the direct transfer (DT) method is often preferred for its ease and practicality of use in the field. Prior to freezing, the embryos are prepared and loaded individually into ¼ cc straws using an ethylene glycol-containing freeze media. The media surrounding the embryos is then seeded with an ice crystal and the straws are cooled slowly in an embryo freezer to -35 C. The embryos are then plunged into liquid nitrogen, where they can be stored for many years until they are thawed for transfer.
Direct transfer of frozen embryos
For embryos frozen using the DT method, thawing for transfer is a simple process. The straw containing the embryo is removed from the liquid nitrogen tank and allowed to air-thaw for a few seconds prior to being placed in a 31 C water bath for 15 seconds. The embryo straw is loaded into an ET gun and the thawed embryo is transferred in exactly the same manner as a fresh embryo is transferred into a recipient.
Important Considerations for Conventional ET
Flush results vary from cow to cow and are dependent upon a number of factors including (but not limited to): cow age, breed, health status and nutritional plane as well as semen quality and the effects of repeated superovulation. Generally speaking, the average number of transferrable embryos obtained per collection is six to seven, although this number can vary widely from zero to 50 or more viable embryos. Some cows may not be suitable donors for conventional ET due to physical scarring or abnormalities of the reproductive tract that prevent embryo production or collection. Donors can be flushed every 45 to 60 days; however, production may decrease over time when cows are continually flushed due to repeated superovulation. In this situation, it can be beneficial to breed the donor and allow her to calve before flushing is resumed.
For fresh-transferred embryos, pregnancy rates of 60-80% can be expected in well-managed recipient herds. As some cellular damage occurs to embryos during the freezing and thawing processes, pregnancy rates for frozen-thawed embryos tend to be approximately 10-15% lower than fresh transfers.
Because a larger-than-normal quantity of oocytes are ovulated over the course of about 24 hours, a larger volume of semen is typically required to inseminate donor cows as compared to breeding by AI or fertilizing oocytes in vitro. Generally speaking, one to three straws of semen are used at each of two or three inseminations. At the first breeding, the quality of the semen is evaluated and the total amount of semen used is adjusted accordingly. Higher quality semen will require fewer straws at each insemination, and lower quality semen will require more straws.