Superovulation of the donor cow
“Superovulation” of the cow is the next step in the embryo transfer process. Superovulation is the release of multiple eggs at a single estrus. Cows or heifers properly treated can release as many as 10 or more viable egg cells at one estrus. Approximately 85% of all normal fertile donors will respond to superovulation treatment with an average of five transferable embryos. Some cows are repeatedly treated at 60-day intervals with a slight decrease in embryo numbers over time. The basic principle of superovulation is to stimulate extensive follicular development through the use of a hormone preparation, which is given intramuscularly or subcutaneously, with follicle stimulating hormone (FSH) activity. Commercially available preparations of FSH are injected twice daily for four days at the middle or near the end of a normal estrous cycle, while a functional corpus luteum (CL) is on the ovary. A prostaglandin injection is given on the third day of the treatment schedule which will cause CL regression and a heat or estrus to occur approximately 48 to 60 hours later.
3) Insemination of the cow
Because of the release of many ova from the multiple follicles on the ovary, there is a greater than normal need to be certain that viable sperm cells reach the oviducts of the superovulated females. Therefore, many embryo transfer technicians will choose to inseminate the cow several times during and after estrus. One scheme that has been used successfully is to inseminate the superovulated cow at 12, 24, and 36 hours after the onset of standing heat. Using high quality semen with a high percentage of normal, motile cells is a very critical step in any embryo transfer program. The correct site for semen placement is in the body of the uterus. This is a small target (1/2 to 1 inch) that is just in front of the cervix. There seems to be a tendency for inseminators to pass the rod too deep and deposit the semen into one of the uterine horns, thereby reducing fertility if ovulations are taking place at the opposite ovary.
Flushing the embryos
To collect the embryos nonsurgically, a small synthetic rubber catheter is inserted through the cervix of the donor cow, and a special medium is flushed into and out of the uterus to harvest the embryos seven or eight days after estrus. This collection procedure is relatively simple and can be completed in 30 minutes or less without harm to the cow. A presterilized stylet is placed in the lumen of the catheter to offer rigidity for passage through the cervix into the body of the uterus. When the tip of the catheter is in the body of the uterus, the cuff is slowly filled with approximately 2 ml of normal saline. The catheter is then gently pulled so that the cuff is seated into the internal os of the cervix. Additional saline is then added to the cuff to completely seal the internal os of the cervix. A Y-connector with inflow and outflow tubes is attached to the catheter. A pair of forceps is attached to each tube to regulate the flow of flushing fluid. The fluid is sequentially added and removed by gravity. The fluid in the uterus is agitated rectally, especially in the upper one-third of the uterine horn. The uterus is finally filled with medium to about the size of a 40 day pregnancy. One liter of fluid is used per donor. Many operators use a smaller volume and flush one uterine horn at a time. Each uterine horn is filled and emptied five to ten times with 30 to 200 ml of fluid each time, according to size of the uterus. The embryos are flushed out with this fluid into a large graduated cylinder. After about 30 minutes, embryos settle and can be located under a stereomicroscope by searching through an aliquot from the bottom of the cylinder.
Evaluation of the embryos
As the individual embryos are located using the microscope, they are evaluated for their quality and classified numerically as to the potential likelihood of success if transferred to a recipient female. The major criteria for evaluation include:
• Regularity of shape of the embryo
• Compactness of the blastomeres (the dividing cells within the boundaries of the embryo)
• Variation in cell size
• Color and texture of the cytoplasm (the fluid within the cell wall)
• Overall diameter of the embryo
• Presence of extruded cells
• Regularity of the zona pellucida (the protective layer of protein and polysaccharides around the single celled embryo)
• Presence of vesicles (small bubble-like structures in the cytoplasm)
Embryos are classified according to these subjective criteria as: Grade
1: Excellent or Good Grade
2: Fair Grade
3: Poor Grade
4: Dead or degenerating
Embryos also are evaluated for their stage of development without regard to quality. These stages are also numbered:
Stage 1: Unfertilized
Stage 2: 2 to 12 cell
Stage 3: Early morula
Stage 4: Morula
Stage 5: Early Blastocyst S
tage 6: Blastocyst
Stage 7: Expanded Blastocyst
Stage 8: Hatched Blastocyst
Stage 9: Expanding Hatched Blastocyst
There is apparently no difference in pregnancy rates of fertilized cells in different stages of development assuming that they are transferred to the recipient female in the appropriate stage of the estrous cycle. Stage 4, 5, and 6 embryos endure the freezing and thawing procedures with the greatest viability. Embryo quality is also of utmost importance in the survival of the freezing and thawing stress. Grade 1 embryos generally are considered the only ones to freeze. Grade 2 embryos can be frozen and thawed, yet pregnancy rates typically are reduced. In a Louisiana study involving 1,116 beef and dairy cows of 15 breeds, 58% of all embryos were transferable, 31% were unfertilized, and 11 % were degenerated.
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