Embryo transplantation, also known as fertilized egg transplantation, involves transplanting a fertilized egg of a female (donor) into the uterus of another female animal (recipient) to make it grow normally, commonly known as "pregnant childbirth." (A) The significance of embryo transfer 1. Give full play to the reproductive potential of excellent cows. Under normal circumstances, an excellent adult cow can only breed one yak per year. Using embryo transfer technology, one can obtain offspring of several to several dozen fine cows a year. Accelerated the establishment and expansion of elite cattle. 2. Inducing twin cows to produce mating cows and then transplanting one embryo to the ovulation contralateral uterine horn. Cows that have not been conceived after such mating may be pregnant by receiving transplanted embryos, while cows that are bred after mating have a twin because of the addition of a transplanted embryo. In addition, an uncommitted cow can also transplant one embryo at each uterine horn and carry a twin, thereby improving production efficiency. 3. The application of embryo transfer can also reduce the number of breeding cows for meat breeding, and can replace the introduction of breeding stocks and preservation of species resources. (B) The physiological basis of embryo transfer 1. The gestational development of the reproductive organs of cows after estrus develops during the first period after estrus (periodic luteal phase). Whether or not they have been fertilized, the cow's reproductive system is under physiological conditions after fertilization. Physiological phenomena, pregnancy and There is no difference in pregnancy. Therefore, the gestational changes of the genital organs of cows after estrus are the main physiology basis for the development of embryos. 2. Early embryonic free state embryos exist independently for a considerable period of time (before attachment). Their development depends essentially on their own storage of nutrients and has not yet established a substantial relationship with the uterus. Therefore, in the case of leaving the living body, it can survive for a short period of time. When it is put back into the same environment as the donor, it can continue to develop. 3. Embryo transplantation does not have immune problems. The genital tract (uterus and fallopian tubes) of the recipient's female reproductive tract (uterus and fallopian tubes) have foreign antigenic material. In general, there is no immune rejection within the same species. It is extremely advantageous to continue the development of an embryo after it has been transplanted from one body to another. 4. Embryos and Recipients Linked Embryos will establish physiological and tissue connections with the receptor's endometrium over a period of time to ensure normal development. In addition, the receptor does not have a genetic effect on the embryo and does not affect the inherently good traits of the embryo. (c) Operational principles of embryo transfer The consistency of the environment before and after the embryo transfer, that is, the living environment after the embryo transfer is compatible with the development stage of the embryo. Including the physiological consistency (ie, the consistency of the donor and the recipient on the time of estrus) and the anatomical consistency (ie, the similarity of the transplanted embryo with the space environment before transplantation) and the species identity. Sex (ie donor and recipient are in the same species, but does not exclude the possibility of successful transplantation between species). 2. Embryo Collection Term The period of embryo collection and transplantation (day of the embryo) cannot exceed the life span of the corpus luteum, and should be transplanted several days before the corpus luteum degeneration. The embryos are usually collected and transplanted within three to one days after the estrous mating of the donor. 3. During all operations, embryos should not be compromised by any adverse factors (physical, chemical, or microbial). Transplanted embryos must be identified and considered normal. (IV) Basic Procedures for Embryo Transfer The basic procedures for embryo transfer include donor superovulation and insemination, simultaneous estrus treatment of recipients, egg collection, ovulation and transplantation. Concerning the process of superovulation and estrus synchronization, it has been described above. This book only describes egg collection, egg retrieval and transplantation. 1. Embryo recovery (Ovicubation) Methods for collecting embryos from donors are surgical and non-surgical. (1) Surgical methods Preoperative preparation is performed according to the requirements of surgical laparotomy. The surgical site was cut at the abdomen white line between the right flank or the lower abdomen to the umbilicus. Stretch your index finger to find the fallopian tubes and uterine horns, leading out of the incision. If the egg is in the 3-4 days after insemination, the fertilized egg has not migrated to the uterine horn, and the fallopian tube may be used for the method of ovulation. A 2-mm-diameter, 10-cm-long polyethylene tube is inserted 2-3 cm from the abdominal cavity of the fallopian tube. The other syringe is used to suck 5-10 ml of egg-soaked liquid at about 30°C, and the 7-gauge needle is connected to puncture the front end of the uterine horn. , And then send people to the isthmus of the fallopian tubes and inject the flushed egg. The puncture needle should be blunt to avoid damage to the lining of the uterus; the rinsing rate should be slow so that the irrigating solution flows continuously. If embryos are harvested 5 days after insemination, uterine horns must also be flushed. That is, 10 to 15 ml of flushed egg is flushed from the upper part of the uterine horn of the uterine tube junction to the uterine horn bifurcation. In order to prevent the oviduct from flowing out of the fallopian tube, a hemostatic forceps can be used to clamp the fallopian tube near the junction of the uterine tube, insert the recovery needle at the bifurcation of the uterine horn, and clamp the uterus with the intestinal clamp to the back of the recovery needle to fix the recovery needle. Do not flush the egg fluid to the uterus. (2) Non-surgical non-surgical harvesting is generally performed 5-7 days after insemination. A two-way catheter may be used for the oocyte. The two-way egg occluder consists of a catheter with a balloon and a single tube. One way is to inflate the airbag, and the other way is to inject and recover the flushing liquid. Insert a metal rod in the catheter to increase the hardness, making it easy to pass through the cervix. The catheter is usually introduced into the uterine horn through the cervix using a rectal grasp method. To prevent contraction of the cervix and impatience of the cows, an epidural anesthesia can be performed with 2% of procaine or lidocaine 5 to 10 ml in the lumbar sacral or coccygeal space at the time of spawning. Wash the genital area before operation and disinfect with alcohol. In order to prevent the catheter from being contaminated in the vagina, a mantle (sold with a product) can be placed over the catheter, and when the catheter is inserted into the cervix, the mantle is pulled away. After the catheter is inserted into the uterine horn of one side, the balloon is inflated from the filling tube, so that the balloon is inflated and touches the inner wall of the uterine horn to prevent backflow of the flushing fluid. Then pull out the pass rod, inject the flushing liquid into the uterine horn through a single tube, each time 15-50 ml, rinse 5-6 times, and collect the flushing liquid in a funnel-shaped container. To recover more of the flushed egg, gently massage the uterine horn in the rectum. Rinse the contralateral uterine horn in the same way. Most of the flushed eggs are tissue culture fluids, such as Ringer's solution, Dulbecco's phosphate buffer solution (PE), Bronsted's solution (BMOC-3) and TCM-199. Commonly used in Dulbecco's phosphate buffer, add 0.4% bovine serum albumin or 1% to 10% calf serum. Oxygen temperature should be used when the 35-37 ° C, to add 1000 international units of penicillin per 1000, streptomycin 500-1000 micrograms, to prevent reproductive tract infection. 2. Embryo Examination (1) Oviposition The collected egg-laying liquid is allowed to stand in a 37°C incubator for 10-15 minutes. After the embryos sink, remove the supernatant. Take a small amount of liquid from the bottom and move it to the plate. After standing, check the number of embryos under a stereoscopic microscope at a low magnification (10 to 20 times), and observe the embryo quality at a large multiple (50 to 100 times). (2) Absorption and absorption of eggs is to remove, clean and process the embryos. The target is accurate, the speed is fast, and the amount of fluid is small and there is no loss. Absorption can be carried out using a 1 ml syringe attached to a special tip, or a homemade suction tube can also be used. (3) Embryo Quality Identification Normally developed embryos, in which the cells (blastomeres) are uniform in shape, uniform in size, evenly distributed, and the outer membrane is intact. No cleavage (unfertilized) and abnormal eggs (rupture of outer membrane, rupture of blastomere, etc.) can be used for transplantation. 3. Embryo Transplantation (1) Surgical Transplantation Recipient cows are prepared preoperatively. Cows have been bred, incised in the right flank, and the non-ovulatory uterine horns have been found. A syringe or oviduct that sucks embryos has been inserted into the uterine horn and injected into the embryo; unmatched cows have been injected into each uterine horn. One embryo; then the uterus is repositioned and the incision is closed. (2) Non-surgical transplantation Non-surgical transplantation is generally performed on the sixth to ninth days after estrus (ie, the blastocyst stage), and premature implantation will affect the conception rate. In the non-surgical transplantation using embryo transfer gun and 0.25 ml fine tube transplantation better. Cut a small amount of the straw, inhale a little of the preservation liquid, suck a gas bubble, and then inhale a little of the liquid containing the embryo, inhale a gas bubble, and finally suck a little of the liquid. The straw containing the embryo was placed in a transplant gun and inserted into the deep part of the uterine horn through the cervix and injected into the embryo. Non-surgical transplants must strictly comply with the aseptic procedures to prevent genital tract infections.
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