动物实验,胚胎移植 2020-08-13 484

　　Embryo transfer technology is one of the most popular animal reproductive technologies, because it is a necessary technical tool for research and development of various embryo biotechnologies (such as cloning, genetic modification, in vitro fertilization and gender identification).

　　Cow donor and acceptor selection

　　The person who provides the embryo is called the donor, and the person who receives the embryo is called the recipient.

　　(1) Selection of Donor Cattle: Principles of Donor Cattle Selection When formulating and applying an embryo transfer plan, you need to carefully select suitable donor cattle, which should be healthy, disease-free and have high reproductive value. It's required. Reproductive function is at a high level. Taking into account the technical and economic aspects, we can refer to the following principles: ①Rich varieties. It meets the standards of this breed, has excellent pedigree, appearance, productivity, early maturity and longevity, genetic stability, clear pedigree and no genetic defects. ② Healthy physique. Physical fitness, strong limbs, normal reproductive function, genetic disease, infection, dystocia, abortion and reproductive system disease, or very low rate of midwifery. There is no obvious estrus cycle, endometritis or long-term fasting reproductive diseases cannot be used as a donor. ③Appropriate age. The suitable age is 15 months to 8 years old. ④Playback quality. It has high fertility, normal reproductive organs and normal estrus cycle. The estrus cycle in infancy is normal (or at least 2 normal estrus before), and there are 2 normal estrus records in 60 days postpartum (1 year good concept, good pregnancy). Conception rate and reproductive index) give birth to calves. ⑤ Ovulation performance. The reproductive organs of cattle, especially the ovaries and uterus, are well-developed and have a good response to superovulation, high ovulation frequency and high quality of fertilized eggs. ⑥Good temperament. Selectively docile cows are used as donors that are easy to handle. Cows expected to be donors must have complete production records (delivery, milking) to determine their reproductive value before being selected as a donor.

　　(2) Selection of recipient cows: recipient cows are only used for pregnancy and do not need genetic characteristics, but the estrus cycle must be normal, reproductive organs are free, large and healthy, cheap and low-yielding young cows or cows As the recipient. Treat donor and recipient cattle simultaneously during estrus

　　The physiological changes of cattle during the estrus cycle are great, so the physiological conditions of the donor and recipient must be kept constant. Otherwise, the transplanted embryo will not survive. .. Therefore, the estrus time of the donor cow and the recipient cow should be the same or similar, and should not exceed 1 day. Therefore, donors and recipients should be treated simultaneously with estrus.

　　in vitro fertilization embryo production

　　Embryo transfer usually uses superovulation to obtain good embryos, but this method is inefficient and expensive. Since the 1980s, developed countries have conducted extensive research on in vitro fertilization embryo production technology and made epoch-making progress. At present, the laboratory production technology of bovine embryos has become a routine technology in many developed countries. Improving laboratory embryo production technology is not only economically important in actual production, but not only can produce a large number of cheap embryos, but also an important technical tool for theoretical research. High-tech technologies such as cloned animal production and transgenic animal production must be based on laboratory embryo culture technology.

　　Currently, there are two main methods for culturing bovine embryos in the laboratory. One is the symbiotic system. The fertilized egg and the adhered cultured epithelial cells are adhered to the cultured oviduct epithelial cells, granulosa cells, etc. on the petri dish, and cultured until a usable embryo is formed. The co-culture system is a complex, time-consuming and laborious embryo culture method. The other is the artificial fallopian tube transfusion (SOF0 culture system, relatively speaking, the operation method is relatively simple, and due to recent research and improvement, the culture effect of SOF culture system is a co-culture system). Meet or exceed

　　Using ultrasonic induction is a method of collecting oocytes from cows and producing embryos through in vitro fertilization. This technology has begun to be applied in production. For cattle that do not have reproductive disorders or superovulation caused by aging, the raw egg collection technology will definitely produce high-quality embryos. The laboratory embryo production technology is a complex system engineering. The whole process includes the collection of oocytes, the maturation of oocytes, the fertilization of sperm, the processing of sperm and eggs, fertilization and fertilization. Culture method, embryo development and culture method In order to obtain the ideal laboratory embryo production effect, it is necessary to improve the efficiency of each step to achieve the most perfect combination of the entire process. Refers to the use of lavage fluid to wash off the early embryos from the uterine horns of the donor cow and collect them in specific blood vessels through surgical and non-surgical methods. There are two methods, and non-surgical methods are currently commonly used: embryos are usually collected within 6 to 7 days after the donor cow is in heat and mating. This stage is simple and easy to implement, the risk of damage to the reproductive tract is low, and the collection effect is good. The front is high and the back is low, which is easy to handle and rinse the embryos, cut off the spine (3-4 tail vertebrae), and disinfect with alcohol, 2% procaine 2-3 ml or 2%. In the case of epidural anesthesia, use 4 to 2 ml of lidocaine hydrochloride (eg 4 to 2%), if the cow is anxious, inject 2% to 4 ml of Jingsongling intramuscularly. Estimate the number of corpus luteum by checking the size of the vulva, clean the vulva, and sterilize the parts with 2-way and 3-way flushing tubes for non-surgical flushing of embryos. The difference is that the flushing fluid is injected and discharged, the two-way type is the same pipe, the three-way type has 2 inlets and 2 outlets, and the other 2 types are fallopian tubes. , There is a duct for injecting air, which can fix the parts when washing the bottom of the uterine horn with water. However, airbags need to be properly inflated and can easily damage the uterus. If the endometrium is too small, the irrigation tube cannot be fixed, leaving a gap in the uterus, and the irrigation fluid will flow out of the vagina. Usually, 500 ml of oocyte wash solution must be used to rinse both sides of the uterine horns at 37°C. The egg liquid should be discharged smoothly and its volume should be the same. Therefore, during the operation, the uterus can be manually lifted through the rectal wall to facilitate infusion. The collected eggs should be placed in a sterile room at 37°C for inspection. Now, the oocyte washing solution used to collect embryos uses modified Dulbecco's phosphate buffered saline (mPBS), which can be mainly used to wash embryos and embryo storage and culture media. The preparation method is simple, low in cost and more suitable. Let the collected egg wash liquid stand for 10 to 30 minutes. After immersing the embryo, aspirate the upper layer and pour the remaining sediment (about 50 ml) into multiple egg test cups. The embryos are detected under a stereo microscope. During the inspection process, the embryos must be carefully and quickly maintained to ensure that they are not lost, and the inspection is completed within 30 minutes to prevent the increase in the temperature of the microscope light source from affecting the quality of the embryos.

　　Place the collected embryos under a high-power microscope to observe their morphology and developmental period. The developing embryo is usually a neat, transparent blastomere, relatively uniform size, uniform and tight distribution, complete zona pellucida and an age-matched development rate. However, unfertilized embryos, zona pellucida and zona pellucida damage are all unusable embryos.

　　Embryo Transfer

　　Bovine embryo transfer methods include surgical transplantation and non-surgical transplantation. Currently, non-surgical transplantation is mainly used. A special embryo transfer device is used to inject embryos into specific parts of the uterine horn through the vagina and cervix. The operation steps are as follows.

　　(1) Carrying and disinfecting the receiving cattle: the same method as the donation cattle.

　　(2) Embryo cleaning and packaging: Wash the identified good embryos with fresh antiseptic solution 2-3 times to remove mucus and adhering impurities, and the embryos are made of 0.25 ml plastic. Encapsulated in a thin tube. .. For encapsulation, first suck the preservation solution into a thin tube about 5 cm long, then form a 0.5 cm air layer in the tube, and then suck the embryo containing the preservation solution about 2.5 cm long. Then an air layer of 0.5 cm is formed. , And finally suck the stock solution to the end of the test tube. After the embryo is loaded, the thin tube should be checked under a microscope to see if the embryo is in the tube.

　　(3) Embryo transfer: Insert the thin tube covering the embryo into the transfer device with the open end (the end away from the cotton core) facing inward. Check the structure of the corpus luteum on the ovulation side of the cow's ovary in the rectum. Grasp the cervix from the rectum, send the transplant to the cervix, grasp the uterine horn of the rectum, and then connect it to the uterine horn on the corpus luteum. Gently push the introducer through the large elbow of the uterine horn, then push the embryo in and slowly remove the syringe. During the operation, the movements should be smooth and fast, the transplantation site should be accurate, and the tubules should be checked for lack of embryos after transplantation. If you drop out of school, you need to replant.

　　Postoperative observation of donors and recipients

　　After embryo transfer, please pay attention to the health of the donor and recipient, and whether they have a fever within the specified time. Donor cows can be raised during the next estrus period, but if a donor is still needed, ovulation usually takes several months. For the recipient cow, estrus after transplantation indicates that the transplantation failed. The embryo may be lost, dead, absorbed or defective, or the uterine environment of the recipient cow may be inappropriate. If the temperature is not high, continue to observe 3-5. During the estrus period, pregnancy diagnosis should be performed at an appropriate time.