In vitro fertilization (IVF) is a technique in which mammalian sperm and egg complete the fertilization process in an artificially controlled environment outside the body. Because it is inseparable from embryo transfer (ET), it is also referred to as IVF-ET. In biology, the animals obtained after IVF embryo transfer to the mother are called test-tubing animals. The study of IVF in rabbits in China began in the 1980s, and the routine process of IVF in rabbits mainly includes several steps, such as supernumerary ovulation, oocyte acquisition, sperm collection and in vitro fertilization.
Operation method
In vitro fertilization in rabbits
Materials and Instruments
Equipment: Move The basic process of in vitro fertilization in rabbits can be divided into the following steps: 2. 12 hours after the last FSH injection, 200 U of human chorionic gonadotropin (hCG) was injected intramuscularly, and then the super-exposed female rabbits were mated with male rabbits in a combined cage. 2. To collect embryos or eggs from the oviductal area, a 3-5 cm incision was made backward along the white line of the abdomen, the abdominal cavity was opened, the oviducts were removed, and a flared opening was identified with a glass probe, and a flushed oviduct with an inner diameter of 1-2.5 mm was inserted and bound with a thread. Then a syringe with 18-22 gauge needle was inserted from the uterine tube junction towards the fallopian tube and 3-5 ml of egg flushing solution was injected for flushing and collected in a 6 cm Petri dish. 3. When embryos or eggs were retrieved from the uterus, the incision was made slightly behind the fallopian tube, about 3 cm, the abdominal cavity was opened, the uterine horns were removed and clamped at the anterior 1/4 of the uterus with a small vascular clip, and the uterine wall was perforated with a veterinary 16-18-gauge syringe needle in front of the clip where there were no large blood vessels. Then the needle was inserted into the uterine cavity from the connection part of the uterine tube, and 5-10 ml of egg flushing fluid was injected into it, so that it flowed out from the small hole and was collected in a 6 cm Petri dish. 2. The experimenter grasped the rabbit's ears and neck skin and held the sperm collector and extended it under the rabbit's belly, so that the opening of the false vagina was close to the underside of the rabbit's vulva, and attention was paid to the fact that the opening of the false vagina was a little low, and the end of the sperm collector was a little high, and the angle of inclination was in line with the angle of the rabbit's penis. 3. When the rabbit's penis was jerked repeatedly, the experimenter should adjust the height and angle of the pseudovagina in time, so that the rabbit's penis could enter the pseudovagina smoothly. 4. Immediately after ejaculation, the open end of the pseudo-vagina should be raised so that the semen can flow into the sperm collection cup to prevent outflow, and then it should be quickly withdrawn from under the abdomen of the rabbit, the sperm collector should be erected, the sperm collection cup should be taken down and the semen sticking to the opening of the external vagina should be introduced into the sperm collection cup, then it should be covered with a lid and labeled, and then it should be sent to artificial insemination room for semen quality check. 2. slowly add 4 ml of DM-heparin solution, and then centrifuge at 1500 r/min for 5 minutes, discard the supernatant. 3. Add 5 ml of HIS solution to suspend the spermatozoa, seal with a rubber stopper and incubate in a water bath at 38 ℃ for 15 min, then centrifuge at 1500 r/min for 5 min, discard the supernatant. 4. The spermatozoa were suspended again with 5 ml of DM-heparin solution and centrifuged at 1000 r/min for 5 min, and 4 ml of the supernatant was aspirated. 5. The remaining 1 ml of DM sperm suspension was gently shaken in a water bath at 38 ℃ for 3 min and then sealed, and incubated at a 45-degree inclination in an incubator at 38.2 ℃ with 5% CO2 for 4 hours. 6. In a 3 cm Petri dish, use a micropipette to make 3-5 drops of 50 μl TCM-199+ rabbit serum culture droplets, put normal oocytes into the droplets, and put about 20 eggs into each droplet, and then add 7.5 μl of in vitro energized semen, and then add 7.5 μl of in vitro energized semen. 7. Cover the droplets with sterilized liquid paraffin and wrap the petri dish with tin foil, place the petri dish in an incubator with 5% CO2 at 38.2 ℃, incubate for 6 hours, and then repeat the fertilization once more. 8. After 12 hours of fertilization, the eggs were detected and washed twice with TCM-199 solution, transferred into newly made TCM-199 + rabbit serum culture droplets, covered with sterilized liquid paraffin, wrapped with tin foil and placed in a 5% CO2 , 38.2 ℃ incubator for incubation. For more product details, please visit Aladdin Scientific website.
① Syringe with 18 to 22 gauge needle
②Glass probe
③Sperm collection cup
④5% CO
2
④5% CO 2 , 38.2 ℃ incubator
⑤5 ml centrifuge tube
⑥Centrifuge
Reagents:
①Material: rabbit
② Follicle stimulating hormone (FSH)
③ Egg flushing fluid:
DPBS (15240-013, Invitrogen) + 0.1% PVA (P-8136, Sigma)
④Human chorionic gonadotropin (hCG)
⑤ Procaine hydrochloride
⑥ DM-heparin solution
⑦HIS solution
⑧TCM-199 + rabbit serum culture droplet
IV. In vitro fertilization