Rabbit artificial insemination technology
1 Semen collection method Semen collection is the primary link of artificial insemination in rabbits. The following is a brief description of how to use fake vaginal sampling and insemination:
1.1 Production of fake vagina: Use a hard plastic tube with a diameter of 5cm or so and a length of about 8cm to dip the two ends. Place a soft sponge of similar internal diameter to the plastic tube. Use a small hole in the direction of the straight axis of the sponge. For 0.5cm, the orifice is umbrella-shaped. Set hardcover can use large condoms instead.
1.2 Equipment preparation before sperm collection: loosen the washed and disinfected condom and insert it into the false vaginal tube so that one end of the opening is turned over and fixed on the edge of the outer shell. The other end of the inner tube with the bladder is exposed to about 1 cm. Inject 50 to 60°C warm water to absorb the sponge, and use a thermometer to take saline or paraffin into the vagina and insert it into the vagina to measure the temperature. When it is reduced to about 43 °C, it can be refined. Temperature measurement should pay attention to external conditions, such as low temperatures, rapid cooling, inner tube temperature higher; on the contrary can be lower. Only 1-2 times for male rabbits are collected, and if two times, the second time the sperm is immature.
1.3 Semen collection: The location of sperm extraction can be in the male rabbit cage. When the male rabbit climbs back, the right hand presents a 43-corner false vagina in the middle of the female rabbit's hind leg and waits for the rabbit to ejaculate. Immediately afterwards, leave the false vaginal orifice upward to prevent loss of semen. 1.4 The fake vaginal weeping has a direct effect on the sperm extraction effect. Male rabbit penis is very sensitive to temperature, less than 40 °C is not easy to cause ejaculation; high temperature extraction will develop a bad habit of psychiatric hyperthermia, but also affect the quality of semen. Since the normal temperature of the female rabbit is between 39 and 40.5°C, the temperature of the pseudo vagina is preferably between 40 and 42°C.
2 Semen inspection and dilution
2.1 Inspection of semen
2.1.1 Appearance check: normal male rabbits' semen is pale milky, with a special smell, but also a few rabbits' semen is not normal, the yellow color mixed with urine has an odor; the genitals have inflammation and reddish bleeding; water-like It is azoospermia. The precision of rabbits is very small, generally between 0.2 and 2 mL, with an average of 0.8 mL. In practical applications, it is generally estimated visually.
2.1.2 Microscopic examination: It is mainly the inspection of sperm density and the assessment of vitality. Sperm density refers to the number of sperm contained within a unit volume. The calculation of sperm count is directly related to the number of live spermatozoa contained in the insemination dose. Visual inspection is commonly used in practice. The visual inspection method is based on the distribution of sperm in the visual field, that is, the distance between sperm and sperm is rated as dense, medium, and lean. Although this method is a subjective assessment, it is simple and easy to use. This method is often used in practical work. The sperm density of rabbits also varies with the seasons.
Sperm activity refers to the percentage of spermatozoa that move straight in semen. The marker 0.90.80.7..., which represents 90%, 80%, 70%,... of spermatozoa with forward motion. This is a subjective assessment method, and the results estimated by experienced technicians are often close to the actual number. The rabbit's sperm motility should be used above 0.5 to insemination. If it is below 0.5, the fertilization rate is very low. The differences in sperm motility in different seasons are also quite significant. The average vigor from September to October is only 0.15; the average vigor from December to February is 0.51; the average vigor from March to May is 0.61.
2.2 Dilution of Semen The purpose of the dilution of semen is to increase the amount of semen and increase the number of inseminating female rabbits. It is also to extend sperm survival time. The following factors should be taken into account when preparing diluted preservation solutions.
2.2.1 Nutrients: Spermatozoa are mainly unilaterally decomposed during metabolism. Mature sperm cannot convert foreign substances into their own components through assimilation. Therefore, in order to supplement the energy consumption of spermatozoa, only the simplest nutrients are currently used: fructose, sorbitol, glycerophosphocholine (GPC) and phospholipids.
2.2.2 Protective ingredients. Buffer substances?? In order to maintain the appropriate pH of semen, sodium citrate, potassium sodium tartrate, potassium dihydrogen phosphate and disodium hydrogen phosphate are commonly used. Anti-cold protection agent?? commonly used lecithin, egg yolk, protein Chen, milk. The bacteriostatic substances are usually penicillin (500 to 1,000 units per 1 mL of dilution) and streptomycin (500 to 1000 μg per mL of dilution).
The ideal dilution for rabbit sperm is widely used in 0.9% sodium chloride or 5% dextrose solution, and the dilution is usually 9 times. The dilution temperature should be isothermal with the semen. Dilution of large bottles (bags) should not be too fast. After the injection, stir gently with a glass rod to make it evenly mixed. Diluted semen should be subjected to a microscopic examination and sperm found to be inactive or dead too much should not be used for insemination.
3 Insemination method Insemination is the injection of semen into the genital tract of a female rabbit and is the last link of artificial insemination. 3.1 The spermatorrhea: glass angiosperm can be used, the small l0cm long hollow glass tube connected with the 2mL syringe, can also be used experimental dropper, each insemination should be replaced once a sterile transfusion device.
3.2 Selection of female rabbits for transmission: The health and disease-free female rabbits should be selected for insemination during estrus, which is of great benefit to improving the conception rate.
3.3 Stimulation of ovulation in the female rabbit: After estrus in the female rabbit, no ovulation occurs if there is no mating or other implication. Artificial insemination lacks the stimulative nature of mating. However, the injection of Hermaphrodite No. 3 in the female rabbits had better ovulation induction effect. Each rabbit was injected with 0.5 μg, and the injection of the rabbit before and after insemination was equally effective.
3.4 Insemination: The assistant grasps the back of the female rabbit so that the female rabbit buttocks are slightly upwards, and the inseminator holds the insemination device (for example, one person can grasp the rabbit with one hand, and the other hand with an insemination device). Diluted semen O.5mL (containing more than 10 million sperm) slowly inserted into the vagina of the female rabbit about 6 ~ 7cm can be inseminated. The insemination device and semen must be warmed to the same temperature as the rabbit's body temperature, and warmed in warm water at 35°C before insemination. When inserting, it should be swiveled gently forward and upward, and it must be swiveled in. It must not be hardened to avoid damaging the genital tract. After insertion, you can gently twitch and rotate several times back and forth to help stimulate the cervix. When the sperma sample is withdrawn, it should be slowly pulled out while rotating to prevent the semen from overflowing with the sperm. After taking out the anaesthetic, the assistant beats the rabbit's buttocks, causing the female rabbit to be frightened so that the sperm can inhale.
0.8 mL of spermatozoa are required for each infusion and dilution. One female rabbit can be fed with 10 female rabbits after dilution. The entire artificial insemination process requires accurate and sterile operation.
1.1 Production of fake vagina: Use a hard plastic tube with a diameter of 5cm or so and a length of about 8cm to dip the two ends. Place a soft sponge of similar internal diameter to the plastic tube. Use a small hole in the direction of the straight axis of the sponge. For 0.5cm, the orifice is umbrella-shaped. Set hardcover can use large condoms instead.
1.2 Equipment preparation before sperm collection: loosen the washed and disinfected condom and insert it into the false vaginal tube so that one end of the opening is turned over and fixed on the edge of the outer shell. The other end of the inner tube with the bladder is exposed to about 1 cm. Inject 50 to 60°C warm water to absorb the sponge, and use a thermometer to take saline or paraffin into the vagina and insert it into the vagina to measure the temperature. When it is reduced to about 43 °C, it can be refined. Temperature measurement should pay attention to external conditions, such as low temperatures, rapid cooling, inner tube temperature higher; on the contrary can be lower. Only 1-2 times for male rabbits are collected, and if two times, the second time the sperm is immature.
1.3 Semen collection: The location of sperm extraction can be in the male rabbit cage. When the male rabbit climbs back, the right hand presents a 43-corner false vagina in the middle of the female rabbit's hind leg and waits for the rabbit to ejaculate. Immediately afterwards, leave the false vaginal orifice upward to prevent loss of semen. 1.4 The fake vaginal weeping has a direct effect on the sperm extraction effect. Male rabbit penis is very sensitive to temperature, less than 40 °C is not easy to cause ejaculation; high temperature extraction will develop a bad habit of psychiatric hyperthermia, but also affect the quality of semen. Since the normal temperature of the female rabbit is between 39 and 40.5°C, the temperature of the pseudo vagina is preferably between 40 and 42°C.
2 Semen inspection and dilution
2.1 Inspection of semen
2.1.1 Appearance check: normal male rabbits' semen is pale milky, with a special smell, but also a few rabbits' semen is not normal, the yellow color mixed with urine has an odor; the genitals have inflammation and reddish bleeding; water-like It is azoospermia. The precision of rabbits is very small, generally between 0.2 and 2 mL, with an average of 0.8 mL. In practical applications, it is generally estimated visually.
2.1.2 Microscopic examination: It is mainly the inspection of sperm density and the assessment of vitality. Sperm density refers to the number of sperm contained within a unit volume. The calculation of sperm count is directly related to the number of live spermatozoa contained in the insemination dose. Visual inspection is commonly used in practice. The visual inspection method is based on the distribution of sperm in the visual field, that is, the distance between sperm and sperm is rated as dense, medium, and lean. Although this method is a subjective assessment, it is simple and easy to use. This method is often used in practical work. The sperm density of rabbits also varies with the seasons.
Sperm activity refers to the percentage of spermatozoa that move straight in semen. The marker 0.90.80.7..., which represents 90%, 80%, 70%,... of spermatozoa with forward motion. This is a subjective assessment method, and the results estimated by experienced technicians are often close to the actual number. The rabbit's sperm motility should be used above 0.5 to insemination. If it is below 0.5, the fertilization rate is very low. The differences in sperm motility in different seasons are also quite significant. The average vigor from September to October is only 0.15; the average vigor from December to February is 0.51; the average vigor from March to May is 0.61.
2.2 Dilution of Semen The purpose of the dilution of semen is to increase the amount of semen and increase the number of inseminating female rabbits. It is also to extend sperm survival time. The following factors should be taken into account when preparing diluted preservation solutions.
2.2.1 Nutrients: Spermatozoa are mainly unilaterally decomposed during metabolism. Mature sperm cannot convert foreign substances into their own components through assimilation. Therefore, in order to supplement the energy consumption of spermatozoa, only the simplest nutrients are currently used: fructose, sorbitol, glycerophosphocholine (GPC) and phospholipids.
2.2.2 Protective ingredients. Buffer substances?? In order to maintain the appropriate pH of semen, sodium citrate, potassium sodium tartrate, potassium dihydrogen phosphate and disodium hydrogen phosphate are commonly used. Anti-cold protection agent?? commonly used lecithin, egg yolk, protein Chen, milk. The bacteriostatic substances are usually penicillin (500 to 1,000 units per 1 mL of dilution) and streptomycin (500 to 1000 μg per mL of dilution).
The ideal dilution for rabbit sperm is widely used in 0.9% sodium chloride or 5% dextrose solution, and the dilution is usually 9 times. The dilution temperature should be isothermal with the semen. Dilution of large bottles (bags) should not be too fast. After the injection, stir gently with a glass rod to make it evenly mixed. Diluted semen should be subjected to a microscopic examination and sperm found to be inactive or dead too much should not be used for insemination.
3 Insemination method Insemination is the injection of semen into the genital tract of a female rabbit and is the last link of artificial insemination. 3.1 The spermatorrhea: glass angiosperm can be used, the small l0cm long hollow glass tube connected with the 2mL syringe, can also be used experimental dropper, each insemination should be replaced once a sterile transfusion device.
3.2 Selection of female rabbits for transmission: The health and disease-free female rabbits should be selected for insemination during estrus, which is of great benefit to improving the conception rate.
3.3 Stimulation of ovulation in the female rabbit: After estrus in the female rabbit, no ovulation occurs if there is no mating or other implication. Artificial insemination lacks the stimulative nature of mating. However, the injection of Hermaphrodite No. 3 in the female rabbits had better ovulation induction effect. Each rabbit was injected with 0.5 μg, and the injection of the rabbit before and after insemination was equally effective.
3.4 Insemination: The assistant grasps the back of the female rabbit so that the female rabbit buttocks are slightly upwards, and the inseminator holds the insemination device (for example, one person can grasp the rabbit with one hand, and the other hand with an insemination device). Diluted semen O.5mL (containing more than 10 million sperm) slowly inserted into the vagina of the female rabbit about 6 ~ 7cm can be inseminated. The insemination device and semen must be warmed to the same temperature as the rabbit's body temperature, and warmed in warm water at 35°C before insemination. When inserting, it should be swiveled gently forward and upward, and it must be swiveled in. It must not be hardened to avoid damaging the genital tract. After insertion, you can gently twitch and rotate several times back and forth to help stimulate the cervix. When the sperma sample is withdrawn, it should be slowly pulled out while rotating to prevent the semen from overflowing with the sperm. After taking out the anaesthetic, the assistant beats the rabbit's buttocks, causing the female rabbit to be frightened so that the sperm can inhale.
0.8 mL of spermatozoa are required for each infusion and dilution. One female rabbit can be fed with 10 female rabbits after dilution. The entire artificial insemination process requires accurate and sterile operation.
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