A method of artificial insemination of sheep

Abstract

The application provides a method for artificial insemination of sheep, which synchronously induces estrus of ewes by implanting a vaginal sponge, promotes follicular development by combined injection of pregnant mare serum when the sponge is removed, and ensures accurate mating time by combining estrus testing and timed insemination scheme; after training by supplemental feeding, semen is collected by using a false vagina with a temperature of 38-40 DEG C, and the motility of the diluted semen is maintained above 0.6; at least one of the two ways of conventional insemination or laparoscopic insemination is selected for insemination; the laparoscopic insemination establishes an operation channel by puncturing the base of the udder of the ewe, and the semen is accurately injected into the first third of the large bend of the uterine horn on both sides under the guidance of the laparoscope, so that the pregnancy rate can reach above 90%; and the pregnancy is checked by using B-ultrasound 25-30 days after mating. The method significantly improves the estrus concentration of ewes, the utilization rate of semen and the pregnancy rate, and is suitable for the reproductive management of large-scale and intensive sheep farms.

Description

Technical Field

[0001] This invention belongs to the technical field of artificial insemination methods for livestock, and specifically relates to a method for artificial insemination of sheep. Background Technology

[0002] The current method of artificial insemination in sheep is laparoscopic insemination. However, this method has drawbacks: it is not recommended for sheep over 4 years of age due to the risk of peritoneal adhesions. For multiparous ewes over 4 years of age, there is currently a lack of a safe and efficient insemination method that can guarantee a high conception rate without causing complications such as peritoneal adhesions due to surgical trauma. This has become a technical bottleneck restricting the full realization of the genetic potential of superior ewes and the improvement of overall flock reproductive efficiency. Therefore, there is an urgent need to develop a new artificial insemination technology suitable for ewes of all ages, especially older ewes. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for artificial insemination of sheep, which addresses the shortcomings of the prior art. The method of the present invention can maximize the utilization rate of excellent breeding rams, accelerate breed improvement, and increase the fertility rate of ewes; overcome the difficulties in mating between rams and ewes due to differences in body and tail size, and control the spread of diseases; and significantly reduce the number of breeding rams required, thus saving feeding costs.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for artificial insemination of sheep, the method comprising the following steps:

[0005] S1, Synchronized estrus

[0006] S101. Plugging: Disinfect the vulva of the non-pregnant ewe with benzalkonium chloride solution, place the tug in the tug-planting device, and then implant the tug.

[0007] S102. Removal of thrombus and injection of pregnant mare serum: The day of thrombus insertion is day zero. The thrombus is removed 10 to 14 days after insertion. At the same time as thrombus removal, pregnant mare serum is injected into each non-pregnant ewe.

[0008] S2, Ram semen collection

[0009] S201. Supplementary feeding and training of breeding rams: One month before semen collection, each breeding ram shall be fed supplementary feed daily and exercised for a certain period of time; training shall be provided to breeding rams that are collecting semen for the first time.

[0010] S202. Adjustment, installation, and semen collection of the artificial vagina: Adjust and disinfect the artificial vagina, and control the internal temperature of the artificial vagina; collect semen from breeding rams using the artificial vagina method, and place the semen in a water bath for testing after collection;

[0011] S3, Semen Quality Inspection and Dilution

[0012] The semen collected from breeding rams in S2 was subjected to quality inspection, and qualified semen was screened. The qualified semen and diluent were placed in a constant temperature water bath and allowed to stand for a period of time before being diluted to obtain diluted semen.

[0013] S4. Insemination: The diluted semen obtained in S3 is used to inseminate non-pregnant ewes using one or both of the conventional insemination and laparoscopic insemination methods.

[0014] S5. Pregnancy Examination

[0015] Pregnancy checks were performed on non-pregnant ewes using veterinary ultrasound 25-30 days after mating.

[0016] Preferably, the mass fraction of the benzalkonium chloride solution in S101 is 0.1%; and the injection volume of the pregnant mare serum in S102 is 330 IU.

[0017] Preferably, the supplementary feed mentioned in S201 consists of 500 grams of carrots, 500 grams of alfalfa hay, 2 eggs, and 300 grams of soybean meal; the exercise time is 1 hour; the training methods are as follows: observation training: allowing the rams collecting semen for the first time to observe other rams mating or collecting semen for imitation and learning; massage training: massaging the testicles of the rams at regular intervals every day for no more than 20 minutes, for one week; scent induction: applying the urine of estrus ewes to the tip of the rams' noses to induce the rams to mount actively; drug assistance: injecting each ram with 1-2 mL of testosterone propionate once a day for 3 consecutive days before starting mounting training.

[0018] Preferably, the internal temperature of the artificial vagina in S202 is 38~40 ℃; the temperature of the water bath is 32~34 ℃.

[0019] Preferably, the quality inspection items in S3 include ejaculate volume, color, odor, cloudiness, motility, and density; the temperature of the constant temperature water bath is 30 ℃, and the standing time is 5 min; the dilution method is as follows: slowly inject the diluent along the bottle wall into the qualified semen and mix evenly; the volume ratio of the qualified semen to the diluent is 1:(2~6), and the diluent is a sheep semen diluent with a concentration of 30 million sperm / mL; the sperm motility in the diluted semen is not less than 0.6.

[0020] Preferably, the conventional insemination method described in S4 is as follows: if the non-pregnant ewe is untied and estrus is tested, the first insemination is performed 12 hours after the non-pregnant ewe comes into estrus, and the second insemination is performed 8 to 10 hours later.

[0021] If a non-pregnant ewe does not test for estrus after the removal of the throttle, the first insemination should be performed 48 hours after the removal of the throttle. At the same time, each non-pregnant ewe should be injected intramuscularly with ovulation induction agent No. 3. The second insemination should be performed 8 to 10 hours later.

[0022] The insemination room and insemination equipment are disinfected, and then the diluted semen obtained in S3 is injected into the uterus of a non-pregnant ewe in estrus using a conventional insemination device; the volume of the diluted semen is 0.1~0.3 mL.

[0023] Preferably, the method for testing estrus after unplugging the ewes is as follows: 24 hours after unplugging, estrus is tested in the morning and evening by a ram for 3-5 days; the dosage of the ovulation induction agent is 12.5 micrograms.

[0024] Preferably, the laparoscopic insemination method described in S4 is as follows:

[0025] Stop feeding non-pregnant ewes 24 hours before insemination and stop drinking water 12 hours before insemination. Use timed insemination or estrus-testing insemination. Remove hair and disinfect the surgical site of the non-pregnant ewe, and inject 12.5 micrograms of A3, 5 mL of aminopyrine and 1 million units of penicillin into the muscle.

[0026] 2-5 cm from the base of the udder of an empty ewe, puncture the left and right anterior sides of the udder respectively, and leave the left and right cannulas.

[0027] Insert the laparoscope into the left cannula and adjust the laparoscope's field of view so that the two uterine horns are in the center of the field of view.

[0028] Insert the insemination gun into the right cannula, and vertically pierce the uterine horn at the anterior third of the greater curvature on both sides, injecting 0.1~0.2 mL of diluted semen into the uterine cavity.

[0029] Preferably, the timed insemination is performed on non-pregnant ewes within 42-50 hours after estrus is removed without estrus testing; the estrus testing insemination is performed on non-pregnant ewes within 14-16 hours after estrus.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1. The artificial insemination method of the present invention has a significantly higher conception rate than vaginal insemination by 15-20%.

[0032] 2. The method of the present invention can avoid the risk of infectious diseases caused by sheep mating and greatly improve the utilization efficiency of breeding rams.

[0033] The present invention will be further described in detail below with reference to the embodiments. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] The method for artificial insemination of sheep in this embodiment includes the following steps:

[0037] S1, Synchronized estrus

[0038] S101, Implantation of tethers: Restrain the non-pregnant ewe participating in mating, disinfect the vulva of the non-pregnant ewe with a 0.1% benzalkonium chloride solution, wipe it clean with tissue paper, and then implant the tether;

[0039] The method of implanting the suture is as follows: place the suture in the suture implanter, apply an appropriate amount of lubricant to the front end, separate the vulva with your fingers, slowly insert the suture implanter into the deep part of the vagina, then push the plunger to leave the suture in the vagina, and finally gently remove the suture implanter, clean and disinfect it, and install the suture for later use.

[0040] S102. Removal of the tether and injection of pregnant mare serum: The day of tether insertion is day zero. The tether is removed 10 to 14 days after insertion. When removing the tether, restrain the non-pregnant ewe, pull the thin thread at the tail of the tether, and slowly pull the tether out. At the same time as removing the tether, inject each non-pregnant ewe with 330 IU of pregnant mare serum (PMSG).

[0041] S2, Ram semen collection

[0042] S201. Supplementary feeding and training of breeding rams: One month before semen collection, each breeding ram should be fed 500 grams of carrots, 500 grams of alfalfa hay, 2 eggs and 300 grams of soybean meal daily, and exercise for 1 hour daily; training should be provided for breeding rams collecting semen for the first time.

[0043] The training methods are as follows: First, observation training: allowing rams collecting semen for the first time to observe other rams mating or collecting semen for imitation and learning; second, massage training: massaging the testicles of rams at regular intervals every day for no more than 20 minutes, for one week; third, scent induction: applying urine from estrus ewes to the tip of the ram's nose to induce the ram to mount; fourth, drug assistance: injecting 1-2 mL of testosterone propionate once a day for 3 consecutive days, followed by mounting training.

[0044] S202. Adjustment, installation, and semen collection of the artificial vagina: Adjust and disinfect the artificial vagina, and control the internal temperature of the artificial vagina to 38~40 ℃; collect semen from breeding rams using the artificial vagina method, and place the semen in a water bath at a temperature of 32~34 ℃ for testing after collection;

[0045] The method for adjusting and disinfecting the artificial vagina is as follows: First, check the outer shell and inner tube of the artificial vagina to ensure that there is no damage or burrs; insert the inner tube into the outer shell with the smooth side of the inner tube facing up, exposing both ends of the outer shell, requiring that the lengths are equal; then fold the two ends of the inner tube over the outer shell so that the outer shell and the inner tube of the artificial vagina are concentric circles, and put rubber rings on both ends of the outer shell to fix the inner tube.

[0046] After disinfecting the inner tube and the collection cup with 75% alcohol, cover them with clean gauze and let them air dry for later use; pour warm water at 41~45℃ into the interlayer between the outer shell and the inner tube through the water injection hole, the water volume being one-third to one-half of the interlayer volume;

[0047] Take a small amount of petroleum jelly with a sterilized glass rod and spread a thin layer evenly from the inside to the outside of the artificial vagina. The depth of application should be about one-third of the length of the artificial vagina. Blow air through the nozzle to make the opening of the artificial vagina at the petroleum jelly-coated end appear as a slit.

[0048] The procedure for using the artificial vagina method is as follows: The semen collector holds the sterilized artificial vagina firmly in their right hand, and uses their index and middle fingers to hold the semen collection cup, with the piston of the artificial vagina facing downwards. They squat on the right rear side of the ram. When the ram mounts the ram and its penis extends, the collector gently supports the ram's foreskin with their left hand, tilting the penis slightly towards the semen collection device. At the same time, the artificial vagina is positioned diagonally downwards and aligned with the penis. The ram then guides its penis into the artificial vagina. When the ram's hindquarters thrust forward rapidly, it indicates that the ram has ejaculated. After the ram has finished mounting, the artificial vagina is removed and quickly erected with the end containing the semen collection bottle facing downwards. The air valve on the piston is opened to release air, the semen collection cup is removed and capped, and the semen is collected.

[0049] S3, Semen Quality Inspection and Dilution

[0050] The semen collected from breeding rams in S2 underwent quality inspection, including: ejaculation volume, color, odor, cloudiness, motility, and density. Qualified semen was screened. The qualified semen and diluent were placed in a constant temperature water bath at 30 ℃ for 5 minutes, then diluted. The dilution method was as follows: the diluent was slowly injected along the bottle wall into the qualified semen, mixed thoroughly, to obtain diluted semen with a sperm motility of not less than 0.6. The volume ratio of qualified semen to diluent was 1:6. The diluent was a sheep semen diluent with a concentration of 30 million sperm per milliliter. It was forbidden to pour qualified semen into the diluent to avoid a decrease in sperm motility due to drastic changes in the liquid environment.

[0051] The quality inspection procedure is as follows: First, observe with the naked eye: the average ejaculation volume of a normal ram is one milliliter, the color is milky white or milky yellow, and it has a slightly fishy smell; under a microscope, the higher the sperm density and the stronger the motility, the more obvious the cloud-like turbulence; and more than 70% of the sperm move in a straight line, which proves that the semen quality meets the requirements for insemination.

[0052] S4. Insemination: Depending on the needs, the diluted semen obtained in S3 is used to inseminate non-pregnant ewes using one or both of the conventional insemination and laparoscopic insemination methods.

[0053] The conventional insemination method is as follows: if the non-pregnant ewe is untied and estrus is tested, the first insemination is performed 12 hours after the non-pregnant ewe comes into estrus, and the second insemination is performed 8 to 10 hours later.

[0054] If a non-pregnant ewe does not test for estrus after the removal of the throttle, the first insemination should be performed 48 hours after the removal of the throttle. At the same time, each non-pregnant ewe should be injected intramuscularly with ovulation induction agent No. 3. The second insemination should be performed 8 to 10 hours later.

[0055] Before insemination, a pedigree survey must be conducted to ensure that the ram providing the semen is not related to the non-pregnant ewe being inseminated; the insemination room should be disinfected to ensure it is clean, dry, and hygienic; if the indoor temperature is low, the heating equipment should be turned on in advance to maintain a suitable indoor temperature;

[0056] Then disinfect the insemination instruments. Disinfect the regular insemination instruments, vaginal dilators, etc. with 75% alcohol, and then rinse with physiological saline three to five times before use. When using continuously, rinse with diluted solution or physiological saline three to five times.

[0057] Then, the insemination procedure is performed. First, the vulva of the non-pregnant ewe is sprayed and rinsed with physiological saline. Take a 1 ml syringe, draw up the diluted semen, connect it to the conventional insemination device, and gently insert the sterilized vaginal speculum into the vagina. Rotate the vaginal speculum slowly to open it and locate the cervix. Insert the tip of the conventional insemination device into the cervix 0.3-0.5 cm deep and gently push the syringe to inject the diluted semen. After insemination, remove the conventional insemination device and vaginal speculum. Each non-pregnant ewe is infused with 0.1-0.3 mL of the diluted semen obtained in S3, which is equivalent to approximately 0.03-0.1 mL of raw semen.

[0058] When performing continuous insemination, the conventional insemination device does not need to be disinfected; when inseminating non-pregnant ewes with endometritis or unclean vaginal mucus, it should be disinfected and rinsed with alcohol and saline solution before continuing insemination.

[0059] The method of laparoscopic insemination is as follows: stop feeding forage 24 hours before insemination, stop drinking water 12 hours before insemination, and use timed insemination or estrus-testing insemination; clean and disinfect the surgical site of the non-pregnant ewe, and inject 12.5 micrograms of A3, 5 mL of aminopyrine and 1 million units of penicillin intramuscularly.

[0060] At the base of the udder of an empty ewe, 2-5 cm from the udder, hold the puncture needle with a cannula in your right hand and the tip of the puncture needle in your left hand. Pierce the abdominal wall on the left front side of the udder, with the puncture depth controlled at 1-2 cm. Pull out the puncture needle and leave the left cannula in place. Repeat the same method to puncture at the opposite point on the right front side of the udder and leave the right cannula in place.

[0061] Insert the laparoscope into the left cannula, adjust the depth and angle of the laparoscope, and ensure that both uterine horns are in the center of the field of view; use the left hand to fix the position and depth of the laparoscope, and use the right hand to insert the insemination gun into the right cannula. After the insemination gun enters the field of view of the laparoscope, retract the insemination gun cover to expose the insemination needle.

[0062] At the anterior third of the greater curvature of each uterine horn, the insemination needle is inserted vertically through the uterine horn to inject 0.1-0.2 mL of diluted semen obtained from S3 into the uterine cavity. During insemination, care should be taken to avoid leakage, air bubbles, or perforation of the uterine horn due to improper insertion depth or angle. If any of these situations occur, the needle should be inserted again at the base of the uterine horn for insemination. After insemination is completed, the laparoscope and insemination gun are removed, the wound is disinfected with povidone-iodine, no sutures are required, and the patient can leave the operating table.

[0063] The scheduled insemination is as follows: for non-pregnant ewes, insemination is performed within 42-50 hours after the removal of the tether without estrus testing; the estrus testing insemination is as follows: for non-pregnant ewes, estrus testing is performed 24 hours after the removal of the tether, and estrus testing is conducted on non-pregnant ewes in the morning and evening by a estrus testing ram for 3-5 days; insemination is performed within 14-16 hours after the non-pregnant ewe comes into estrus.

[0064] S5. Pregnancy Examination

[0065] 25-30 days after mating, veterinary ultrasound is used to check the pregnancy of non-pregnant ewes. Before the ultrasound examination, the breeder restrains the non-pregnant ewe to ensure that she does not move during the ultrasound. Spray 0.1% benzalkonium chloride disinfectant on the rectal probe, and insert it into the ewe's anus with the reflective side facing downwards. The movement should be gentle. The probe is inserted 5-10 cm into the anus. First, locate the bladder, and then go a little deeper to locate the uterus. The uterus is located on the dorsal side behind the bladder. Based on the image, pregnancy is determined.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for artificial insemination of sheep, characterized in that, The method includes the following steps: S1, Synchronized estrus S101. Plugging: Disinfect the vulva of the non-pregnant ewe with benzalkonium chloride solution, place the tug in the tug-planting device, and then implant the tug. S102. Removal of thrombus and injection of pregnant mare serum: The day of thrombus insertion is day zero. The thrombus is removed 10 to 14 days after insertion. At the same time as thrombus removal, pregnant mare serum is injected into each non-pregnant ewe. S2, Ram semen collection S201. Supplementary feeding and training of breeding rams: One month before semen collection, each breeding ram shall be fed supplementary feed daily and exercised for a certain period of time; training shall be provided to breeding rams that are collecting semen for the first time. S202. Adjustment, installation, and semen collection of the artificial vagina: Adjust and disinfect the artificial vagina, and control the internal temperature of the artificial vagina; collect semen from breeding rams using the artificial vagina method, and place the semen in a water bath for testing after collection; S3, Semen Quality Inspection and Dilution The semen collected from breeding rams in S2 was subjected to quality inspection, and qualified semen was screened. The qualified semen and diluent were placed in a constant temperature water bath and allowed to stand for a period of time before being diluted to obtain diluted semen. S4, Insemination The diluted semen obtained in S3 was used to inseminate non-pregnant ewes using one or both of the conventional insemination and laparoscopic insemination methods. S5. Pregnancy Examination Pregnancy checks were performed on non-pregnant ewes using veterinary ultrasound 25-30 days after mating.

2. The method for artificial insemination of sheep according to claim 1, characterized in that, The mass fraction of the benzalkonium chloride solution in S101 is 0.1%; the injection volume of the pregnant mare serum in S102 is 330 IU.

3. The method for artificial insemination of sheep according to claim 1, characterized in that, The supplementary feed mentioned in S201 consists of 500 grams of carrots, 500 grams of alfalfa hay, 2 eggs, and 300 grams of soybean meal; the exercise time is 1 hour; the training method is: observation training: allowing rams collecting semen for the first time to observe other rams mating or collecting semen for imitation and learning. Massage training: Massage the testicles of the rams regularly every day for no more than 20 minutes, for a period of one week; Scent induction: Apply urine from estrus ewes to the tip of the rams' noses to induce them to mount; Drug assistance: Inject each ram with 1-2 mL of testosterone propionate once a day for 3 consecutive days, and then begin mounting training.

4. The method for artificial insemination of sheep according to claim 3, characterized in that, The internal temperature of the artificial vagina described in S202 is 38~40 ℃; the temperature of the water bath is 32~34 ℃.

5. The method for artificial insemination of sheep according to claim 1, characterized in that, The quality inspection items described in S3 include ejaculate volume, color, odor, cloudiness, motility, and density; the constant temperature water bath temperature is 30 ℃, and the standing time is 5 min; the dilution method is as follows: slowly inject the diluent along the bottle wall into the qualified semen and mix evenly; the volume ratio of the qualified semen to the diluent is 1:(2~6), and the diluent is a sheep semen diluent with a concentration of 30 million sperm / mL; the sperm motility in the diluted semen is not less than 0.

6.

6. The method for artificial insemination of sheep according to claim 1, characterized in that, The conventional insemination method described in S4 is as follows: if the non-pregnant ewe is untied and estrus is tested, the first insemination is performed 12 hours after the non-pregnant ewe comes into estrus, and the second insemination is performed 8 to 10 hours later. If a non-pregnant ewe does not test for estrus after the removal of the throttle, the first insemination should be performed 48 hours after the removal of the throttle. At the same time, each non-pregnant ewe should be injected intramuscularly with ovulation induction agent No.

3. The second insemination should be performed 8 to 10 hours later. The insemination room and insemination equipment are disinfected, and then the diluted semen obtained in S3 is injected into the uterus of a non-pregnant ewe in estrus using a conventional insemination device; the volume of the diluted semen is 0.1~0.3 mL.

7. A method for artificial insemination of sheep according to claim 6, characterized in that, The method for testing estrus in non-pregnant ewes after untying the tether is as follows: 24 hours after untying, estrus is tested in the morning and evening by a ram for 3-5 days; the dosage of the ovulation induction agent No. 3 is 12.5 micrograms.

8. A method for artificial insemination of sheep according to claim 1, characterized in that, The laparoscopic insemination method described in S4 is as follows: Stop feeding non-pregnant ewes 24 hours before insemination and stop drinking water 12 hours before insemination. Use timed insemination or estrus-testing insemination. Remove hair and disinfect the surgical site of the non-pregnant ewe, and inject 12.5 micrograms of A3, 5 mL of aminopyrine and 1 million units of penicillin into the muscle. 2-5 cm from the base of the udder of an empty ewe, puncture the left and right anterior sides of the udder respectively, and leave the left and right cannulas. Insert the laparoscope into the left cannula and adjust the laparoscope's field of view so that the two uterine horns are in the center of the field of view. Insert the insemination gun into the right cannula, and vertically pierce the uterine horn at the anterior third of the greater curvature on both sides, injecting 0.1~0.2 mL of diluted semen into the uterine cavity.

9. A method for artificial insemination of sheep according to claim 8, characterized in that, The scheduled insemination refers to inseminating non-pregnant ewes within 42-50 hours after estrus is removed without testing for estrus. The estrus-testing insemination refers to inseminating non-pregnant ewes within 14-16 hours after estrus is tested for estrus.