Non-surgical embryo collection method for sheep

Abstract

The invention discloses a non-surgical embryo collection method for sheep. The method mainly comprises the following steps of preparation work, embryo collection and ending work. According to embryo collection, an ovum flushing tube penetrates through the cervix through the vagina, enters the uterus and reaches the uterine horn on one side, and ovum flushing is carried out. The non-surgical embryo collection method for the sheep, disclosed by the invention, is different from a conventional surgical operation used at present for collecting eggs from an abdomen opening, but enters the uterus body through the vagina to flush the eggs and recover the embryos, so that the donor sheep can be prevented from being damaged, and different types of egg flushing tubes are selected according to the tightness of the cervical opening of the donor sheep, so that the embryo collection efficiency is improved. Damage to the cervix can be avoided, and the ovum flushing tube can accurately enter uterine horns on the two sides.

Description

Technical Field

[0001] The invention relates to the technical field of embryo transplantation, in particular to a non-surgical embryo collection method for sheep. Background Art

[0002] Embryo transplantation technology is the most technologically advanced rapid breeding technology in the modern sheep industry. In recent years, this technology has been mainly used for the breeding of precious and rare breeds. It has played a great role in the rapid expansion of excellent breeds introduced from the international market. The current conventional embryo transplantation requires the collection of embryos through surgery, which may cause damage to the donor sheep, making the application of embryo transplantation technology risky and making the promotion of this technology in actual production not high. Surgical embryo collection often causes irreversible damage to the reproductive system tissues of the breeding sheep, and the direct consequence is the loss of reproductive ability. At the same time, the damage to the expensive breeding sheep will cause serious economic losses, which is also difficult for ranchers to bear, thus greatly inhibiting the widespread application of this technology.

[0003] The current sheep embryo transplantation technology mainly uses surgical methods to collect embryos. The basic process is: anesthesia, abdominal shaving, disinfection, scalpel incision of 3-5 cm in the abdomen, insert fingers into the abdomen to touch the uterine horns, pull out two uterine horns (even ovaries), use a small hemostatic forceps to poke a small hole with a diameter of 3-5 mm on one side of the uterine horn 2-3 cm above the bifurcation of the uterine horns, and then insert the fallopian tube, inflate the balloon at the head of the fallopian tube with 3-5 ml of air to block the channel in the uterine horn; insert a trocar into the thinner end of the uterine horn on that side and inject the egg flushing solution, which is recovered by the fallopian tube, and then repeat the same method to collect embryos from the other side of the uterine horn. After the embryo collection is completed, the uterine horn is returned to the abdominal cavity, and the abdominal peritoneum, muscles and skin are sutured. This series of actions will cause adhesion of the sheep's reproductive organs and abdominal tissues due to bleeding, foreign matter, infection, etc. The direct result of this adhesion and other injuries caused by improper operation is a decrease in the sheep's reproductive capacity and difficulty in conception. Repeated embryo collection operations will result in unfertilized eggs, and even fertilized embryos will have serious degradation of embryo quality, which will significantly reduce the conception rate. The biggest risk of using surgical embryo collection technology is the damage to the donor sheep. The donors are often expensive breeding sheep. For example, the cost of an imported breeding sheep currently ranges from 15,000 to tens of thousands or even higher. At present, the breeding sheep injury rate of the team that can implement this technology in my country is generally between 10-30%, which is the main reason for the high cost of using this technology and its inability to be widely used.

[0004] After being gradually recognized by breeding companies, the injury rate of donor sheep has basically refused to implement this technology. People have begun to actively study ways to reduce surgical trauma. At present, some people have tried to use non-surgical embryo recovery technology established by laparoendoscopy to prevent postoperative adhesion after embryo collection, but this technology still has many problems: first, the operation still requires surgery and is more difficult than existing conventional surgery; second, the embryo recovery rate is reduced. In essence, this method still cannot do without surgery, but the wound is smaller, and it does not fundamentally solve the problem of breeding sheep injury. In addition, due to the lack of corresponding commercial equipment, its standardization process is severely restricted, so it is unlikely to be applied on a large scale in a short period of time, and surgical collection of embryos still dominates. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide a non-surgical method for embryo collection from sheep, which can solve the problem of damage to sheep during the embryo collection process in the prior art.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] A method for non-surgical embryo collection from sheep comprises the following steps:

[0008] S1. Preparation;

[0009] S2. Embryo Collection:

[0010] S21. Insert the oviduct with a steel core through the vagina into the cervix of the donor sheep, slowly and tentatively pass through the cervix, enter the uterine body, and then deviate to one side to allow the oviduct to enter one uterine horn. Continue to insert the oviduct to a depth of about 2-3 cm. When you feel the resistance increase, pull out the steel core from the oviduct.

[0011] S22. 100 ml of oviduct flushing solution is injected through the oviduct, and then the oviduct flushing reflux valve is opened to access the embryo sieve to recover the oviduct flushing solution;

[0012] S23. Repeat step S22 2-3 times;

[0013] S24. Gently pull back the fallopian tube to withdraw from one uterine horn, then insert the steel core into the fallopian tube, and then insert the fallopian tube into the other uterine horn, and repeat steps S22 and S23.

[0014] S3. Post-embryo processing;

[0015] S4. Embryo processing.

[0016] Preferably, S1 preparation work includes:

[0017] S11. Perform synchronization, superovulation and breeding treatment on the donor sheep according to the embryo transfer procedure. Deprive the donor sheep of water and food for 24 hours one day before embryo collection.

[0018] S12. On the day of embryo collection, the donor sheep is routinely anesthetized, placed on a conventional embryo transfer operating cart in a side-lying position, and a fixable vaginal opener is inserted into the vagina to fix it and expose the vagina and cervical opening.

[0019] Preferably, the post-processing of S3 embryo collection includes:

[0020] S31. After the egg flushing solution is recovered, gently draw out the oviduct;

[0021] S32. Remove the vaginal opener, untie the donor sheep, inject the anesthetic, and the embryo collection is completed.

[0022] Preferably, the S4 embryo processing comprises:

[0023] S41. The oviduct flushing fluid recovered is connected to the embryo sieve;

[0024] S42. Excess liquid is drained through the embryo sieve, and the remaining liquid is 15-20 ml, and the liquid containing the embryo is transferred to the embryo picking cup;

[0025] S43. Place the egg cup under a microscope to search for embryos, and transfer the embryos into the egg collection cup containing culture medium using an embryo suction tube;

[0026] S44. Evaluate embryo quality and select effective embryos for transplantation into recipient sheep.

[0027] Preferably, the step S11 comprises:

[0028] S111. Synchronous estrus of donor sheep: placing vaginal plug in the vagina of donor sheep;

[0029] S112. Donor sheep superovulation: FSH 400 IU was injected 8 times using the decreasing method; the thrombus was removed at the 7th FSH injection;

[0030] S113. Breeding of donor sheep: natural mating or artificial insemination can be used;

[0031] S114. The donor sheep shall be deprived of water and food one day before embryo collection;

[0032] S115. Time for collecting embryos from donor sheep: On the 6th day after mating, when the embryonic development stage is at the morula stage, the eggs are flushed and the embryos are collected.

[0033] Preferably, the oviduct is an 8# model with a diameter of 3 mm.

[0034] Preferably, the oviduct is a 9# model with a diameter of 3.5 mm.

[0035] Preferably, the oviduct is a 10# model with a diameter of 4 mm.

[0036] Preferably, the oviduct is a 11# model with a diameter of 4.5 mm.

[0037] Preferably, the diameter of the fallopian tube flushing steel core is 2 mm.

[0038] The non-surgical method for sheep embryo collection of the present invention has the following beneficial effects:

[0039] (1) Non-surgical embryo collection is performed through the vagina, instead of conventional abdominal surgical embryo collection.

[0040] (2) No more surgical operations are used. The oviduct passes through the vagina, through the cervix, into the uterus, and finally reaches the uterine horn. The oviduct is flushed and the embryo is recovered with oviduct fluid. There will be no adhesion of the abdomen and reproductive organs like surgical embryo collection, and there will be no wound infection, wound non-healing, intestinal fistulas that often occur due to poor peritoneal suture, and no stress. The donor sheep recovers quickly after embryo collection and can be repeated again after a short period of time. It can also reduce the cost of surgical instruments, antibacterial drugs, sutures and surgical auxiliary materials. The biggest benefit is zero damage to precious donor sheep. It also reduces the difficulty of implementing embryo transplantation technology and speeds up the expansion of high-quality, high-yield and precious breeds of sheep.

[0041] (3) During the embryo collection process, different types of oviducts can be selected according to the tightness of the donor sheep's cervical opening, which can avoid damage to the cervical opening and allow the oviduct to accurately enter the uterine horns on both sides. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 The present invention is a flow chart of a method for non-surgical embryo collection from sheep according to one embodiment of the present invention. DETAILED DESCRIPTION

[0044] The method of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0046] See also Figure 1 , the present invention provides a technical solution:

[0047] The non-surgical embryo collection method for sheep comprises the following steps:

[0048] S1. Preparation:

[0049] S11. Perform synchronization, superovulation and breeding treatments on the donor sheep according to the embryo transfer procedure, and stop water and food one day before embryo collection.

[0050] S111. Synchronous estrus of donor sheep: placing vaginal plug in the vagina of donor sheep;

[0051] S112. Donor sheep superovulation: 8 injections of FSH (follicle stimulating hormone) 400 IU were performed using the decreasing method; the thrombus was removed at the 7th FSH injection;

[0052] S113. Breeding of donor sheep: natural mating or artificial insemination can be used;

[0053] S114. The donor sheep shall be deprived of water and food one day before embryo collection;

[0054] S115. Time for collecting embryos from donor sheep: On the 6th day after mating, when the embryonic development stage is at the morula stage, the eggs are flushed and the embryos are collected.

[0055] S12. On the day of embryo collection, the donor sheep is routinely anesthetized, placed on a conventional embryo transfer operating cart in a side-lying position, and a fixable vaginal opener is inserted into the vagina to fix it and expose the vagina and cervical opening.

[0056] S2. Embryo Collection:

[0057] S21. Insert the oviduct with a thin steel core through the vagina into the cervix of the donor sheep, slowly and tentatively pass through the cervix, and after entering the uterine body, deviate to one side so that the oviduct enters one uterine horn, and continue to insert it to a depth of about 2-3cm. After feeling the resistance increase, pull out the steel core in the oviduct; the models of oviduct are: 8# with a diameter of 3mm, 9# with a diameter of 3.5mm, 10# with a diameter of 4mm, and 11# with a diameter of 4.5mm. Choose oviducts of different thicknesses according to the tightness of the cervix. The material of the oviduct can be one of silicone, soft plastic, and soft rubber.

[0058] S22. 100 ml of egg flushing solution is injected through the oviduct each time. Emcare egg flushing solution can be used for egg flushing solution. Then, the oviduct reflux valve is opened to connect the embryo sieve to recover the egg flushing solution.

[0059] S23. Repeat step S22 2-3 times to recover as much of the egg flushing solution as possible;

[0060] S24. Gently pull back the fallopian tube to withdraw from one uterine horn, then insert the steel core into the fallopian tube, and then insert the fallopian tube into the other uterine horn, and repeat S22 and S23.

[0061] The whole embryo collection process is non-invasive and non-bleeding, so there will be no adhesion of the abdominal cavity and reproductive organs like surgical embryo collection, no wound infection, wound non-healing, intestinal fistulas often caused by poor peritoneal suture, no stress, and the donor sheep will recover quickly after surgery. It can also reduce the cost of surgical instruments, antibacterial drugs, sutures and surgical accessories. The biggest advantage is zero damage to the precious donor sheep, reducing the difficulty of embryo transplantation and accelerating the breeding of high-quality, high-yield and precious breed sheep.

[0062] S3. Post-embryo collection processing:

[0063] S31. After the egg flushing solution is recovered, gently draw out the oviduct;

[0064] S32. Remove the vaginal opener, untie the donor sheep, inject the anesthetic, and the embryo collection is completed.

[0065] S4 embryo processing:

[0066] S41. The oviduct flushing fluid recovered is connected to the embryo sieve;

[0067] S42. Drain excess liquid through the embryo sieve, leaving 15-20 ml of liquid, and transfer the liquid containing the embryo into the embryo picking cup;

[0068] S43. Place the egg collection cup under a microscope to search for embryos, and use an embryo pipette to transfer the embryos into an egg collection cup containing culture medium, which can be Emcare culture medium;

[0069] S44. Evaluate embryo quality and select effective embryos for transplantation into recipient sheep.

[0070] In order to investigate the embryo recovery rates of non-surgical embryo collection and surgical embryo collection, a comparative experiment was conducted as shown below.

[0071] Both the donor sheep for surgical embryo collection and the donor sheep for non-surgical embryo collection were fertilized with laparoscopic artificial insemination technology during breeding. The number of corpora lutea on the ovaries of each donor sheep was counted. The sheep were then divided into two groups, one of which was performed using the surgical embryo collection method and the other using the non-surgical embryo collection method disclosed in the present application. The number of embryos recovered from each donor sheep and the overall embryo recovery rate were obtained. The results are shown in Table 1.

[0072] Table 1 Number of embryos obtained by surgical and non-surgical embryo collection

[0073]

[0074] A total of 21 donor sheep were used in the experiment, 11 of which were used for embryo collection by the non-surgical embryo collection method disclosed in the present application, and 10 of which were used for embryo collection by surgical method. 151 embryos were recovered by the non-surgical embryo collection method, and 210 corpora lutea were found in the 11 donor sheep, with a recovery rate of 71.9%. 169 embryos were recovered by surgical embryo collection, and 229 corpora lutea were found in the 10 donor sheep, with a recovery rate of 73.8%.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. A method for non-surgical embryo collection from sheep, characterized in that: The following steps are involved: S1. Preparation; S2. Embryo Collection: S21. Insert the oviduct with a steel core through the vagina into the cervix of the donor sheep, slowly and tentatively pass through the cervix, enter the uterine body, and then deviate to one side to allow the oviduct to enter one uterine horn. Continue to insert the oviduct to a depth of about 2-3 cm. When you feel the resistance increase, pull out the steel core from the oviduct. S22. 100 ml of oviduct flushing solution is injected through the oviduct, and then the oviduct flushing reflux valve is opened, and the embryo sieve is connected to recover the oviduct flushing solution; S23. Repeat step S22 2-3 times; S24. Gently withdraw the oviduct, withdraw from one side of the uterine horn, and then insert the steel core into the oviduct, and then insert the oviduct into the other side of the uterine horn, and repeat steps S22 and S23; S3. Post-embryo processing; S4. Embryo processing.

2. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The S1 preparation work includes: S11. Perform synchronization, superovulation and breeding treatment on the donor sheep according to the embryo transfer procedure, and stop water and food on the day before embryo collection; S12. On the day of embryo collection, the donor sheep is routinely anesthetized, placed on a conventional embryo transfer operating cart in a side-lying position, and a fixable vaginal opener is inserted into the vagina to fix it and expose the vagina and cervical opening.

3. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The post-processing of S3 embryo collection includes: S31. After the egg flushing solution is recovered, gently draw out the oviduct; S32. Remove the vaginal opener, untie the donor sheep, inject the anesthetic, and the embryo collection is completed.

4. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The S4 embryo processing includes: S41. The oviduct flushing fluid recovered is connected to the embryo sieve; S42. Excess liquid is drained through the embryo sieve, and the remaining liquid is 15-20 ml, and the liquid containing the embryo is transferred to the embryo picking cup; S43. Place the egg cup under a microscope to search for embryos, and use an embryo pipette to transfer the embryos into an egg collection cup filled with culture medium; S44. Evaluate embryo quality and select effective embryos for transplantation into recipient sheep.

5. The method for non-surgical embryo collection from sheep according to claim 2, characterized in that: The step S11 comprises: S111. Synchronous estrus of donor sheep: placing vaginal plug in the vagina of donor sheep; S112. Donor sheep superovulation: FSH 400 IU was injected 8 times using the decreasing method; the thrombus was removed at the 7th FSH injection; S113. Breeding of donor sheep: natural mating or artificial insemination can be used; S114. The donor sheep shall be deprived of water and food one day before embryo collection; S115. Time for collecting embryos from donor sheep: On the 6th day after mating, when the embryonic development stage is at the morula stage, the eggs are flushed and the embryos are collected.

6. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The oviduct flushing tool is 8# model with a diameter of 3mm.

7. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The oviduct flushing tube is a 9# model with a diameter of 3.5mm.

8. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The oviduct flushing tool is a 10# model with a diameter of 4mm.

9. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The oviduct flushing tube is 11# model with a diameter of 4.5mm.

10. The method for non-surgical embryo collection from sheep according to claim 1, characterized in that: The diameter of the oviduct flushing steel core is 2 mm.

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