阅读说明：本技术 一种马的胚胎移植方法 (Horse embryo transplantation method ) 是由 邹敬清 邹志钢 张凤 张建军 盛俊 荆海涛 郭海鹏 周群 于 2021-10-08 设计创作，主要内容包括：本发明涉及胚胎移植技术领域,具体涉及一种马的胚胎移植方法；包括以下步骤：S1、供体和受体的选择,S2、器材准备,S3、供体和受体的冲洗,S4、胚胎采集,S5、对胚胎清洗,S6、胚胎检测,S7、胚胎装管,S8、将胚胎移入受体母马；本发明选择优良的供体母马以及国产受体母马,经过供体和受体的冲洗、胚胎采集、对胚胎清洗、胚胎检测、胚胎装管以及将胚胎移入受体母马过程,将供体母马的胚胎移植给受体母马,采用的非手术胚胎移植方法,胚胎移植平均成功率能够达到92%,有效的提高了胚胎移植成功率,同时,能够有效的提高每匹供体母马的生崽产量。(The invention relates to the technical field of embryo transplantation, in particular to a horse embryo transplantation method; the method comprises the following steps: s1, selection of donor and recipient, S2, equipment preparation, S3, washing of donor and recipient, S4, embryo collection, S5, embryo cleaning, S6, embryo detection, S7, embryo tubulation, S8, transfer of embryo into recipient mare; the method selects excellent donor mares and domestic receptor mares, and transplants the embryos of the donor mares to the receptor mares through the processes of washing the donor mares and the receptor mares, embryo collection, embryo cleaning, embryo detection, embryo tubing and embryo transfer to the receptor mares.)

1. A method of equine embryo transfer comprising the steps of:

s1, selection of donor and acceptor: selecting donor mares with high breeding value and robust recipient mares, and performing intravenous injection of chorionic gonadotropin when the diameter of follicles of the donor mares is more than or equal to 40mm in the estrus of the donor mares, wherein the injection amount is 1000-;

s2, equipment preparation: preheating reagents and consumables which are in contact with embryos in a 37 ℃ sterile environment for 6-8 hours, and wiping alcohol and sterilizing ultraviolet rays on a laboratory, a sterile operating platform and experimental instruments;

s3, rinsing of donor and acceptor: flushing the anesthetized donor mare and the anesthetized recipient mare, and keeping the vulva and the periphery of the donor mare and the recipient mare clean, sterile and anhydrous;

s4, embryo collection: collecting embryos from donor mares;

s5, embryo cleaning: searching embryos, and cleaning the embryos by adopting an embryo washing tube;

s6, embryo detection: placing the washed embryos under a stereomicroscope with the size of more than 50 times, performing embryo quality identification and classification according to detection standards, and selecting excellent embryos for later use;

s7, embryo tubulation: transferring the excellent embryo into an embryo transfer tube;

s8, transfer of embryo into recipient mares: embryos were transferred to recipient mares using a embryo transfer gun.

2. The method for embryo transfer in equine of claim 1, wherein said step S1 for selecting a donor mare with high breeding value is to select a mare with high productivity, stable inheritance, and no inherited or infectious diseases as a donor mare.

3. The equine embryo transfer method of claim 1 wherein, in said step S3, the washing of the donor and recipient includes the steps of:

s301, anesthetizing donor mares and recipient mares which are not subjected to water feeding for 10-24 hours by using 2ml of 2% procaine;

s302, vulva and periphery of the donor mare and the receptor mare are treated by 2% ammonium chloride solution.

4. The equine embryo transfer method of claim 1 wherein, in said step S4, embryos are collected from donor mares, comprising the steps of:

s401, a technician wears sterile gloves to send a collecting tube into a mare uterus;

s402, inflating the air bag of the embryonal tube to clamp the embryonal tube in the cervix;

s403, after the embryo collecting tube is placed, injecting embryo collecting liquid into the mare uterus and gently softening the uterus;

s404, leading out the embryo collecting liquid to wash the uterus, repeatedly washing the uterus for 5-7 times, collecting the embryo collecting liquid by using an embryo collecting vessel, and finishing washing.

5. The equine embryo transfer method according to claim 1, wherein in said step S5, embryos are searched and washed by using embryo-washing tubes, comprising the steps of:

s501, standing the embryo collecting vessel for 10-20min, removing supernatant liquid when the embryo is lowered to the bottom of the embryo collecting vessel, and then placing the embryo collecting vessel under a 10-20 ploid visual microscope to search for the embryo;

s502, moving the embryo into the first dish hole of the embryo washing dish from the embryo collecting dish by using the embryo washing pipe, replacing a new embryo washing pipe, moving the embryo into the second dish hole from the first dish hole of the embryo collecting dish, and repeatedly cleaning for 7 times.

6. The method for transplanting embryos of claim 5, wherein in the step S501, embryos are searched along an S-shaped path from top to bottom and from left to right, and coordinate positions of each embryo are recorded according to the horizontal and vertical lines at the bottom of the embryo collecting vessel.

7. The method for embryo transfer of horse in claim 1, wherein in step S7, excellent embryos are transferred into embryo transfer tubes in the order of liquid-gas-liquid-embryo-liquid-gas-liquid.

8. The method for embryo transfer in horses according to claim 1, wherein in step S8, embryo transfer gun is used to transfer embryo into recipient mare, comprising the following steps:

s801, placing the embryo transfer tube filled with the embryo into an embryo transfer gun;

s802, an operator wears sterile gloves to send a embryo transfer gun into the uterus of a recipient mare;

and S803, slowly extending the transplantation gun into the uterine horn great bend, gently inserting the inner core and pushing out the embryo, and taking out the transplantation gun to finish embryo transplantation.

Technical Field

The invention relates to the technical field of embryo transplantation, in particular to a horse embryo transplantation method.

Background

Embryo transplantation, also called fertilized egg transplantation, refers to a technique of transplanting an early embryo in a female animal or an embryo obtained by in vitro fertilization or other means into another female animal of the same species and having the same physiological condition, and continuing to develop into a new individual. The traditional embryo transplantation is generally carried out by operation, but the success rate of transplantation is not high, and the embryo transplantation requirement of horses can not be met due to trauma.

In summary, the development of a horse embryo transplantation method remains a key problem to be solved urgently in the technical field of embryo transplantation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a horse embryo transplantation method, which is characterized in that excellent donor mares and domestic receptor mares are selected, and the embryos of the donor mares are transplanted to the receptor mares through the processes of washing of the donor mares and the receptor, embryo collection, embryo cleaning, embryo detection, embryo tubulation and embryo transfer to the receptor mares.

In order to achieve the purpose, the invention provides the following technical scheme:

a method of equine embryo transfer comprising the steps of:

s1, selection of donor and acceptor: selecting donor mares with high breeding value and robust recipient mares, and performing intravenous injection of chorionic gonadotropin when the diameter of follicles of the donor mares is more than or equal to 40mm in the estrus of the donor mares, wherein the injection amount is 1000-;

s2, equipment preparation: preheating reagents and consumables which are in contact with embryos in a 37 ℃ sterile environment for 6-8 hours, and wiping alcohol and sterilizing ultraviolet rays on a laboratory, a sterile operating platform and experimental instruments;

s3, rinsing of donor and acceptor: flushing the anesthetized donor mare and the anesthetized recipient mare, and keeping the vulva and the periphery of the donor mare and the recipient mare clean, sterile and anhydrous;

s4, embryo collection: collecting embryos from donor mares;

s5, embryo cleaning: searching embryos, and cleaning the embryos by adopting an embryo washing tube;

s6, embryo detection: placing the washed embryos under a stereomicroscope with the size of more than 50 times, performing embryo quality identification and classification according to detection standards, and selecting excellent embryos for later use;

s7, embryo tubulation: transferring the excellent embryo into an embryo transfer tube;

s8, transfer of embryo into recipient mares: embryos were transferred to recipient mares using a embryo transfer gun.

The invention is further configured to: in said step S1, selecting a donor mare of high breeding value means selecting a mare with high productivity, stable inheritance, no genetic and infectious diseases as the donor mare.

The invention is further configured to: in the step S3, the rinsing of the donor and the recipient includes the following steps:

s301, anesthetizing donor mares and recipient mares which are not subjected to water feeding for 10-24 hours by using 2ml of 2% procaine;

s302, vulva and periphery of the donor mare and the receptor mare are treated by 2% ammonium chloride solution.

The invention is further configured to: in said step S4, collecting an embryo from a donor mare, comprising the steps of:

s401, a technician wears sterile gloves to send a collecting tube into a mare uterus;

s402, inflating the air bag of the embryonal tube to clamp the embryonal tube in the cervix;

s403, after the embryo collecting tube is placed, injecting embryo collecting liquid into the mare uterus and gently softening the uterus;

s404, leading out the embryo collecting liquid to wash the uterus, repeatedly washing the uterus for 5-7 times, collecting the embryo collecting liquid by using an embryo collecting vessel, and finishing washing.

The invention is further configured to: in step S5, searching for an embryo and washing the embryo with an embryo washing tube, including the following steps:

s501, standing the embryo collecting vessel for 10-20min, removing supernatant liquid when the embryo is lowered to the bottom of the embryo collecting vessel, and then placing the embryo collecting vessel under a 10-20 ploid visual microscope to search for the embryo;

s502, moving the embryo into the first dish hole of the embryo washing dish from the embryo collecting dish by using the embryo washing pipe, replacing a new embryo washing pipe, moving the embryo into the second dish hole from the first dish hole of the embryo collecting dish, and repeatedly cleaning for 7 times.

The invention is further configured to: in step S501, when searching for embryos, the embryos are searched along an S-shaped route from top to bottom and from left to right, and coordinate positions of each embryo are recorded according to the horizontal and vertical lines at the bottom of the embryo collecting vessel.

The invention is further configured to: in the step S7, excellent embryos are transferred into the embryo transfer tube in the order of liquid-gas-liquid-embryo-liquid-gas-liquid.

The invention is further configured to: in step S8, the embryo is transferred to a recipient mare by using a embryo transfer gun, comprising the following steps:

s801, placing the embryo transfer tube filled with the embryo into an embryo transfer gun;

s802, an operator wears sterile gloves to send a embryo transfer gun into the uterus of a recipient mare;

and S803, slowly extending the transplantation gun into the uterine horn great bend, gently inserting the inner core and pushing out the embryo, and taking out the transplantation gun to finish embryo transplantation.

Advantageous effects

Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:

the method selects excellent donor mares and domestic receptor mares, and transplants the embryos of the donor mares to the receptor mares in a non-operative embryo transplantation mode, and specifically, through the processes of washing the donor mares and the receptor, collecting the embryos, cleaning the embryos, detecting the embryos, loading the embryos into tubes and transferring the embryos to the receptor mares, the average success rate of embryo transplantation can reach 92 percent, the success rate of embryo transplantation is effectively improved, meanwhile, the yield of piglets of each donor mare can be effectively improved, and the method has wider market prospect and is more suitable for popularization.

Drawings

FIG. 1 is a flow chart of a method of equine embryo transfer.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will be further described with reference to the following examples.

Example 1

Referring to fig. 1, a method for embryo transfer of a horse includes the following steps:

step one, selection of a donor and an acceptor: selecting donor mare with high breeding value and robust recipient mare, and intravenously injecting chorionic gonadotropin at estrus stage of donor mare when the diameter of follicle is greater than or equal to 40mm, wherein the injection amount is 1000 units/piece.

Selecting a donor mare with high breeding value means selecting a mare with high productivity, stable inheritance and no genetic and infectious diseases as a donor mare.

Step two, equipment preparation: placing the reagent and consumables in contact with embryo in 37 deg.C sterile environment, preheating for 6 hr, and performing alcohol wiping and ultraviolet sterilization on laboratory, sterile operation table, and experimental apparatus.

Step three, rinsing the donor and the receptor: the anesthetized donor and recipient mares were rinsed and the vulva and surrounding area of the donor and recipient mares were kept clean, sterile, and anhydrous.

Washing of the donor and the recipient, comprising the steps of:

301) donor and recipient mares, which had been fasted for 10 hours, were anesthetized with 2ml of 2% procaine.

302) And vulva and periphery of donor mare and recipient mare were treated with 2% ammonium chloride solution.

Step four, embryo collection: embryos were collected from donor mares.

Collecting an embryo from a donor mare comprising the steps of:

401) the technician wears sterile gloves to send the collecting tube into the mare uterus.

402) And inflating the air bag of the collecting tube to clamp the collecting tube in the cervix.

403) And after the embryo collecting tube is placed, injecting embryo collecting liquid into the mare uterus and gently softening the uterus.

404) And leading out the embryo collecting liquid to wash the uterus, repeatedly washing the uterus for 5 times, collecting the embryo collecting liquid by an embryo collecting vessel, and finishing washing.

Step five, embryo cleaning: searching for embryos, and cleaning the embryos by adopting an embryo washing tube.

Searching embryos, and cleaning the embryos by adopting an embryo washing tube, wherein the method comprises the following steps:

501) standing the embryo collecting vessel for 10min, removing supernatant when the embryo is lowered to the bottom of the embryo collecting vessel, and then placing the embryo collecting vessel under a 10-fold visual microscope to search for the embryo.

502) The embryo is moved into the first dish hole of the embryo-washing dish from the embryo-collecting dish by the embryo-washing tube, the new embryo-washing tube is replaced, and the embryo is moved into the second dish hole from the first dish hole of the embryo-collecting dish, so that the washing is repeated for 7 times.

When searching for embryos, searching for the embryos according to an S-shaped route from top to bottom and from left to right, and recording the coordinate position of each embryo according to the transverse and longitudinal lines at the bottom of the embryo collecting vessel.

Step six, embryo detection: and (3) placing the washed embryos under a stereomicroscope with the magnification of more than 50 times, carrying out embryo quality identification and classification according to detection standards, and selecting excellent embryos for standby, wherein the detection standards are shown in table 1.

Table 1: embryo quality identification and classification

Rank of	Standard of merit
		Class A	Is excellent. The embryo cell mass is in a uniform and symmetrical spherical shape, the size, the color and the density of a single blastomere are consistent, irregular cells are relatively few, at least 85% of the cell mass was intact and viable embryonic cell masses. The transparent band is smooth and complete.
Class B	Is good. There is some irregularity in the size and shape of the embryo cell mass and the color and density of the individual cells. At least 50% of the cells The structure is a perfect, active embryo cell mass.
		Class C	The quality is poor. There are severe irregularities in the size and shape of the embryo cell mass as well as the color and density of the individual cells. At least 25% of the cells The structure is a perfect, active embryo cell mass.
Class D	Death or degeneration. A degenerated embryo, oocyte or single cell embryo, without viability.

Seventhly, loading the embryos into a tube: excellent embryos were transferred into embryo transfer tubes.

Excellent embryos were transferred into embryo transfer tubes in the order of liquid-gas-liquid-embryo-liquid-gas-liquid.

Step eight, transferring the embryo into a receptor mare: embryos were transferred to recipient mares using a embryo transfer gun.

Transferring embryos to recipient mares using a embryo transfer gun, comprising the steps of:

801) and placing the embryo transfer tube filled with the embryo into an embryo transfer gun.

802) And the operator wears sterile gloves to send the embryo transfer gun into the uterus of the recipient mare.

803) Slowly extending the transplantation gun into the uterine horn great bend, gently inserting the inner core and pushing out the embryo, and taking out the transplantation gun to finish embryo transplantation.

Example 2

The method for embryo transfer of horses provided in this example is substantially the same as in example 1, with the main differences:

in step one, the injection amount of chorionic gonadotrophin is 2000 units/p;

in the second step, preheating for 7 hours in a sterile environment at 37 ℃;

in step three, the water deprived fasting time for the donor mare and the recipient mare was 17 hours;

in step four, the uterus is washed for 6 times;

in the fifth step, the embryo collecting vessel is kept still for 15min and is placed under a 15-fold visual microscope to search embryos.

Example 3

The method for embryo transfer of horses provided in this example is substantially the same as in example 1, with the main differences:

in the first step, the injection amount of chorionic gonadotrophin is 3000 units/piece;

in the second step, preheating for 8 hours in a sterile environment at 37 ℃;

in step three, the water deprived fasting time for the donor mare and the recipient mare was 24 hours;

in step four, the uterus is washed 7 times;

in the fifth step, the embryo collecting vessel is kept still for 20min and is placed under a 20-fold visual microscope to search for embryos.

Embryo transfer test

Selecting a horse: 2 ice island horses;

selection of donor mares: 5 Mars in the Iceland;

selection of recipient mares: 100 domestic mares;

recipient mares were divided into 20 groups, and embryo transfer tests were performed on each group using the method of the present invention according to example 1, and the test results are shown in table 2.

Table 2: embryo transfer test data statistical table

	A group of	Two groups are	Three groups	Four groups	Five groups
						Number of successful transplants	18	20	19	18	17
Success rate	90%	100%	95%	90%	85%

By analyzing the relevant data in the table, the average success rate of the horse embryo transplantation is 92% by the horse embryo transplantation method provided by the invention, the horse embryo transplantation success rate is good, and the horse reproductive capacity can be effectively improved. Therefore, the horse embryo transplantation method provided by the invention has wider market prospect and is more suitable for popularization.

The method selects excellent donor mares and domestic receptor mares, and transplants the embryos of the donor mares to the receptor mares in a non-operative embryo transplantation mode, and specifically, through the processes of washing the donor mares and the receptor, collecting the embryos, cleaning the embryos, detecting the embryos, loading the embryos into tubes and transferring the embryos to the receptor mares, the average success rate of embryo transplantation can reach 92 percent, the success rate of embryo transplantation is effectively improved, meanwhile, the yield of piglets of each donor mare can be effectively improved, and the method has wider market prospect and is more suitable for popularization.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.