阅读说明：本技术 一种活体采集猪体内1、2、4、8细胞期胚胎的收集方法 (Method for collecting embryos at 1, 2, 4 and 8 cell stages in live pigs ) 是由 华再东 郭帅 朱喆 顾浩 陈帆 毕延震 任红艳 张立苹 肖红卫 于 2021-10-25 设计创作，主要内容包括：本发明公开了一种活体采集猪体内1、2、4、8细胞期胚胎的收集方法,该方法主要包括以下流程：母猪发情鉴定、人工输精、手术冲胚、挑胚及体外胚胎培养等步骤。本发明方法提出了一种活体收集猪体内1-细胞、2-细胞、4-细胞、8-细胞期胚胎的收集方法及专用设备,该方法能从供体母猪输卵管内获得不同发育阶段的胚胎,供胚胎发育、基因原核注射、嵌合体动物制备等研究,有效解决了体外授精胚胎质量差、移植受孕率低的问题,从而提高转基因或基因编辑猪的生产效率。(The invention discloses a method for collecting embryos at 1, 2, 4 and 8 cell stages in a live body collected pig, which mainly comprises the following steps: the method comprises the steps of sow oestrus identification, artificial insemination, embryo flushing by operation, embryo picking, in-vitro embryo culture and the like. The method can obtain embryos at different development stages from the oviduct of a donor sow for research on embryo development, gene prokaryotic injection, chimeric animal preparation and the like, and effectively solves the problems of poor quality of in vitro inseminated embryos and low transplanting and conception rate, thereby improving the production efficiency of transgenic or gene-edited pigs.)

1. A method for collecting embryos at 1, 2, 4 and 8 cell stages in a live pig body is characterized by comprising the following steps: the method utilizes the difference of time and space to judge the development stage of the embryo and the approximate position in the body, and mainly comprises the following aspects:

firstly, collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination;

secondly, collecting 2-cell-stage embryos, wherein the optimal time is 26-32 hours after artificial insemination;

thirdly, collecting 4-cell-stage embryos, wherein the optimal time is 34-40 hours after artificial insemination;

fourthly, collecting 8-cell-stage embryos, wherein the optimal time is 42 to 48 hours after artificial insemination;

the method mainly comprises the following steps:

preparation of embryos prior to Collection

1. Preparation of the apparatus

(1) Egg flushing device

(2) Ovum collecting device

(3) Embryo transfer device

(4) Egg collecting dish (watch dish)

(5) Surgical instrument

2. Preparation of egg-washing liquid and culture liquid

(1) Egg-washing liquid

(2) Culture solution

(II) preparation of operating theatre

1. Time of acquisition

2. Anesthesia and restraint of Donor pigs

3. Surgical site and disinfection

4. Surgical method

5. Embryo Collection

(1) Embryo collecting of fallopian tube

The method mainly comprises the following steps:

s1: inserting one end of the egg collecting device into the position of about 1-2cm from the bell mouth of the umbrella part of the fallopian tube, fixing a sucker by using a thumb and a forefinger, connecting an injector with an interface of a suction disc, pumping out air until the umbrella of the fallopian tube is tightly fixed on the sucker, and connecting the other end of the ovum collecting device with a surface dish;

s2: filling a liquid storage tank of the egg flushing device with egg flushing liquid, covering a cover tightly, placing one end with a puncture needle at a uterine tube joint part, inserting a needle head towards the direction of an oviduct (the needle head is not connected with an inflating device at the moment), opening a micro inflating pump switch, selecting proper impact strength of the egg flushing liquid by an adjusting valve, starting flushing the egg by connecting the needle head after the impact strength is adjusted, and paying attention to balance of a watch glass when flushing the egg, wherein the watch glass is full of liquid and needs to be replaced by a new watch glass;

s3: then the other side of the oviduct is led out of the incision, and the embryo is washed by the same method;

(2) uterine horn embryo collection

The method mainly comprises the following steps:

s1: according to the difference of the egg punching time, or the distance, cutting a small hole by an ophthalmologic scissors at a position 30-70cm away from the uterine horn junction part and without a blood vessel on the back of the uterine horn, inserting a uterine horn egg punching device, wherein the opening is towards the direction of the uterine tube junction part, and the opening at the other end is connected with a surface dish or a flat dish;

s2: injecting 30-50ml of ovum-flushing liquid into uterine horn from the uterine tube junction by using an ovum-flushing device;

s3: the ovum-flushing liquid flows through the uterine horn and brings the embryo into a surface dish or a plate for receiving the ovum;

s4: in view of the overlong uterine horn, a method of segmented ovum flushing can also be adopted;

s5: then the uterine horn on the other side is led out of the incision, and the embryo is washed by the same method;

s6: picking eggs;

s7: postoperative treatment of donor sows.

2. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: the embryo transfer device preparation in various preparation works before embryo collection comprises the following manufacturing steps:

a. igniting the alcohol blast burner, and placing a glass tube with the outer diameter of about 4mm above the flame of the alcohol blast burner;

b. continuously rotating the glass tube to ensure that the glass tube is uniformly heated and is burnt for about 4-5 seconds;

c. when the glass tube is burned to be red, the glass tube is stretched by light force, and the length is 8-9 cm;

d. after the elongated glass tube is cooled, the middle of the elongated glass tube is broken into two glass suction tubes by a sand wheel;

e. connecting the glass suction tube with a silicone tube with a length of 20-30cm, paying attention to tight connection to prevent air leakage, connecting the other end of the silicone tube of the scalp needle with the front end of an infusion apparatus, cutting off the redundant infusion apparatus, inserting a suction head into the other end, and completing the manufacturing of the embryo transfer device.

3. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: anesthesia and restraint of donor pigs in preparation for operating theatres, which mainly comprises the following aspects:

(1) donor pig palate traction, limiting activity, and performing general anesthesia by 20-30mg/kg (per kilogram body weight milligram) sodium pentobarbital (2.5%) or chloral magnesium sulfate hydrate injection (8% chloral hydrate, 5% magnesium sulfate) 150-200ml ear vein injection;

(2) because of different sensitivity to narcotics among individuals, the state of the pig needs to be observed at any time during injection, the injection speed needs to be slow, the excessive injection is prevented, and the pig can be used when the eyelid just loses reaction and the stimulation of the hoof has no obvious reflection;

(3) the anesthesia is put on the operation retaining frame in place and the four limbs are fixed;

(4) in order to prevent the pig from struggling and bouncing in the operation process, the limbs and the head are firmly bound by ropes;

(5) in order to lead the abdominal cavity viscera to incline forwards and facilitate the operation, the operating table needs to be low in front (low in head) and high in back (high in tail);

(6) the operation process comprises intramuscular or intravenous instillation of a proper amount of ketamine hydrochloride according to the requirement.

4. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: the main steps for the operation site and disinfection in the preparation of the operating room are as follows:

s1: washing the operation part with soap water and a brush, and cleaning the operation part with a towel after washing;

s2: the surgical site is generally selected between the 2 nd and 3 rd nipples, hair is cut at the surgical site by electric scissors or hair scissors, the hair stubble is cut off, the surgical site is cleaned by clear water, wiped dry, then coated with 2-4% iodine tincture, and then deiodinated by 70-75% alcohol cotton;

s3: the disinfection procedure is as follows: iodine tincture cotton ball → alcohol cotton ball from inside to outside;

s4: covering the wound towel on the operation part to expose the predetermined incision in the middle of the opening of the wound towel.

5. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: the operation method in the preparation of the operation room mainly comprises the following steps:

s1: cutting skin 5-8cm away from blood vessel along midline of abdomen, separating fat and muscle layer to expose peritoneum, and cutting peritoneum with blunt scissors;

s2: the operator stretches the index finger and the middle finger out of the abdominal cavity of the patient from the incision, touches the uterus or the uterine horn at the front and back positions of the junction with the pelvic cavity, clamps the uterus or the uterine horn with the two fingers after touching the uterus or the uterine horn, and draws the uterus or the uterine horn to the surface of the wound;

s3: and (5) guiding out the oviduct and the ovary along one uterine horn, and observing the ovulation point on the surface of the ovary and the development of the follicle.

6. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: for egg picking in preparation of an operating room, the main steps are as follows:

s1: placing the surface dish for receiving the ovum under a solid microscope, and finding out the embryo in the ovum flushing liquid by using the large visual field of a low-power lens;

s2: the diameter of the pig embryo is about 150 μm, the size of the pig embryo is only the size of a needle point when the pig embryo is observed by naked eyes, the pig embryo is a spherical body and is circular under a mirror, and the outer layer of the pig embryo is a transparent belt;

s3: the fallopian tubes were pipetted with a small amount of PBS followed by human eggs, and the washing was repeated in a second petri dish, then all the eggs were pooled in one petri dish, and one donor egg was placed in one dish and operated at room temperature of 20-25 ℃.

7. The method for collecting embryos at 1, 2, 4 and 8 cell stages in pigs collected in vivo according to claim 1, which comprises the following steps: the postoperative treatment of donor sows in preparation for operating rooms mainly comprises the following aspects:

s1: after the embryo collection of the donor is finished, the uterus is moistened by sterilized normal saline at 37 ℃, the blood clot is washed away, and the organ is reset;

s2: after the peritoneum and the muscle are sutured, anti-inflammatory and antiseptic drugs such as sulfanilamide and the like are smeared;

s3: after the skin was sutured, iodine tincture, intramuscular injection of penicillin and streptomycin were applied around the wound.

Technical Field

The invention relates to the technical field of pig embryo transplantation, in particular to a method for collecting embryos at 1, 2, 4 and 8 cell stages in a live body collected pig.

Background

The success of porcine embryo transfer trials was first reported by Kvashickii in 1951, and by the 60's of the 20 th century, surgical approaches to collecting and transferring embryos have been largely established. After the 70 s, north america and europe began to apply embryo transfer techniques to the introduction of extravasated blood and international introduction to locked herds. The emergence of transgenic animal technology in the 80 s promoted the application and development of embryo transfer technology. The good embryo collection technology can obtain the embryos with the maximum quantity from the reproductive tract of the donor sow for the operation of transgenosis; the high-efficiency embryo transfer technology can ensure that the recipient sow can obtain higher conception rate and more litter size, thereby improving the production efficiency of the transgenic pig. The transgenic pig can be produced by any method through embryo transfer technology in the last step of the preparation of the transgenic embryo. Some transgenic embryos prepared by very complicated procedures may perform well in the early stages, but because embryo transfer techniques are not well-known, transgenic pigs are not available, or are obtained with low efficiency, which is very invaluable. Embryo collection and transplantation are key basic technologies for preparing transgenic pigs.

Disclosure of Invention

The application aims at solving the technical problem of efficiently collecting embryos at 1, 2, 4 and 8 cell stages in a pig body in a living body, and related creative inventions are carried out: 1. self-control of the uterine tube; 2. controlling the egg flushing time; 3. the key technology in the process of washing eggs.

In order to achieve the purpose, the invention provides the following technical scheme: a method for collecting 1, 2, 4 and 8 cell-stage embryos in live pigs, which utilizes the difference of time and space to judge the development stage and the approximate position of the embryos in the bodies, mainly comprises the following steps:

firstly, collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination;

secondly, collecting 2-cell-stage embryos, wherein the optimal time is 26-32 hours after artificial insemination;

thirdly, collecting 4-cell-stage embryos, wherein the optimal time is 34-40 hours after artificial insemination;

and fourthly, collecting 8-cell-stage embryos, wherein the optimal time is 42-48 hours after artificial insemination.

The method mainly comprises the following steps:

preparation of embryos prior to Collection

1. Preparation of the apparatus

(1) Egg flushing device

(2) Ovum collecting device

(3) Embryo transfer device

(4) Egg collecting dish (watch dish)

(5) Surgical instrument

2. Preparation of egg-washing liquid and culture liquid

(1) Egg-washing liquid

(2) Culture solution

(II) preparation of operating theatre

1. Time of acquisition

2. Anesthesia and restraint of Donor pigs

3. Surgical site and disinfection

4. Surgical method

5. Embryo Collection

(1) Embryo collecting of fallopian tube

The method mainly comprises the following steps:

s1: inserting one end of the egg collecting device into the position of about 1-2cm from the bell mouth of the umbrella part of the fallopian tube, fixing a sucker by using a thumb and a forefinger, connecting an injector with an interface of a suction disc, pumping out air until the umbrella of the fallopian tube is tightly fixed on the sucker, and connecting the other end of the ovum collecting device with a surface dish;

s2: filling a liquid storage tank of the egg flushing device with egg flushing liquid, covering a cover tightly, placing one end with a puncture needle at the uterine tube joint part, inserting the needle head towards the oviduct direction (the needle head is not connected with the inflating device at the moment), opening a micro inflating pump switch, selecting proper impact strength of the egg flushing liquid by an adjusting valve, and starting to flush eggs by connecting the needle head after the micro inflating pump switch is adjusted. When eggs are washed, the surface dish is kept balanced, and the surface dish needs to be replaced when the liquid is full;

s3: the other fallopian tube was then pulled out of the incision and the embryo was washed in the same manner.

(2) Uterine horn embryo collection

The method mainly comprises the following steps:

s1: according to the difference of the egg punching time, or the distance, a small hole is cut by an ophthalmologic scissors at a position 30-70cm away from the uterine horn junction part and without blood vessels on the back of the uterine horn, the uterine horn egg collecting device is inserted, the opening is towards the direction of the uterine tube junction part, and the opening at the other end is connected with a surface dish or a flat dish;

s2: injecting 30-50ml of ovum-flushing liquid into uterine horn from the uterine tube junction by using an ovum-flushing device;

s3: the ovum-flushing liquid flows through the uterine horn and brings the embryo into a surface dish or a plate for receiving the ovum;

s4: in view of the overlong uterine horn, a method of segmented ovum flushing can also be adopted;

s5: the other uterine horn was then pulled out of the incision and the embryo was rinsed in the same manner.

6. Egg picking

7. Post-operative treatment of donor sows

Preferably, the embryo transfer device in each preparation before embryo collection is prepared by the following steps:

a. igniting the alcohol blast burner, and placing a glass tube with the outer diameter of about 4mm above the flame of the alcohol blast burner;

b. continuously rotating the glass tube to ensure that the glass tube is uniformly heated and is burnt for about 4-5 seconds;

c. when the glass tube is burned to be red, the glass tube is stretched by light force, and the length is 8-9 cm;

d. after the elongated glass tube is cooled, the middle of the elongated glass tube is broken into two glass suction tubes by a sand wheel;

e. the glass suction tube is connected with a silicone tube with the length of 20-30cm, and the connection is tight to prevent air leakage.

Preferably, the anesthesia and restraint of the donor pig in preparation for the operating room essentially comprises the following aspects:

(1) donor pig palate traction, limiting activity, and performing general anesthesia by 20-30mg/kg (per kilogram body weight milligram) sodium pentobarbital (2.5%) or chloral magnesium sulfate hydrate injection (8% chloral hydrate, 5% magnesium sulfate) 150-200ml ear vein injection;

(2) because of different sensitivity to narcotics among individuals, the state of the pig needs to be observed at any time during injection, the injection speed needs to be slow, the excessive injection is prevented, and the pig can be used when the eyelid just loses reaction and the stimulation of the hoof has no obvious reflection;

(3) the anesthesia is put on the operation retaining frame in place and the four limbs are fixed;

(4) in order to prevent the pig from struggling and bouncing in the operation process, the limbs and the head are firmly bound by ropes;

(5) in order to lead the abdominal cavity viscera to incline forwards and facilitate the operation, the operating table needs to be low in front (low in head) and high in back (high in tail);

(6) the operation process comprises intramuscular or intravenous instillation of a proper amount of ketamine hydrochloride according to the requirement.

Preferably, the preparation of the operating room for the operation site and the disinfection comprises the following main steps:

s1: washing the operation part with soap water and a brush, and cleaning the operation part with a towel after washing;

s2: the surgical site is generally selected between the 2 nd and 3 rd nipples, hair is cut at the surgical site by electric scissors or hair scissors, the hair stubble is cut off, the surgical site is cleaned by clear water, wiped dry, then coated with 2-4% iodine tincture, and then deiodinated by 70-75% alcohol cotton;

s3: the disinfection procedure is as follows: iodine tincture cotton ball → alcohol cotton ball from inside to outside;

s4: covering the wound towel on the operation part to expose the predetermined incision in the middle of the opening of the wound towel.

Preferably, the operation method in preparation of the operation room comprises the following main steps:

s1: cutting skin 5-8cm away from blood vessel along midline of abdomen, separating fat and muscle layer to expose peritoneum, and cutting peritoneum with blunt scissors;

s2: the operator stretches the index finger and the middle finger out of the abdominal cavity of the patient from the incision, touches the uterus or the uterine horn at the front and back positions of the junction with the pelvic cavity, clamps the uterus or the uterine horn with the two fingers after touching the uterus or the uterine horn, and draws the uterus or the uterine horn to the surface of the wound;

s3: and (5) guiding out the oviduct and the ovary along one uterine horn, and observing the ovulation point on the surface of the ovary and the development of the follicle.

Preferably, for egg picking in preparation of an operating room, the main steps are as follows:

s1: placing the surface dish for receiving the ovum under a solid microscope, and finding out the embryo in the ovum flushing liquid by using the large visual field of a low-power lens;

s2: the diameter of the pig embryo is about 150 μm, the size of the pig embryo is only the size of a needle point when the pig embryo is observed by naked eyes, the pig embryo is a spherical body and is circular under a mirror, and the outer layer of the pig embryo is a transparent belt;

s3: the fallopian tubes were pipetted with a small amount of PBS followed by human eggs, and the washing was repeated in a second petri dish, then all the eggs were pooled in one petri dish, and one donor egg was placed in one dish and operated at room temperature of 20-25 ℃.

Preferably, the postoperative treatment of the donor sow in preparation for the operating room mainly comprises the following aspects:

s1: after the embryo collection of the donor is finished, the uterus is moistened by sterilized normal saline at 37 ℃, the blood clot is washed away, and the organ is reset;

s2: after the peritoneum and the muscle are sutured, anti-inflammatory and antiseptic drugs such as sulfanilamide and the like are smeared;

s3: after the skin was sutured, iodine tincture, intramuscular injection of penicillin and streptomycin were applied around the wound.

Compared with the prior art, the invention has the beneficial effects that:

the method can obtain the embryos in the reproductive tract of donor sows in the maximum quantity for transgenic operation, and can ensure that recipient sows obtain higher conception rate and more litter size, thereby improving the production efficiency of transgenic pigs and effectively solving the problems that the traditional pig embryo transplantation technology is not closed, and the transgenic pigs cannot be obtained or are obtained with low efficiency.

Drawings

FIG. 1 is a schematic structural view of an ovum-flushing device for flushing ovum in fallopian tube according to the present invention;

FIG. 2 is a schematic structural view of the egg collecting device for pigs in the invention;

FIG. 3 is a schematic view of an embryo transfer device of the present invention;

FIG. 4 is a statistical table of embryo collection time and embryo development stage.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.

Example 1:

referring to fig. 1-4, the present invention provides a technical solution: a method for collecting 1, 2, 4 and 8 cell-stage embryos in live pigs, which utilizes the difference of time and space to judge the development stage and the approximate position of the embryos in the bodies, mainly comprises the following steps:

firstly, collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination;

secondly, collecting 2-cell-stage embryos, wherein the optimal time is 26-32 hours after artificial insemination;

thirdly, collecting 4-cell-stage embryos, wherein the optimal time is 34-40 hours after artificial insemination;

and fourthly, collecting 8-cell-stage embryos, wherein the optimal time is 42-48 hours after artificial insemination.

The method mainly comprises the following steps:

preparation of embryos prior to Collection

1. Preparation of the apparatus

(1) An egg flushing device: as shown in figure 1, the connecting part of the ovum flushing device is a hose transformed from an infusion apparatus, when a needle head is inserted into the oviduct, the limit of direct connection between the needle head and an injector is overcome, the ovum flushing device is free to operate, and the needle head can be really inserted into the inner cavity of the oviduct. In the process of flushing ova, even if an operator shakes slightly or a pig shakes slightly, the phenomenon that the needle head scratches, scratches or falls off the oviduct can not occur due to the buffering effect of the hose, so that the success rate of flushing ova is greatly improved. In addition, the storage tank for the egg-washing liquid is particularly arranged, so that the use of the egg-washing liquid can be better controlled, and the complicated operation of manually pushing and pulling the injector by the miniature inflating pump is more time-saving and labor-saving.

(2) An egg collecting device: the ovum collecting device for washing ovum of the oviduct is two glass tubes with the length of 130mm, the wall thickness of 0.8mm and the inner diameter of 3mm, and the connecting part is connected by a universal connector; the connecting part of the suction cup and the fallopian tube is provided with a horn-shaped suction cup which is convenient for fixing the fallopian tube umbrella; an egg collecting device for uterine horn egg flushing, a glass tube with the outer diameter of 10mm and the length of 130 mm; the two kinds of glass tubes are bent to 120 degrees in the middle of the alcohol blast lamp, and both ends are fired and ground smoothly;

because this collection ovum device intermediate junction is crooked, the position that collection ovum ware can be put increases, and the limitation is little, even connect the ovum carelessly sometimes or the pig is disturbed slightly, collection ovum ware can remove thereupon, can not cause towards the phenomenon that the ovum liquid flows outside collection ovum ware, or towards the ovum liquid of flowing in the collection ovum ware and spill.

(3) An embryo transfer device: the embryo transfer device is a tool for collecting eggs or embryos from one container and transferring the eggs or embryos to another container (such as collecting the eggs from a surface dish under a dissecting mirror after flushing the eggs and transferring the eggs to a culture dish) and is also a tool for transferring the embryos, the embryo transfer device is formed by drawing a suction pipe (shown in figure 3) from a glass tube with the outer diameter of about 4mm, then connecting the suction pipe with a scalp needle hose with a needle removed head, and splicing a liquid outlet end with a suction head (shown in figure 4) which is sequentially provided with an infusion apparatus.

The manufacturing steps are as follows:

a. igniting the alcohol blast burner, and placing a glass tube with the outer diameter of about 4mm above the flame of the alcohol blast burner;

b. continuously rotating the glass tube to ensure that the glass tube is uniformly heated and is burnt for about 4 seconds;

c. when the glass tube is burned to be red, the glass tube is pulled to be long by light force, and the length is 8 cm;

d. after the elongated glass tube is cooled, the middle of the elongated glass tube is broken into two glass suction tubes by a sand wheel;

e. the glass suction tube is connected with a silicone tube with the length of 20cm, and the connection is tight to prevent air leakage.

(4) Egg collection dish (watch dish): a8 cm diameter petri dish was prepared for receiving the egg wash solution during egg washing. Each donor pig needs 2 dishes, numbers are written, and the donor pigs are placed in an incubator for standby.

(5) Surgical equipment: sterilizing the instruments (such as scalpel, surgical scissors, hemostatic forceps, needle holder, tissue forceps, suture needle, suture thread, wound towel, gauze, watch glass, oviduct, and oviduct) under high pressure, and soaking the metal instruments in 0.1% benzalkonium bromide solution before operation.

2. Preparation of egg-washing liquid and culture liquid

(1) Washing the egg liquid: finished PBS or DPBS powder is purchased from a reagent company and prepared by double distilled water according to the instruction.

(2) Culture solution: the temporary culture can be performed by using an egg-washing solution, but 10% of calf serum is added, and the obtained product is filtered and sterilized by using a 0.22 mu m filter membrane, and then is subpackaged and stored for later use.

(II) preparation of operating theatre

Before each operation, the operating room is fumigated with formalin, and irradiated with ultraviolet lamp for 30min after ventilation.

1. Time of acquisition

According to the different embryo collecting parts, the embryo collecting method can be divided into oviduct embryo collecting and uterine horn embryo collecting. The distribution of early embryo in reproductive tract of sow is observed by fan Junhua et al (1995), and 7-8 months old Hubei white pig is bred after oestrus, and embryo collection is performed in oviduct or uterine horn by respectively 24 and 48 … … 192h with the time of finishing the last breeding as 0, and then microscopic examination is performed, and the result is shown in figure 4;

as can be seen from FIG. 4, if embryo collection from the oviduct should be performed 18 hours after the last mating, embryos of corresponding period are collected in different periods according to the standing reflex performance of different sows, and if time is delayed, the embryos enter the uterus, and the difficulty of embryo collection is greatly increased.

If 1-cell stage embryos are collected, the optimal time is 18-24 hours after artificial insemination; collecting embryos at 2-cell stage, wherein the optimal time is 26-32 hours after artificial insemination; collecting 4-cell stage embryos, optimally 34-40 hours after artificial insemination; the 8-cell stage embryos are collected, optimally 42-48 hours after artificial insemination.

2. Anesthesia and restraint of Donor pigs

It mainly includes the following several aspects:

(1) donor pig palate traction, limiting activity, 20mg/kg (per kilogram body weight) sodium pentobarbital (2.5%) or chloral magnesium sulfate hydrate injection (chloral hydrate 8%, magnesium sulfate 5%) 150ml ear vein injection, general anesthesia;

(2) because of different sensitivity to narcotics among individuals, the state of the pig needs to be observed at any time during injection, the injection speed needs to be slow, the excessive injection is prevented, and the pig can be used when the eyelid just loses reaction and the stimulation of the hoof has no obvious reflection;

(3) the anesthesia is put on the operation retaining frame in place and the four limbs are fixed;

(4) in order to prevent the pig from struggling and bouncing in the operation process, the limbs and the head are firmly bound by ropes;

(5) in order to lead the abdominal cavity viscera to incline forwards and facilitate the operation, the operating table needs to be low in front (low in head) and high in back (high in tail);

(6) the operation process comprises intramuscular or intravenous instillation of a proper amount of ketamine hydrochloride according to the requirement.

3. Surgical site and disinfection

The method mainly comprises the following steps:

s1: washing the operation part with soap water and a brush, and cleaning the operation part with a towel after washing;

s2: the surgical site is generally selected between the 2 nd and 3 rd nipples, hair is cut at the surgical site by electric scissors or hair scissors, the hair stubble is cut off, the surgical site is cleaned by clear water, wiped dry, then coated with 2% iodine tincture, and then deiodinated by 70% alcohol cotton;

s3: the disinfection procedure is as follows: iodine tincture cotton ball → alcohol cotton ball from inside to outside;

s4: covering the wound towel on the operation part to expose the predetermined incision in the middle of the opening of the wound towel.

4. Surgical method

The method mainly comprises the following steps:

s1: cutting skin 5cm along the midline of abdomen to avoid blood vessel, separating fat and muscle layer, exposing peritoneum, and cutting peritoneum with blunt scissors;

s2: the operator stretches the index finger and the middle finger out of the abdominal cavity of the patient from the incision, touches the uterus or the uterine horn at the front and back positions of the junction with the pelvic cavity, clamps the uterus or the uterine horn with the two fingers after touching the uterus or the uterine horn, and draws the uterus or the uterine horn to the surface of the wound;

s3: and (5) guiding out the oviduct and the ovary along one uterine horn, and observing the ovulation point on the surface of the ovary and the development of the follicle.

5. Embryo Collection

(1) Embryo collecting of fallopian tube

The method mainly comprises the following steps:

s1: inserting one end of the oviduct from the bell mouth of the umbrella part of the fallopian tube to the depth of about 1cm, fixing the oviduct by using a thumb and a forefinger, and connecting the other end of the oviduct with a watch;

s2: sucking 15ml of ovum-flushing liquid at 37 ℃ by using an injector, inserting a needle head of an ovum-flushing device in the uterine tube combining part towards the direction of an oviduct, pushing the injector in the uterine tube combining part, and enabling the ovum-flushing liquid to flow into the oviduct from the uterine tube combining part and flow to a watch glass through the oviduct;

s3: the other fallopian tube was then pulled out of the incision and the embryo was washed in the same manner.

(2) Uterine horn embryo collection

The method mainly comprises the following steps:

s1: according to the difference of the egg punching time, or the distance, a small hole is cut by an ophthalmologic scissors at the position 30cm away from the uterine horn junction part, which is not provided with blood vessels, and is inserted into a uterine horn fallopian tube, the opening of the small hole faces the direction of the uterine tube junction part, and the opening at the other end of the small hole is connected with a surface dish or a flat dish;

s2: injecting 30ml of ovum-flushing liquid into uterine horn from the uterine tube junction by using an injector;

s3: the ovum-flushing liquid flows through the uterine horn and brings the embryo into a surface dish or a plate for receiving the ovum;

s4: in view of the overlong uterine horn, a method of segmented ovum flushing can also be adopted;

s5: the other uterine horn was then pulled out of the incision and the embryo was rinsed in the same manner.

6. Egg picking

The method mainly comprises the following steps:

s1: placing the surface dish for receiving the ovum under a solid microscope, and finding out the embryo in the ovum flushing liquid by using the large visual field of a low-power lens;

s2: the diameter of the pig embryo is about 150 μm, the size of the pig embryo is only the size of a needle point when the pig embryo is observed by naked eyes, the pig embryo is a spherical body and is circular under a mirror, and the outer layer of the pig embryo is a transparent belt;

s3: the fallopian tubes were pipetted with a small amount of PBS followed by human eggs, and the washing was repeated in a second petri dish, then all the eggs were pooled in one petri dish, and one donor egg was placed in one dish and operated at room temperature of 20 ℃.

7. Post-operative treatment of donor sows

It mainly includes the following several aspects:

s1: after the embryo collection of the donor is finished, the uterus is moistened by sterilized normal saline at 37 ℃, the blood clot is washed away, and the organ is reset;

s2: after the peritoneum and the muscle are sutured, anti-inflammatory and antiseptic drugs such as sulfanilamide and the like are smeared;

s3: after the skin was sutured, iodine tincture, intramuscular injection of penicillin and streptomycin were applied around the wound.

Example 2:

referring to fig. 1-4, the present invention provides a technical solution: a method for collecting 1, 2, 4 and 8 cell-stage embryos in live pigs, which utilizes the difference of time and space to judge the development stage and the approximate position of the embryos in the bodies, mainly comprises the following steps:

firstly, collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination;

secondly, collecting 2-cell-stage embryos, wherein the optimal time is 26-32 hours after artificial insemination;

thirdly, collecting 4-cell-stage embryos, wherein the optimal time is 34-40 hours after artificial insemination;

and fourthly, collecting 8-cell-stage embryos, wherein the optimal time is 42-48 hours after artificial insemination.

The method mainly comprises the following steps:

preparation of embryos prior to Collection

1. Preparation of the apparatus

(1) An egg flushing device: as shown in figure 1, the ovum flushing device for flushing ovum of the oviduct is characterized in that a connecting part of the ovum flushing device is a hose formed by reforming an infusion apparatus, when a needle head is inserted into the oviduct, the limitation that the needle head is directly connected with an air charging and liquid charging device is overcome, the operation is free, and the needle head can be really inserted into the inner cavity of the oviduct. In the process of flushing ova, even if an operator shakes slightly or a pig shakes slightly, the phenomenon that the needle head scratches, scratches or falls off the oviduct can not occur due to the buffering effect of the hose, so that the success rate of flushing ova is greatly improved. In addition, the storage tank for the egg-washing liquid is particularly arranged, so that the use of the egg-washing liquid can be better controlled, and the complicated operation of manually pushing and pulling the injector by the miniature inflating pump is more time-saving and labor-saving.

(2) An egg collecting device: the ovum collecting device for washing ovum of the oviduct is two glass tubes with the length of 130mm, the wall thickness of 0.8mm and the inner diameter of 3mm, and the connecting part is connected by a universal connector; be equipped with loudspeaker form washing dish sucking disc with oviduct connecting portion and be convenient for fixed oviduct umbrella, because this collection ovum device intermediate junction is universal, the position increase that collection ovum ware can be put, the limitation is little, even connect sometimes that the ovum carelessly or the pig is disturbed slightly, collection ovum ware can remove thereupon, can not cause towards the phenomenon that the ovum liquid spills outside the collection ovum ware, or the interior towards the ovum liquid of collection ovum ware.

(3) An embryo transfer device: the embryo transfer device is a tool for collecting eggs or embryos from one container and transferring the eggs or embryos to another container (such as collecting the eggs from a surface dish under a dissecting mirror after flushing the eggs and transferring the eggs to a culture dish) and is also a tool for transferring the embryos, the egg transfer device is firstly formed by drawing a suction pipe (shown in figure 3) from a glass tube with the outer diameter of about 4mm, then is connected with a scalp needle hose with a needle removed head, and is formed by splicing a liquid outlet end with a suction head (shown in figure 4) which is sequentially provided with a transfusion device.

The manufacturing steps are as follows:

a. igniting the alcohol blast burner, and placing a glass tube with the outer diameter of about 4mm above the flame of the alcohol blast burner;

b. continuously rotating the glass tube to ensure that the glass tube is uniformly heated and is burnt for about 4.5 seconds;

c. when the glass tube is burned to be red, the glass tube is pulled to be long by light force, and the length is 8.5 cm;

d. after the elongated glass tube is cooled, the middle of the elongated glass tube is broken into two glass suction tubes by a sand wheel;

e. the glass suction tube is connected with a silicone tube with the length of 25cm, and the connection is tight to prevent air leakage.

(4) Egg collection dish (watch dish): a petri dish with a diameter of 9cm was prepared for receiving the egg-washing solution during washing of the eggs. Each donor pig needs 3 dishes, numbers are written, and the donor pigs are placed in an incubator for standby.

(5) Surgical equipment: sterilizing the instruments (such as scalpel, surgical scissors, hemostatic forceps, needle holder, tissue forceps, suture needle, suture thread, wound towel, gauze, watch glass, oviduct, and oviduct) under high pressure, and soaking the metal instruments in 0.1% benzalkonium bromide solution before operation.

2. Preparation of egg-washing liquid and culture liquid

(1) Washing the egg liquid: finished PBS or DPBS powder is purchased from a reagent company and prepared by double distilled water according to the instruction.

(2) Culture solution: the temporary culture can be performed by using an egg-washing solution, but 10% of calf serum is added, and the obtained product is filtered and sterilized by using a 0.22 mu m filter membrane, and then is subpackaged and stored for later use.

(II) preparation of operating theatre

Before each operation, the operating room is fumigated with formalin, and irradiated with ultraviolet lamp for 30min after ventilation.

1. Time of acquisition

According to the different embryo collecting parts, the embryo collecting method can be divided into oviduct embryo collecting and uterine horn embryo collecting. Researches find the distribution of early embryos in the reproductive tract of the sow, and for 7-8-month old white pigs, after oestrus, mating is carried out, the time for finishing the last mating is 0, the embryos are collected in different time periods respectively, and then microscopic examination is carried out, wherein the result is shown in figure 4;

as can be seen from FIG. 4, 1-cell stage embryos were collected, optimally 18-24 hours after artificial insemination; collecting embryos at 2-cell stage, wherein the optimal time is 26-32 hours after artificial insemination; collecting 4-cell stage embryos, optimally 34-40 hours after artificial insemination; the 8-cell stage embryos are collected, optimally 42-48 hours after artificial insemination.

2. Anesthesia and restraint of Donor pigs

It mainly includes the following several aspects:

(1) donor pig palatal traction, limiting activity, and general anesthesia by 25mg/kg (mg per kg body weight) sodium pentobarbital (2.5%) or chloral magnesium sulfate hydrate injection (chloral hydrate 8%, magnesium sulfate 5%) 175ml in ear vein;

(2) because of different sensitivity to narcotics among individuals, the state of the pig needs to be observed at any time during injection, the injection speed needs to be slow, the excessive injection is prevented, and the pig can be used when the eyelid just loses reaction and the stimulation of the hoof has no obvious reflection;

(3) the anesthesia is put on the operation retaining frame in place and the four limbs are fixed;

(4) in order to prevent the pig from struggling and bouncing in the operation process, the limbs and the head are firmly bound by ropes;

(5) in order to lead the abdominal cavity viscera to incline forwards and facilitate the operation, the operating table needs to be low in front (low in head) and high in back (high in tail);

(6) the operation process comprises intramuscular or intravenous instillation of a proper amount of ketamine hydrochloride according to the requirement.

3. Surgical site and disinfection

The method mainly comprises the following steps:

s1: washing the operation part with soap water and a brush, and cleaning the operation part with a towel after washing;

s2: the surgical site is generally selected between the 2 nd and 3 rd nipples, hair is cut at the surgical site by electric scissors or hair scissors, the hair stubble is cut off, the surgical site is cleaned by clear water, wiped dry, then coated with 3% iodine tincture, and then deiodinated by 72% alcohol cotton;

s3: the disinfection procedure is as follows: iodine tincture cotton ball → alcohol cotton ball from inside to outside;

s4: covering the wound towel on the operation part to expose the predetermined incision in the middle of the opening of the wound towel.

4. Surgical method

The method mainly comprises the following steps:

s1: cutting skin 7cm along the midline of abdomen to avoid blood vessel, separating fat and muscle layer, exposing peritoneum, and cutting peritoneum with blunt scissors;

s2: the operator stretches the index finger and the middle finger out of the abdominal cavity of the patient from the incision, touches the uterus or the uterine horn at the front and back positions of the junction with the pelvic cavity, clamps the uterus or the uterine horn with the two fingers after touching the uterus or the uterine horn, and draws the uterus or the uterine horn to the surface of the wound;

s3: and (5) guiding out the oviduct and the ovary along one uterine horn, and observing the ovulation point on the surface of the ovary and the development of the follicle.

5. Embryo Collection

(1) Embryo collecting of fallopian tube

The method mainly comprises the following steps:

s1: inserting one end of the egg collecting device into the position of about 1-2cm from the bell mouth of the umbrella part of the fallopian tube, fixing a sucker by using a thumb and a forefinger, connecting an injector with a sucker interface for washing a dish, pumping out air until the umbrella of the fallopian tube is tightly fixed on the sucker, and connecting the other end of the ovum collecting device with a surface dish;

s2: filling a liquid storage tank of the egg flushing device with egg flushing liquid, tightly covering a cover, enabling one end with a puncture needle to be located at a uterine tube joint part, inserting a needle head towards the direction of an oviduct (the needle head is not connected with an inflating device at the moment), opening a micro inflating pump switch, selecting proper impact strength of the egg flushing liquid by an adjusting valve, and starting to flush eggs at a connecting needle head after the impact strength is adjusted. When eggs are washed, the surface dish is kept balanced, and the surface dish needs to be replaced when the liquid is full;

s3: the other fallopian tube was then pulled out of the incision and the embryo was washed in the same manner.

(2) Uterine horn embryo collection

The method mainly comprises the following steps:

s1: according to the difference of the egg punching time, or the distance, a small hole is cut by an ophthalmologic scissors at a position 30-70cm away from the uterine horn junction part and without blood vessels on the back of the uterine horn, the uterine horn egg collecting device is inserted, the opening is towards the direction of the uterine tube junction part, and the opening at the other end is connected with a surface dish or a flat dish;

s2: injecting 30-50ml of ovum-flushing liquid into uterine horn from the uterine tube junction by using an ovum-flushing device;

s3: the ovum-flushing liquid flows through the uterine horn and brings the embryo into a surface dish or a plate for receiving the ovum;

s4: in view of the overlong uterine horn, a method of segmented ovum flushing can also be adopted;

s5: the other uterine horn was then pulled out of the incision and the embryo was rinsed in the same manner.

6. Egg picking

The method mainly comprises the following steps:

s1: placing the surface dish for receiving the ovum under a solid microscope, and finding out the embryo in the ovum flushing liquid by using the large visual field of a low-power lens;

s2: the diameter of the pig embryo is about 150 μm, the size of the pig embryo is only the size of a needle point when the pig embryo is observed by naked eyes, the pig embryo is a spherical body and is circular under a mirror, and the outer layer of the pig embryo is a transparent belt;

s3: the embryo transfer device aspirates a small amount of PBS followed by human eggs, and repeats the washing in a second petri dish in sequence, then all the eggs are collected in one petri dish, and one donor egg is placed in one dish and operated at 25 ℃.

7. Post-operative treatment of donor sows

It mainly includes the following several aspects:

s1: after the embryo collection of the donor is finished, the uterus is moistened by sterilized normal saline at 37 ℃, the blood clot is washed away, and the organ is reset;

s2: after the peritoneum and the muscle are sutured, anti-inflammatory and antiseptic drugs such as sulfanilamide and the like are smeared;

s3: after the skin was sutured, iodine tincture, intramuscular injection of penicillin and streptomycin were applied around the wound.

Example 3:

referring to fig. 1-4, the present invention provides a technical solution: a method for collecting 1, 2, 4 and 8 cell-stage embryos in live pigs, which utilizes the difference of time and space to judge the development stage and the approximate position of the embryos in the bodies, mainly comprises the following steps:

firstly, collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination;

secondly, collecting 2-cell-stage embryos, wherein the optimal time is 26-32 hours after artificial insemination;

thirdly, collecting 4-cell-stage embryos, wherein the optimal time is 34-40 hours after artificial insemination;

and fourthly, collecting 8-cell-stage embryos, wherein the optimal time is 42-48 hours after artificial insemination.

The method mainly comprises the following steps:

preparation of embryos prior to Collection

1. Preparation of the apparatus

(1) An egg flushing device: as shown in figure 1, the ovum-flushing device for flushing ovum of the oviduct, which is developed by the invention, overcomes the limitation of direct connection between a needle head and an injector when the needle head is inserted into the oviduct, has free operation and can ensure that the needle head is inserted into the inner cavity of the oviduct. In the process of flushing ova, even if an operator shakes slightly or a pig shakes slightly, the phenomenon that the needle head scratches, scratches or falls off the oviduct can not occur due to the buffering effect of the hose, so that the success rate of flushing ova is greatly improved.

(2) An egg collecting device: the ovum collecting device developed by the invention is shown in figure 2, and the ovum collecting tube used for washing ovum of the oviduct is a glass tube with the length of 140mm, the wall thickness of 1.1mm and the inner diameter of 4 mm; a uterine tube for collecting uterine horn ovum, a glass tube with the outer diameter of 10mm and the length of 140 mm; the two kinds of glass tubes are bent at an angle of 140 degrees in the middle of the alcohol blast lamp, and both ends are fired and ground smoothly;

because the middle of the egg collecting device is bent, the position where the egg collecting dish can be placed is increased, the limitation is small, even if the egg is not received carelessly or the pig slightly harasses, the egg collecting dish can move along with the egg collecting device, and the phenomenon that the egg flushing liquid flows out of the egg collecting dish or the egg flushing liquid in the egg collecting dish is poured can not be caused.

(3) An embryo transfer device: the embryo transfer device is a tool for collecting eggs or embryos from one container and transferring the eggs or embryos to another container (such as collecting the eggs from a surface dish under a dissecting mirror after flushing the eggs and transferring the eggs to a culture dish) and is also a tool for transferring the embryos, the embryo transfer device is formed by drawing a suction pipe (shown in figure 3) from a glass tube with the outer diameter of about 4mm, then connecting the suction pipe with a scalp needle hose with a needle removed head, and splicing a liquid outlet end with a suction head (shown in figure 4) which is sequentially provided with an infusion apparatus.

The manufacturing steps are as follows:

a. igniting the alcohol blast burner, and placing a glass tube with the outer diameter of about 4mm above the flame of the alcohol blast burner;

b. continuously rotating the glass tube to ensure that the glass tube is uniformly heated and is burnt for about 5 seconds;

c. when the glass tube is burned to be red, the glass tube is pulled to be long by light force, and the length is 9 cm;

d. after the elongated glass tube is cooled, the middle of the elongated glass tube is broken into two glass suction tubes by a sand wheel;

e. the glass suction tube is connected with a silicone tube with the length of 30cm, and the connection is tight to prevent air leakage.

(4) Egg collection dish (watch dish): a10 cm diameter petri dish was prepared for receiving the egg wash solution during egg washing. Each donor pig needs 4 dishes, numbers are written, and the donor pigs are placed in an incubator for standby.

(5) Surgical equipment: sterilizing the instruments (such as scalpel, surgical scissors, hemostatic forceps, needle holder, tissue forceps, suture needle, suture thread, wound towel, gauze, watch glass, oviduct, and oviduct) under high pressure, and soaking the metal instruments in 0.1% benzalkonium bromide solution before operation.

2. Preparation of egg-washing liquid and culture liquid

(1) Washing the egg liquid: finished PBS or DPBS powder is purchased from a reagent company and prepared by double distilled water according to the instruction.

(2) Culture solution: the temporary culture can be performed by using an egg-washing solution, but 10% of calf serum is added, and the obtained product is filtered and sterilized by using a 0.22 mu m filter membrane, and then is subpackaged and stored for later use.

(II) preparation of operating theatre

Before each operation, the operating room is fumigated with formalin, and irradiated with ultraviolet lamp for 30min after ventilation.

1. Time of acquisition

Collecting embryos at 1-cell stage, wherein the optimal time is 18-24 hours after artificial insemination; collecting embryos at 2-cell stage, wherein the optimal time is 26-32 hours after artificial insemination; collecting 4-cell stage embryos, optimally 34-40 hours after artificial insemination; the 8-cell stage embryos are collected, optimally 42-48 hours after artificial insemination. Performing surgical operation at different time periods, collecting embryos, and performing microscopic examination, wherein the result is shown in fig. 4;

as can be seen from FIG. 4, if embryos are collected from the fallopian tubes, they should be performed within 48 hours of the last mating; if embryos are collected from the uterine horn, it should be done 48 hours after the last mating until the embryo is implanted, and care should be taken to place the embryo at the site of the uterine horn at different developmental times for the purpose of orientation. The longer the period after 48 hours, the more scattered the distribution of the embryos in the uterine horn, and the greater the difficulty in collecting the embryos.

2. Anesthesia and restraint of Donor pigs

It mainly includes the following several aspects:

(1) donor pig palate traction, limiting activity, according to 30mg/kg (per kilogram body weight) sodium pentobarbital (2.5%) or chloral magnesium sulfate hydrate injection (chloral hydrate 8%, magnesium sulfate 5%) 200ml ear vein injection, general anesthesia;

(2) because of different sensitivity to narcotics among individuals, the state of the pig needs to be observed at any time during injection, the injection speed needs to be slow, the excessive injection is prevented, and the pig can be used when the eyelid just loses reaction and the stimulation of the hoof has no obvious reflection;

(3) the anesthesia is put on the operation retaining frame in place and the four limbs are fixed;

(4) in order to prevent the pig from struggling and bouncing in the operation process, the limbs and the head are firmly bound by ropes;

(5) in order to lead the abdominal cavity viscera to incline forwards and facilitate the operation, the operating table needs to be low in front (low in head) and high in back (high in tail);

(6) the operation process comprises intramuscular or intravenous instillation of a proper amount of ketamine hydrochloride according to the requirement.

3. Surgical site and disinfection

The method mainly comprises the following steps:

s1: washing the operation part with soap water and a brush, and cleaning the operation part with a towel after washing;

s2: the surgical site is generally selected between the 2 nd and 3 rd nipples, hair is cut at the surgical site by electric scissors or hair scissors, the hair stubble is cut off, the surgical site is cleaned by clear water, wiped dry, then coated with 4% iodine tincture, and then deiodinated by 75% alcohol cotton;

s3: the disinfection procedure is as follows: iodine tincture cotton ball → alcohol cotton ball from inside to outside;

s4: covering the wound towel on the operation part to expose the predetermined incision in the middle of the opening of the wound towel.

4. Surgical method

The method mainly comprises the following steps:

s1: cutting skin 8cm along the midline of abdomen to avoid blood vessel, separating fat and muscle layer, exposing peritoneum, and cutting peritoneum with blunt scissors;

s2: the operator stretches the index finger and the middle finger out of the abdominal cavity of the patient from the incision, touches the uterus or the uterine horn at the front and back positions of the junction with the pelvic cavity, clamps the uterus or the uterine horn with the two fingers after touching the uterus or the uterine horn, and draws the uterus or the uterine horn to the surface of the wound;

s3: and (5) guiding out the oviduct and the ovary along one uterine horn, and observing the ovulation point on the surface of the ovary and the development of the follicle.

5. Embryo Collection

(1) Embryo collecting of fallopian tube

The method mainly comprises the following steps:

s1: inserting one end of an egg collecting device into the range of about 1-2cm from a bell mouth of an umbrella part of the fallopian tube, fixing a sucker by using a thumb and a forefinger, connecting and connecting a dish washing sucker interface by using an injector in a linking manner, pumping out air until the fallopian tube umbrella is tightly fixed on the sucker, and connecting the other end of the fallopian tube umbrella with a surface dish;

s2: filling a liquid storage tank of the egg flushing device with egg flushing liquid, tightly covering a cover, enabling one end with a puncture needle to be located at a uterine tube joint part, inserting a needle head towards the direction of an oviduct (the needle head is not connected with an inflating device at the moment), opening a micro inflating pump switch, selecting proper impact strength of the egg flushing liquid by an adjusting valve, and starting to flush eggs at a connecting needle head after the impact strength is adjusted. When eggs are washed, the surface dish is kept balanced, and the surface dish needs to be replaced when the liquid is full;

s3: the other fallopian tube was then pulled out of the incision and the embryo was washed in the same manner.

(2) Uterine horn embryo collection

The method mainly comprises the following steps:

s1: according to the difference of the egg punching time, or the distance, a small hole is cut by an ophthalmologic scissors at a position 30-70cm away from the uterine horn junction part and without blood vessels on the back of the uterine horn, the uterine horn egg collecting device is inserted, the opening is towards the direction of the uterine tube junction part, and the opening at the other end is connected with a surface dish or a flat dish;

s2: injecting 30-50ml of ovum-flushing liquid into uterine horn from the uterine tube junction by using an ovum-flushing device;

s3: the ovum-flushing liquid flows through the uterine horn and brings the embryo into a surface dish or a plate for receiving the ovum;

s4: in view of the overlong uterine horn, a method of segmented ovum flushing can also be adopted;

s5: the other uterine horn was then pulled out of the incision and the embryo was rinsed in the same manner.

6. Egg picking

The method mainly comprises the following steps:

s1: placing the surface dish for receiving the ovum under a solid microscope, and finding out the embryo in the ovum flushing liquid by using the large visual field of a low-power lens;

s2: the diameter of the pig embryo is about 150 μm, the size of the pig embryo is only the size of a needle point when the pig embryo is observed by naked eyes, the pig embryo is a spherical body and is circular under a mirror, and the outer layer of the pig embryo is a transparent belt;

s3: the embryo transfer device aspirates a small amount of PBS followed by human eggs, and repeats the washing in a second petri dish in sequence, then all the eggs are collected in one petri dish, and one donor egg is placed in one dish and operated at 25 ℃.

7. Post-operative treatment of donor sows

It mainly includes the following several aspects:

s1: after the embryo collection of the donor is finished, the uterus is moistened by sterilized normal saline at 37 ℃, the blood clot is washed away, and the organ is reset;

s2: after the peritoneum and the muscle are sutured, anti-inflammatory and antiseptic drugs such as sulfanilamide and the like are smeared;

s3: after the skin was sutured, iodine tincture, intramuscular injection of penicillin and streptomycin were applied around the wound.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.