CN113749045A - Method for improving cow breeding efficiency - Google Patents
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- CN113749045A CN113749045A CN202111072107.3A CN202111072107A CN113749045A CN 113749045 A CN113749045 A CN 113749045A CN 202111072107 A CN202111072107 A CN 202111072107A CN 113749045 A CN113749045 A CN 113749045A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- Environmental Sciences (AREA)
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- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A method for improving the reproductive efficiency of cows comprises the following steps: step 1: obtaining an embryo; step 2: receptor cow in-phase estrus PG method; and step 3: loading the embryo into a tube; and 4, step 4: gun loading and transplanting: and 5: and (5) managing the transplanted recipient cow. The invention can fully explore the breeding potential of breeding cows, improve the breeding efficiency of cows, and improve the breeding potential of cows to 1 year, 1 fetus and 1 calf in natural state to 1 year, 1 fetus and 1 calf in 1 fetus, even 1 fetus and two calves by purchasing frozen embryos, carrying out superovulation, artificial insemination and living embryo flushing (in-vivo embryos) on donor cows, in-vitro fertilization and in-vitro culture (in-vitro embryos) of embryos and combining with double embryo transplantation on recipient cows.
Description
Technical Field
The invention relates to a method for improving cow breeding efficiency.
Background
Cattle are single-fetus animals, and have longer breeding cycle and lower breeding rate in a natural state. Usually 2 fetuses and 1 calf in 3 years, and 1 fetuses and two calves and 1 fetuses and 3 calves are occasionally observed. If calculated according to the reproductive age of 10 years of cow, the cow can only leave 7-8 offspring at most in one birth. For cows, only 3-4 female calves are left. The primary matching age of a replacement cow with normal development is generally 1.5-2 years old, the oestrus cycle is generally 21 days, and the pregnancy period is generally 280 days. From the reproductive characteristics and the reproductive cycle of the cattle, the cattle have the basic conditions of 1 birth and more calves in 1 year. Therefore, the research and development of the method for improving the breeding efficiency of the cows and realizing 1-birth and more calves in 1 year has very important significance for improving the cow breeding benefit.
The prior art mainly has two kinds: firstly, Artificial Insemination (AI) + Embryo Transfer (ET) technique; secondly, an exogenous hormone induction method: the use of gonadotropins such as: pregnant Mare Serum (PMSG), Human Chorionic Gonadotropin (HCG), trihexyphenidyl hormone (ITC), anti-gonadotropin serum (anti-PMSG) and the like. Two prior art techniques.
The technical scheme of the prior art I is as follows:
the technical scheme of Artificial Insemination (AI) and Embryo Transplantation (ET) is that artificial insemination is carried out on the 1 st day after the cow is in heat (the 0 th day on the heat day) to ensure that the cow is pregnant, and 1 embryo (both fresh embryo and frozen embryo) is transplanted on the opposite side of the uterine horn on the pregnant side (namely, the uterine horn on the opposite side of the corpus luteum) on the 7 th day so as to achieve the purpose of improving the reproductive rate of the cow.
The first prior art has the following defects:
the technical scheme of Artificial Insemination (AI) + Embryo Transfer (ET) has the following disadvantages: firstly, artificial insemination can not ensure the pregnancy of cows; secondly, rectal examination is needed when embryo transfer is carried out on the 7 th day to judge whether the artificial insemination is pregnant and the position of pregnancy (the left side or the right side of the uterine horn) so as to determine the position of embryo transfer (the opposite side of the uterine horn on the pregnant side, namely, the uterine horn on the opposite side of the corpus luteum). This procedure is susceptible to abortion in pregnant cows; thirdly, if the artificial insemination is pregnant, the cervix of the cow is in a contraction and closing state, at the moment, a transplantation gun is not easy to pass through the cervix during embryo transplantation, the transplantation difficulty is increased, bacteria are easy to invade the uterus, and further abortion is caused, so that the abortion is avoided.
The technical scheme of the prior art II is as follows:
the exogenous hormone induction method mainly utilizes injection of gonadotropins such as: pregnant Mare Serum (PMSG), Human Chorionic Gonadotropin (HCG), triplet hormone (ITC), anti-gonadotropin serum (anti-PMSG) and the like induce the cow to discharge a plurality of eggs in one ovulation period, thereby achieving the purpose of improving the high reproduction rate of the cow.
The second prior art has the following defects:
the exogenous hormone induction method has the following defects: firstly, exogenous hormones are various in types, different in components and different in effects, so that the selection is difficult; secondly, due to the existence of individual differences of cows, the dosage cannot be accurately controlled, namely the use effect cannot be ensured; thirdly, the specific ovulation number cannot be accurately known after the exogenous hormone is used, and once the ovulation number exceeds 2 (most of the ovulation number exceeds 2), the probability of pregnancy abortion is greatly improved.
Disclosure of Invention
The invention aims to effectively solve the problems of high operation difficulty and easy artificial abortion in the prior art; the second prior art has the technical problems that the hormone types are difficult to select, the dosage is difficult to control, and multiple abortion occurs.
In order to solve the technical problems, the invention adopts a double-embryo transfer technical scheme, namely: the technical proposal of transplanting 2 embryos to a recipient cow at a time by using an embryo transplantation technology is utilized, thereby achieving the purpose of improving the breeding efficiency of cows.
A method for improving the reproductive efficiency of cows comprises the following steps:
step 1: obtaining an embryo;
step 2: receptor cow in-phase estrus PG method: the contemporaneous estrus of the recipient cow is kept consistent with that of the donor cow in time, namely PG is injected intramuscularly at a dose of 2/head in the morning on the 12 th day, the 26 th day and the 27 th day respectively, and the estrus is observed and recorded on the 28 th to 29 th days;
and step 3: loading the embryo into a tube;
and 4, step 4: gun loading and transplanting:
1) loading the tubule filled with 2 embryos into a transplantation gun, wherein a sleeve is externally arranged outside the transplantation gun, and a soft plastic outer sleeve is externally arranged outside the sleeve;
2) 2 embryos are transplanted into a recipient cow which is processed by estrus synchronization by using a rectal grasping method, wherein the recipient cow is located at a position which is 1/2-1/4 deep from the top of a uterine horn. The action is gentle during the transplantation, so as to avoid the bleeding caused by the puncture of the cervix and the uterus. The transplantation time is controlled to be completed within 5 min as much as possible;
and 5: managing a recipient cow after transplantation:
factors such as the nutritional level and the stress of the transplanted recipient cattle can influence the conception rate. Therefore, in addition to the well-done regular feeding management, the following points should be noted:
1) the transplanted receptor cattle and other cattle are bred in groups;
2) the nutrition requirement is ensured, and the successful pregnancy and calving after transplantation are ensured;
3) and (5) paying attention to the condition of returning to the emotion and carrying out pregnancy check in time. The pregnancy and the fetal number of the receptor cows can be confirmed by adopting a B ultrasonic or rectal examination method within 28 to 60 days after transplantation;
4) the non-pregnant recipient cattle determined by pregnancy examination should be bred or transplanted in time.
The embryo acquisition requires, for purchase of frozen embryos: grade a embryos.
The method for obtaining the A-grade and/or B-grade embryo by flushing the donor cow living body embryo at 7 days after superovulation and artificial insemination comprises the following steps:
step 1: selection of donor cows: selecting young cows with age of 2-5 years, strong constitution, complete development of reproductive organs and no reproductive system diseases or cows with 1 birth as donors;
step 2: donor cows PG method of estrus synchronization, superovulation and artificial insemination procedure: PG was injected intramuscularly at 2/head dose on day 1. On day 12 PG was again given intramuscularly at 2/head dose; FSH was injected into each muscle at 3 ml/head for 1 time at 7 am and 7 pm on day 24; on day 25, FSH was administered 1 time by 2.5 ml/head 7 o' clock in the morning and at the evening. FSH is injected for 1 time at 7 points in the morning of 26 days according to the volume of 2 ml/head muscle; FSH is injected 1 time per head muscle at 2 ml/2M/head, and PG is injected 1 time per head at 7 pm. FSH is injected 1 time and PG is injected 1 time at 7 points of the morning at 1 ml/head muscle and 2 times per head on day 27; FSH was injected 1 time per 1 ml/head muscle at 7 o' clock night. Observing estrus and performing artificial insemination on the estrus cows 28-29 days, and recording the day of artificial insemination as 0 day; intramuscular injection of 20-30 μ g/head promoting row III on day 29;
and step 3: and (3) embryo punching: on day 35, i.e., 7 days after artificial insemination, in vivo embryo flushing was performed.
The embryo is obtained by in vitro fertilization culture; the method comprises the following steps: a grade a embryo comprising the steps of:
step 1: and (3) processing the sperms:
1) and (3) unfreezing frozen semen: water bath at 37 deg.c for about 5 sec;
2) adding 200 mul of double antibody into 15 ml of the semen to be mixed evenly;
3) adding 7 ml of semen into a centrifugal tube, cutting one end of the frozen semen without a cotton plug, immersing the section into the semen washed by the centrifugal tube, cutting one end of the cotton plug, completely blowing the frozen semen into the tube, uniformly mixing and centrifuging for 5 min at the rotating speed of 1500 r/min;
4) after the centrifugation is finished, removing the supernatant, adding 7 ml of washing liquid, and centrifuging for 5 min at the rotating speed of 1500 r/min, and removing the supernatant after the centrifugation is finished;
5) measuring the amount of the semen washing solution in the residual centrifuge tube, adding the semen washing solution to 750 mu l, adding 750 mu l of the ovum washing solution, and uniformly mixing;
6) making 100 mul of liquid drop fertilization plate cover oil, and balancing a warm box for standby;
step 2: and (3) processing the oocyte:
1) after the oocyte is mature, gently blowing and beating the physical degranulation cells in a washing tray by using a 100 mu l pipette gun, and completely removing the physical degranulation cells as far as possible;
2) cleaning the oocyte after the granular cells are removed by using an egg washing liquid;
and step 3: fertilization:
1) putting the cleaned oocyte into a fertilization drip tray;
2) dropping 15-20 oocytes in 100 mul;
3) after the fertilization time reaches 8 h, transferring fertilized eggs from the fertilization plate to a culture plate as soon as possible;
and 4, step 4: in vitro culture:
1) making one IVC drip washing plate, culturing with four-hole plate (500 μ l per hole), and covering with oil for balancing overnight;
2) removing the adhering impurities outside the fertilized eggs as clean as possible;
3) after washing, the cells were transferred to a culture dish, and the number of cells per well was not more than 50.
When the embryo is frozen, the embryo is taken out from a liquid nitrogen tank and slowly waved in the air for 10s, namely air bath, then put into water at 37 ℃ for thawing, taken out after ice crystals are completely thawed, the end without the foam plug is cut by a tubule shear, the embryo is transferred into a protective solution, the procedure is repeated to thaw another embryo, the quality of the embryo is checked under a 100-fold microscope, and the embryo which meets the A-level embryo is re-loaded into tubules according to 2 tubules per tubule. And (3) loading the tube in a mode of 'protective solution + air section + protective solution + embryo + protective solution' during reloading.
The embryo tubulation adopts the embryo which is fresh embryo obtained by living embryo flushing and in vitro culture embryo A grade and/or B grade obtained by in vitro fertilization, and directly tubulates according to the mode of 'protective solution + air section + protective solution + embryo + protective solution'.
The embryo punching comprises the following steps:
step 1: donor cattle baoding: driving the donor cattle to a fixing frame for fixing;
step 2: rectal examination and vulvar washing: cleaning up the feces of the rectum of the well-defined donor cattle, checking the development condition of the ovary corpus luteum at two sides, and recording the number of the ovaries; thoroughly cleaning the vulva of the donor cattle with toilet paper or clear water, soapy water, physiological saline and the like, and then spraying alcohol with an alcohol watering can for disinfection;
and step 3: and (3) dilating cervix: for young cows, because the cervix is tightly closed, a dilating rod is used to dilate the cervix in order to draw mucus and the vas deferens into the uterus. For a multiparous cow, this step may be optional depending on rectal examination. The principle is that a mucus pump and a embryo washing tube can smoothly pass through the cervix to reach the corresponding position of the uterus;
and 4, step 4: extracting mucus: an operator slowly inserts the clean mucus pump into the cervix of a donor cow through the vagina, and an assistant rapidly pumps out the handle of the mucus pump, which is similar to the handle of an injector, so that the tube of the mucus pump instantly forms vacuum negative pressure, mucus in the cervix and the uterus is sucked into the tube of the mucus pump, and the mucus pump is slowly pumped out of the cervix. The steps can be repeated for 1-2 times according to the mucus extraction condition until the mucus is basically extracted completely;
and 5: anesthesia: 4ml of procaine hydrochloride or other anesthetic is injected into the cavities of the second caudal vertebra and the third caudal vertebra to perform local anesthesia on the donor cow. The judgment standard of anesthesia success is as follows: after a few minutes of anesthetic injection, the cow tail is gently shaken left and right without resistance, which indicates that the anesthesia is successful. If resistance exists, the anesthetic dosage needs to be increased until the anesthesia succeeds;
step 6: inserting a blank punching tube: the special embryo-washing tube is slowly passed through cervix via rectum and inserted into uterine horn. When the uterine horn is near the thin part of the uterine horn, the guide steel core in the embryo washing pipe is slowly drawn out by 10-15cm, so that the rubber pipe at the front section of the embryo washing pipe becomes flexible to adapt to the natural bending of the uterine horn. When the embryo-washing tube reaches the corresponding position, the more the embryo-washing tube goes deep into the uterine horn, the better the embryo-washing tube is, 10-15 ml of air is injected by a 20ml injector through the air hole of the embryo-washing tube, the specific injected air quantity is determined according to the thickness of the cervix, the operation aim is to enable the balloon at the front end of the embryo-washing tube to be full, the outward outlet of the cervix is blocked, the embryo-washing liquid can be discharged into the funnel only through the embryo-washing tube, and the embryo is convenient to collect. Then slowly drawing out the blank punching pipe to guide the steel core;
and 7: connecting a blank punching pipe and a funnel by using a three-way pipe: one end of the three-way pipe is connected with a blank punching pipe, one end of the three-way pipe is connected with a blank collecting funnel, the other end of the three-way pipe is reserved with injection blank punching liquid, and joints of all parts are screwed;
and 8: and (3) embryo punching: after the connection between the embryo punching tube and the three-way tube is finished, firstly, the hemostatic forceps are used, and the sawtooth part at the tip is bound by medical white tape so as to avoid damaging the transparent hose and clamp the hose at the end of the embryo collecting funnel; at the moment, 100 ml of a large-size syringe is used for extracting 37.6 ℃ constant-temperature water bath and 60 ml of embryo-flushing liquid, the embryo-flushing liquid is connected to the opening of the connecting hose, the embryo-flushing liquid is slightly pumped back, and then the embryo-flushing liquid is slowly injected into the uterus until the injection resistance is slightly existed; then the hemostatic forceps are loosened, the hose connected with the injector is clamped, and embryo washing liquid in the uterus flows back to the embryo collecting funnel, the operation is repeated for a plurality of times, the injection speed at the later stage can be accelerated, the uterus is washed by the embryo washing liquid, so that the free embryo is the best embryo washing time about 7 days after the hybridization, and the embryo is not implanted in the uterus at this time; injecting 30-50 ml each time with the embryo flushing liquid flowing back to the embryo collecting funnel; 500 ml of embryo flushing liquid is used in the whole process; after the completion, 20ml of the injector is connected to the blast hole of the embryo-washing tube, so that the air at the front end of the embryo-washing tube flows back, the injector is slightly drawn out to facilitate the expansion of the balloon at the front end of the embryo-washing tube, the embryo-washing tube is slowly drawn out, the front end of the embryo-washing tube is lifted, and the embryo-washing liquid in the tube completely enters the embryo collecting funnel. Taking down the embryo collecting funnel, and marking the donor cow number, the left and right embryo-flushing uterine horn and the embryo-checking position by using a marking pen on a funnel cover;
repeating the steps 6, 7 and 8 to punch the uterine horn on the other side;
and step 9: injection of P: injecting 4ml PG intramuscularly after embryo flushing to promote luteal lysis and enter the next estrus cycle;
unbinding the donor cattle, and replacing the donor cattle, and repeating the steps 1 to 9.
The PG is chloroprostenol.
The semen washing liquid comprises 15 ml of SOF, 0.3 mg of heparin and 0.03183g of Caffeine.
The double-resistant is penicillin and streptomycin.
The egg washing solution comprises 8 ml of SOF, 0.16 mg of heparin and 0.048 g of BSA.
The protective solution is 10% 199 solution, namely 1ml FBS +9ml 199 base solution.
The beneficial effect of adopting above-mentioned technical scheme is:
the invention can fully explore the breeding potential of breeding cows, improve the breeding efficiency of cows, and improve the breeding potential of cows to 1 year, 1 fetus and 1 calf in natural state to 1 year, 1 fetus and 1 calf in 1 fetus, even 1 fetus and two calves by purchasing frozen embryos, carrying out superovulation, artificial insemination and living embryo flushing (in-vivo embryos) on donor cows, in-vitro fertilization and in-vitro culture (in-vitro embryos) of embryos and combining with double embryo transplantation on recipient cows.
Detailed Description
The invention is described in further detail below:
a method for improving cow breeding efficiency comprises the following specific operation steps:
first, embryo acquisition
The embryo can be obtained by the following 3 ways:
(one) purchase of frozen embryos, requiring: grade a embryos.
And (II) adopting a grade A embryo and/or a grade B embryo obtained by flushing the donor cow living body embryo on the 7 th day after superovulation and artificial insemination.
The operation procedure is as follows:
1. selection of donor cows: young cows with age of 2-5 years, strong constitution, complete development of reproductive organs and no reproductive system diseases or cows with 1 birth are selected as donors.
2. Donor cow estrus synchronization (PG method), superovulation and artificial insemination procedures: PG was injected intramuscularly at 2/head dose on day 1. PG (chloroprostenol) was again injected intramuscularly at 2/head dose on day 12. FSH (gonadotropin, produced in Canada, size 20ml, 700 mg) was injected 1 time per 3 ml/head muscle at 7 am and 7 pm on day 24. On day 25, FSH was administered 1 time by 2.5 ml/head 7 o' clock in the morning and at the evening. FSH is injected for 1 time at 7 points in the morning of 26 days according to the volume of 2 ml/head muscle; FSH is injected 1 time per head muscle at 2 ml/2M/head, and PG is injected 1 time per head at 7 pm. FSH is injected 1 time and PG is injected 1 time at 7 points of the morning at 1 ml/head muscle and 2 times per head on day 27; FSH was injected 1 time per 1 ml/head muscle at 7 o' clock night. On days 28-29, estrus was observed and artificial insemination was performed on the estrus cows (day of artificial insemination scored as 0 day). Intramuscular injection was performed on day 29 with a third 20-30. mu.g/head.
3. And (3) embryo punching: viable embryo flushing was performed on day 35 (day 7 after artificial insemination).
(1) Donor cattle baoding: the donor cattle were driven to a holding rack for holding.
(2) Rectal examination and vulvar washing: cleaning up the feces of the fixed donor cattle rectum, checking the development condition of the ovary corpus luteum on two sides, and recording the number (aiming at conveniently judging the number of embryos when the embryos are washed and checked). The vulva of the donor cattle is thoroughly cleaned by toilet paper or clear water (soap water), physiological saline and the like, and then is disinfected by spraying alcohol with an alcohol watering can.
(3) And (3) dilating cervix: for young cows, because the cervix is tightly closed, a dilating rod is used to dilate the cervix in order to draw mucus and the vas deferens into the uterus. For a multiparous cow, this step may be optional depending on rectal examination. The principle is that the mucus pump and the embryo washing tube can smoothly pass through the cervix to reach the corresponding position of the uterus.
(4) Extracting mucus: an operator slowly inserts the clean mucus pump into the cervix of a donor cow through the vagina, and an assistant rapidly pumps out the handle of the mucus pump (similar to the handle of an injector), so that the mucus pump hose instantly forms vacuum negative pressure, mucus in the cervix and the uterus is sucked into the mucus pump hose, and the mucus pump is slowly pumped out of the cervix. This step can be repeated 1-2 times according to mucus extraction condition until mucus is substantially extracted.
(5) Anesthesia: 4ml of procaine hydrochloride (or other anesthetic) was injected into the second and third caudal vertebra cavities to locally anesthetize the donor cows. The judgment standard of anesthesia success is as follows: after a few minutes of anesthetic injection, the cow tail is gently shaken left and right without resistance, which indicates that the anesthesia is successful. If there is resistance, the anesthetic dose needs to be increased until the anesthesia succeeds.
(6) Inserting a blank punching tube: the special embryo-washing tube is slowly passed through cervix via rectum and inserted into uterine horn. When the uterine horn is near the thin part of the uterine horn, the guide steel core in the embryo washing pipe is slowly drawn out by 10-15cm, so that the rubber pipe at the front section of the embryo washing pipe becomes flexible to adapt to the natural bending of the uterine horn. When the embryo-washing tube reaches the corresponding position (the deeper the embryo-washing tube reaches the thinner part of uterine horn, the better), 10-15 ml of air is injected through the air hole of the embryo-washing tube by using a 20ml injector (the specific injected air quantity is determined according to the thickness of cervix), the operation aim is to enable the balloon at the front end of the embryo-washing tube to be full, the outward outlet of the cervix is blocked, the embryo-washing liquid can be discharged into the funnel only through the embryo-washing tube, and the embryo is convenient to collect. Then the blanking tube is slowly drawn out to guide the steel core.
(7) Connecting a blank punching pipe and a funnel by using a three-way pipe: one end of the three-way pipe is connected with a blank punching pipe, one end of the three-way pipe is connected with a blank collecting funnel, the other end of the three-way pipe is reserved for injecting blank punching liquid, and the joints of all parts are screwed down.
(8) And (3) embryo punching: after the connection between the embryo punching tube and the three-way tube is finished, the tube at the end of the embryo collecting funnel is clamped by hemostatic forceps (the sawtooth part at the tip is tied up by medical white tape so as to avoid damaging the transparent tube). At this time, a large-size syringe (100 ml) is used for extracting the embryo-flushing liquid (constant-temperature water bath at 37.6 ℃ and about 60 ml) to be connected with the opening of the connecting hose, the liquid is slightly pumped back (aiming at exhausting the air in the embryo-flushing pipe as much as possible), and then the embryo-flushing liquid is slowly injected into the uterus until the injection resistance is slightly reached. Then the hemostatic forceps are loosened, the hose connected with the injector is clamped, the embryo flushing liquid in the uterus is enabled to flow back to the embryo collecting funnel, and the operation is repeatedly carried out for a plurality of times (the injection speed in the later period can be accelerated, which is similar to the process of flushing the uterus with the embryo flushing liquid, so that the free embryos (the best embryo flushing time is about 7 days after the hybridization, the embryos are not implanted in the uterus at this time) flow back to the embryo collecting funnel along with the embryo flushing liquid, and 30-50 ml is injected each time). About 500 ml of embryo-flushing liquid is used in the whole process. After the completion, an injector (20 ml) is connected to the blast hole of the embryo-washing tube to lead the air at the front end of the embryo-washing tube to flow back, the injector is slightly drawn out to facilitate the expansion of the balloon at the front end of the embryo-washing tube, the embryo-washing tube is slowly drawn out, the front end of the embryo-washing tube is lifted, and the embryo-washing liquid in the tube completely enters the embryo collecting funnel. The embryo collecting funnel is taken down, the donor cow number and the embryo uterine horn (left and right) are marked on the funnel cover by a marking pen, and then the embryo is sent to be detected.
Repeating the steps (6), (7) and (8) to make the uterine horn on the other side.
(9) PG (chloroprostenol) injection: 4ml of PG is injected into muscles after embryo flushing is finished, so that luteal lysis is promoted, and the next estrus cycle is started.
Unbinding the donor cattle, and replacing the donor cattle to repeat the steps (1) to (9).
And (III) culturing the obtained embryo by In Vitro Fertilization (IVF). The method comprises the following steps: grade a embryos.
The operation procedure is as follows:
1. processing sperm
(1) And (3) unfreezing frozen semen: water bath at 37 deg.c for about 5 sec.
(2) To 15 ml of the eluate (SOF 15 ml + heparin 0.3 mg + Caffeine 0.03183 g) was added 200. mu.l of a diabody (penicillin + streptomycin) and mixed well.
(3) Adding 7 ml of semen into a centrifugal tube, cutting one end of the frozen semen without a cotton plug, immersing the section into the semen washed by the centrifugal tube, cutting one end of the cotton plug, completely blowing the frozen semen into the tube, uniformly mixing and centrifuging for 5 min (rotating speed 1500 r/min).
(4) And after the centrifugation is finished, removing the supernatant, adding 7 ml of washing liquid, and centrifuging for 5 min (the rotating speed is 1500 r/min), thus removing the supernatant.
(5) The amount of the sperm in the remaining centrifuge tube was measured and added to 750. mu.l, and 750. mu.l of an egg wash (8 ml of SOF, 0.16 mg of heparin, 0.048 g of BSA) was added thereto and mixed well.
(6) Making 100 mul of liquid drop fertilization plate cover oil, and balancing the incubator for later use.
2. Processing oocytes
(1) After maturation of the oocytes, the physically degranulated cells were gently blown in a wash dish using a 100. mu.l pipette and removed as completely as possible.
(2) The oocytes from which the granulocytes had been removed were washed with an egg wash (SOF 8 ml + heparin 0.16 mg + BSA 0.048 g).
3. Fertilization of
(1) The washed oocytes were placed in a fertilization drip tray.
(2) 15-20 oocytes were dropped in 100. mu.l drops.
(3) After 8 h fertilization time, the fertilized eggs were transferred from the fertilization tray to the culture tray as soon as possible.
4. In vitro culture
(1) One IVC drip plate was made and the plates were incubated with 500. mu.l of four well plates per well and equilibrated overnight with oil.
(2) The conglutination impurities outside the fertilized eggs are removed as clean as possible.
(3) After washing, the cells were transferred to a culture dish, and the number of cells per well was not more than 50.
Second, the recipient cow estrus in synchronization (PG method)
The estrus of the recipient cows was kept consistent with that of the donor cows in time, i.e., PG was injected intramuscularly at a dose of 2/head on day 12, 26 and 27 am, respectively, and the estrus was observed and recorded on days 28-29.
Third, embryo tubulation
1. For frozen embryos, the embryos were removed from the liquid nitrogen tank and gently waved in air for 10s (air bath), then thawed in water at 37 ℃ and removed after the ice crystals were completely thawed, the non-foam plug end was cut with a pair of fine tube scissors and the embryos were transferred to a protective solution (10% 199 solution, i.e. 1ml FBS +9ml 199 base solution). Repeating the above procedures to thaw another embryo, checking the quality of the embryo under a 100-fold microscope, and reloading into tubules according to 2 embryos per tubule if the quality of the embryo meets the A-level embryo. And (3) loading the tube in a mode of 'protective solution + air section + protective solution + embryo + protective solution' during reloading.
2. For fresh embryos obtained by embryo washing of living bodies and in-vitro cultured embryos (A grade and/or B grade) obtained by in-vitro fertilization, directly tubing according to the mode of 'protective solution + air section + protective solution + embryos + protective solution'.
Transplanting in gun
1. The tubule filled with 2 embryos is loaded into a transplantation gun, a sleeve is additionally arranged outside the transplantation gun, and a soft plastic coat is additionally arranged outside the sleeve.
2. 2 embryos are transplanted into a recipient cow which is processed by estrus synchronization by using a rectal grasping method, wherein the recipient cow is located at a position which is 1/2-1/4 deep from the top of a uterine horn. The action is gentle during the transplantation, so as to avoid the bleeding caused by the puncture of the cervix and the uterus. The transplantation time is controlled to be completed within 5 min as much as possible.
Management after transplantation of recipient cows
Factors such as the nutritional level and the stress of the transplanted recipient cattle can influence the conception rate. Therefore, in addition to the well-done regular feeding management, the following points should be noted:
1. the transplanted receptor cattle are bred with other cattle in groups.
2. Ensure the nutritional requirement and ensure the successful pregnancy and calving after transplantation.
3. And (5) paying attention to the condition of returning to the emotion and carrying out pregnancy check in time. Pregnancy and fetal number confirmation may be performed on the recipient cows 28-60 days after transplantation using B-ultrasound or rectal examination.
4. The non-pregnant recipient cattle determined by pregnancy examination should be bred or transplanted in time.
The cattle donor superovulation and recipient synchronization estrus treatment scheme (PG method) is shown in the table 1 in detail:
table 1: cattle donor superovulation and recipient synchronization estrus processing scheme (PG method)
Remarking: (1) PG: 0.2mg/2 ml/count produced by Shanghai Mingsheng. (2) FSH: canada, 20ml, 400 mg.
In addition to the PG method, the method of embedding pessaries (CIDR method) can be adopted for the simultaneous estrus treatment of donor cow superovulation and recipient cow of the invention, and the details are shown in Table 2;
table 2: cattle donor superovulation and recipient synchronization estrus processing scheme (CIDR method)
Remarking: (1) PG: 0.2mg/2 ml/count produced by Shanghai Mingsheng. (2) FSH: canada, 20ml, 400 mg.
Claims (10)
1. A method for improving the breeding efficiency of cows is characterized in that: it comprises the following steps:
step 1: obtaining an embryo;
step 2: receptor cow in-phase estrus PG method: the contemporaneous estrus of the recipient cow is kept consistent with that of the donor cow in time, namely PG is injected intramuscularly at a dose of 2/head in the morning on the 12 th day, the 26 th day and the 27 th day respectively, and the estrus is observed and recorded on the 28 th to 29 th days;
and step 3: loading the embryo into a tube;
and 4, step 4: gun loading and transplanting:
1) loading the tubule filled with 2 embryos into a transplantation gun, wherein a sleeve is externally arranged outside the transplantation gun, and a soft plastic outer sleeve is externally arranged outside the sleeve;
2) transplanting 2 embryos to a receptor cow which is processed by estrus synchronization and is far from the top 1/2-1/4 of a uterine horn by adopting a rectum holding method; the action is gentle during transplantation, so that the bleeding caused by puncturing the cervix and the uterus is avoided; the transplantation time is controlled to be completed within 5 min as much as possible;
and 5: managing a recipient cow after transplantation:
factors such as the nutritional level, the stress magnitude and the like of the transplanted recipient cattle can influence the conception rate; therefore, in addition to the well-done regular feeding management, the following points should be noted:
1) the transplanted receptor cattle and other cattle are bred in groups;
2) the nutrition requirement is ensured, and the successful pregnancy and calving after transplantation are ensured;
3) paying attention to the situation of returning emotion and carrying out pregnancy check in time; the pregnancy and the fetal number of the receptor cows can be confirmed by adopting a B ultrasonic or rectal examination method within 28 to 60 days after transplantation;
4) the non-pregnant recipient cattle determined by pregnancy examination should be bred or transplanted in time.
2. The method of increasing reproductive efficiency of cows according to claim 1, wherein: the embryo acquisition requires, for purchase of frozen embryos: grade a embryos.
3. The method of increasing reproductive efficiency of cows according to claim 1, wherein: the method for obtaining the A-grade and/or B-grade embryo by flushing the donor cow living body embryo at 7 days after superovulation and artificial insemination comprises the following steps:
step 1: selection of donor cows: selecting young cows with age of 2-5 years, strong constitution, complete development of reproductive organs and no reproductive system diseases or cows with 1 birth as donors;
step 2: donor cows PG method of estrus synchronization, superovulation and artificial insemination procedure: on day 1, PG was injected intramuscularly at 2/head dose; on day 12 PG was again given intramuscularly at 2/head dose; FSH was injected into each muscle at 3 ml/head for 1 time at 7 am and 7 pm on day 24; on the 25 th day, 2.5 ml/head of FSH is reduced and injected into each muscle at 7 o' clock in the morning and at the evening for 1 time; FSH is injected for 1 time at 7 points in the morning of 26 days according to the volume of 2 ml/head muscle; FSH is injected for 1 time and PG is injected for 1 time at 7 o' clock according to 2 ml/head muscle at night; FSH is injected 1 time and PG is injected 1 time at 7 points of the morning at 1 ml/head muscle and 2 times per head on day 27; FSH is injected for 1 time according to 1 ml/head muscle at 7 o' clock in the evening; observing estrus and performing artificial insemination on the estrus cows 28-29 days, and recording the day of artificial insemination as 0 day; intramuscular injection of 20-30 μ g/head promoting row III on day 29;
and step 3: and (3) embryo punching: on day 35, i.e., 7 days after artificial insemination, in vivo embryo flushing was performed.
4. The method of increasing reproductive efficiency of cows according to claim 1, wherein: the embryo is obtained by in vitro fertilization culture; the method comprises the following steps: a grade a embryo comprising the steps of:
step 1: and (3) processing the sperms:
1) and (3) unfreezing frozen semen: water bath at 37 deg.c for about 5 sec;
2) adding 200 mul of double antibody into 15 ml of the semen to be mixed evenly;
3) adding 7 ml of semen into a centrifugal tube, cutting one end of the frozen semen without a cotton plug, immersing the section into the semen washed by the centrifugal tube, cutting one end of the cotton plug, completely blowing the frozen semen into the tube, uniformly mixing and centrifuging for 5 min at the rotating speed of 1500 r/min;
4) after the centrifugation is finished, removing the supernatant, adding 7 ml of washing liquid, and centrifuging for 5 min at the rotating speed of 1500 r/min, and removing the supernatant after the centrifugation is finished;
5) measuring the amount of the semen washing solution in the residual centrifuge tube, adding the semen washing solution to 750 mu l, adding 750 mu l of the ovum washing solution, and uniformly mixing;
6) making 100 mul of liquid drop fertilization plate cover oil, and balancing a warm box for standby;
step 2: and (3) processing the oocyte:
1) after the oocyte is mature, gently blowing and beating the physical degranulation cells in a washing tray by using a 100 mu l pipette gun, and completely removing the physical degranulation cells as far as possible;
2) cleaning the oocyte after the granular cells are removed by using an egg washing liquid;
and step 3: fertilization:
1) putting the cleaned oocyte into a fertilization drip tray;
2) dropping 15-20 oocytes in 100 mul;
3) after the fertilization time reaches 8 h, transferring fertilized eggs from the fertilization plate to a culture plate as soon as possible;
and 4, step 4: in vitro culture:
1) making one IVC drip washing plate, culturing with four-hole plate (500 μ l per hole), and covering with oil for balancing overnight;
2) removing the adhering impurities outside the fertilized eggs as clean as possible;
3) after washing, the cells were transferred to a culture dish, and the number of cells per well was not more than 50.
5. The method of increasing reproductive efficiency of cows according to claim 1, wherein: when the adopted embryos are frozen embryos, the embryos are taken out from a liquid nitrogen tank and slowly waved in the air for 10s, namely air bath, then put into water at 37 ℃ for unfreezing, taken out after ice crystals are completely melted, the end without a foam plug is cut by a tubule shear, the embryos are transferred into a protective solution, the procedure is repeated for unfreezing another embryo, the quality of the embryo is checked under a 100-fold microscope, and the embryos conforming to the A-level embryo are re-loaded into the tubule according to 2 small embryos per tubule; and (3) loading the tube in a mode of 'protective solution + air section + protective solution + embryo + protective solution' during reloading.
6. The method of increasing reproductive efficiency of cows according to claim 1, wherein: the embryo tubulation adopts the embryo which is fresh embryo obtained by living embryo flushing and in vitro culture embryo A grade and/or B grade obtained by in vitro fertilization, and directly tubulates according to the mode of 'protective solution + air section + protective solution + embryo + protective solution'.
7. The method of increasing reproductive efficiency of cows according to claim 3, wherein: the embryo punching comprises the following steps:
step 1: donor cattle baoding: driving the donor cattle to a fixing frame for fixing;
step 2: rectal examination and vulvar washing: cleaning up the feces of the rectum of the well-defined donor cattle, checking the development condition of the ovary corpus luteum at two sides, and recording the number of the ovaries; thoroughly cleaning the vulva of the donor cattle with toilet paper or clear water, soapy water, physiological saline and the like, and then spraying alcohol with an alcohol watering can for disinfection;
and step 3: and (3) dilating cervix: for young cows, because the cervix is tightly closed, the cervix needs to be dilated by a dilating rod so as to draw mucus and a germ tube into the uterus; for the multiparous cows, the step can be determined according to rectal examination conditions and can be used or not used; the principle is that a mucus pump and a embryo washing tube can smoothly pass through the cervix to reach the corresponding position of the uterus;
and 4, step 4: extracting mucus: an operator slowly inserts a clean mucus pump into a cervix of a donor cow through a vagina, an assistant rapidly pumps out a handle of the mucus pump, the handle is similar to a handle of an injector, vacuum negative pressure is instantly formed in a mucus pump rubber tube, mucus in the cervix and a uterus is sucked into the mucus pump rubber tube, and the mucus pump is slowly pumped out of the cervix; the steps can be repeated for 1-2 times according to the mucus extraction condition until the mucus is basically extracted completely;
and 5: anesthesia: injecting 4ml of procaine hydrochloride or other anesthetic into the cavities of the second caudal vertebra and the third caudal vertebra to perform local anesthesia on the donor cow; the judgment standard of anesthesia success is as follows: after the injection of the anesthetic for a few minutes, the success of the anesthesia is indicated by slightly shaking the oxtail left and right without resistance; if resistance exists, the anesthetic dosage needs to be increased until the anesthesia succeeds;
step 6: inserting a blank punching tube: a special embryo washing tube slowly passes through the cervix and is inserted into the uterine horn through the rectum; when the uterine horn is near the thin part of the uterine horn, the guide steel core in the embryo washing pipe is slowly drawn out by 10-15cm, so that the rubber pipe at the front section of the embryo washing pipe becomes flexible to adapt to the natural bending of the uterine horn; after the embryo-washing tube reaches the corresponding position, the more the embryo-washing tube goes deep into the uterine horn, the better the embryo-washing tube is, 10-15 ml of air is injected by a 20ml injector through the air hole of the embryo-washing tube, the specific injected air quantity is determined according to the thickness of the cervix, the operation aim is to enable a balloon at the front end of the embryo-washing tube to be full, the outward outlet of the cervix is blocked, the embryo-washing liquid can be discharged into a funnel only through the embryo-washing tube, and the embryo is convenient to collect; then slowly drawing out the blank punching pipe to guide the steel core;
and 7: connecting a blank punching pipe and a funnel by using a three-way pipe: one end of the three-way pipe is connected with a blank punching pipe, one end of the three-way pipe is connected with a blank collecting funnel, the other end of the three-way pipe is reserved with injection blank punching liquid, and joints of all parts are screwed;
and 8: and (3) embryo punching: after the connection between the embryo punching tube and the three-way tube is finished, firstly, the hemostatic forceps are used, and the sawtooth part at the tip is bound by medical white tape so as to avoid damaging the transparent hose and clamp the hose at the end of the embryo collecting funnel; at the moment, 100 ml of a large-size syringe is used for extracting 37.6 ℃ constant-temperature water bath and 60 ml of embryo-flushing liquid, the embryo-flushing liquid is connected to the opening of the connecting hose, the embryo-flushing liquid is slightly pumped back, and then the embryo-flushing liquid is slowly injected into the uterus until the injection resistance is slightly existed; then the hemostatic forceps are loosened, the hose connected with the injector is clamped, and embryo washing liquid in the uterus flows back to the embryo collecting funnel, the operation is repeated for a plurality of times, the injection speed at the later stage can be accelerated, the uterus is washed by the embryo washing liquid, so that the free embryo is the best embryo washing time about 7 days after the hybridization, and the embryo is not implanted in the uterus at this time; injecting 30-50 ml each time with the embryo flushing liquid flowing back to the embryo collecting funnel; 500 ml of embryo flushing liquid is used in the whole process; after the completion, 20ml of the injector is connected to the blast hole of the embryo-washing tube, so that the air at the front end of the embryo-washing tube flows back, the light pumping of the injector is convenient for the expansion of the balloon at the front end of the embryo-washing tube, the embryo-washing tube is slowly pumped out, the front end of the embryo-washing tube is lifted, and the embryo-washing liquid in the tube completely enters the embryo collecting funnel; taking down the embryo collecting funnel, and marking the donor cow number, the left and right embryo-flushing uterine horn and the embryo-checking position by using a marking pen on a funnel cover;
repeating the steps 6, 7 and 8 to punch the uterine horn on the other side;
and step 9: and (3) PG injection: injecting 4ml PG intramuscularly after embryo flushing to promote luteal lysis and enter the next estrus cycle;
unbinding the donor cattle, and replacing the donor cattle, and repeating the steps 1 to 9.
8. The method of increasing reproductive efficiency of cows according to claim 3, wherein: the PG is chloroprostenol.
9. The method of increasing reproductive efficiency of cows according to claim 4, wherein: the semen washing solution is 15 ml of SOF, 0.3 mg of heparin and 0.03183g of Caffeine; the double-antibody is penicillin and streptomycin; the egg washing solution comprises 8 ml of SOF, 0.16 mg of heparin and 0.048 g of BSA.
10. Method for increasing the reproductive efficiency of cows according to claim 5 or 6, characterized in that: the protective solution is 10% 199 solution, namely 1ml FBS +9ml 199 base solution.
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| CN102743240A (en) * | 2012-08-08 | 2012-10-24 | 贵州省畜牧兽医研究所 | Method and device for performing artificial insemination on cows |
| CN113180878A (en) * | 2021-04-23 | 2021-07-30 | 广西壮族自治区畜牧研究所 | Method for improving double-birth rate of cows |
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| CN102743240A (en) * | 2012-08-08 | 2012-10-24 | 贵州省畜牧兽医研究所 | Method and device for performing artificial insemination on cows |
| CN113180878A (en) * | 2021-04-23 | 2021-07-30 | 广西壮族自治区畜牧研究所 | Method for improving double-birth rate of cows |
Non-Patent Citations (1)
| Title |
|---|
| 郭伟婷等: "胚胎移植受体牛选择与饲养管理", 《今日畜牧兽医》 * |
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