CN113180878A - Method for improving double-birth rate of cows - Google Patents
Method for improving double-birth rate of cows Download PDFInfo
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- CN113180878A CN113180878A CN202110439002.0A CN202110439002A CN113180878A CN 113180878 A CN113180878 A CN 113180878A CN 202110439002 A CN202110439002 A CN 202110439002A CN 113180878 A CN113180878 A CN 113180878A
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- A—HUMAN NECESSITIES
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- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
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- Veterinary Medicine (AREA)
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Abstract
The invention discloses a method for improving the double-birth rate of cows, which comprises the following steps: the method comprises the steps of insemination, egg collection, embryo selection, pretreatment of a receiving cow, transplantation, production and the like, wherein the high-quality cow is used as a donor cow, a plurality of embryos are obtained by a superovulation treatment method, other cows are used as receiving cows to produce offspring of the high-quality cow, the offspring of the high-quality cow can be improved by multiple times, the propagation speed of the high-quality cow is accelerated, the twins rate of the cow is improved by adopting a double-embryo transplantation method, the probability of producing two calves by the cow in one year is increased, the reproduction rate of the cow is improved, and the health degree of the calves is ensured.
Description
Technical Field
The invention relates to the technical field of mammal embryo transplantation, in particular to a method for improving the double-birth rate of cows.
Background
Embryo transplantation, also called fertilized egg transplantation, refers to a technique of transplanting an early embryo of a female animal or an embryo obtained by in vitro fertilization or other means into the body of the female animal to continue to develop into a new individual. In the related technology, pregnant cows with multiple fetuses are easy to abortion, and the born calves are weak, small and difficult to live, and the breeding efficiency and the effect are poor.
Disclosure of Invention
The invention mainly aims to provide a method for improving the double-birth rate of cows, aiming at improving the success rate and efficiency of cow embryo transplantation.
In order to achieve the purpose, the method for improving the double-birth rate of the cows comprises the following steps:
insemination: carrying out superovulation treatment on a donor cattle, and carrying out artificial insemination;
egg collection: sterilizing the donor cattle, extracting fertilized eggs by adopting a perfusion method, and performing static culture on the extracted fertilized eggs;
embryo selection: culturing the fertilized eggs subjected to static culture to obtain embryos, selecting two embryos according to a preset condition, and performing fresh embryo transplantation on the two selected embryos;
pretreating a receptor cow; nursing the recipient cattle before transplantation, pre-embedding progesterone suppository and performing estrus synchronization treatment on the recipient cattle so as to enable the date difference of estrus of the recipient cattle and the donor cattle to be less than or equal to 1 day;
transplanting: selecting two embryos, transplanting the embryos into the uterus of the recipient cattle, and observing pregnancy reaction;
production: and (4) carrying out feeding management on the pregnant recipient cattle until production.
Preferably, in the step of insemination, the step of superovulation processing includes:
intramuscular injection of follicle stimulating hormone at a preset frequency 9-13 days after pre-burying of the progesterone suppository in the donor cattle, and intramuscular injection of prostaglandin at the same time 3 days after intramuscular injection of follicle stimulating hormone in the donor cattle, wherein the mass range of the prostaglandin is 2.0-2.4 mg.
Preferably, in the step of superovulation, the predetermined frequency is set to be in the range of 12h to 14h in the morning and evening.
Preferably, the step of static culturing the extracted fertilized egg further comprises:
and putting fertilized eggs of the same donor cattle into the same culture dish, and cleaning and removing impurities from the fertilized eggs.
Preferably, in the step of embryo selection, the preset conditions include morphology, hue, split sphere size, uniformity, cell density, zona pellucida barrier, and denaturation.
Preferably, the embryo selection step is followed by an embryo containment step comprising:
and (3) sucking the preservation solution, the air bubbles, the freezing solution containing the embryos, the air bubbles and the preservation solution in sequence by using a suction pipe, and heating and sealing the suction pipe.
Preferably, in the step of pre-treating the recipient cattle;
the recipient cattle are 3-8 years old and have normal reproductive performance, and have more than 2 normal natural oestrus records and no more than two non-pregnancy records after being delivered for more than 60 days.
Preferably, in the step of pretreating the recipient cattle, the number of pairs of the recipient cattle and the number of pairs of the donor cattle are 3: 1.
preferably, in the transplanting step, the diameter of the follicle in the recipient cow is in the range of 10mm to 20mm when the recipient cow is in estrus, and ovulation occurs within 36 hours after estrus.
Preferably, the step of transferring said selected embryo into the uterus of said recipient cow comprises:
the recipient cattle was baoding, 2% pinocembrin was injected intramuscularly, and embryo transplantation was performed using a transplanter.
The method for improving the double-birth rate of the cow in the technical scheme of the invention comprises the following steps: the method comprises the steps of insemination, egg collection, embryo selection, pretreatment of a receiving cow, transplantation, production and the like, wherein the high-quality cow is used as a donor cow, a plurality of embryos are obtained by a superovulation treatment method, other cows are used as receiving cows to produce offspring of the high-quality cow, the offspring of the high-quality cow can be improved by multiple times, the propagation speed of the high-quality cow is accelerated, the twins rate of the cow is improved by adopting a double-embryo transplantation method, the probability of producing two calves by the cow in one year is increased, the reproduction rate of the cow is improved, and the health degree of the calves is ensured.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a flow chart of an embodiment of the method of the present invention for increasing the birthing rate of cows;
fig. 2 is a flow time profile of the insemination step.
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.
Referring to fig. 1, the invention provides a method for improving the double-birth rate of cows.
In the embodiment of the invention, the method for improving the double-birth rate of the cows comprises the following steps:
s10: insemination: carrying out superovulation treatment on a donor cattle, and carrying out artificial insemination;
the specific steps of insemination comprise:
s11: selection of donor cattle. The method can select cows of cow varieties to be bred as donor cows, the donor cows should have high production performance, the age of the donor cows is generally 3-8 years old, young donor cows are about 18 months old, the body is robust, genetic diseases do not exist, breeding functions are normal, the oestrus cycle is normal, and the fat condition is good.
S12: feeding and managing donor cattle: before superovulation treatment, the donor cattle are ensured to have enough high-quality hay, silage or succulent feed, concentrate, vitamins and minerals, and are supplied with clean drinking water and salt, so that reasonable feeding and careful management are realized.
S13: superovulation treatment: taking out the pre-embedded progesterone suppository 9-13 days after pre-embedding the progesterone suppository in the donor cattle, injecting follicle stimulating hormone (FSH folliculture-stimulating hormone) in an intramuscular injection mode at a preset frequency, wherein the preset frequency is once every morning and night, the time interval is 12-14 h, the injection is reduced in 4 days, and injecting follicle stimulating hormone (PG prostagladin) in an intramuscular injection mode at the preset frequency simultaneously after 3 days after the donor cattle injects the follicle stimulating hormone in an intramuscular injection mode, and the mass range of the prostaglandin is 2.0-2.4 mg.
S14: oestrus observation and artificial insemination: after 40-48 h after PGF2 alpha treatment, the majority of the treated donor cattle begin to estrate, at the moment, the estrus condition of the donor cattle is observed from the day 2 and the evening of intramuscular PGF2 alpha injection, the observation is carried out at least once in the morning and evening, the first artificial insemination is carried out 4-6 h after the donor cattle is observed to receive climbing, the second artificial insemination is carried out after 12h, the amount of the artificial insemination is increased, and the ovary of the donor cattle is touched during the artificial insemination.
Referring to fig. 2, in an embodiment of the present invention, when FSH injection is started on day 9 after progesterone suppository pre-implantation, the pre-implanted progesterone suppository is removed on day 11 and PGF2 α injection is started until day 12, FSH injection is stopped, estrus observation is performed on the donor cow after day 13, artificial insemination is performed after day 14, and embryo collection is started on day 20.
S20: collecting eggs, sterilizing the donor cattle, extracting fertilized eggs by adopting a perfusion method, and performing static culture on the extracted fertilized eggs, wherein the egg collection specifically comprises the following steps:
s21: the egg collecting time is that the oestrus day of a donor cow is taken as the 0 th day, the ovary is checked on the 5 th to 6 th days to determine the corpus luteum number so as to arrange the number of the heads of the receptor cows, and the egg is recovered in a non-operation way on the 7 th day.
S22: preparation of instruments and egg wash: and (3) flushing the sterilized uterine tube with Phosphate Buffer Saline (PBS) solution for 2-3 times, inserting a steel core, and checking the balloon. The PBS solution is put into a water bath with 37 ℃ for pre-warming for standby, and necessary anesthetic, syringe, alcohol cotton, flushing disinfectant and the like are prepared.
S23: after the fixing of donor cattle and the fixing of anaesthetized donor cattle are finished, alcohol is used for disinfection between the first and the second tail vertebrae, and 3 ml-5 ml of 2% procaine is used for epidural anaesthesia.
S24: and (3) carrying out sterilization treatment on donor cattle: after anesthesia, the oxtail of the donor cattle is vertically bound on a fixing frame, excrement is removed, the perineum and the vulva are washed by clear water, then potassium permanganate solution is used for washing and disinfection, sterilized toilet paper is used for wiping, and finally the vulva is disinfected by an alcohol cotton ball.
S25: extracting fertilized eggs by a perfusion method, inserting the uterine tubes and flushing egg parts: the prepared uterine tube is handed to the right hand of an operator, the left index finger and the thumb of the operator open the vulva to insert the uterine tube, the left hand is stretched into the rectum, the uterine tube is carefully induced to enter one uterine horn to the large bend through the cervix, a little steel core is drawn out, and the uterine tube is pushed forwards to enable the tip of the uterine tube to be close to the deep part of the uterine horn. A30 ml syringe is held by the assistant and 10ml of air is pushed in at a time, and then inflated with 1ml counts as required by the surgeon. The inflation quantity is determined according to the size of the uterus and the position of the uterine tube in the uterus, generally about 14ml to 16ml of the air is inflated for young donor cattle, and about 18ml to 25ml of the air is inflated for multiparous donor cattle. After the tubal irrigation is fixed, the steel core is drawn out, and perfusion is started. Injecting 30 ml-50 ml of PBS egg-washing liquid into a 50ml syringe each time, washing uterine horns by times, and injecting into a covered egg-collecting cup after recovery. The other side of the uterine horn is washed by pulling the straight fallopian tube with the right hand of the operator, and the assistant carefully inserts the steel core. After the left hand of the operator induces the stable insertion of the steel core in the rectum, the balloon is deflated. The operator then inserts the uterine tube into the uterine horn on the other side and irrigates the uterine horn in the same manner.
S26: after the uterine horn on two sides is treated after operation, the uterine tube is extracted, a terramycin solution is prepared by dissolving 3g of terramycin powder in 100ml of sterilized double distilled water or normal saline, or 10ml of penicillin and streptomycin are injected into uterus, and finally PGF2 alpha is injected intramuscularly.
S30: embryo selection: culturing the fertilized eggs subjected to static culture to obtain embryos, selecting two embryos according to a preset condition, and performing fresh embryo transplantation on the two selected embryos; wherein, in this step, specifically include:
s31: and (3) filtering and egg collecting cup method, after recovery, washing the filter screen for 2-3 times by using an injector, sucking off foams in the cup, and placing under a solid microscope for inspection.
S32: before static culture, fertilized eggs of the same donor cattle are placed in the same culture dish, and the fertilized eggs are cleaned to remove impurities. The specific steps of cleaning and impurity removing are as follows: and removing mucus and impurities on the periphery of the fertilized eggs by using a glass rod. And transferring the fertilized eggs to the liquid drops of the culture dish by using a suction pipe, and putting the fertilized eggs of the same donor cattle into the same liquid drops. And then sucking a small amount of PBS (phosphate buffer solution) of the second liquid drop to the first liquid drop by using a suction pipe, sucking all the fertilized eggs, preventing mucus and impurities from being sucked, moving to the second liquid drop, and flushing the fertilized eggs for 3-5 times in sequence.
S33: identification and selection of embryos: the morphology, color tone, size and uniformity of the blastomeres, cell density, zona pellucida barrier and degeneration of the fertilized eggs were observed under a 40-50 times physical dissection microscope, and in this example, embryos were classified into A, B, C grades.
Specifically, the grade A embryo is set to have complete shape, clear outline, spherical shape, uniform size of split sphere, compact structure, moderate color tone and transparency, and no attached cells or vacuoles. The B level has clear outline and good color tone and cell density, some attached cells and vacuoles can be seen, and the denatured cells account for about 10-30%. The C level has unclear profile, dark color, loose structure, more free cells or vacuoles and 30 to 50 percent of denatured cells. The grading of the embryos should also take into account the degree of development of the zygotes. Fertilized eggs recovered on day 7 after estrus should be placed in the stage from dense morula to blastocyst during normal development. The A, B grade embryo can be used for fresh embryo transplantation, except that fertilized eggs below 16 cells and more than half of embryos with degenerated cells belong to the same class. That is, two A, B grade embryos can be selected for fresh embryo transfer.
S34: and (3) embryo sealing: and (3) sucking the preservation solution, the air bubbles, the preservation solution containing the embryos and the air bubbles in sequence by using a suction pipe, and heating and sealing the suction pipe. That is, A, B-grade embryos were aspirated in the following order using a 0.25ml plastic pipette, and the storage solution, air bubbles, storage solution containing embryos, and air bubbles were sealed by heating, and one pipette was attached to each embryo.
S35: and (3) embryo observation: observing the shape and position of embryo with special plastic suction tube observation mirror or microscope eyepiece, sterilizing with 70% alcohol cotton ball, cutting off the upper layer of suction tube by 1cm, and transplanting fresh embryo.
S40: pretreating a receptor cow; nursing, pre-embedding progesterone suppository and performing estrus synchronization treatment on the receptor cattle before transplantation so as to enable the date difference of estrus of the receptor cattle and the donor cattle to be less than or equal to 1 day, and specifically, the method comprises the following steps:
s41: selecting a receptor cow which is healthy, disease-free, large in physique, good in fat condition, normal in reproductive performance, more than 60 days after delivery, has more than two times of normal natural oestrus records, good in lactation performance, free of dystocia history and free of artificial fertilization for two times, wherein the age of the receptor cow is 3-8 years.
S42: the method is used for breeding and managing the recipient cattle, and has the advantages of careful management, reasonable breeding, clean environment, fresh forage grass, feed and drinking water, and salt and necessary additives supplement. A certain number of cattle flocks are formed, and the herds are prevented from being extruded excessively so as to be convenient for oestrous observation. The long term starved recipient cattle are concerned with enhancing exercise. Preventing stress reaction caused by changes of feed and environment before and after transplantation.
S43: and (3) carrying out estrus synchronization treatment on the recipient cattle, wherein the number of the recipient cattle and the number of the donor cattle are calculated according to the following ratio of 3: 1, namely performing superovulation on one donor cow and performing simultaneous estrus treatment on 3 acceptor cows. Burying the progesterone suppository in the same day as that of the donor cattle, injecting 2.0-2.4 mg of PGF2 alpha on the 11 th day after burying the progesterone suppository, and withdrawing the progesterone suppository. The superovulation schedule of the recipient cattle should match that of the donor cattle.
S44: estrus observation is carried out by confirming estrus according to the fact that recipient cattle receive climbing in principle and observing the estrus condition for 30min respectively in the morning and in the evening. The oestrous recipient cattle are examined directly to confirm the follicles and to determine ovulation the next morning or evening. If a cow ovulating 36 hours after estrus cannot be used as a recipient cow.
S45: when the estrus of the donor cattle and the recipient cattle is poor and the embryo is transferred, if the estrus cycles of the donor cattle and the recipient cattle are not the same, the donor cattle and the recipient cattle are difficult to be born. The uterine environments of both are required to be consistent. The difference between the estrus days of the receptor cattle and the donor cattle is less than or equal to 1 day.
S50: transplanting: transplanting the selected embryo into the uterus of the recipient cattle, and observing pregnancy reaction; the method specifically comprises the following steps:
s51: when the transplanted acceptor cattle is examined, the diameter of the follicle which is directly examined when the acceptor cattle is in estrus is about 10 mm-20 mm, and ovulation occurs within 36 hours after estrus. The corpus luteum is examined one day before transplantation, the diameter of the corpus luteum is more than 11 mm-15 mm, the hand feeling of the corpus luteum is good, and the quality is soft and full.
S52: selecting the day difference of estrus of a recipient cow according to the embryo development condition, transplanting the compact morula to the recipient cow in estrus for 6 days, transplanting the early blastula to the recipient cow in estrus for 7 days, and transplanting the blastula and the expanded blastula to the recipient cow in estrus for 8 days. If more embryos are produced than the number of recipient cattle, the excess embryos can be used for cryopreservation; if an insufficient number of embryos are produced, the partially preserved frozen embryos can be used to transplant recipient cattle.
S53: preparing the apparatus, namely wrapping the transplanting gun, the gun head and the metal protective sleeve with tinfoil respectively and sterilizing under high pressure. When transplanting, the plastic suction tube filled with embryo is sterilized by 70% alcohol cotton ball, then one end of the seal is cut off, the plastic suction tube is put into a transplanting gun, and a protective outer sleeve is sleeved on the plastic suction tube, so that the plastic suction tube can be used for transplanting.
S54: preparing a receptor cattle, fixing the receptor cattle, removing excrement, injecting 2% pinosylvin or using 3ml of 2% procaine to break the vulva between the first and second caudal vertebras of the receptor cattle, washing and disinfecting with potassium permanganate water, wiping with sterile paper, and finally disinfecting with an alcohol cotton ball.
S55: the transplantation method comprises opening vulva with left hand, and inserting the transplantation device into vagina with right hand. Then the left hand is extended into rectum, when the transplanter reaches the cervical orifice, the transplanting gun is clamped by the ring finger and the middle finger of the right hand, the soft outer sleeve is pulled outwards by the thumb and the forefinger, the outer sleeve is poked through the transplanting gun, and the transplanting gun is inserted into the cervix. The left hand is induced in the rectum to ensure that the gun head is gently and stably inserted into the uterine horn of the lateral corpus luteum to the large bend, the embryo is pushed out by holding the right hand of the transplanting device, and the transplanting gun is slowly and rotatably drawn out. Another embryo was transferred to the uterine horn on the other side in the same manner.
S60: and (4) production, namely, carrying out feeding management on the pregnant recipient cattle until production.
S61: pregnancy diagnosis, usually beginning three months after transplantation. When the fetus is pregnant for 90 days, the uterus continues to grow and falls into the abdominal cavity, the fetus can be sensed to float, the pregnancy horn has pregnancy veins, and the uterine wall can touch cotyledons, so that the fetus can be determined.
S62: the donor cattle is produced, the donor cattle is fed and managed according to pregnant cows, and the twins rate is counted according to the calving condition after the calving period.
The method for improving the twins rate of the cow can improve the twins rate of the cow, increase the probability of producing two calves of the cow in one year, improve the reproduction rate of the cow, and because the embryo transplantation method is adopted, the excellent cow is used as a donor, a plurality of embryos are obtained by the superovulation method, other cows are used as receptors to produce offspring of the excellent cow, the offspring of the excellent cow can be improved by times, the propagation speed of the excellent cow is accelerated, furthermore, the sex-controlled frozen semen is subjected to artificial insemination on the donor cow, the sex of the offspring of the calves can be controlled according to production requirements, for example, a fattening cattle farm needs a bull, the artificial insemination can be carried out by using Y semen, the artificial insemination can be carried out by using X semen, the artificial insemination can be carried out for producing the calves, and the production efficiency is improved.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for increasing the double-birth rate of a cow is characterized by comprising the following steps:
insemination: carrying out superovulation treatment on a donor cattle, and carrying out artificial insemination;
egg collection: sterilizing the donor cattle, extracting fertilized eggs by adopting a perfusion method, and performing static culture on the extracted fertilized eggs;
embryo selection: culturing the fertilized eggs subjected to static culture to obtain embryos, selecting two embryos according to a preset condition, and performing fresh embryo transplantation on the two selected embryos;
pretreating a receptor cow; nursing the recipient cattle before transplantation, pre-embedding progesterone suppository and performing estrus synchronization treatment on the recipient cattle so as to enable the date difference of estrus of the recipient cattle and the donor cattle to be less than or equal to 1 day;
transplanting: selecting two embryos, transplanting the embryos into the uterus of the recipient cattle, and observing pregnancy reaction;
production: and (4) carrying out feeding management on the pregnant recipient cattle until production.
2. The method of increasing the twins rate of a cow as claimed in claim 1, wherein in said step of inseminating, said step of superovulation handling includes:
intramuscular injection of follicle stimulating hormone at a preset frequency 9-13 days after pre-burying of the progesterone suppository in the donor cattle, and intramuscular injection of prostaglandin at the same time 3 days after intramuscular injection of follicle stimulating hormone in the donor cattle, wherein the mass range of the prostaglandin is 2.0-2.4 mg.
3. The method for increasing the twins rate of a cow as claimed in claim 2, wherein said superovulation procedure is performed at a predetermined frequency of 12h to 14h in the morning and evening.
4. The method for increasing the twins rate of cows according to claim 1, further comprising, before the step of subjecting the extracted fertilized eggs to static culture:
and putting fertilized eggs of the same donor cattle into the same culture dish, and cleaning and removing impurities from the fertilized eggs.
5. The method of claim 1, wherein in the step of embryo selection, the predetermined conditions include morphology, hue, split sphere size, uniformity, cell density, zonal barrier, and degeneration.
6. The method of increasing the twins rate of a cow according to claim 1, further comprising an embryo containment step after the embryo selection step, the embryo containment step comprising:
and (3) sucking the preservation solution, the air bubbles, the freezing solution containing the embryos, the air bubbles and the preservation solution in sequence by using a suction pipe, and heating and sealing the suction pipe.
7. The method of increasing twin rate in a cow of claim 1, wherein in the step of pretreating the recipient cow;
the recipient cattle are 3-8 years old and have normal reproductive performance, and have more than 2 normal natural oestrus records and no more than two non-pregnancy records after being delivered for more than 60 days.
8. The method for increasing twin rate in a cow according to claim 1, wherein in the step of pretreating the recipient cow, the number of pairs of the recipient cow and the number of pairs of the donor cow are 3: 1.
9. the method for increasing the twins rate of a cow as set forth in claim 1, wherein the diameter of the follicle in the recipient cow in the step of transplantation ranges from 10mm to 20mm, and ovulation occurs within 36 hours after estrus.
10. The method of claim 1, wherein the step of selecting two of said embryos and transferring said embryos to the uterus of said recipient cow comprises:
the recipient cattle was baoding, 2% pinocembrin was injected intramuscularly, and embryo transplantation was performed using a transplanter.
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