CN111588834B - Method for improving bovine embryo transplantation estrus synchronization effect and used medicine - Google Patents
Method for improving bovine embryo transplantation estrus synchronization effect and used medicine Download PDFInfo
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Abstract
The invention discloses a method for improving the estrus synchronization effect of bovine embryo transplantation and a used medicament, in particular provides an application of luteinizing hormone releasing hormone A3 in preparing a medicinal composition for improving the estrus synchronization effect of bovine embryo transplantation, and the method for improving the estrus synchronization effect of bovine embryo transplantation comprises the following steps: providing a donor bovine; injecting medicine into the muscle of the donor cattle; continuously observing the oestrus and ovulation conditions of the cows within 3d from the time of injecting the medicaments, recording the oestrus starting time and the oestrus ending time of the cows, observing the oestrus in the morning and at night every day, determining the oestrus time, the oestrus program and the ovulation condition by combining rectal examination according to the appearance oestrus symptoms of the cows, determining the oestrus in the 3d after injecting the medicaments as oestrus synchronization, and carrying out artificial insemination on the cows with the oestrus and the ovulation according to a freezing and matching technical operation rule at proper time, wherein the semen is the frozen semen of the cows of the same donor; and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, and feeding the cow in the pregnancy period according to a conventional method.
Description
Technical Field
The invention belongs to the field of animal husbandry, relates to a cattle breeding technology, and particularly relates to a method for improving the estrus synchronization effect of cattle embryo transplantation. The present invention achieves the technical effect of one or more aspects of the present invention by injecting luteinizing hormone releasing hormone A3, LHRH-A3, into cows. The invention also relates to a medicament for the method for improving the estrus synchronization effect of bovine embryo transplantation. The result of the invention shows that the medicament has obvious effect of inducing estrus synchronization treatment on cows which have no estrus for a long time or have slow estrus after delivery, and is an ideal estrus-promoting medicament.
Background
At present, when a recipient cow is selected in production, the recipient cow has two schemes of natural estrus or synchronous estrus by injecting medicaments, and the latter scheme plays a very important role in improving the production efficiency.
Estrus synchronization refers to the use of certain hormone preparations to artificially control and regulate the progress of the estrus cycle of a group of dam animals so that they will have a concentrated estrus within a predetermined time. Oestrogen inhibiting agents such as pregnenones. The control method includes prostaglandin method and progestogen method. Generally, estrus synchronization refers to a technique of regulating the estrus cycle of a dam using a hormone preparation to make it estrus in a relatively concentrated time. Commonly used estrus-synchronization drugs, one type is agents that inhibit estrus, such as pregnenones, during their treatment, increase progesterone levels in the dams at follicular stage, inhibit the maturation of the follicles, and stop natural estrus, actually prolonging the estrus cycle. After a certain period of time, the drug treatment was stopped at the same time, so that progesterone levels were reduced and follicles in the treated dams began to grow rapidly and appeared at the same time.
Estrus synchronization is a new technology for controlling estrus. The research of all countries in the world is started from the 60 s, and the research also shows the power of the research on breeding and improving cattle in all countries along with the deep research. Therefore, China also starts research in seventy-eight years, and a plurality of units carry out tests and obtain certain results. A group of cows is treated by medicinal hormones, the natural oestrus cycle of the group of cows is consciously adjusted and changed, the dispersed oestrus of each cow is adjusted to a certain time, the whole group of cows centrally and uniformly oestrus, centralized breeding is realized, and the production efficiency can be greatly improved.
The importance of estrus synchronization is manifold. For example, 1, estrus, mating, pregnancy and delivery of a herd can be adjusted to be performed simultaneously within a certain time, so that the advantages that calves are born simultaneously, management and cultivation in the lactation period can be unified, scientific feeding management of the calves and the grown-up calves can be intensively done on a large scale, and the defects that a plurality of devices are utilized and the work of personnel is uneven throughout the year are overcome; 2. the frozen semen and the artificial insemination are technical works, and the efficiency and the working quality can be improved by concentrating the strength. Timing insemination can be carried out without estrus catching identification; 3. meanwhile, oestrus, superovulation and embryo transplantation are several important links of a new propagation technology, but only by the way, the embryo transplantation can be realized only when a donor cow and a receptor cow oestrate simultaneously and genitals are in the same physiological state.
The synchronous estrus is based on the physiological mechanism of animals as a theoretical basis. The shape and function of the ovary play an important role in the reproductive physiology of cows, and the ovary passes through two stages, namely a follicular stage and a luteal stage in the oestrus cycle of cows. The two phases are alternated and appear repeatedly to form an estrus cycle. Control of the luteal phase of both phases is critical to the control of the estrus cycle. Each cow in the cow group is in different stages of the estrus cycle, and the control of estrus is to control the life of corpus luteum in the corpus luteum through hormone or drug treatment, so that the estrus cycles of all cows are adjusted to the same stage, and the purpose of synchronous estrus is achieved. There are two main approaches to luteal control, one is the administration of progestins and the other is the administration of prostaglandins to interrupt the natural estrous cycle of the cow. The effect of using the progestogen progesterone and analogues thereof is that the continuous use of the progestogen in the blood maintains a certain level, inhibits the growth and development and the estrus of follicles on ovaries, and is always in the artificial corpus luteum stage, sometimes the corpus luteum on the ovaries has disappeared, which means that the endogenous hormone level is reduced, but as the exogenous hormone is still acting, the cows will not estre, which is equivalent to prolonging the estrus cycle and postponing the estrus. The second approach is to use prostaglandins such as prostaglandin F2. And the like. Aims to dissolve corpus luteum, reduce the level of progesterone, stop the luteal phase, promote the release of pituitary gonadotropin and ensure the estrus to come. Thus, the estrus cycle is shortened and the estrus comes ahead.
There are several references to methods of improving reproductive performance in animals using luteinizing hormone releasing hormone A3. For example, CN101711513A (application No. 200910272961.7) discloses a method for increasing the ovulation number of sows and an injection thereof, belonging to the technical field of agricultural biological products. The method is characterized in that PG (sodium chlorophrostate) and PMSG (serum gonadotropin) are injected simultaneously before estrus, or a mixture of PG and PMSG is injected, HCG (chorionic gonadotropin) or LHRH-A3 (luteinizing hormone releasing hormone A3) is injected during estrus, the effect of promoting the ovulation number of young sows or multiparous sows to reach 2-3 times of the normal egg discharge amount is achieved, the reproductive performance of the sows is improved, the number of sows is obviously increased, and the method is stable in technology and obvious in effect; does not induce ovarian cyst, and can achieve the same superovulation effect on replacement gilts by using the method at any time before sexual maturity.
CN103083110A (application No. 201210578058.5) discloses a method for improving the estrus synchronization rate and pregnancy rate of embryo transplantation receptor cattle, vitamin ADE10 ml is injected into the receptor cattle with or without cycle on any day, a progestogen vaginal suppository is placed for 9-12 days, 1000 units of progestogen are injected into the pregnant horse 1 day before the suppository is taken, and 0.5 mg of chloroprostenol is injected into the suppository at the time of the suppository is taken. Taking out and oestrus 2-3 days after the embolism, injecting 2000 units of chorionic hormone or 25 micrograms of No. 3 chorionic hormone into the cattle, injecting an anti-inflammatory drug into the uterus of a receptor cattle which has oestrous and has slight metritis after treatment, purifying the uterus on the same day, and injecting 10 milliliters of vitamin ADE into the uterus, so that the intrauterine environment of the receptor cattle is more favorable for pregnancy. Embryo transfer is carried out on the 7 th to 7.5 th day of estrus, and the recipient cattle with insufficient corpus luteum firmness at the time of transfer are injected with 100 units of progesterone or 25 micrograms of luteinizing hormone. The estrus is believed to be greater than 90% for this estrus regimen.
CN107372349A (application number 201710791995.1) relates to a cross breeding method of a cool red bull and a jungle bull, wherein the jungle red bull is taken as a male parent and the cool red bull is taken as a female parent; after the heat of the cold red cow is confirmed, the cold red cow is frozen with the frozen semen of the bull, vitamin ADE is injected before freezing, and luteinizing hormone releasing hormone A3 is injected during freezing; performing concentrated feed supplement regulation and control according to body conditions in the perinatal period of the cold red cow, and supplementing calcium and magnesium preparations; the calf is separated from cow immediately after birth, and the calf diarrhea prevention bag is continuously taken for one week after the calf is fed with clean colostrum. The bred plain red bull and the bred cattle interspecific hybrid F1 generation have strong stress resistance, good fattening production performance and strong intramuscular fat deposition capability, and provide scientific basis for further improvement, reasonable utilization of resources and superior commercial value of the hybrid F1 generation of local fine cattle in China.
CN109964878A (application No. 201910384234.3) relates to a cross breeding method of Xinjiang Kazakh and Angus cattle, wherein the Angus cattle are used as male parents and the Xinjiang Kazakh are used as female parents; after the estrus of the cow of the Kazakh cattle in Xinjiang is confirmed, the cow is frozen with frozen semen of the bull of the Angus cattle, vitamin ADE is injected before freezing, and luteinizing hormone releasing hormone A3 is injected during freezing; performing concentrated feed supplement regulation and control according to body conditions during the perinatal period of the cow of Kazakh cattle in Xinjiang, and supplementing vitamin A and calcium-magnesium preparations; the calf is separated from cow immediately after birth, and the calf diarrhea prevention bag is continuously taken for one week after the calf is fed with clean colostrum. The hybrid between Xinjiang Kazakh cattle and Angus cattle bred by the method has strong stress resistance of F1 generation, has good fattening production performance, and provides scientific basis for further improvement of local improved Xinjiang Kazakh cattle in China, reasonable utilization of resources and commercial value of hybrid F1 generation superior medium-grade beef.
However, there is still a need in the art for new methods for improving estrus, ovulation, and conception in animals such as cattle, and for improving the estrus synchronization of bovine embryo transfer in order to improve the efficiency of animal husbandry.
Disclosure of Invention
The invention aims to provide a novel method for improving estrus, ovulation and conception of animals such as cattle, and is expected to improve the synchronous estrus effect of cattle embryo transplantation so as to improve the efficiency of animal husbandry production. It has been surprisingly found that the injection of luteinizing hormone releasing hormone a3 to achieve estrus synchronization in cattle and thereby ovulation and conception in test animals provides unexpected results and the present invention has been completed based on such findings.
The invention makes the cow realize estrus synchronization by injecting luteinizing hormone releasing hormone A3 into the cow, and further makes the test animal ovulate and conception, essentially, the invention is a method for improving the estrus synchronization effect of cow embryo transplantation, and essentially can improve the availability of the receptor cow in the cow embryo transplantation.
To this end, the present invention provides in a first aspect the use of luteinizing hormone releasing hormone a3 for the manufacture of a pharmaceutical composition for improving the estrous effects of bovine embryo transfer synchronization.
The use according to the first aspect of the invention, wherein the composition comprises luteinizing hormone-releasing hormone a 3.
Use according to the first aspect of the invention wherein the composition comprises luteinizing hormone releasing hormone a3, arginine hydrochloride, triethyl citrate.
The use according to the first aspect of the invention, wherein the composition comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, wherein the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mu g: 10-200 mg: 1-20 mg.
The use according to the first aspect of the invention, wherein the composition comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, and the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mug: 20-100 mg: 2-10 mg.
The use according to the first aspect of the invention, wherein the composition comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, and the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mug: 50 mg: 6 mg.
The use according to the first aspect of the invention, wherein the composition is administered by injection after dissolution in water.
The use according to the first aspect of the invention, wherein the composition is a freeze-dried powder injection.
The use according to the first aspect of the invention, wherein the composition is prepared according to a process comprising the steps of: dissolving luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate in appropriate amount of water for injection (such as LHRH-A3 concentration of 1-20 μ g/ml, such as 2-10 μ g/ml, such as 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, packaging into penicillin bottles, and freeze drying.
The use according to the first aspect of the present invention, wherein the composition is administered by injection, diluted with water for injection to a concentration of LHRH-A3 of 1 to 20 μ g/ml, such as 2 to 10 μ g/ml, such as 5 μ g/ml, and administered by injection in prescribed doses.
The use according to the first aspect of the present invention, wherein the method for improving the estrus synchronization of bovine embryo transfer comprises the steps of:
(1) providing a donor bovine: the donor cattle are nonpregnant and non-estrualized multiparous cows, and the anogenital tract diseases are confirmed through rectal examination; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of luteinizing hormone releasing hormone A3 to donor cattle;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus time and the oestrus ending time once every morning and evening for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the appearance oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, carrying out artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule, wherein the semen is the frozen semen of the same donor cow;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The use according to the first aspect of the invention, wherein the donor bovine is selected from: and cattle, yellow cattle, cold red cattle, Kazakh cattle, and Angus cattle.
The use according to the first aspect of the present invention, wherein the donor cattle are cattle more than 4 months post-partum.
The use according to the first aspect of the present invention, wherein the donor cattle are cattle aged 3 to 8 years old.
The use according to the first aspect of the invention, wherein the dose of the luteinizing hormone-releasing hormone A3 is 1-20 mug per 100kg body weight.
The use according to the first aspect of the present invention, wherein the dose of the luteinizing hormone-releasing hormone A3 is 2-10 μ g/100kg body weight.
The use according to the first aspect of the invention, wherein the dose of luteinizing hormone-releasing hormone a3 is 5 μ g per 100kg body weight.
The use according to the first aspect of the invention, wherein the luteinizing hormone-releasing hormone a3 is administered by injection in a composition thereof.
Further, the second aspect of the present invention provides a method for improving the estrus synchronization of bovine embryo transfer, which comprises the following steps:
(1) providing a donor bovine: the donor cattle are nonpregnant and unrelieved multiparous cows, and the anogenital tract disease is confirmed through rectal examination; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of luteinizing hormone releasing hormone a3 in donor cattle;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus time and the oestrus ending time once every morning and evening for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the appearance oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, carrying out artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule, wherein the semen is the frozen semen of the same donor cow;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The method according to the second aspect of the present invention, wherein the donor cattle are selected from: and cattle, yellow cattle, cold red cattle, Kazakh cattle, and Angus cattle.
The method according to the second aspect of the present invention, wherein the donor cattle are cattle more than 4 months postpartum.
The method according to the second aspect of the present invention, wherein the donor cattle are cattle aged 3 to 8 years old.
The method according to the second aspect of the present invention, wherein the dose of the luteinizing hormone-releasing hormone A3 is 1 to 20 μ g/100kg body weight.
The method according to the second aspect of the present invention, wherein the dose of the luteinizing hormone-releasing hormone A3 is 2 to 10 μ g/100kg body weight.
The method according to the second aspect of the invention, wherein the dose of luteinizing hormone-releasing hormone A3 is 5 μ g/100kg body weight.
The method according to the second aspect of the invention, wherein said luteinizing hormone-releasing hormone a3 is administered by injection in a composition thereof.
The method according to the second aspect of the invention wherein the composition comprises luteinizing hormone-releasing hormone a3 and arginine hydrochloride, triethyl citrate.
The method according to the second aspect of the present invention, wherein the composition comprises luteinizing hormone releasing hormone a3 and arginine hydrochloride, triethyl citrate in a weight ratio of 5 μ g: 10-200 mg: 1-20 mg.
The method according to the second aspect of the present invention, wherein the composition comprises luteinizing hormone releasing hormone a3 and arginine hydrochloride, triethyl citrate in a weight ratio of 5 μ g: 20-100 mg: 2-10 mg.
The method according to the second aspect of the present invention, wherein the composition comprises luteinizing hormone releasing hormone a3 and arginine hydrochloride, triethyl citrate in a weight ratio of 5 μ g: 50 mg: 6 mg.
The method according to the second aspect of the present invention, wherein the composition is administered by injection after dissolution in water.
The method according to the second aspect of the present invention, wherein the composition is a freeze-dried powder injection.
The method according to the second aspect of the present invention, wherein the composition is prepared according to a method comprising the steps of: dissolving luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate in appropriate amount of water for injection (such as LHRH-A3 concentration of 1-20 μ g/ml, such as 2-10 μ g/ml, such as 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, packaging into penicillin bottles, and freeze drying.
The method according to the second aspect of the present invention, wherein the composition is administered by injection, diluted with water for injection to a LHRH-A3 concentration of 1 to 20. mu.g/ml, such as 2 to 10. mu.g/ml, such as 5. mu.g/ml, and administered by injection in a prescribed dose.
Further, the third aspect of the present invention provides a composition comprising luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate.
The composition according to the third aspect of the invention comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, wherein the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mug: 10-200 mg: 1-20 mg.
The composition according to the third aspect of the invention comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, wherein the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mug: 20-100 mg: 2-10 mg.
The composition according to the third aspect of the invention comprises the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate, wherein the weight ratio of the luteinizing hormone releasing hormone A3 to the arginine hydrochloride to the triethyl citrate is 5 mug: 50 mg: 6 mg.
The composition according to the third aspect of the present invention, which is dissolved in water and administered by injection.
The composition according to the third aspect of the present invention, which is a freeze-dried powder injection.
A composition according to the third aspect of the present invention, which is prepared according to a process comprising the steps of: dissolving luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate in appropriate amount of water for injection (such as LHRH-A3 concentration of 1-20 μ g/ml, such as 2-10 μ g/ml, such as 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, packaging into penicillin bottles, and freeze drying.
The composition according to the third aspect of the present invention is diluted with water for injection to a concentration of LHRH-A3 of 1 to 20. mu.g/ml, for example, 2 to 10. mu.g/ml, for example, 5. mu.g/ml, and is administered by injection in a prescribed dose.
Any feature of any aspect of the invention or any embodiment of any aspect thereof is equally applicable to any other embodiment or any embodiment of any other aspect thereof, provided that they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
In the present invention, the luteinizing hormone-releasing hormone a3, english name: luteinizing Hormone Releasing Hormone A3, also called ovulation promoting NO. 3, ovulation promoting NO. three, ovulation promoting Hormone NO. three, polyembryon, etc., abbreviated as LHRH-A3 in English.
Luteinizing hormone releasing hormone A3(LHRH-A3) as a hormone medicine can promote the release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) from anterior pituitary of animals to promote ovarian follicle maturation and ovulation, so that the pituitary can be immediately released as synthesized hormone, and hormone synthesis can be stimulated. Used for treating cow ovulation retardation, ovary quiescence, corpus luteum persistence, ovarian cyst and early pregnancy diagnosis: it can also be used for inducing ovulation in fish. For male animals, spermatogenesis can be promoted.
Luteinizing hormone releasing hormone a3 is commonly used in the field of animal husbandry: 1. increasing the number born. 2. Improve the conception rate in the estrus. 3. Treating follicular maturity difference, ovulation delay, and follicular cyst. 4. Enhance the effect of superovulation. 5. Strengthen the function of corpus luteum. 6. Can be used for inducing ovulation in fish.
The application of the luteinizing hormone releasing hormone A3 in the breeding of cattle is mainly as follows:
promoting the restoration of ovarian function, shortening calving interval, and carrying out intramuscular injection of 5-15 microgram within 8-18 days after delivery for 1 time every day for 3 days;
and (3) the tire receiving rate is improved: a. when in mating, 25 mu g is injected intramuscularly, which can obviously improve the estrus conception rate; b. 25 mu g of glaprost sodium (carbendazim) is injected intramuscularly about 40 days after delivery, 0.4-0.6 mg of glaprost sodium (carbendazim) is injected intramuscularly at intervals of 6 days, and 20 mu g of glaprost sodium (carbendazim) is injected intramuscularly at intervals of 1 day, so that the conception rate within 60 days after delivery can be obviously improved;
treating follicular maldevelopment, ovulation retardation: 25-30 mu g of intramuscular injection is carried out during or 2 hours before the hybridization;
treatment of multiple follicular development: intramuscular injection of 25 μ g per day for 3 consecutive days;
treating ovarian cyst: intramuscular injection of 25 μ g for 5 consecutive days and 100mg progesterone daily for 3 months after pregnancy should be repeated to prevent recurrence.
The application of the luteinizing hormone releasing hormone A3 in pig breeding mainly comprises the following steps:
the conception rate and the litter size of the sows in the estrus are improved: the sows are injected with 25-50 mu g of intramuscular in the afternoon of the 2 nd day of estrus, and are bred for 1 time in the morning and afternoon of the 3 rd day, and the average litter size of the sows is increased by 1.5-2;
the boar has weak libido, and 25 mu g of boar is taken every day for 3-5 days continuously.
The application of the luteinizing hormone releasing hormone A3 in sheep reproduction mainly comprises the following steps:
after the ewes normally estre, 5-10 mu g of the goat feed is injected into the ewes by muscle injection, and after 4-6 hours, hybridization is carried out (if the ewes do not estre, 0.5-1 ml of three-in-one hormone can be used for promoting estrus first), so that the twins rate of the goats is obviously increased, and the number of the sheep born is increased.
The application of luteinizing hormone releasing hormone A3 in other domestic animals mainly comprises the following steps:
increasing conception rate and litter size, and intramuscular injection at the same time of or several hours before mating: deer 15 microgram/head, rabbit 0.2-0.5 microgram/head, dog fox 15 microgram/head, and hide animal 5 microgram/head.
The application of the luteinizing hormone releasing hormone A3 in aquatic products mainly comprises the following steps: ovulation induction: intraperitoneal injection is carried out, wherein each time of the intraperitoneal injection is one time for each fish, the amount of grass carp is 2-5 mu g, and the amount of silver carp and bighead carp is 3-5 mu g.
The invention has surprisingly found that the heat, ovulation and conception effect of the animals can be obviously improved by using the method of the invention.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention generally and/or specifically describes the materials used in the tests, as well as the test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1: test of estrus synchronization effect
The estrus synchronization effect test was performed as follows.
(1) Providing a donor bovine: the method comprises the following steps of (1) confirming the diseases of the genital tract by rectal examination of south-yang yellow cattle, which are born for more than 4 months, aged 3-8 years, barren and unhappy multiparous cows; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of luteinizing hormone-releasing hormone A3(LHRH-A3) to donor cattle at a dose of 5 μ g/100kg body weight;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus once in the morning and at night every day for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the external oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, performing artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule at a proper time, wherein the semen is the frozen semen of the Nanyang yellow cattle;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The preparation of the drug used in step (2) of this example: dissolving commercially available raw material medicine (> 99%) of luteinizing hormone releasing hormone A3 with water for injection (5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, subpackaging in penicillin bottles, and freeze-drying for use; when in use, the injection is diluted to 5 mu g/ml by water for injection and is injected according to the regulated dose.
The results of some embodiments of the present application are as follows:
note: in the table, ovulatory animals are counted on the basis of estrus in the same period, estrus pregnancies are counted on the basis of ovulatory animals, and total pregnancies are counted on the basis of the total number of test animals. It has been surprisingly found that the injection of both arginine hydrochloride and triethyl citrate together with the injection of luteinizing hormone releasing hormone A3 can significantly increase the conception rate.
Example 2: test of estrus synchronization
The estrus synchronization effect test was performed as follows.
(1) Providing a donor bovine: the method comprises the following steps of (1) checking rectum of a plurality of multiparous cows with age of 3-8 years and no estrus of more than 4 months postpartum yellow cattle to confirm the anogenital tract disease; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of a composition comprising luteinizing hormone releasing hormone A3(LHRH-A3) to donor cattle at a dose of 5 μ g/100kg body weight in LHRH-A3;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of cows within 1-3 d from the day of 0d of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus once in the morning and at night every day for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the external oestrus symptoms of the cows by combining rectal examination, determining the oestrus synchronization according to the judgment of the oestrus appearing in 3d after the medicine injection, and carrying out artificial insemination on the oestrus and the ovulation cows according to a freeze-matching technical operation procedure at the right time, wherein the semen is the frozen semen of the Nanyang yellow cattle;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The composition administered in step (2) of this example contains LHRH-a3, arginine hydrochloride, triethyl citrate in a weight ratio of 5 μ g: 50 mg: 6mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug of luteinizing hormone releasing hormone A3 (> 99%) and arginine hydrochloride and triethyl citrate in proportion with water for injection (to LHRH-A3 concentration of 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, subpackaging in penicillin bottles, and freeze drying for use; when in use, the injection is diluted by water for injection until the concentration of LHRH-A3 is 5 mug/ml, and the injection is administered according to the regulated dose.
Example 3: test of estrus synchronization effect
The estrus synchronization effect test was performed as follows.
(1) Providing a donor bovine: the method comprises the following steps of (1) checking rectum of a plurality of multiparous cows with age of 3-8 years and no estrus of more than 4 months postpartum yellow cattle to confirm the anogenital tract disease; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of a composition comprising luteinizing hormone releasing hormone A3(LHRH-A3) to donor cattle at a dose of 5 μ g/100kg body weight in LHRH-A3;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus once in the morning and at night every day for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the external oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, performing artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule at a proper time, wherein the semen is the frozen semen of the Nanyang yellow cattle;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The composition administered in step (2) of this example comprises LHRH-a3, arginine hydrochloride in a weight ratio of 5 μ g: 50mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug (> 99%) of luteinizing hormone releasing hormone A3 and arginine hydrochloride in water for injection according to a certain proportion (the concentration of LHRH-A3 is 5 mu g/ml), filtering and sterilizing by using a 0.22 mu m microporous filter membrane, subpackaging into a penicillin bottle, and freeze-drying for later use; when in use, the injection is diluted by water for injection until the concentration of LHRH-A3 is 5 mug/ml, and the injection is administered according to the regulated dosage.
Example 4: test of estrus synchronization
The estrus synchronization effect test was performed as follows.
(1) Providing a donor bovine: the method comprises the following steps of (1) checking rectum of a plurality of multiparous cows with age of 3-8 years and no estrus of more than 4 months postpartum yellow cattle to confirm the anogenital tract disease; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of a composition comprising luteinizing hormone releasing hormone A3(LHRH-A3) at a dose of 5 μ g/100kg body weight, LHRH-A3, to donor cattle;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus once in the morning and at night every day for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the external oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, performing artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule at a proper time, wherein the semen is the frozen semen of the Nanyang yellow cattle;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
The composition administered in step (2) of this example comprises LHRH-a3 and triethyl citrate in a weight ratio of 5 μ g: 6mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug (> 99%) of luteinizing hormone releasing hormone A3 and triethyl citrate in water for injection according to a certain proportion (the concentration of LHRH-A3 is 5 mu g/ml), filtering and sterilizing by using a 0.22 mu m microporous filter membrane, subpackaging into penicillin bottles, and freeze-drying for later use; when in use, the injection is diluted by water for injection until the concentration of LHRH-A3 is 5 mug/ml, and the injection is administered according to the regulated dose.
Example 5: test of estrus synchronization
The estrus synchronization effect test was performed on 44 Japanese pure blood and cattle according to the method of example 2, and the results showed that: the estrus count is 41 (the synchronization rate is 93.2%), the ovulation count is 38 (the ovulation rate is 92.7%), the conception count is 33 (the estrus conception rate is 86.8%), and the total conception rate is 75.0%.
Example 6: preparation of the composition
The weight ratio of LHRH-A3, arginine hydrochloride and triethyl citrate is 5 mug: 100 mg: 2mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug (> 99%) of luteinizing hormone releasing hormone A3 and arginine hydrochloride and triethyl citrate in proportion with water for injection (the concentration of LHRH-A3 is 5 mu g/ml), filtering and sterilizing with a 0.22 mu m microporous filter membrane, subpackaging in penicillin bottles, and freeze-drying to obtain the composition. When the composition is used, the composition can be diluted with water for injection to LHRH-A3 concentration of 5 μ g/ml, and injected into a predetermined dosage for administration.
Example 7: preparation of the composition
The weight ratio of LHRH-A3, arginine hydrochloride and triethyl citrate is 5 mug: 20 mg: 10mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug of luteinizing hormone releasing hormone A3 (> 99%) and arginine hydrochloride and triethyl citrate in proportion with water for injection (LHRH-A3 concentration is 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, subpackaging in penicillin bottles, and freeze drying to obtain the composition. When the composition is used, the composition can be diluted with water for injection to LHRH-A3 concentration of 5 μ g/ml, and injected into a predetermined dosage for administration.
Example 8: preparation of the composition
The weight ratio of LHRH-A3 to arginine hydrochloride to triethyl citrate is 5 mug: 200 mg: 20mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug (> 99%) of luteinizing hormone releasing hormone A3 and arginine hydrochloride and triethyl citrate in proportion with water for injection (the concentration of LHRH-A3 is 5 mu g/ml), filtering and sterilizing with a 0.22 mu m microporous filter membrane, subpackaging in penicillin bottles, and freeze-drying to obtain the composition. When the composition is used, the composition can be diluted by water for injection until the concentration of LHRH-A3 is 5 μ g/ml, and the composition can be injected according to the regulated dosage.
Example 9: preparation of the composition
The weight ratio of LHRH-A3 to arginine hydrochloride to triethyl citrate is 5 mug: 10 mg: 1mg, the preparation method of the composition comprises the following steps: dissolving commercially available bulk drug of luteinizing hormone releasing hormone A3 (> 99%) and arginine hydrochloride and triethyl citrate in proportion with water for injection (LHRH-A3 concentration is 5 μ g/ml), filtering with 0.22 μm microporous membrane for sterilization, subpackaging in penicillin bottles, and freeze drying to obtain the composition. When the composition is used, the composition can be diluted with water for injection to LHRH-A3 concentration of 5 μ g/ml, and injected into a predetermined dosage for administration.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for increasing the estrus effect of bovine embryo transfer synchronization, said method being a non-therapeutic method comprising the steps of:
(1) providing a donor bovine: the donor cattle are nonpregnant and non-estrualized multiparous cows, and the anogenital tract diseases are confirmed through rectal examination; during the whole test period, grazing is mainly performed in the daytime, and forage is supplemented at night;
(2) administration: intramuscular injection of a composition comprising luteinizing hormone-releasing hormone A3 to a donor bovine at a dose of 5 μ g per 100kg body weight calculated as luteinizing hormone-releasing hormone A3; the composition comprises lutein releasing hormone A3, arginine hydrochloride and triethyl citrate, wherein the weight ratio of the three is 5 mu g: 50 mg: 6 mg;
(3) estrus and insemination: continuously observing the oestrus and ovulation conditions of the cows within 1-3 d from 0d on the day of medicine injection, recording the oestrus time and the oestrus ending time of the cows, observing the oestrus time and the oestrus ending time once every morning and evening for 2-3 h each time, determining the oestrus time, the oestrus program and the ovulation condition according to the appearance oestrus symptoms of the cows, determining oestrus synchronization according to rectal examination, carrying out artificial insemination on the oestrus and ovulation cows according to a freeze-matching technical operation rule, wherein the semen is the frozen semen of the same donor cow;
(4) and (3) pregnancy judgment: and (5) determining whether the cow is pregnant according to the rectal examination result 90 days after mating, calculating the estrus conception rate, and feeding the cow in the pregnancy according to a conventional method.
2. The method of claim 1, wherein the donor bovine is selected from the group consisting of: and cattle, yellow cattle, cold red cattle, Kazakh cattle, and Angus cattle.
3. The method of claim 2, wherein the donor bovine is a 4 month post-partum bovine.
4. The method of claim 2, wherein the donor cattle are cattle aged 3-8 years.
5. The method of claim 1, wherein the composition is prepared according to a process comprising the steps of: dissolving the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate by using a proper amount of water for injection according to a certain proportion until the concentration of the luteinizing hormone releasing hormone A3 is 5 microgram/ml, filtering and sterilizing by using a microporous filter membrane of 0.22 microgram, subpackaging in penicillin bottles, and freeze-drying to obtain the medicine.
6. A composition for use in the method for improving the estrus synchronization of bovine embryo transfer according to any of claims 1-5, wherein the composition comprises lutein releasing hormone A3, arginine hydrochloride and triethyl citrate in a weight ratio of 5 μ g: 50 mg: 6 mg.
7. The composition of claim 6, which is a freeze-dried powder injection.
8. The composition of claim 7, prepared according to a process comprising the steps of: dissolving the luteinizing hormone releasing hormone A3, arginine hydrochloride and triethyl citrate by using a proper amount of water for injection according to a proportion until the concentration of the luteinizing hormone releasing hormone A3 is 5 mug/ml, filtering and sterilizing by using a 0.22μm microporous filter membrane, subpackaging in a penicillin bottle, and freeze-drying to obtain the luteinizing hormone releasing hormone A3 injection.
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| CN109964878A (en) * | 2019-05-09 | 2019-07-05 | 新疆三农草原牧业有限公司 | The cross breeding method of Xinjiang Kazakhstan Plate and Angus |
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