RU2709176C1 - Method for induction of superovulation as per a combined scheme in embryo transplantation technology - Google Patents

Abstract

FIELD: agriculture; animal breeding.

SUBSTANCE: invention relates to agriculture, in particular to a method for production of bovine embryos. Method is characterized by that on the first day of sexual cycle intramuscularly introduced is Surfagon in amount of 10 ml, on seventh day – Estrofan 3 ml and Tetravit 10 ml, on tenth day – Oluvine 5 ml, followed by artificial insemination. On the seventh day after insemination, progesterone is introduced in amount of 2 ml, then embryos are washed out.

EFFECT: use of the invention will make it possible to increase quantity of high-quality embryos.

1 cl, 2 tbl

Description

The invention relates to agriculture, in particular cattle breeding for cattle breeding by embryo transplantation for further genomic editing and selection of healthy livestock.

The basis of embryo transplantation is six consecutive technological processes: preparation of donors, recipients, obtaining embryos, morphological evaluation of their quality, freezing and transplantation to recipients.

The first step in the implementation of the embryo transplantation method is the selection of donor cows. The transplantation method is suitable for a limited number of individuals that can become breed improvers by one or more significant characteristics. For the role of donors, pedigree heifers or cows with high-value qualities are selected in order to obtain from them a significant number of calves having the same properties. Donors can be animals belonging to any desired valuable breed.

Cows with a violated sexual cycle, ovarian cysts are not suitable for use as donors.

The ovaries of healthy cows contain hundreds of thousands of immature germ cells (oocytes), but, as a rule, one follicle matures during one sexual cycle. Therefore, only one egg is fertilized and only one calf is born.

Superovulation refers to the isolation of many eggs from the ovaries of a cow during one sexual cycle, which is one of the most important stages in the technology of embryo transplantation. For these purposes, there are certain methods that allow maximum use of the biological reproductive potential of the ovaries of cows. The basis of this method is the synchronous growth of many follicles, their maturation with subsequent ovulation.

According to the known method, according to the Instructions for transplantation of cattle embryos, hormonal processing of an animal for superovulation in donors, which includes 8-10-fold administration of a drug containing follicle-stimulating hormone (FSH) and luteinizing hormone (LH) every 12 hours in for 4-5 days (4 or 5-day regimen for the administration of gonadotropic drugs). This period of time (4-5 days) is sufficient for the complete maturation of many follicles with the formation of full-fledged high-quality eggs. One of the main disadvantages of this method is the short period of inactivation of FGS, therefore, in order to sustainably maintain an effective level of the hormone in the blood, which is necessary for the growth and maturation of a large number of follicles, it is necessary to repeatedly inject drugs into the body of the animal, which leads to an increase in the complexity of the process. In addition, such repeated administration of the drug causes a variation in the concentration of the administered gonadotropins in the blood serum of the animal, which reduces the chances of obtaining high results of superovulation.

A known method (patent BY 12490), where as a prolongator use a powdered polymer of polyvinyl alcohol (PVA), which is diluted in physiological saline with a temperature of 50-60 ° C to a concentration of 12% suspension, which is cooled to 40-42 ° C and in quantity 5-7 ml are mixed with 50 mg of natural FSH. The resulting working solution is once administered subcutaneously to donor cows in the scapula region at 5-7 ml / head. All animal donors were treated with natural hormone preparation of the Russian production of the FSH-Super trademark according to its instructions. The disadvantage of this method is the low efficiency of the prolonged action of polyvinyl alcohol on FSH, used in accordance with the prototype method for the induction of superovulation in donor cows in the form of a drug brand "FSH-super." As a result, there is not enough synchronous growth, maturation and ovulation of many follicles, which leads to a low percentage of quality embryos.

According to a method for stimulating FSH prolongation for superovulation in donor cows (prototype patent BY 12490), which involves a single administration of the FSH super preparation containing pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (luteinizing hormone) into the luteal phase of the sexual cycle LH), in combination with a prolongator, followed by administration of prostoglandin after 48 hours twice with an interval of 12 hours at doses of 500 and 250 μg / head for cloprostenol. Animals are artificially inseminated twice with sperm from a proven bull 48 and 60 hours after the first injection of prostaglandin F2α. The extraction of embryos is carried out on the 7th day of the genital cycle. The resulting embryos are counted, assessed for their viability and suitability for embryo transfer.

A known method of prolonging the action of a single injection of FSH using carboxy methyl cellulose and polyvinylpyrrolidone, a 3.2% solution of gelatin as a prolongator (Smith LE, Sitton GD, Vincent C.K. Limited inqection of follicle stimulating hormone for multiple births in beef cattle. // J. Anim. Sci., 1973, 37, 523-527). A known prolongator is a prolongon, which allows for a single injection of FSH to cause superovulation in 75% of animals with an ovulation number of 9.9 to 10.7 (using various FSH preparations) and high-quality embryos from 2.9 to 3.5 per donor. At the same time, a higher level of engraftment of embryos obtained from cows treated with FSH with prolongon was noted. (Dovgopol V.F., Ostashko F.I., Isachenko I.I. Improvement of the non-surgical method of embryo transplantation. // Zootechny, 1992, 2, 14-17). However, the aforementioned prolongators do not provide the optimal time for the effective action of follicle-stimulating hormone (FSH), necessary to induce multiple growth of follicles, or provide a high number of ovulations, but the number of suitable embryos obtained is lower by 50% of the average, or cause polycystic ovaries, which can lead to forced culling of the animal.

The objective of the invention is the creation of superovulation in donor cows with prolonging the action of pituitary gonadotropins, which allows to increase the number of synchronously mature follicles with their subsequent ovulation and the release of high-quality mature eggs suitable for fertilization, and after their fertilization - to increase the number of quality embryos obtained. The quantity and quality of the obtained embryos is the main indicator of the effectiveness of the induction of superovulation in embryo donor cows.

In this regard, in the technology of embryo transplantation, it becomes expedient to develop and implement a method of inducing superovulation according to a combined scheme. Thanks to the developed scheme, the time spent by exogenous gonadotropin in the body increases and its release is uniform, leading to the maintenance of the necessary level of this hormone in the blood, which makes it possible to maximize the biological reproductive potential of the ovaries of cows, synchronizing the growth of many follicles and their maturation with subsequent ovulation.

The technical result of the invention is to increase the number of quality embryos obtained from donor cows as a result of superovulation.

The proposed method of stimulation of superovulation consists of two stages:

1. The preparatory phase (which in turn consists of two blocks):

- Monitoring the level of feeding and completeness of the diet of the animal donor.

- Compliance with daily hourly exercise, or rectogenital massage.

2. The main stage. Scheme using hormonal drugs with follicle stimulating activity.

The drugs are administered to the animal donor according to the scheme shown in table 1.

Surfagon. A drug in 1 ml as an active substance contains 5 or 10 μg of surfon, excipients: 9 mg of sodium chloride, 0.5 mg of nipagin, as well as water for injection up to 1 ml.

Surfagon is a synthetic analogue of gonadotropin-releasing hormone (GnRH) - luliberin. Competitively binds to cell receptors of the anterior pituitary gland, causing, like other GnRH analogues, a short-term increase in the level of sex hormones in the blood. The increased content of gonadotropins in the blood persists for 3-4 hours after administration, then the content drops rapidly. The half-life of the surfer is the same as that of natural luliberin. During this time, the peptide breaks down into amino acids and is excreted from the body.

Ovulin (Global-Vet) The hormonal drug in 1 vial contains the human chorionic gonadotropin (hCG) - 1000 IU / vial as an active substance, and 0.002 g / vial as an auxiliary component of mannitol.

Gonadotropin, which is part of Ovulin, has a gonadotropic, mainly luteinizing effect.

Estrofan. In 1 ml of the drug, the active substance contains cloprostenol -250 μg and auxiliary components: citric acid - 9.55 mg, chlorcresol - 1 mg, sodium hydroxide - 5 mg and water for injection up to 1 ml.

Cloprostenol. Synthetic hormone prostaglandin PGF2a. It has a luteolytic effect on the corpus luteum of the ovaries, relieves the inhibitory effect of progesterone on the hypothalamic-pituitary complex and promotes the growth of follicles in the ovaries and, as a result, increases the level of estrogen in the blood, the manifestation of sexual hunting and subsequent ovulation of mature follicles.

Estrofan. To induce and synchronize sexual hunting, the drug is administered to cows and heifers at a dose of 2 ml twice with an interval of 10 days.

Progesterone. The drug has a biological and therapeutic effect of the natural hormone of the corpus luteum: it reduces the excitability and contraction of the muscles of the uterus, creates the necessary conditions for fertilization, reduces the sensitivity of the uterus to oxytocin, and stimulates the functional activity of the mammary gland.

Tetravit is released in the form of a clear, oily injection solution of light yellow color. The following vitamins are contained in 1 ml of solution: F (5 mg), E (20 mg), D3 (25,000 ME), A (50,000 ME).

Scientific and economic experience was carried out on cows of black-motley breed. Evaluation of the effectiveness of the superovulation program was carried out according to the number of prepared animals according to this methodology, as a percentage of all animals that underwent artificial insemination, and full embryos received from them.

To carry out the induction of superovulation of animals, the embryo transplantation technique was issued a technical assignment in writing with the inclusion of information about cow numbers and dates of drug administration. 3 similar donor cows were processed with the same feeding and keeping conditions. The effectiveness of the method was determined by counting the number of embryos suitable for embryo transplantation. The results of the experiment are shown in table 2.

Cows donor embryos of black-and-white breed under the numbers No. 1, 2, 3, at the age of 5 years, carried out the induction of superovulation in accordance with the patented method.

The work on the introduction of drugs was carried out according to Scheme 1. On the first day, 10 ml of Surfagon was injected intramuscularly into the croup. On the seventh day, Estrofan 3 ml + Tetravit 10 ml was administered. On the tenth day, Ovulin was introduced 5 ml, after which they carried out artificial insemination. Used frozen semen of high quality with a high percentage of normal, motile sperm. On the seventh day after insemination of the donor cow, 2 ml of progesterone was administered, after which embryos were extracted.

The first donor cow received 18 embryos, of which 14 were of high quality and suitable for transplantation or cryopreservation.

15 embryos were obtained from the second donor cow, of which 11 were high-quality and suitable for transplantation or cryopreservation.

From the third donor cow, 20 fetuses were obtained, of which 16 were qualitative and suitable for transplantation or cryopreservation.

Thus, the application of the proposed method allows the cow or heifer to isolate more than 10 viable eggs in one sexual period.

The possibility of implementing this invention is the introduction on the first day - Surfagon 10 ml. On the seventh day - Estrofan 3 ml + Tetravit 10 ml. On the tenth day - Ovulin 5 ml, after which they carried out artificial insemination. On the seventh day after insemination of the donor cow - progesterone 2 ml, after which the embryos are removed.

Sources of information

1. Instructions for transplantation of cattle embryos. - M. - Gosagroprom of the USSR. - 1987.

2. Copyright certificate No. 199990346, cl. A61D 19/04.

3. Conditions for the application of biotechnological methods of artificial insemination of pedigree cows and heifers. - M.: FSINI "Rosinformagroteh", 2017. - 24 p.

4. Order of the Ministry of Agriculture of Russia dated March 18, 2016 No. 102. “On the approval of the conditions for the application of biotechnological methods of artificial insemination of breeding cows and heifers.”

5. Patent BY 12490 C1 2009.10.30

6. GOST 28424-2014 Means of reproduction "Embryos of cattle" T / U Moscow. - Standartinform. - 2015.

7. Dovgopol V.F., Ostashko F.I., Isachenko I.I. Improving the non-surgical method of embryo transplantation. // Zootechny, 1992, 2, 14-17.

8. Smith L.E., Sitton G. D., Vincent C.K. Limited inqection of follicle stimulating hormone for multiple births in beef cattle. // J. Anim. Sci., 1973, 37, 523-527.

Claims (1)

A method for producing cattle embryos, characterized in that on the first day of the sexual cycle, Surfagon is administered intramuscularly in an amount of 10 ml, on the seventh day - Estrofan 3 ml and Tetravit 10 ml, on the tenth day - Oluvin 5 ml, after which artificial insemination is carried out, on the seventh day after insemination, progesterone is introduced in an amount of 2 ml, then the embryos are washed out.