RU2750208C1 - Method for obtaining embryos from heifers-donors of holstein breed - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. The invention is a method of obtaining embryos from donor heifers of the Holstein breed, including the selection of donor heifers of the Holstein breed at the age of 13-15 months, clinically healthy, having a good conformation, excellent pedigree, the productivity of the mother for the highest lactation of at least 9000 kg with a fat content in milk not less than 3.9%, protein - 3.3%, induction of superovulation according to the scheme by multiple injections of hormonal preparations containing follicle-stimulating and luteinizing hormones, and hormonal preparations for synchronizing sexual desire and regulating reproductive function, which are administered in different dosage from 1 to 5 ml per 1 head, repeated insemination at intervals, washing out of embryos after insemination and assessment of the quality of embryos, according to the invention, the induction of superovulation is carried out according to the scheme: on the 1st day in the morning, the drug "Estrofantin" is administered at a dose of 3 ml, in the morning on the 2nd day, drugs are administered: "CIDR" in a dose of one capsule, "Carolin" in a dose of 20 ml and "Sedimin" - 10 ml, on the 3rd and 11th day, also in the morning, in a dose of 5 ml, the drug "Fertagil" is injected, then on the 6th, 7th, and 8th days, the drug "Folligon" is injected in the morning and evening with a daily decreasing dose of 1 ml, 0.7 ml and 0.5 ml, respectively, on the 9th day in the morning the heifers are injected with drugs: "Folligon" at a dose of 0.4 ml, "Dinolytic" - 5 ml and from the vagina of the donor heifer remove the capsule "CIDR", and in the evening the preparations: "Folligon" and "Dinolytic" in the same doses, then on the 11th day in the morning after the introduction of the drug "Fertagil" carry out the first insemination in the amount of 2 doses and after 8-9 hours the second insemination is carried out with one dose, then on the 18th day, 7 days after re-insemination, in the morning the embryos are washed out and the "SIDR" drug is administered to the donor heifer.EFFECT: invention improves quality of embryos and increases their number.1 cl, 5 tbl

Description

The invention relates to agriculture, namely to dairy farming.

A known method of obtaining calves transplants of different genotypes on the paternal line from cows-donors of embryos (RF, patent No. 2112368), including the entire technology of embryo transplantation. The selection is carried out from at least one genetically valuable embryo donor cow for heterosperm insemination with mixed sperm of improving bulls, and then an immunogenetic analysis of the origin of the transplanted calves by parental blood groups is performed.

The disadvantage of this method is the impossibility of using the method in livestock breeding.

A method for producing cattle embryos is known from the prior art, including the selection of clinically healthy donor heifers with good conformation, excellent pedigree, mother's productivity for the highest lactation of at least 9000 kg, with a fat content in milk of at least 3.9%, protein - 3, 3%, induction of superovulation according to the scheme, by repeated administration in the form of injections, by repeated administration in the form of injections of hormonal preparations containing follicle-stimulating and luteinizing hormones and hormonal preparations for synchronization of sexual desire and regulation of reproductive function, repeated insemination at intervals, washing out of embryos after insemination and assessment of the quality of embryos (Guidelines for obtaining and transplanting cattle embryos / D.V. Popov, A.V. Brigida, G.Yu. Kosovskiy. M .: KlabPrint, 2017. - 55 p., 4, 8- 12 s.).

The closest technical solution is (see Nimbona K, et al., Obtaining embryos from cows and donor heifers, Scientific support of the agro-industrial complex, Collection of articles based on the materials of the 73rd scientific-practical conference of students on the basis of research work for 2017, Krasnodar, 25.04 .2018, p. 312-314) - D1, including the selection of donor heifers of the Holstein breed at the age of 13-15 months, clinically healthy, with a good conformation, excellent pedigree, the mother's productivity for the highest lactation is not less than 9000 kg, with a fat content in milk not less than 3.9%, protein - 3.3%, induction of superovulation, through multiple administration of hormonal drugs and insemination, stimulate superovulation using hormonal drugs that are administered for 10 days in a row at different dosages from 1 to 5 ml per 1 head ., multiple insemination and washing out of embryos.

The disadvantage of the known technical solution lies in the fact that heifers aged 15 months and older are used to obtain embryos, which does not sufficiently ensure the accelerated receipt of embryos and their quality.

The technical result is to improve the quality of embryos and increase their number.

The technical result is achieved by the fact that in the method of obtaining embryos from heifers - donors of the Holstein breed, including the selection of donor heifers of the Holstein breed at the age of 13-15 months, clinically healthy, having a good conformation, excellent pedigree, the productivity of the mother for the highest lactation is not less than 9000 kg, with a fat content in milk of at least 3.9%, protein - 3.3%, induction of superovulation according to the scheme, by multiple injection in the form of injections of hormonal preparations containing follicle-stimulating and luteinizing hormones and hormonal preparations to synchronize sexual activity and regulate reproductive function , which are administered in different dosages from 1 to 5 ml per 1 head, repeated insemination at intervals, washing out of embryos after insemination and assessment of the quality of embryos according to the invention, the induction of superovulation is carried out according to the scheme: on the 1st day, in the morning, the drug "Estrofantin" is injected into dose of 3 ml, in the morning, on the 2nd day, drugs are administered: "CIDR" in a dose of one capsule, "Karol in "in a dose of 20 ml and" Sedimin "- 10 ml, on the 3rd and 11th day, also in the morning, in a dose of 5 ml, the drug" Fertagil "is administered, then on the 6th, 7th, and 8th day in the morning and in the evening, the drug "Folligon" is administered with a daily decreasing dose of 1 ml; 0.7 ml and 0.5 ml, on the 9th day in the morning the heifers are injected with drugs: "Folligon" at a dose of 0.4 ml, "Dinolytic" - 5 ml and the capsule "CIDR" is removed from the vagina of the donor heifer, and in the evening preparations: "Folligon" and "Dinolytic" in the same doses, then on the 11th day, in the morning after the introduction of the drug "Fertagil", the first insemination is carried out in the amount of 2 doses and after 8-9 hours the second insemination is carried out with one dose, then on the 18th day, after 7 days, after re-insemination, in the morning the embryos are washed out and the "SIDR" drug is administered to the donor heifer.

The novelty of the proposed proposal is due to the fact that, due to the ultra-early insemination of donor heifers, namely at 13-15 months, using hormonal stimulation with certain drugs, providing superovulation, the reproductive abilities of heifers increase, ensuring their intensive and long-term use and the number of inseminations is reduced, which affects the economic feasibility of growing embryos.

The method of obtaining embryos from heifers - donors of the Holstein breed is as follows.

On the farm, from the zootechnical documentation, heifers are selected at the age of 13-145 months, origin (mother and father of the Holstein breed), with a live weight of 350 - 400 kg, mothers' milk yield is more than 7500 kg. In the selected heifers, the condition of the genitals is assessed. In rectal examination, the uterus is located in the pelvic cavity, the cervix is clearly expressed, the length of the uterine horns is 20-35 cm, without adhesions and neoplasms, the ovaries are oval in shape and dense consistency, 2-1.5 cm in size, the duration of previous cycles should be 19- 22 days, active corpus luteum of the ovary 1-1.5 cm, animals are not used if there is a corpus luteum and a follicle at the same time. Clinically healthy animals: animal fatness 2.75-3.5 points, animals without diseases of the limbs, gastrointestinal tract, respiratory organs.

After assessing the health status of donor heifers, they are carried out for 10 days according to the superovulation induction scheme presented in Table 1.

For the induction of superovulation, the following hormonal drugs are used:

The drug "Estrofantin" is a veterinary drug for the treatment and regulation of reproductive function in female farm animals. The drug has luteolytic properties, i.e. causes morphological regression of the corpus luteum; reduces the level of progesterone, which promotes the release of gonadotropic hormones (FSH and LH), the development of folliculogenesis in the ovaries in female animals and causes full estrus and ovulation; has a significant effect on the transformation of proliferative endometrium into secretory, thereby increasing the number of uterine glands, has uterotonic activity; regulates the inflammatory response by affecting vascular tone (https://veterinarka.ru/vetmedicaments/estrofantin.html).

CIDR is a gestagenic drug. When it is introduced into the vagina, progesterone is released from the capsule at a constant rate and penetrates through the vaginal mucosa into the bloodstream. Progesterone inhibits the hypothalamic-pituitary system, as a result of which there is no release of gonadotropic hormones - follicle stimulating hormone (FSH) and luteinizing hormone (LH), and as a result, follicle maturation and ovulation of eggs do not occur. After removing the drug from the vagina, the level of progesterone in the blood decreases within 4-6 hours, as a result, follicles mature and ovulate.

In the claimed method, the preparation is used to synchronize the hunting of cattle; for this purpose, CIDR is injected into the vagina of heifers for a period of 7 to 12 days. Immediately after removing the drug, the animal is injected with Dinolytic in a dose of 5 ml. Animals come into the hunt 36-96 hours after the extraction of the CIDR, the bulk of the cows and heifers come into the hunt after 72 hours. (Https://www.vidal.ru/veterinar/cidr-28048)

The drug "Carolin" is used to normalize metabolism, prevent retention of placenta and postpartum endometritis, normalize reproductive functions. The drug has a synergistic effect on the body of animals, which is expressed in the normalization of metabolism, the prevention of hypo- and avitaminosis A, an increase in the reproductive functions of the breeding stock of agricultural animals, a decrease in morbidity and mortality, an increase in the productivity of newborn young animals (https://ultravet.ru/karolin -inekcionnyy-100ml-20fl-upak).

When administered intramuscularly, the drug "Sedimin" is absorbed and deposited in the liver and hematopoietic organs, replenishing the deficiency of iron, iodine and selenium in the body. The use of Sedimin eliminates iron deficiency (stimulates erythropoiesis and hemoglobin synthesis), iodine deficiency (prevents the formation of goiter) and selenium, which causes white muscle disease in calves and liver dystrophy in piglets; normalizes metabolic processes, accelerates the growth and development of animals, increases the body's resistance to various diseases, improves the general condition.

In addition, the use of Sedimin helps to increase the reproductive capacity of the animal's uterus and obtain a viable offspring; normalizes and stimulates the intrauterine development of the fetus, ensures the prevention of postpartum pathology and diseases (retention of afterbirth, endometritis); causes an increase in the general resistance of the organism of farm animals (https://www.vidal.ru/veterinar/sedimin-27638).

The drug "Fertagil" is a hormonal drug designed to increase the reproductive function of cattle and rabbits. The pharmacological properties of Fertagil are associated with the combined action of these hormones, which have a direct effect on the ovaries: FSH stimulates the growth and development of follicles, the synthesis and secretion of estrogens, and LH induces ovulation and controls the function of the corpus luteum. After intramuscular administration, gonadorelin is rapidly absorbed from the injection site, remaining in the blood plasma for no more than 20 minutes. Gonadorelin is metabolized to amino acids, peptides and carbohydrates. Gonadorelin is excreted in the urine and exhaled air. The drug is administered at a dose of 5 ml, which is standard and recommended by the drug's instructions, in addition, unlike the instructions, it is administered simultaneously with the first insemination (https://veterinarka.ru/vetmedicaments/fertagil.html)

The drug "Folligon" contains 1000 or 5000 IU of gonadotropin from the serum of pregnant mares as an active ingredient, as auxiliary substances: 5 mg of mannitol, 0.315 mg of sodium hydrogen phosphate dihydrate, 0.33 mg of sodium dihydrogen phosphate dihydrate. Pharmacological properties Pharmacotherapeutic group of the drug: hormones and their antagonists. The serum of pregnant mares contains a gonadotropic hormone with follicle stimulating (FSH) and luteinizing (LH) activity. When ingested, it binds to LH and FSH receptors in target organs. FSH causes the growth and development of follicles, LH stimulates ovulation and the formation of the corpus luteum. When injected, the maximum concentration of gonadotropin is reached after 8 hours in pigs and sheep and after 16 hours in cattle. The half-life of the hormone from the body of animals, depending on their species, is 34-150 hours.

Folligon is used to increase reproductive function, both separately and in combination with gestagens and prostaglandins in female cattle, for the induction and synchronization of sexual heat and to induce superovulation in embryo donor cows (https://veterinarka.ru/vetmedicaments/folligon .html).

Dinolitik (dinolytic) is a hormonal drug for synchronizing sexual activity and regulating reproductive function. This drug is administered simultaneously with the drug "Folligon" (dose 0.4 ml, also designed to maintain superovulation) in the evening of the 6th day and on the 7th day, in the morning at dose of 5 ml for the formation of homogeneous groups of animals, which do not need to be monitored daily for their state of sexual activity and inseminated in a short time, and it is also better to plan and control production processes. The dose of 5 ml is standard and is recommended by the instructions of the drug (https://www.vidal.ru/veterinar/dinolytic-28050)

In the claimed method, 13-15 month-old heifers are used as donors, to which on the 1st day, in the morning, the drug "Estrofantin" is administered at a dose of 3 ml. The introduction of the drug promotes the release of gonadotropic hormones (FSH and LH), the development of folliculogenesis in the ovaries in heifers and causes full ovulation. At a dose of less than 3 ml, the release of ganotropic drugs will pass more slowly, at a higher dose, hormonal levels may be disturbed.

In the morning, on the 2nd day, heifers are injected with a composition of drugs: "CIDR" in a dose of one capsule, "Carolin" in a dose of 20 ml and "Sedimin" - 10 ml.

On the 3rd and 11th day, also in the morning, the drug Fertagil is administered at a dose of 5 ml.

Then, on the 6th day, in the morning and in the evening, a loading dose of 1 ml is injected to stimulate the growth and maturation of follicles in the ovaries in heifers, increase the secretion of estrogen, ensure ovulation and the formation of a yellow body, as well as the release of steroid hormones, both in the follicles and in the cells of the yellow body. At a lower dose, the induction of superovulation will take place more slowly, at a higher dose, hormonal levels may be disrupted.

On the 7th and 8th day, the drug "Folligon" is administered with a decreasing dose of 0.7 ml and 0.5 ml, respectively, to maintain superovulation, with a deviation from the dose, there may be disturbances in the process, to maintain superovulation, with a deviation from the dose, there may be disturbances in the process.

On the 9th day, in the morning, the heifers are injected with drugs: "Folligon" at a dose of 0.4 ml, "Dinolytic" - 5 ml, and the capsule "CIDR" is removed from the vagina, and in the evening the drugs: "Folligon" and "Dinolytic" in the same doses, also to maintain superovulation, with a deviation from the dose, there may be disruptions in the process for the formation of homogeneous groups of animals, which do not need to be monitored daily for their state of sexual activity and inseminated in a short time, as well as better plan and control production processes. The dose of the drug "Dinolytic" 5 ml is standard and is recommended by the instructions of the drug.

Then on the 11th day, in the morning after the introduction of the drug "Fertagil", the first insemination is carried out in the amount of 2 doses, and after 8-9 hours the second insemination is carried out with one dose. The drug is administered at a dose of 5 ml, which is standard and recommended by the drug's instructions, in addition, unlike the instructions, it is administered simultaneously with the first insemination

Then, on the 18th day, after 7 days, after re-insemination, in the morning at 10.30 am, embryos are washed out with PBS liquid enriched with 2% fetal bovine serum, for each washout 0.5 liters at three times repetition and the drug "CIDR" is injected into the vagina.

After 10 days, the process of induction of superovulation is repeated according to the proposed scheme.

The selection of a bull - a producer for insemination of donors is carried out individually for each animal. Criteria for the selection of bulls: exclusion of inbreeding, there should be the first breeding category in terms of milk yield and fat content of milk, high productivity of the mother and mother of the bull's father, ease of calving for bulls 7% or less to exclude complications during calving.

Insemination of donor heifers is carried out twice with 3 doses. The first insemination in the amount of 2 doses on the 8th day from the beginning of the hormonal regimen in the morning, the second insemination with one dose on the same day in the evening, with an interval of 8-9 hours. Three doses of semen were used per animal: bovine semen was used for insemination according to individual selection.

An example of a specific implementation of the proposed method for obtaining embryos from donor heifers of the Holstein breed

To prove the effect of the claimed method of obtaining embryos from donor heifers of the Holstein breed, two groups of donor heifers of the Holstein breed were formed: 1 control and 2 experimental with a high level of genetic potential (Table 2).

In mothers of donor heifers (groups 1 and 2), the difference in milk productivity was insignificant: in terms of milk yield 69 kg (0.7%), fat and protein content in milk, respectively, 0.07% and 0.03% (in absolute value) ...

The future productivity of embryos obtained from selected heifers and breeding bulls with sexed semen, which were inseminated by donors, was calculated (Table 3).

The embryos were washed in accordance with the recommendations of the International Embryo Technology Society (IETS).

On the 18th day, embryos were washed and evaluated in donor heifers by inserting a balloon catheter into the uterus and washing the embryos using PBS liquid enriched with 2% fetal calf serum for each washout of 0.5 liters in triplicate. In the process of washing out, a transrectal massage was performed, allowing the injected fluid to diffuse into the two horns of the uterus. Within 15 minutes, flasks with washed media from each donor heifer were placed in the laboratory, ambient temperature 25 ° C.

The embryos were examined under a microscope at a magnification of 100; 60 times, taking into account the stages of development of blastocytes: stages of morula - 5 days; compact stage - 6 days; young stage of blastocytes - 7 days; blastocyte stage - 7 days - 8 days and titrated blastocyte stage - 9 days.

The embryo assessment was influenced by the following indicators: number, development, color and compactness of cells; the size of the perivitelline space; the number of degenerate cells.

In the course of research, the quality of embryos was ranked according to 4 classes.

Grade I - an excellent grade was assigned if the ideal embryo for its washout period, spherical shape, symmetrical with cells of the same size, color and texture, or the embryo is slightly late development.

Class II - good development of the embryo, if they look 1-2 days younger, or have defects - screened cells in the perivitellian space, blastomeres of various development, some degenerated cells and individual vesicles are found.

III class - the assessment is unsatisfactory, if there are clearly visible anomalies and many defects, cells are evacuated, degenerate, of different sizes, as well as large and numerous vesicles.

IV class - marriage, dead or degenerated, severe abnormalities, growth retardation at an early stage with degenerated cells.

To prove the effectiveness of the method, 2 groups of analogs of 6 heifers were created: group 1 - with ovarian superovulation according to the prototype, group 2 - with ovarian superovulation according to the claimed method.

Table 4 presents data on the number and quality of washed embryos from donor heifers:

Thus, analyzing the data in the table, it can be argued that the claimed method of obtaining embryos is 3-4% more efficient than the prototype.

The results of the experiment are shown in more detail in Table 5. The resulting embryos in different groups differ in quantity and quality:

- The first group - the number from 3 to 28 embryos, on average - 9.17 per head, of which 45 embryos were transplanted, on average 7.50 embryos per head.

- The second group - from 1 to 16 embryos, on average - 8.33 per head, of which 36 (6 embryos per head) are transplanted embryos

- The third group - from 3 to 35 embryos (21.5 embryos per head) and there are 113 embryos (18.83 first class)

- The fourth group - from 3 to 19 embryos (10.33 on average), of which 6.67 are embryos of the first class

- The fifth group - from 13 to 42 embryos, on average 20.17 embryos from 12 transplanted embryos

Table 5 presents data on the number and quality of washed embryos from donor heifers:

Thus, analyzing the data in Table 5, it can be argued that the use of the proposed method for obtaining embryos allowed to obtain from 1 to 42 embryos from each donor cow. 35 embryos of the first class were obtained from one cow, and from the other 33, 26, 18, respectively, of the first class.

The solution to this problem is possible with the introduction of new biotechnological methods in cattle breeding, including the receipt and transplantation of embryos from highly productive animals. In connection with an increase in the milk productivity of cows, the lactation dominant suppresses the reproductive functions in animals, which makes it difficult to obtain calves from highly productive animals annually, to carry out effective selection in herds in dairy cattle herds and to achieve expanded reproduction.

Claims (1)

A method of obtaining embryos from donor heifers of the Holstein breed, including the selection of heifers donors of the Holstein breed at the age of 13-15 months, clinically healthy, having a good conformation, excellent pedigree, the mother's productivity for the highest lactation of at least 9000 kg with a fat content in milk of at least 3 , 9%, protein - 3.3%, induction of superovulation according to the scheme by repeated administration in the form of injections of hormonal preparations containing follicle-stimulating and luteinizing hormones, and hormonal preparations for synchronization of sexual desire and regulation of reproductive function, which are administered for 10 consecutive days in different dosages from 1 to 5 ml per 1 head, repeated insemination with an interval, washing out of embryos after insemination and assessment of the quality of embryos, characterized in that the induction of superovulation is carried out according to the scheme: on the 1st day in the morning, the drug "Estrofantin" is administered at a dose of 3 ml, in the morning on the 2nd day, drugs are administered: "CIDR" in a dose of one capsule, "Karelia" in a dose of 20 ml and "Sedimin" - 10 m l, on the 3rd and 11th day, also in the morning in a dose of 5 ml, the drug "Fertagil" is administered, then on the 6th, 7th and 8th day, in the morning and in the evening, the drug "Folligon" is injected with a daily dose of 1 ml decreasing accordingly, 0.7 ml and 0.5 ml, on the 9th day in the morning the heifers are injected with drugs: "Folligon" at a dose of 0.4 ml, "Dinolytic" 5 ml and the capsule of the drug "CIDR" is removed from the vagina of the donor heifer, and in the evening preparations: "Folligon" and "Dinolytic" in the same doses, then on the 11th day in the morning after the introduction of the drug "Fertagil" the first insemination is carried out in the amount of 2 doses and after 8-9 hours the second insemination is carried out with one dose, then on On the 18th day, 7 days after re-insemination, the embryos are washed out in the morning and the "SIDR" drug is administered to the donor heifer.