CN111202837B

CN111202837B - A preparation for controlling ovulation of equine animal and method for controlling ovulation

Info

Publication number: CN111202837B
Application number: CN201811399191.8A
Authority: CN
Inventors: 曾申明; 杨峰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2022-02-08
Anticipated expiration: 2038-11-22
Also published as: CN111202837A

Abstract

本发明提供一种用于控制马属动物排卵的制剂及控制排卵的方法，所述制剂包含促性腺激素释放激素类似物和动物体内可降解的缓释载体。通过将促性腺激素释放激素制备为缓释制剂，有效延长了其在体内的半衰期，使得制剂能够长时间、高效稳定地发挥促进排卵的作用。通过注射本发明的制剂，96％以上的母马和母驴在48小时以内排卵，并且其中96％以上动物的排卵时间控制在8小时内，实现了马属动物的高效排卵控制，显著提高了马属动物的配种妊娠率。本发明的排卵控制方法操作简便、成本低廉、控制效果显著，对动物本身及其产品不产生任何有害影响，可以在马属动物本交配种、人工授精、胚胎移植和定时输精中广泛应用，具有广阔的市场前景。The present invention provides a preparation for controlling ovulation in an equine animal and a method for controlling ovulation, the preparation comprising a gonadotropin-releasing hormone analog and a degradable slow-release carrier in the animal. By preparing the gonadotropin-releasing hormone into a sustained-release preparation, its half-life in the body is effectively prolonged, so that the preparation can play the role of promoting ovulation efficiently and stably for a long time. By injecting the preparation of the present invention, more than 96% of the mares and female donkeys ovulate within 48 hours, and the ovulation time of more than 96% of the animals is controlled within 8 hours, thus realizing the high-efficiency control of ovulation in horses, and significantly improving the Breeding Pregnancy Rates in Equines. The ovulation control method of the invention has the advantages of simple operation, low cost, remarkable control effect, no harmful effects on the animal itself and its products, and can be widely used in the mating species of horses, artificial insemination, embryo transfer and timed insemination, Has broad market prospects.

Description

A preparation for controlling ovulation of equine animal and method for controlling ovulation

Technical Field

The invention relates to the field of animal reproduction, in particular to a preparation for controlling ovulation of an equine animal and a method for controlling ovulation.

Background

With the development of national economy and the improvement of the living standard of people, the industries of raising, training, competition, entertainment and the like of horses develop rapidly, the market potential is huge, the output value of horses in recent years is in a straight-line rising trend, and the total output value of the Chinese horse industry reaches trillion scale by 2030. At present, the quantity of stocked Chinese horses is about 600 ten thousand, but the quality is uneven, special speed and excellent endurance performance are lacked, and horse riding and leisure riding varieties cannot meet the current market demand. Therefore, breeding of elite breeds is one of the problems to be solved at present. Meanwhile, in recent 10 years, along with the improvement of health consciousness of people, health care values of donkey meat, donkey milk, donkey bones, donkey placentas, donkey hide gelatin and the like are more and more valued, the market demand of donkey products is rapidly increased, and the donkey industry is expected to reach the scale of hundreds of billions in 2020. At present, the demand of live donkeys is increasingly expanded, only the market of donkey-hide gelatin needs 400 pieces of donkey hide, the total stock of Chinese donkeys is less than 500 ten thousand, the supply is not in demand, and the donkeys have to be imported abroad, so that the price of the international donkey hide is greatly increased. The occurrence of illegal vendors hunting and the private slaughter of agricultural donkeys in the major donkey-keeping countries in the middle east and africa severely affects local agricultural production and animal welfare. Donkey skin export has been banned in a number of countries, which has had a great impact on the donkey-hide industry in China. Therefore, Chinese has very urgent need for propagation technology of elite horses and donkeys. Because of late sexual maturity, single birth and longer gestation period (horse, 340 days; donkey, 365 days) in equine animals, fertility is lower compared to other livestock. In most areas of china, mares or donkeys basically have one birth in two years or two births in three years. This directly affects the development of the donkey-related industry. Therefore, how to improve the breeding efficiency of mares and donkey is a problem which needs to be solved urgently at present.

The biggest problems of equine reproduction are long oestrus duration (5-8 days for mares and 6-9 days for donkey), most of ovulation occurs on the 5 th-9 th day of oestrus, but the ovulation time is irregular and is influenced by various factors such as weather, colony effect, sunshine time, temperature, nutritional environment and the like. This makes it difficult to determine the optimal mating or insemination time for mares and mules, especially for frozen semen insemination, resulting in low estrus conception rates. In production, in order to improve the conception rate, only multiple times of insemination or repeated inspection of follicle development dynamics can be adopted to accurately predict ovulation time, but the labor and technical cost is high, and the conception rate has a direct relation with the subjective judgment of technicians, thus seriously restricting the economic benefit of intensive farms.

In recent years, the research on the ovulation control method of mares is rapidly progressed in developed countries of the mare industry, and the method mainly adopted is as follows: when mare follicles reach a diameter of 35mm or more, ovulation is induced by intramuscular injection of human chorionic gonadotropin (hCG), gonadotropin releasing hormone (GnRH) and analogues thereof, of which hCG is most commonly used at once. According to the study by Barbacini et al, marbles with follicle diameters above 35mm were treated with 2000IU of hCG, and 75% of the treated marbles ovulated within 25-48 hours, with an overall ovulatory rate of 90% at 48 hours (Barbacini S, Zavaglia G, Gulden P, Marchi V, Necchi D.retrospecific study on the impact of hCG in an equivalent injury promotion using a free section. fluid vector discovery.2010, 12:312 317.). However, hCG is a foreign glycoprotein, and repeated injections induce mares to produce anti-hCG antibodies, resulting in a gradual decrease in ovulation effect, and some mares do not even have an ovulation response after injection, so that the number of uses in one mating season is generally not more than twice. In 1975, GnRH was first reported to be successfully used for inducing ovulation in animals, and analogues of GnRH have been widely used in animals such as cattle, pigs, sheep and fish. However, GnRH and its analogues generally have half-lives in vivo of around a few minutes, and although some analogues have half-lives that can be extended to tens of minutes, their use in equine animals is still limited. Because the equine animal has a long estrus period and the ovulation time is not fixed, a plurality of repeated injections are needed to obtain a good ovulation effect, but the labor cost and the animal stress response are increased in production, and the popularization and the application are difficult (Camillo F, Pacini M, Panzani D, Vannozzi I, Rota AL, Asia G.clinical use of wireless data injections of fastener acetate to inductor infection in the mark.2004, Vet Res Commun 28, 169-. Therefore, new methods of ovulation control need to be explored.

The physiological characteristics of donkey reproduction are greatly different from those of horses. Researches at home and abroad find that the donkey can ovulate in four seasons, and the ovulation rate is only high or low in different seasons; and the horse is basically in a poor situation from late autumn to early spring. In addition, the normal estrus of donkeys is 2-3 days longer than that of horses, and the ovulation time is more difficult to grasp, which is also the most important reason for the lower reproduction of the mother donkeys than that of horses. At present, few researches on ovulation control of the female donkey are conducted at home and abroad, and the ovulation control method for the female horse is difficult to be applied to the female donkey. Therefore, the ovulation control and timing hybridization technology of the donkey has important significance for rapidly improving the fertility of the donkey and promoting the development of the donkey industry.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a preparation for controlling the ovulation of an equine animal and a method for controlling the ovulation.

The invention provides a formulation for controlling ovulation in an equine animal comprising a gonadotropin releasing hormone (GnRH) analogue and a degradable sustained release carrier in the animal.

In the invention, the degradable sustained-release carrier in the animal body comprises one or more of poly (DL-lactide-co-glycolide), polysucrose, polylactide, polyglycolide, polyethylene glycol, polyacetic acid, polyglycolic acid, sucrose acetate isobutyrate, polyanhydride, polylysine and alginate;

preferably, the degradable slow-release carrier in the animal body is selected from one of poly (DL-lactide-co-glycolide), polysucrose, polylactide, polyglycolide, polyethylene glycol, polyglycolic acid, sucrose acetate isobutyrate, polyanhydride, polylysine and alginate.

In the invention, the gonadotropin releasing hormone analogue comprises one or more of triptorelin, buserelin, gonadorelin, dessertraline, leuprorelin, goserelin and natural gonadotropin releasing hormone;

preferably, the gonadotropin releasing hormone is selected from one of triptorelin, buserelin, gonadorelin, dessertrin, natural gonadotropin releasing hormone.

In the present invention, when different gonadotropin releasing hormone analogues are selected for effective ovulation control in equine animals, the formulations differ in their content of gonadotropin releasing hormone analogues: when the gonadotropin releasing hormone is selected from one of triptorelin, buserelin, gonadorelin, dessertrin and natural gonadotropin releasing hormone, the mass percentage of the gonadotropin releasing hormone analogue is as follows: 0.005-1.0% of triptorelin, 0.0002-0.2% of buserelin, 0.001-0.08% of gonadorelin, 0.01-0.2% of deserelin, 0.001-0.5% of leuprorelin, 0.0001-0.05% of goserelin and 0.005-0.01% of natural gonadotropin releasing hormone.

In the invention, the mass percentage content ratio of the gonadotropin releasing hormone analogue to the degradable sustained release carrier in the animal body is 1: 300-1: 14000;

preferably, in order to realize better sustained-release effect and simultaneously enable the gonadotropin releasing hormone analogue to effectively exert the effect of controlling ovulation, the mass percentage content ratio of the gonadotropin releasing hormone analogue to the degradable sustained-release carrier in the animal body is 1: 1400-1: 14000.

in the invention, in order to realize a better slow release effect and simultaneously enable the gonadotropin releasing hormone analogue to effectively play a role in controlling ovulation, the degradable slow release carrier in the animal body has a mass percentage content of 60-85%; the mass percentage content of the gonadotropin releasing hormone analogue is 0.005-0.2%;

as a preferred embodiment of the invention, when the degradable slow-release carrier in the animal body is polysucrose and the gonadotropin-releasing hormone analogue is Deserelin, the mass percentage content ratio of the Deserelin to the polysucrose is 1: 3500-1: 7500;

wherein the mass percent of the polysucrose is 70-75%, and the mass percent of the Deserelin is 0.01-0.2%.

In the invention, the preparation takes one or a mixture of more of water, dimethyl sulfoxide, ethanol, ethyl lactate, ethyl acetate and propylene carbonate as a solvent.

The formulation further comprises auxiliary additives; the auxiliary additive comprises one or two of sucrose and trehalose.

Preferably, the auxiliary additive is trehalose; the mass percentage of the trehalose is 0.01-0.5%.

As a preferred embodiment of the present invention, the formulation for controlling ovulation in an equine animal comprises:

further, the invention also provides a preparation method of the preparation, which comprises the following steps:

(1) fully dissolving gonadotropin releasing hormone analogue and auxiliary additive in a solvent to prepare a solution A;

(2) fully dissolving the degradable sustained-release carrier in the animal body into a solvent to prepare a solution B;

(3) mix solution A and B well.

In the invention, the preparation is filled into a glass bottle after being prepared, sealed and sterilized by irradiation; or filtering the two solutions with hydrophobic filter membrane with pore diameter of 0.22 μm for sterilization before mixing, aseptically mixing, filling into glass bottle, and sealing.

Furthermore, the invention also provides a method for controlling the ovulation of the equus animals, which is to inject the preparation into the equus animals in estrus.

Specifically, the method for controlling the ovulation of the equine animal comprises any one of the following steps:

(1) natural mating is carried out after the follicle is mature, and the preparation is injected after the mating is finished or is injected before the mating;

(2) injecting the preparation after the follicle is mature, and preserving semen with fresh semen or low temperature for insemination after 8-12 hours;

(3) injecting the preparation after the follicle is mature, and after 30-34 hours, using frozen semen for insemination;

the judgment standard of follicle maturity is as follows: when the animal is a horse, the diameter of the follicle is more than or equal to 30-35 mm; when the animal is donkey, the diameter of the follicle is more than or equal to 25-30 mm.

Preferably, the criteria for judging follicular maturation are: when the animal is a horse, the follicle is mature, and the diameter of the follicle is more than or equal to 35 mm; when the animal is donkey, the follicle is mature, and the diameter of the follicle is more than or equal to 30 mm.

In the present invention, if ovulation is not detected within 48 hours and the follicle develops normally in (1) and (2) above, the preparation may be optionally re-injected once, and then re-bred or inseminated until ovulation is detected; if ovulation does not occur within 12 hours and the follicle develops normally as in (3) above, another injection of the preparation can be selected, followed by another insemination until ovulation is detected.

In the invention, the injection dosage of the preparation is 0.1-10.0 mL, preferably 0.5-2.5 mL.

The invention has the beneficial effects that:

(1) the invention realizes the slow and controllable release of the gonadotropin releasing hormone by preparing the gonadotropin releasing hormone into a slow release preparation, thereby effectively prolonging the half-life period of the gonadotropin releasing hormone in vivo; by compounding a specific slow-release carrier and gonadotropin-releasing hormone and optimizing the content ratio of each component, the ovulation lotion can efficiently and stably play a role in promoting ovulation for a long time, so that the efficient ovulation control of the equus animals is realized (more than 96% of mares and mullets ovulate within 48 hours, and more than 96% of the mares ovulate within 8 hours), the pregnancy rate of natural hybridization and artificial hybridization of the equus animals is further improved, and the reproduction efficiency of the equus animals is effectively improved.

(2) The preparation for controlling the ovulation of the equus animals, provided by the invention, has the advantages of simple preparation process, low raw material price and low production cost; does not have any harmful effect on the animals and the products thereof.

(3) By adopting the ovulation control method provided by the invention, the optimal mating time is determined by accurately controlling the ovulation time, and the conception rate of mating or artificial insemination can be obviously improved: the mare is bred at one time, and the conception rate reaches 81%; after artificial insemination is carried out for one time by using fresh sperms, the conception rate reaches more than 70 percent; the estrus conception rate of semen deposition by using the frozen semen can reach more than 60 percent; the female donkey can give off sperms once, and the conception rate can reach more than 50%; the estrus conception rate after the semen is transfused by the frozen semen can reach more than 40 percent.

(4) The ovulation control method provided by the invention obviously reduces the mating times, improves the use efficiency of the semen of the improved male livestock, reduces the mating cost of the pregnant female livestock and greatly improves the economic benefit of a pasture.

Drawings

FIG. 1 is a graph showing the distribution of ovulation time of the donkey in example 6.

FIG. 2 shows the change of reproductive hormones of the female donkey after injection of the ovulatory fluid in example 9. Wherein A is estrogen (E)₂) B is the profile of insulin-like growth factor-1 (IGF-1) and C is the profile of progestogen (P); g1, G2 and G3 are respectively 3 donkey heads injected with oviposition liquid, and CN1, CN2 and CN3 are respectively 3 donkey heads naturally ovulated.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention, which is still within the purview of this patent.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of an egg-discharging solution for controlling ovulation in animals

1. Ovulation fluid containing different slow release carriers and application effect thereof

(1) Preparation of ovulatory solution containing different slow-release carriers

The following a to J provide the ovulation solutions of the present example containing different sustained release carriers, respectively:

A. weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of poly (DL-lactide-co-glycolide), adding ethanol, and fully dissolving to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

B. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions, and supplementing to 1000g with sterile ultrapure water to obtain 0.01% Deserelin egg discharging solution.

C. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polyethylene glycol, adding ethanol, and fully dissolving to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

D. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polylactide, adding ethanol, and fully dissolving to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

E. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polyglycolide, adding ethanol, and fully dissolving to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

F. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polyglycolic acid, adding ethanol, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

G. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of sucrose acetate isobutyrate, adding ethanol, and fully dissolving to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution. .

H. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polyanhydride, and adding ethanol to fully dissolve to obtain a slow-release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% Deserelin egg discharging solution.

I. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding sterile ultrapure water to fully dissolve the dessertraline and the trehalose to obtain a gonadotropin releasing hormone solution; weighing 700g of polylysine, and adding water to fully dissolve to obtain a slow release carrier solution; mixing the two solutions, and supplementing to 1000g with sterile ultrapure water to obtain 0.01% Deserelin egg discharging solution.

J. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding sterile ultrapure water to fully dissolve the dessertraline and the trehalose to obtain a gonadotropin releasing hormone solution; weighing 700g of alginate, and adding sterile ultrapure water to fully dissolve to obtain a slow-release carrier solution; mixing the two solutions, and supplementing to 1000g with sterile ultrapure water to obtain 0.01% Deserelin egg discharging solution.

(2) Analysis of ovulation control effect of different ovulation injections

In inner Mongolia region, hybrid donkey 3-8 years old Dezhou is selected to count 300 heads, the fat condition is more than moderate, the health and the fertility are normal, the hybrid donkey is randomly divided into 10 groups, and each group has 30 heads. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) blocking the rectal wall by using a B ultrasonic probe at 8: 00-10: 00 in the morning of 2-8 months every day, and checking the conditions of follicular development and endometrial morphological change. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is crossed vertically, a maximum value is measured in the transverse direction, and the two values are added to obtain an average value, namely the diameter of the follicle. When the diameter of the follicle reaches more than 30mm, 1.0mL of the ovulation injection prepared from the A to J is respectively injected into the hip muscle of each group of donkey at 10:00 a.m. the day. The results of B-ultrasonic examination of ovulation 24 hours and 48 hours after injection are shown in Table 1, and the data in Table 1 are the average values of the ovulation detection results after the ovulation liquid is injected in different oestrus cycles of the same group, and the results show that the ovulation control effect is the best by using the ficoll as the slow release carrier, and 96.0% of the donkey can ovulate within 48 hours. (data in Table 1 are A-J groups each group of ova-excreting fluids injected several times in different estrus cycles of the same group, average value was taken)

TABLE 1 Effect of different sustained Release vehicles on ovulation control Effect of donkey

Note: the numerical superscripts a, b in the same column indicate significant differences (P < 0.05).

2. Ovulation lotion containing different GnRH analogues and application effect thereof

(1) Preparation of oviposition solutions containing different GnRH analogues

The following a to E provide the ovulatory solutions containing different GnRH analogues prepared in this example, respectively:

A. weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution. .

B. Weighing 2.0g of buserelin and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.2% buserelin egg discharging solution. .

C. Weighing 0.4g of gonadorelin and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.04% gonadorelin ovum-discharging solution.

D. Weighing 0.2g of triptorelin and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.02% triptorelin eggbeaction solution.

E. Weighing 0.1g of natural GnRH and 2.0g of trehalose, and adding ethanol for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of polysucrose, and adding ethanol to fully dissolve to obtain a slow release carrier solution; mixing the two solutions, and adding ethanol to 1000g to obtain 0.01% natural GnRH egg discharging solution.

(2) Analysis of ovulation control effect of different ovulation injections

The ovulation detection method is as described in (2) in the above 1. The results are shown in table 2, and show that the combination of the dessertraline and the polysucrose has the best effect on ovulation control of the donkey.

TABLE 2 Effect of different GnRH analogues on ovulation control Effect of donkey

3. Ovulation lotion prepared from slow release carrier and gonadotropin releasing hormone in different proportions and application effect thereof

(1) Preparation of oviposition solution containing different proportions of slow release carrier and gonadotropin releasing hormone

The following A to I provide the ovulation solution containing the sustained-release carrier and gonadotropin releasing hormone of the embodiment respectively in different proportions:

A. weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 850g of polysucrose, and adding ultrapure sterile water for full dissolution to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

B. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 800g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

C. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 750g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

D. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

E. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 650g of polysucrose, and adding ultrapure sterile water for full dissolution to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

F. Weighing 0.1g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 600g of polysucrose, and adding ultrapure sterile water to fully dissolve to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.01% of Deserelin egg discharging solution.

G. Weighing 0.05g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.005% Deserelin egg discharging solution.

H. Weighing 0.2g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.02% of Deserelin egg discharging solution.

I. Weighing 0.5g of dessertraline and 2.0g of trehalose, and adding ultrapure sterile water for full dissolution to obtain a gonadotropin releasing hormone solution; weighing 700g of ficoll, adding ultrapure sterile water, and fully dissolving to obtain a slow-release carrier solution; mixing the two solutions uniformly, and supplementing to 1000g with ultrapure sterile water to obtain 0.05% of the desertraline eggbeater solution.

(2) Analysis of ovulation control effect of different ovulation injections

The ovulation detection method is as described in (2) in the above 1. The results are shown in Table 3, and show that the concentration of the ficoll as a sustained release carrier has a certain influence on the ovulation control effect, wherein the ovulation control effect is best when the goserelin is in the ficoll solution containing 75% (C) and 70% (D)

TABLE 3 content ratio of different sustained-release carriers and gonadotropin-releasing hormone for controlling ovulation of donkey

Influence of (2)

Example 2 ovulation control and Natural hybridization effects of Alagilin (sweat blood) horses

In the vicinity of the inner Mongolia call and Haote city, 32 Alhagiki mares of 3-10 years old are selected, the fat condition is more than moderate, the health is realized, and the fecundity is normal. Raising in a colony house with an outdoor playground, freely drinking water, and feeding green hay (Chinese wildrye and alfalfa) twice. In 4-6 months, a male horse is used for trying at 8: 00-10: 00 in the morning every day, if the male horse is close to the female horse, the male horse stands still, the tail lifts, the female horse opens frequently, and urine is discharged, which indicates that the female horse can be bred. At the same time, the ultrasound probe was used to examine follicular and uterine edema in the rectal wall. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is crossed vertically, a maximum value is measured in the transverse direction, and the two values are added to obtain an average value, namely the diameter of the follicle. When the diameter of the follicle reaches more than 35mm and the endometrial edema is obvious, the male horse is selected for hybridization. After successful mating, 1.0mL of the ovulation injection (composition of the ovulation solution: 0.01% of Deserelin, 0.2% of trehalose, 70% of polysucrose, water as a solvent, and the content of each component in percentage by mass) prepared in group D of 3 of example 1 was injected intramuscularly to the buttocks. After 24 hours intervals, ovulation was checked. If ovulation occurs within 48 hours, pregnancy tests are carried out 14 days later using type B ultrasonic waves with a rectal probe. If ovulation does not occur within 48 hours and the follicle develops normally, another mating is required and another ovulation injection is made in the same manner as above until ovulation is detected. The results of ovulation control and conception are shown in table 4, and show that only 25% of test horses ovulate 24 hours after the injection of the ovulatory injection, and that 32 test horses ovulate at the time of 48 hours of test, with the ovulatory rate being 100%. After 14 days, 26 patients can be detected to have been pregnant, and the conception rate is 81.3%.

TABLE 4 ovulation control and conception rate results after mating for Alhagepin mares

Example 3 ovulation control and effects of breeding with frozen semen of Mongolian horses

In the Shenyang city of Liaoning, 35 mares of Mongolian horses of 3-8 years old were selected, were healthy and normal in fertility with moderate or more temperament. Raising in a colony house with an outdoor playground, freely drinking water, and feeding green hay (Chinese wildrye and alfalfa) twice. And (3) examining the development condition of the follicles of the horse group by using a B ultrasonic rectum at 8: 00-10: 00 in the morning of 6-8 months every day. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is vertically crossed, a maximum value is measured in the transverse direction, and the two values are added and then averaged to obtain the diameter of the follicle. When the diameter of the follicle reached more than 35mm and the edema of the endometrium was significant, 1.0mL of the ovulation injection solution (the composition of the ovulation solution is as follows: 0.01% of dessertraline, 0.2% of trehalose, 70% of ficoll, water as a solvent, and the content of each component is in mass percent) prepared in group D of example 1 was intramuscularly injected to the buttocks. After 30 hours, B-ultrasonography was performed and the ovulated mares were discarded. 4 frozen sperms (0.5mL plastic tubules, thawed in 37 ℃ water bath for 15 seconds) were taken out from the liquid nitrogen tank, and 2.0mL sperms (about 1.4 hundred million effective sperms) were delivered to the uterine horn on the large follicle side. After 8 hours intervals, the same dose of frozen semen was used once for insemination. After 14 days, the pregnancy status was checked by B-ultrasound. Ovulation control and conception results are shown in table 5, and show that only 8.6% of the test horses ovulate 30 hours before the injection of the ovulatory injection. 85.7% of the horses tested ovulate between 32 and 48 hours. After 14 days, 17 patients can be detected to have been pregnant, and the conception rate is 56.7%.

TABLE 5 ovulation control and conception rate after mating of frozen semen of Mongolian horses

EXAMPLE 4 Effect of different injection times on ovulation control Effect of donkey

In inner Mongolia and Shandong areas, total 213 heads of Dezhou donkey of 3-8 years old and local hybrid female donkey are selected, the fat condition is more than moderate, and the health and the fertility are normal. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) blocking the rectal wall by using a B ultrasonic probe at 8: 00-10: 00 in the morning of 2-8 months every day, and checking the conditions of follicular development and endometrial morphological change. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is crossed vertically, a maximum value is measured in the transverse direction, and the two values are added to obtain an average value, namely the diameter of the follicle. Donkey groups were divided into three groups according to the size of the diameter of the follicles at the time of injection of the ovarial fluid: 25-30mm, 31-35mm, 35-40 mm. Each group of control and experimental group was examined 10:00 the same day as the morning, and the control group was injected intramuscularly with 1.0mL of a blank injection containing no GnRH analogue at the hip, and the experimental group was injected with 1.0mL of an ovulation injection (composition of ovulation-inducing solution: Deserelin 0.01%, trehalose 0.2%, and sucrose 70%, water as a solvent, and the content of each composition in% by mass) prepared in group D of example 1 at 3. Ovulation was checked with B-

ultrasound

24 and 48 hours after injection. Results of ovulation control are shown in table 6. The results show that after the donkey with the diameter of 25-30mm follicle diameter injects the ovulation fluid, both the control group and the experimental group do not ovulate for 24 hours, the control group still does not ovulate for 48 hours, and the experimental group ovulate 72.2 percent of the donkey; after the female donkey with the follicle diameter of 31-35mm is injected with the ovulation-discharging liquid, the control group does not ovulate for 24 hours, the experimental group has 5.4 percent of the female donkey ovulate, the control group still does not ovulate for 48 hours, and the experimental group has 96.0 percent of the female donkey ovulate; after the injection of the ovulatory fluid into the donkey with the follicle diameter of 36-40mm, 11.1% of the donkey in the control group ovulate 24 hours, 25.9% of the donkey in the experimental group ovulate, 50.0% of the donkey in the control group ovulate 48 hours, and 100.0% of the donkey in the experimental group ovulate. The result shows that the ovulation liquid injection can obviously promote the ovulation of the donkey; when the diameter of the follicle of the donkey reaches more than 30mm, the ovulation promoting effect is good when the ovulation liquid is injected, and under the condition, ovulation is detected in more than 96% of the donkey after the ovulation liquid is injected for 48 hours.

TABLE 6 Effect of ovulation injection on ovulation control Effect of donkey at different follicular development stages

Note: the values in the same column, superscript a, B, indicate significant difference (P < 0.05), superscript A, B indicate very significant difference (P < 0.01).

EXAMPLE 5 Effect of injection dose of ovulation injection solution on ovulation control Effect of donkey

In inner Mongolia and Shandong areas, total 142 donkey of Dezhou donkey of 3-8 years old and local hybrid female donkey are selected, the fat condition is more than moderate, the health is realized, and the fertility is normal. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) blocking the rectal wall by using a B ultrasonic probe at 8: 00-10: 00 in the morning of 2-8 months every day, and checking the conditions of follicular development and endometrial morphological change. On a B-ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is crossed vertically, a maximum value is measured in the transverse direction, and the two values are added to obtain the average value, namely the diameter of the follicle. Selecting donkey with follicle diameter above 30mm, and dividing into three groups. Each group of buttocks muscles was injected with oviposition solutions prepared in group D of example 1 of the present invention (composition of oviposition solution: 0.01% of Deserelin, 0.2% of trehalose, 70% of polysucrose, water as solvent, content of each component in mass%) at three different doses of 2.0mL, 1.6mL and 0.8 mL. Ovulation was examined by B-ultrasonography at 24 hours and 48 hours after injection, and the ovulation effects are shown in Table 7. The result shows that the ovulation promoting effect is better when the injection dosage of the ovulation lotion is 1.6mL, and ovulation can be detected by all the female donkeys 48 hours after injection.

TABLE 7 Effect of different injection doses on ovulation Effect of donkey

Example 6 control of ovulation time of donkey

In inner Mongolia and Shandong areas, total 268 heads of Dezhou donkeys of 3-8 years old and local hybrid mother donkeys are selected, the fat condition is more than moderate, the health is realized, and the fertility is normal. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) in 3-8 months, blocking the rectal wall by using a B ultrasonic probe at 8: 00-10: 00 in the morning every day, and checking the conditions of follicular development and endometrial morphological change. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is crossed vertically, a maximum value is measured in the transverse direction, and the two values are added to obtain an average value, namely the diameter of the follicle. The ovulatory control effect of the ovulatory control method of the present invention is shown in Table 8 by selecting a donkey having a follicle diameter of 30mm or more and injecting 0.8mL of the ovulatory fluid prepared in group D of example 1 (the ovulatory fluid comprises 0.01% of Deserelin, 0.2% of trehalose, and 70% of ficoll, and the content of each component is in mass percentage, using water as a solvent) into the hip muscle, and examining the ovulatory control effect at 24 hours, 32 hours, and 48 hours after the injection by means of B-ultrasonography. The results showed that 94.8% of the donkey mothers could detect ovulation 48 hours after the injection of the ovation fluid.

TABLE 8 distribution of ovulation time of donkey after injection of ovulation fluid of the present invention

Furthermore, in order to grasp the distribution of ovulation between 32 hours and 48 hours after the ovulation fluid injection, 30 donkey females in Texas of 3-8 years old were selected, and the feeding management was performed as described in example 4. When the diameter of the donkey follicle reaches more than 30mm, 0.8mL of the oviposition discharging liquid is injected. Ovulation was checked with ultrasound B at 32 hours post-injection (18:00) and every two hours until 48 hours post-injection. The distribution of ovulation is shown in FIG. 1, where 96.67% of the ovulation rate is from 34 to 42 hours and the peak of ovulation occurs 38 hours after injection (24: 00).

Example 7 conception effect of female donkey after timed artificial insemination using refrigerated semen

In inner Mongolia and Shandong areas, 189 heads of Dezhou donkeys of 3-8 years old and local hybrid mother donkeys are selected, the fat condition is more than moderate, and the health and the fertility are normal. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) examining the development condition of the follicles of the horse group by using a B ultrasonic rectum at 8: 00-10: 00 in the morning every day in 3-8 months. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is vertically crossed, a maximum value is measured in the transverse direction, and the two values are added and then averaged to obtain the diameter of the follicle. When the diameter of the follicle reached 30mm or more and endometrial edema was significant, 1.0mL of the ovulation solution (composition of the ovulation solution: 0.01% of Deserelin, 0.2% of trehalose, 70% of ficoll, water as a solvent, and content of each component in mass%) prepared in group D in example 1 was intramuscularly injected at the buttocks. After 8-10 hours, the uterus is transfused with 10mL of refrigerated (4 ℃) semen (containing 6 hundred million effective sperm). After 48 hours intervals, ovulation was checked. If ovulation occurs within 48 hours, 14 days later, pregnancy tests are performed using type B ultrasound with a rectal probe. If not ovulatory within 48 hours and the follicles were developing normally, the ovulatory injection prepared in example 1 was re-injected and the sperms were inseminated once in the same procedure as above. Ovulation and conception results are shown in table 9. The results show that 95.8% of the female donkeys ovulate 48 hours after the injection of the egg discharging liquid, and 51.3% of the female donkeys are pregnant 14 days later.

TABLE 9 conception rate of frozen semen for female donkey after artificial insemination at regular time

Example 8 conception effect of the female donkey after the timed Artificial insemination Using frozen semen

In inner Mongolia areas, 96 donkey of Dezhou donkey of 3-8 years old and local hybrid female donkey are selected, the fat condition is more than moderate, the health is realized, and the fertility is normal. Feeding in a colony house with an outdoor playground, freely drinking water, and feeding with the special compound feed twice a day. And (3) examining the development condition of the follicles of the horse group by using a B ultrasonic rectum at 8: 00-10: 00 in the morning of 6-8 months every day. On a B ultrasonic instrument, the diameter of the follicle is measured by a cross method, namely, a maximum value is measured in the longitudinal direction, then the follicle is vertically crossed, a maximum value is measured in the transverse direction, and the two values are added and then averaged to obtain the diameter of the follicle. When the diameter of the follicle reaches more than 30mm and the endometrial edema is obvious, 1.0mL of the ovulation solution prepared in group D in example 1 (the composition of the ovulation solution is as follows: 0.01% of dessertraline, 0.2% of trehalose, 70% of ficoll, water as a solvent, and the content of each component in percentage by mass) is injected into the buttock muscle and divided into three groups: group A adopts 10 frozen sperms for one-time semen deposition; group B adopts twice semen deposition, 5 frozen semen each time; group C adopted twice semen deposition, 3 frozen semen each time. After 30-34 hours, 1.5-5 mL of frozen semen (containing 1.0-5.0 hundred million effective sperms) is used for the uterine horn of the large follicle side to perform insemination. B. After 8 hours of interval of the two groups C, the same dose of frozen semen is used for insemination, and pregnancy examination is carried out by B ultrasonic after 14 days. If ovulation does not occur within 24 hours and the follicle develops normally, the same procedure is followed to inject the ovate drainage solution and insemination once again. The results of the conception are shown in Table 10. The results show that the conception rates of A, B, C groups adopting one-time insemination and two-time insemination with different dosages are respectively 42.42%, 35.48% and 34.38%, and the effect of one-time insemination is slightly good.

TABLE 10 conception rate of frozen semen for female donkey after timed artificial insemination

Example 9 comparison of hormone level changes in ovulation controlled donkey with naturally ovulating donkey

6 donkey heads with follicle diameter of 30-35mm were selected, wherein 3 of the 6 donkey heads were injected with the ovulation fluid (the composition of the ovulation fluid is: 0.01% of dessertraline, 0.2% of trehalose, 70% of ficoll, water as solvent, the content of each component is mass%) prepared in group D of 3 of example 1 in 1.0mL (the 3 donkey heads are respectively designated as G1, G2 and G3), and the other 3 donkey heads were injected with physiological saline (the 3 donkey heads are respectively designated as CN1, CN2 and CN 3). The injection time was recorded as 0 hour, and blood was collected every 8 hours until ovulation of the donkey was detected by B-ultrasonography. Blood samples were collected, centrifuged to obtain serum, stored at-20 ℃ and subjected to hormone testing. The results are shown in FIG. 2, and the results show that E₂And IGF-1 is decreased before ovulation and the level of progestogen is gradually increased before ovulation, and the ovulation fluid is injectedAccelerates this process, promotes follicular maturation and ovulation, and the anovulatory fluid is very stable throughout ovulation for more than 40 hours.

In conclusion, the sustained-release preparation prepared from the gonadotropin releasing hormone realizes the slow and controllable release of the gonadotropin releasing hormone, and effectively prolongs the half-life period of the gonadotropin releasing hormone in vivo; by compounding a specific slow-release carrier and gonadotropin-releasing hormone and optimizing the content ratio of each component, the ovulation lotion can efficiently and stably play a role in promoting ovulation for a long time, so that the efficient ovulation control of the equine animals is realized (more than 96% of the equine animals can ovulate in 48 hours), the pregnancy rate of natural mating and artificial mating of the equine animals is further improved, and the reproduction efficiency of the equine animals is effectively improved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. a method for controlling female donkey ovulation for non-therapeutic purposes, is characterized in that,

The method comprises the following steps (1)-(3):

(1) After the follicles are mature, natural breeding is performed first, and the preparations for ovulation control are injected after the breeding is completed, or the preparations for ovulation control are injected before breeding;

(2) After the follicles mature, inject the preparations used to control ovulation, and 8-12 hours later, use fresh semen or cryopreserved semen for artificial insemination;

(3) After the follicles mature, inject preparations for ovulation control, and 30 to 34 hours later, use frozen semen for artificial insemination;

The criteria for determining the maturity of the follicles are: the diameter of the female donkey follicles is greater than or equal to 30 mm;

The preparation for controlling ovulation consists of the following components by mass percentage:

Deserelin 0.01%;

Ficoll 70%;

Trehalose 0.2%;

water balance;

The injection dosage of the preparation for controlling ovulation is 0.8-2.0 mL.