Use of GnRH antagonists in the regulation of estrus in mammals

Abstract

In the field of mammalian reproduction, the use of GnRH antagonists in regulating mammalian estrus is disclosed. GnRH antagonists can suppress estrus for longer periods with less frequent use and can be used for estrus synchronization and batch production in livestock, or for estrus regulation in pets.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of Chinese Patent Application No. 202211376492.5 filed on November 4, 2022, the content of which is incorporated herein by reference.

[0002] The present invention relates to the field of mammalian reproduction, and in particular to the use of GnRH antagonists in the regulation of estrus in mammals, pharmaceutical compositions, the use of GnRH antagonists and gonadotropins in the regulation of estrus synchronization in mammals, methods for suppressing estrus in mammals, and methods for controlling estrus synchronization in mammals.

Background Art

[0003] GnRH antagonists are GnRH analogs. GnRH is a decapeptide secreted from the hypothalamus, which can bind to GnRH receptors, activate downstream signaling pathways, and promote the secretion of mammalian gonadotropins. It is an important regulatory factor for the hypothalamus - pituitary - gonad axis. GnRH antagonists are GnRH analogs with modified and substituted amino acids. They can competitively bind to GnRH receptors and block downstream signaling pathways, thereby suppressing the secretion of gonadotropins in animals. Based on the physiological function of GnRH, currently, GnRH antagonists are mainly used clinically for the treatment of male prostate cancer, female infertility, endometriosis, etc., including Abarelix, Cetrorelix, and Degarelix.

[0004] As living standards improve, consumption of livestock products continues to rise, and the demand for high-quality, safe livestock products continues to increase. Currently, in the livestock breeding process, methods such as estrus synchronization are used to improve reproductive efficiency and increase reproductive profits and product supply. In cattle and sheep breeding, vaginal suppositories containing progestins are mainly used to synchronize the estrus of female animals, while in pig breeding, altenologists are administered for 18 consecutive days to synchronize the estrus of prospective sows. Livestock treated with vaginal suppositories or altenologists can achieve estrus synchronization, and batch production can then be carried out to improve reproductive efficiency and economic profits. Both vaginal suppositories and altenologists are essentially synthetic progestins and pose a risk of environmental pollution and health impacts. Cattle and sheep use vaginal suppositories to regulate estrus, but prolonged use of vaginal suppositories can lead to suppository dislodgement and premature estrus, as well as vaginal adhesions and infections that can affect the animal's health. Altrenostists need to be paid for 18 consecutive days, which is a cumbersome procedure, increases labor costs, and multiple payouts increase the risk of disease transmission.

[0005] There are many methods to suppress estrus in pets, which can be divided into surgical and non-surgical methods. Currently, surgical methods are the most widely used among those in practical use. However, sterilization surgery has several problems. Firstly, sterilization is irreversible for pets, and they cannot regain their reproductive capacity after the surgery. Secondly, sterilization surgery also poses certain risks to the pet's health and can potentially threaten their life. Postoperative complications such as bleeding and infection also affect the pet's health. Most importantly, sterilization surgery increases the incidence of diseases such as cruciate ligament rupture, hemangioendothelioma, lymphoma, bladder cancer, and excessive obesity in pets. Furthermore, pets may suffer from urinary incontinence after sterilization, which can seriously affect their health and quality of life. In addition, a small number of pets use estrus suppressants. For example, Suprelorin, a product of Virbac in France, can be used to suppress estrus in male dogs, but in male dogs, it can cause side effects such as aggression, increased libido, and urine marking behavior in the initial stages of use. On the other hand, when used in female dogs, the side effects are too severe, inducing estrus in female animals and causing uterine disease, ovarian cysts, pseudopregnancy, urinary incontinence, cystitis, weight gain, and changes in coat and behavior, making clinical use in female dogs impossible. [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to overcome the problems of the prior art and to provide the use of a GnRH antagonist in the regulation of estrus in mammals. The GnRH antagonist can suppress estrus for a longer period with less frequent use and can be used for estrus synchronization and batch production in livestock, or for estrus regulation in pets. [Means for solving the problem]

[0007] To achieve the above objective, a first aspect of the present invention provides the use of a GnRH antagonist in the regulation of estrus in mammals.

[0008] Preferably, the use is to suppress estrus or to regulate estrus synchronization.

[0009] Preferably, the GnRH antagonist is at least one selected from Cetrorelix, Abarelix, and Degarelix, preferably Degarelix.

[0010] A second aspect of the present invention provides a pharmaceutical composition comprising a GnRH antagonist and a gonadotropin.

[0011] Preferably, the gonadotropin is at least one selected from follicle-stimulating hormone (FSH) and its analogues, pregnant mare serum gonadotropin (PMSG) and its analogues, and human chorionic gonadotropin (HCG) and its analogues.

[0012] A third aspect of the present invention provides the use of GnRH antagonists and gonadotropins in the regulation of estrus synchronization in mammals.

[0013] A fourth aspect of the present invention provides a method for suppressing estrus in mammals, comprising administering an effective amount of a GnRH antagonist to mammals that are not in estrus, thereby suppressing estrus in the mammals.

[0014] A fifth aspect of the present invention provides a method for regulating estrus synchronization in mammals, comprising administering a GnRH antagonist to a non-estrus mammal to suppress estrus, and then administering an effective amount of gonadotropin to achieve estrus synchronization. [Effects of the Invention]

[0015] The GnRH antagonist used in this invention to suppress estrus in mammals can be administered much less frequently than conventional estrus inhibitors, and a single injection can maintain the inhibitory effect for a longer period. In particular, Degarelix can achieve a superior inhibitory effect at the same dosage, can reverse the inhibitory effect, does not cause permanent estrus suppression, and is especially suitable for estrus suppression in pets.

[0016] The pharmaceutical composition described in the present invention is particularly suitable for the breeding and production of livestock, and achieves the objective of estrus synchronization by suppressing estrus in female animals for a long period of time using a GnRH antagonist, and then inducing estrus in the animals using a gonadotropin. [Brief explanation of the drawing]

[0017] [Figure 1] The figure shows the estrus synchronization of female mice injected with Degarelix 1.0 mg / kg in Example 2 of the present invention. [Figure 2] The diagram shows the estrus synchronization of reserve sows treated with Altenologist (a) and Degarelix (b) in Example 3 of the present invention. [Figure 3] The curve diagrams show the changes in testosterone concentration (a) and scrotal circumference (b) in male dogs treated with Degarelix in Example 4 of the present invention, where ▲ represents the control group and ■ represents the Degarelix-treated group. [Modes for carrying out the invention]

[0018] The endpoints and values ​​of the ranges disclosed herein are not limited to these precise ranges or values, and these ranges or values ​​should be understood to include values ​​that approximate these ranges or values. In the case of numerical ranges, one or more new numerical ranges can be obtained by combining the endpoint values ​​of each range, the endpoint values ​​of each range with individual point values, and the individual point values ​​with each other, and these numerical values ​​should be considered as specifically disclosed herein.

[0019] Biological products of hormones often use biological experimental methods to compare with standard products in order to test their efficacy, because their chemical components may not be constant or their quality specifications cannot be tested by physical and chemical methods. Through biological identification, the minimum potency unit with specific biological efficacy is called unit (U), and the standard unit determined through international agreement is called international unit (IU). The conversion between units and weights of different drugs is not the same. Therefore, in the present invention, the units used are based on the measurement units provided by the purchased products. Among them, the measurement units of GnRH antagonists use "mg / kg" or "μg / mg", and the measurement units of FSH, PMSG, and HCG all use "unit".

[0020] The first aspect of the present invention provides the use of GnRH antagonists in regulating estrus of mammals.

[0021] Preferably, the use is to suppress estrus or regulate estrus synchronization.

[0022] The type of the mammal is not particularly limited. Generally, it is a cat, dog, mouse, rabbit, horse, cow, sheep, or pig. Among them, in the case of livestock such as horses, cows, sheep, or pigs, GnRH antagonists can be used to regulate estrus synchronization as needed to achieve batch production, and the administration target is usually female animals. When suppressing estrus, the gender of the mammal is not restricted, and both males and females can be used, but the mechanism of action may be different.

[0023] Preferably, the GnRH antagonist is at least one selected from Cetrorelix, Abarelix, and Degarelix, and more preferably Degarelix.

[0024] Those skilled in the art can adjust the dosage of the GnRH antagonist according to the specific type of mammal and the purpose of administration. Generally, the dosage of the GnRH antagonist is 1 to 5000 μg per kg of animal body weight. For example, in the case of rodents such as mice and rabbits, the dosage of the GnRH antagonist is 1000 to 2000 μg per kg of body weight; in the case of medium and large livestock such as pigs, cows, sheep, and horses, the dosage of the GnRH antagonist is 1 to 100 μg per kg of body weight; in the case of pets such as dogs and cats, the dosage of the GnRH antagonist is 1000 to 5000 μg per kg of body weight.

[0025] The present invention also relates to the use of a GnRH antagonist in the manufacture of a product for regulating the estrus of mammals. The product may be provided in the form of a liquid, solid, capsule, injection, etc., and serves as an estrus inhibitor.

[0026] The GnRH antagonist may be used in combination with other substances to regulate the estrus of mammals. This includes, but is not limited to, using the GnRH antagonist in combination with gonadotropins, steroid hormones, prostaglandins, and their analogs.

[0027] The second aspect of the present invention provides a pharmaceutical composition comprising a GnRH antagonist and a gonadotropin.

[0028] For the description of the GnRH antagonist, refer to the first aspect.

[0029] Preferably, the gonadotropin is at least one selected from follicle-stimulating hormone (FSH) and its analogs, pregnant mare serum gonadotropin (PMSG) and its analogs, and human chorionic gonadotropin (HCG) and its analogs.

[0030] Those skilled in the art can adjust the dosage of gonadotropins according to the specific type of mammal, but generally, the dosage of gonadotropins is 5,000 to 6,000 units. Specifically, for example, in the case of mice, the dosage of follicle-stimulating hormone (FSH) and its analogues is 5 to 10 units, the dosage of PMSG and its analogues is 5 to 10 units, and the dosage of hCG and its analogues is 5 to 10 units. In the case of sows, the dosage of follicle-stimulating hormone (FSH) and its analogues is 1,000 to 2,000 units, the dosage of PMSG and its analogues is 500 to 1,500 units, and the dosage of hCG and its analogues is 500 to 1,000 units.

[0031] The GnRH antagonist and gonadotropin in the aforementioned pharmaceutical composition can be stored independently and used to achieve different stages of estrus synchronization, thereby achieving the effect of estrus synchronization.

[0032] A third aspect of the present invention provides the use of GnRH antagonists and gonadotropins in the regulation of estrus synchronization in mammals.

[0033] For a description of GnRH antagonists, refer to the first aspect; for a description of gonadotropins, refer to the second aspect; and for specific administration methods, refer to the fifth aspect.

[0034] A fourth aspect of the present invention provides a method for suppressing estrus in mammals, comprising administering an effective amount of a GnRH antagonist to mammals that are not in estrus, thereby suppressing estrus in the mammals.

[0035] In the present invention, the effective dose of a GnRH antagonist means an amount that can appropriately and effectively suppress estrus in this situation, or control the duration of estrus suppression within a certain range. It should be understood that the effective dose of a GnRH antagonist may vary considerably depending on the mammalian species and specific needs, and further details may be found in the first embodiment.

[0036] The aforementioned administration method is not particularly limited and may be a conventional administration method in the art, such as at least one selected from intravenous injection, intramuscular injection, subcutaneous injection, subcutaneous implantation, and vaginal suppository.

[0037] Those skilled in the art should understand that the frequency of administration can be selected according to the type, efficacy, dosage, and animal species of the GnRH antagonist. For example, a single dose can be administered at intervals to selectively adjust the duration of estrus suppression. Generally, estrus suppression in mammals is also applicable to pets such as cats and dogs. In the case of pets, estrus synchronization is not necessary, and estrus suppression is the primary use. Depending on the injection dose, the suppression period can be extended to more than three months.

[0038] A fifth aspect of the present invention provides a method for controlling estrus synchronization in mammals, comprising administering a GnRH antagonist to a mammal that is not in estrus to suppress estrus in the mammal, and then administering an effective amount of gonadotropin to achieve estrus synchronization.

[0039] For an explanation of the effective dose, see the fourth aspect. In the case of gonadotropins, the effective dose is the amount that can appropriately and effectively control estrus synchronization in this situation. For further details, see the second aspect.

[0040] A method for administering the GnRH antagonist may be described in the fourth embodiment. To achieve estrus synchronization, the dose of the GnRH antagonist can be adjusted so that the estrus suppression time of the mammal is within a certain range. Preferably, to achieve estrus synchronization, 5,000 to 6,000 units of gonadotropin are administered 7 to 35 days after GnRH antagonist administration (while the animal is still in a state of estrus suppression).

[0041] The method of administering the gonadotropin may be a conventional method in the art. Preferably, the method of administering the gonadotropin is to administer an effective amount of gonadotropin (at least one selected from FSH and its analogues, PMSG and its analogues, and HCG and its analogues, more preferably PMSG or HCG) to a mammal at once, or to first administer an effective amount of at least one of FSH and its analogues and PMSG and its analogues (more preferably PMSG) to a mammal, and then administer HCG or its analogue 24 to 96 hours later. Those skilled in the art can select an appropriate administration method depending on the type of animal.

[0042] The method of administering the gonadotropin is not particularly limited and may be a conventional method in the art, such as intramuscular injection, subcutaneous injection, oral administration, subcutaneous implantation, and vaginal suppository.

[0043] The method of administering gonadotropins and GnRH antagonists may be the same or different.

[0044] Preferably, the mammal is a horse, a cow, a sheep, or a pig. It should be understood that the use of estrus synchronization is often applied to female animals such as sows, cows, or sheep.

[0045] A preferred embodiment of the present invention provides a method for regulating estrus synchronization in sows, comprising administering 10-20 μg / kg of Degarelix to a reserve sow, and then administering 500-1500 units of PMSG or an analogue 18-35 days later to achieve estrus synchronization in the sow.

[0046] A preferred embodiment of the present invention provides a method for controlling estrus synchronization in female mice, comprising administering 0.5 to 2 mg / kg of Degarelix to female mice, administering 5 to 10 units of PMSG or its analogue 7 to 35 days later, and then administering 5 to 10 units of HCG or its analogue 46 to 48 hours later to achieve estrus synchronization in the female mice.

[0047] Those skilled in the art can perform estrus synchronization procedures on other mammals by referring to the methods described above, but these will not be described in detail here.

[0048] In this invention, GnRH antagonists and gonadotropins are commercially available and can be used in accordance with the instructions for use of the specific product.

[0049] The present invention will be described in detail below with reference to examples.

[0050] Abarelix, Cetrorelix, and Degarelix were all purchased from Acmec. PMSG was purchased from Ningbo Sansheng Biotechnology Co., Ltd. HCG was purchased from Ningbo Sansheng Biotechnology Co., Ltd.

[0051] Example 1 This embodiment describes the screening of GnRH antagonists for suppressing estrus in female mice. The estrus-suppressing effects of three types of GnRH antagonists (Abarelix, Cetrorelix, and Degarelix) were compared. Twenty-four six-week-old female mice were selected and divided into four groups of six. Female mice in the negative control group were subcutaneously injected with saline solution, female mice in the Abarelix group were subcutaneously injected with a dose of 2 mg / kg / day for eight consecutive days, female mice in the Cetrorelix group were subcutaneously injected with a dose of 2 mg / kg / day for eight consecutive days, and female mice in the Degarelix group were subcutaneously injected with a single dose of 2 mg / kg. After administration, vaginal smear experiments were performed daily on the female mice to detect the stage of the estrous cycle in each group. The results of the vaginal smears showed that all three GnRH antagonists could suppress estrus in the female mice, and that estrus could be restored after a predetermined suppression period. As shown in Table 1, continuous injection of Abarelix for 8 days effectively suppressed estrus in female mice for 11 days or more, continuous injection of Cetrorelix for 8 days effectively suppressed estrus in female mice for 12 days or more, and a single injection of Degarelix suppressed estrus in female mice for a long period of 28 days or more.

[0052] [Table 1]

[0053] Example 2 This embodiment describes the use of Degarelix in suppressing estrus and achieving estrus synchronization in female mice. Experiments were conducted on the duration of estrus suppression in female mice with various doses of Degarelix, and estrus synchronization treatment was performed on female mice whose estrus was suppressed. (1) Estrus suppression Thirty-two six-week-old female mice were selected and divided into four groups of eight. Female mice in the negative control group were subcutaneously injected with saline, while the three Degarelix groups received a single subcutaneous injection of different doses of Degarelix (0.4, 0.6, and 1.0 mg / kg). After administration, vaginal smears were performed daily to detect estrus. The results of the vaginal smears showed that female mice treated with Degarelix remained in the interestrus period for a certain time, and then returned to a normal estrus cycle over time. The interestrus period differed in female mice from different dose groups, with higher doses resulting in a longer interestrus period. This indicates that a single injection of Degarelix can effectively suppress estrus in female mice, and the duration of suppression is positively correlated with the dose (Table 2).

[0054] [Table 2]

[0055] (2) Estrus synchronization In estrus suppression experiments, it was found that a dose of 1.0 mg / kg could suppress estrus in female mice for a long period of at least 15 days. Further estrus synchronization experiments were conducted using six 6-week-old female mice. The specific method involved subcutaneously injecting 1.0 mg / kg of Degarelix into the six female mice, intraperitoneally injecting 5 units of PMSG 14 days after administration, and intraperitoneally injecting 5 units of hCG 46 hours later to detect estrus in the female mice. As shown in Figure 1, the results indicate that the female mice went into estrus concentrated on days 17 and 18, demonstrating that Degarelix can suppress estrus in female mice, and that estrus synchronization can be achieved by subsequently treating them with exogenous hormones.

[0056] Example 3 This embodiment describes the use of Degarelix in suppressing estrus and achieving estrus synchronization in sows. Experiments were conducted to determine the duration of estrus suppression in sows treated with different doses of Degarelix, and estrus synchronization treatment was performed on sows whose estrus was suppressed. (1) Estrus suppression Thirty healthy prospective female pigs were selected and divided into a control group and a Degarelix experimental group, with six prospective female pigs in each group. The four Degarelix experimental groups received intramuscular injections of Degarelix at doses of 10, 20, 40, and 160 μg / kg body weight, respectively. The control group of female pigs received intramuscular injections of saline solution. The appearance of the female pigs' vulvas and signs of estrus, such as their orthostatic response, were observed, and whether the female pigs were in estrus was determined and recorded. The recovery time from estrus after administration was also recorded. The results showed that the estrus time after administration in the Degarelix experimental group of sows was significantly later than in the control group, and that the time required to recover from estrus increased with higher injection doses. The estrus suppression time in the 160 μg / kg dose group reached over 90 days, indicating that Degarelix can effectively suppress estrus in sows, and that the suppression time is positively correlated with the dose (Table 3).

[0057] [Table 3]

[0058] (2) Estrus synchronization This study compared experiments using Degarelix and altrenost to achieve estrus synchronization in prospective sows. Twelve healthy prospective sows were selected and divided into two groups: an altrenost group and a Degarelix group. The altrenost group sows were given 20 mg of altrenost daily for 18 consecutive days, while the Degarelix group sows received 15 μg / kg body weight of Degarelix intramuscularly on the first day only. Twenty days after the initial administration, 1000 units of PMSG were intramuscularly injected into prospective sows in both groups to detect whether estrus synchronization could be achieved in prospective sows treated in different ways. Figure 2 shows the results of estrus synchronization after estrus suppression with Degarelix and altrenost, followed by treatment with PMSG. The experimental results showed that all reserve sows in both the altrenost-treated and Degarelix-treated groups went into estrus within one week of exogenous hormone administration. Sows in the altrenost-treated group experienced concentrated estrus within a total of four days, from day 3 to day 6 after PMSG injection, while reserve sows in the Degarelix-treated group experienced concentrated estrus within a total of three days, from day 3 to day 5 after PMSG injection (Figure 2). This indicates that after estrus suppression in reserve sows by Degarelix and altrenost, the use of the exogenous hormone PMSG effectively promotes estrus synchronization, and that Degarelix achieves estrus synchronization in reserve sows with a single intramuscular injection, while also improving the degree of estrus concentration to some extent.

[0059] Example 4 This embodiment describes the use of Degarelix in suppressing estrus in dogs. (1) Inhibitory effect of Degarelix on male dogs Healthy adult male dogs with normal reproductive capacity were selected and divided into an experimental group and a control group. Animals in each group were treated by subcutaneous injection. The control group was injected with physiological saline, and the experimental group was injected with Degarelix at a dose of 1.5 mg / kg body weight. After treatment, animals in each group were kept and observed in the same manner. Blood was periodically collected from the animals, and the testosterone concentration in the serum was measured and recorded (blood was collected before drug injection, and at 1, 2, 4, 6, 8, 24, 48, 72, 120, and 168 hours after injection, and thereafter every 168 hours (1 week) until the end of the experiment). In addition, the scrotal circumference of the male animals was periodically measured and recorded, and the sexual behavior of the male animals was observed and recorded and compared with that of the control group males. The results showed that, compared to the control group, male dogs treated with Degarelix had significantly lower testosterone levels, significantly lower scrotal circumference after a predetermined treatment time (as shown in Figure 3, ▲ represents the control group, ■ represents the experimental group), and significantly reduced sexual behavior. Before treatment, male dogs in the experimental group exhibited normal erection and ejaculation behavior, and semen could be collected. However, male dogs in the experimental group treated with Degarelix exhibited abnormal erections, and semen could not be collected. Male dogs in the control group exhibited normal erection and ejaculation behavior before and after administration of saline, and semen could be collected. The above results indicate that Degarelix can significantly suppress estrus in male dogs. (2) Inhibitory effect of Degarelix on female dogs Healthy female dogs in the proestrus phase (less than 3 days) were selected and divided into an experimental group and a control group. Animals in each group were treated with subcutaneous injection. The control group received saline solution, while the experimental group received Degarelix at a dose of 1.5 mg / kg body weight. After treatment, animals in each group were kept and observed in the same manner, and estrus in the female dogs was detected. The results showed that, three days after injection, the estrus symptoms in female dogs treated with Degarelix gradually disappeared, the redness and swelling of the vulva gradually decreased, and vaginal mucus discharge also gradually decreased. Vaginal smear results indicated that the female dogs in the experimental group rapidly transitioned from proestrus to interestrus. The female dogs in the control group were in normal estrus, with redness and swelling of the vulva and vaginal mucus discharge. This indicates that treatment with Degarelix during proestrus can effectively suppress estrus in female dogs.

[0060] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical idea of ​​the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in other appropriate ways. These simple modifications and combinations are also considered to be within the scope of disclosure of the present invention and fall within the scope of protection of the present invention.

Claims

1. The use of GnRH antagonists in the regulation of estrus in mammals.

2. The use according to claim 1, wherein the use is to suppress estrus or to regulate estrus synchronization.

3. The use according to claim 1 or 2, wherein the GnRH antagonist is at least one selected from Cetrolex, Abarelix, and Degarelix, preferably Degarelix.

4. The use according to any one of claims 1 to 3, wherein the mammal is a cat, dog, mouse, rabbit, horse, cow, sheep, or pig.

5. The use according to any one of claims 1 to 4, wherein the dose of the GnRH antagonist is 1 to 5000 μg per 1 kg body weight of the animal.

6. A pharmaceutical composition, The aforementioned pharmaceutical composition comprises a GnRH antagonist and a gonadotropin. Preferably, the GnRH antagonist is at least one selected from Cetrolex, Abarelix, and Degarelix, more preferably Degarelix. Preferably, the gonadotropin is at least one selected from follicle-stimulating hormone (FSH) and its analogues, pregnant mare serum gonadotropin (PMSG) and its analogues, and human chorionic gonadotropin (HCG) and its analogues. Preferably, the dosage of the GnRH antagonist is 1 to 100 μg / kg body weight, and the dosage of the gonadotropin is 5 to 6,000 units, characterized in the pharmaceutical composition.

7. The use of GnRH antagonists and gonadotropins in the regulation of estrus synchronization in mammals.

8. A method for suppressing estrus in mammals, comprising administering an effective amount of a GnRH antagonist to a mammal that is not in estrus, thereby suppressing estrus in the mammal. Preferably, the mammal is a cat or a dog.

9. A method for regulating estrus synchronization in mammals, comprising administering a GnRH antagonist to a mammal that is not in estrus to suppress estrus, and then administering an effective amount of gonadotropin to achieve estrus synchronization, Preferably, the mammal is a horse, a cow, a sheep, or a pig.

10. The method of administering the aforementioned gonadotropin is: The method according to claim 9, wherein an effective amount of gonadotropin is administered to the mammal at once, or an effective amount of at least one of FSH and its analogues and PMSG and its analogues is first administered to the mammal, and then hCG or its analogue is administered 24 to 96 hours later.

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