Animal castration method

Abstract

The present invention relates to the technical field of biology, and particularly to an animal castration method. The present invention provides a non-surgical animal castration method, comprising administering a GnRH-I-AP205 virus-like particle subunit vaccine to an animal. The method is good in safety and non-irritant, does not cause anaphylaxis, and is still safe under overdosage. Moreover, the required dosage is small, and 0.25 ml per animal is able to take effect.

Description

Animal castration methods Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an animal castration method. Background Art

[0002] Castration is the indirect removal of an animal's reproductive system or the loss of sexual function. In animal husbandry, castration is a common procedure performed on animals of varying ages. It aims to reduce feed loss during the fattening phase due to sexual activity, reduce injuries and deaths caused by estrus and aggressive behavior in male animals, improve meat quality (e.g., reduce mutton and off-flavor flavors), and leverage the growth characteristics of castrated animals to achieve greater fattening. Therefore, castration is a crucial technical step in the livestock fattening process.

[0003] Methods of castration include chemical, medical, and surgical procedures. Surgery is the most commonly used method and involves the removal of the testicles, ovaries, or gonads. Chemical methods involve the use of drugs to suppress sex hormone secretion. Medical castration involves injecting drugs, such as luteinizing hormone-releasing hormone analogs, to suppress testicular androgen production.

[0004] For centuries, most male livestock, with the exception of breeding stock, have been surgically castrated. This is a very stressful procedure for the animals, typically requiring 7-10 days to recover, and can sometimes lead to death due to wound infection. Furthermore, surgical castration of female animals carries a high risk of infection and requires significant skill and physical strength from the operator. Furthermore, in many countries and regions, animal welfare activists have been exerting pressure to halt surgical castration. Therefore, a safe, harmless, and inexpensive method for castrating animals is highly desirable in both food animal production and the pet sector.

[0005] Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a non-surgical animal castration method.

[0007] The animal castration method of the present invention comprises administering GnRH-I-AP205 virus-like particle subunit vaccine.

[0008] In the embodiment of the present invention, the administration method is injection.

[0009] In some embodiments, the injection is intramuscular, intravenous, subcutaneous, or intradermal.

[0010] In some specific embodiments, the subcutaneous injection site is between the shoulder blades at the back of the neck, the back, the base of the ear, the abdomen, the outer side of the leg or the inner side of the leg. Preferably, the subcutaneous injection in the method of the present invention is a subcutaneous injection in the neck behind the ear.

[0011] In an embodiment of the present invention, the injection dose is 0.25 mL / animal to 1.0 mL / animal. Preferably, the injection dose is 0.25 mL / animal to 0.5 mL / animal, or 5 mL / animal to 1.0 mL / animal. More preferably, the injection dose is 0.25 mL / animal, 0.5 mL / animal, or 1.0 mL / animal.

[0012] In some embodiments, the injections are administered 1 to 3 times, for example, 1, 2, or 3 times. Preferably, the injections are administered 2 times. The interval between the two injections is 3 to 5 weeks. In some embodiments, the interval between the two injections is 3, 4, or 5 weeks.

[0013] Preferably, the injection procedure is as follows: an initial injection of 0.25 mL to 1.0 mL / animal and an additional injection of 0.25 mL to 1.0 mL / animal 4 weeks later.

[0014] The dosage of the two injections may be equal or unequal, and the present invention is not limited thereto. For example, the dosage of the first injection and the second injection are independently selected from 0.25 ml / animal, 0.5 ml / animal, and 1.0 ml / animal. In a specific embodiment, the dosage of the first injection and the second injection are equal. Preferably, the dosage of both injections is 0.5 ml / animal.

[0015] In the present invention, the immune production period of castration is 5 weeks after the first injection, and the immune duration period is 12 months.

[0016] In the present invention, the animal is a mammal, for example, a feline, a canine, or a rodent.

[0017] Based on safety considerations, the animals are non-pregnant animals.

[0018] In the present invention, the feline animals include lions, tigers, leopards or cats; the canine animals include dogs, foxes, hyenas, wolves or jackals; and the rodents include mice, squirrels or rabbits.

[0019] In some embodiments, the animal is a cat, and the cat is a cat between 4 and 24 months old. For example, the cat is a cat between 4 and 12 months old, or a cat between 12 and 18 months old, or a cat between 18 and 24 months old. Specifically, the cat is a 4-month-old cat, a 5-month-old cat, a 6-month-old cat, a 7-month-old cat, an 8-month-old cat, a 9-month-old cat, a 10-month-old cat, an 11-month-old cat, a 12-month-old cat, a 13-month-old cat, a 14-month-old cat, a 15-month-old cat, a 16-month-old cat, a 17-month-old cat, an 18-month-old cat, a 19-month-old cat, a 20-month-old cat, a 21-month-old cat, a 22-month-old cat, a 23-month-old cat, or a 24-month-old cat.

[0020] The present invention does not impose any restrictions on animal weight; animals within the reproductive period are within the scope of the present invention. In some embodiments, the cat weighs 2 to 6 kg. In the present embodiments, the vaccine dose is calculated based on an animal weight of 2 to 6 kg. If the animal weighs less than 2 kg or more than 6 kg, the dose is proportionally converted based on the relationship between animal weight and dose described in the embodiments.

[0021] In the present invention, the aluminum hydroxide adjuvant content in the GnRH-I-AP205 virus-like particle subunit vaccine is 400 μg / ml. The mass ratio of antigen (GnRH-I-AP205) to adjuvant is 1:(0.1-10). Preferably, the mass ratio of antigen to adjuvant in the subunit vaccine is 1:0.1, 1:0.5, 1:1, 1:2, 1:5, or 1:10.

[0022] Furthermore, the present invention provides the use of the aforementioned method in castration of male animals.

[0023] In some embodiments, the castration comprises: increasing GnRH protein antibody titer, reducing testosterone levels, reducing testicular volume or reducing testicular quality, reducing sperm motility and / or inhibiting mounting behavior.

[0024] Furthermore, the present invention provides use of the aforementioned method in castration of female animals.

[0025] In some embodiments, the castration comprises: increasing GnRH protein antibody titer, reducing estradiol levels and / or preventing pregnancy.

[0026] The present invention provides a non-surgical method for animal castration, comprising administering a GnRH-I-AP205 virus-like particle subunit vaccine to animals. This method is safe, non-irritating, and non-allergic, and remains safe even in overdoses. Furthermore, the required dosage is very small, requiring only 0.25 ml per animal to be effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the serum GnRH antibody levels in each group;

[0028] Figure 2 shows the serum testosterone levels in each group;

[0029] Figure 3 shows the serum estradiol levels in each group;

[0030] Figure 4 shows the changes in serum GnRH antibody levels in each group over time. DETAILED DESCRIPTION

[0031] The present invention provides methods for animal castration. Those skilled in the art can refer to the contents of this document and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0032] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0033] "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0034] "Include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0035] "Injection" refers to the process and method of injecting liquid or gas into the body using medical devices such as syringes, steel needles, indwelling needles, catheters, etc. to achieve diagnosis, treatment and other purposes.

[0036] The test materials used in the present invention are all common commercially available products. In the examples, the test substance involved in the experiment, referred to as "preparation" in some examples, is a GnRH virus-like particle subunit vaccine. Specifically, its carrier is AP205 phage capsid protein. Its preparation method is referred to in Chinese Patent Application No. 202011399282.9.

[0037] The present invention will be further described below in conjunction with the embodiments:

[0038] Example 1 Minimum inoculation dose test

[0039] Grouping: 30 healthy cats (15 female cats and 15 male cats) were divided into 5 groups, with 3 female cats and 3 male cats in each group. One group was the control group, and the other 4 groups were injection groups.

[0040] Inoculation: Inject cats subcutaneously behind the ears and neck at doses of 0.13 ml / cat, 0.25 ml / cat, 0.5 ml / cat, and 1.0 ml / cat, respectively. Inject one cat per dose group. 28 days later, inject a second dose using the same route. The control group receives no injection and is housed and observed under the same conditions.

[0041] GnRH protein antibody titer: 14 days after the second injection, the serum GnRH protein antibody titer of cats in the control group was <1:300; the serum GnRH protein antibody titer of cats in the 0.13 ml / cat dosage group was ≤1:600; the serum GnRH protein antibody titer of cats in the 0.25 ml / cat dosage group was between 1:900 and 1:1500; the serum GnRH protein antibody titer of cats in the 0.5 ml / cat and 1.0 ml / cat dosage groups was ≥1:1500.

[0042] Testicular volume of male cats: The testicular volume of the test male cats in the 0.13 ml / cat dosage group and the control group increased, the testicular volume of the three test male cats in the 0.25 ml / cat dosage group decreased, and the testicular volume of all the test male cats in the 0.5 ml / cat dosage group and the 1.0 ml / cat dosage group decreased.

[0043] Cage test:

[0044] Mounting behavior of male cats: The male cats in the control group and the 0.13ml / cat dosage group showed obvious mounting behavior, while the male cats in the 0.25ml / cat, 0.5ml / cat and 1.0ml / cat dosage groups did not show mounting behavior.

[0045] Pregnancy status of female cats: 3 test female cats and 3 test male cats in the control group caused the female cats paired in the same cage to become pregnant, and 6 / 6 cats were in estrus; 3 test female cats in the 0.13ml / cat dosage group were all pregnant, and the female cats paired in the same cage with the 3 test male cats were all pregnant, and 6 / 6 cats were in estrus; 3 test female cats in the 0.25ml / cat dosage group were not pregnant, and the female cats paired in the same cage with the 3 test male cats were not pregnant, and 6 / 6 were protected; 3 test female cats in the 0.5ml / cat and 1.0ml / cat dosage groups and the female cats paired in the same cage with the 3 test male cats were not pregnant, and 6 / 6 were protected.

[0046] Example 2 Inoculation procedure determination test

[0047] A total of 96 cats were tested. They were given two and three injections, respectively. Twelve cats in each of the two- and three-injection groups were vaccinated, while 12 cats in the control group were vaccinated with saline solution. The remaining 60 cats were used for breeding. The results showed that:

[0048] The serum GnRH protein antibody titers of the control cats were all less than 1∶300. The testicles of the 6 test male cats were enlarged. All 6 test male cats showed mounting behavior after being paired with female cats in the same cage.

[0049] The serum GnRH protein antibody titers of cats in the two-dose group ranged from 1:600 ​​to 1:900. Four male cats showed increased testicular volume and four male cats exhibited mounting behavior after being cage-paired with female cats.

[0050] The serum GnRH protein antibody titers of cats in the three-injection group were all ≥1:1500, the testicular volume of the six test male cats was reduced, and the six test male cats did not show mounting behavior after being paired with female cats in the same cage.

[0051] 30 days after the cage experiment, all 6 female cats in the control group were pregnant, and 6 / 6 cats were in estrus. All 6 female cats paired with the 6 male cats in the control group were pregnant, and 6 / 6 cats were in estrus. The 4 female cats in the two-injection group were pregnant, and 2 / 6 cats were protected. The 4 female cats in the two-injection group were pregnant, and 2 / 6 cats were protected. The 6 female cats in the three-injection group were not pregnant, and 6 / 6 cats were protected. The 6 female cats in the two-injection group were not pregnant, and 6 / 6 cats were protected.

[0052] According to the above test results, the vaccination program was determined as follows: the first injection for healthy cats aged 4 to 24 months, the second injection after 28 days, and the third injection after 56 days, each time 0.5 ml.

[0053] Example 3 Efficacy Test

[0054] A total of 128 cats were tested, and the cats were inoculated with 3 batches of products. 28 days later, the second injection was given with the same dose and route. The results showed that:

[0055] 1. Antibody titer:

[0056] Fourteen days after the second injection, the serum GnRH protein antibody titers of cats in the three product-injected groups were all ≥1:1500, while those in the control group were all <1:300. There were highly significant differences in the antibody levels between the three product-injected groups and the control group (all P < 0.01) (Figure 1).

[0057] 2. Testicular volume:

[0058] On days 42 and 56 after the first injection, the testicular volumes of the male cats in the three product injection groups decreased, while those in the control group increased. There was no significant difference in testicular volume between the three product injection groups and the control group (all P>0.05).

[0059] 70 days after the first injection, the testicular volume of male cats in the three product injection groups decreased, while that in the control group increased. There were significant differences in the testicular volume between the three product injection groups and the control group (all P < 0.05).

[0060] 3. Testosterone levels:

[0061] 42 days after the first injection, the testosterone levels of male cats in the three injection groups decreased compared with those in the control group, all below 5ng / ml. There were extremely significant differences in the testosterone levels of male cats in the three injection groups and the control group (all P < 0.01).

[0062] Figure 2 shows the changes: 28 days after the first injection, serum testosterone levels in male cats in all groups (each receiving one batch of product) were significantly lower than those in the control group (P < 0.05). 42 days after the first injection, serum testosterone levels were extremely significantly lower than those in the control group (P < -0.01).

[0063] 4. Estradiol levels:

[0064] 42 days after the first injection, compared with the estradiol levels of the female cats in the control group, the estradiol levels of the female cats in the three batches of product injection groups were significantly decreased, all below 100pg / ml. There were extremely significant differences in the estradiol levels of the female cats in the three batches of product injection groups and the control group (all P < 0.01).

[0065] Figure 3 shows the changes in serum estradiol concentration: 28 days after the first injection, the serum estradiol concentration of female cats in each group (each group was injected with one batch of product) was significantly lower than that in the control group (P < -0.05). 42 days after the first injection, the serum estradiol concentration was extremely lower than that in the control group (P < 0.01).

[0066] 5. Sperm motility:

[0067] 42 days after the first injection, the sperm motility of the male cats in the three product injection groups decreased, while the sperm motility of the male cats in the control group was normal.

[0068] 6. Same cage test:

[0069] Fourteen days after the second injection, a cage-sharing experiment was conducted, followed by 21 consecutive days of observation. None of the male cats in the three product-injected groups exhibited mounting behavior, indicating no estrus, with 6 / 6 cats in protective estrus. All six male cats in the control group exhibited multiple mounting behaviors, with 6 / 6 cats in estrus. Thirty days after the cage-sharing experiment, none of the female cats paired with the male cats in the three product-injected groups became pregnant, with 6 / 6 cats in protective estrus. All six female cats in the control group became pregnant, with 6 / 6 cats in estrus. All six female cats paired with the male cats in the control group became pregnant, with 6 / 6 cats in estrus.

[0070] The above results show that when the product is inoculated into healthy cats aged 4 to 24 months at a dose of 0.5 ml / cat, and then injected for the second time with the same dose and route 28 days later, the cats produce GnRH protein antibodies. 14 days after the second injection, the serum GnRH protein antibody titer of the injected group cats is ≥1:1500, which inhibits the reproductive ability of the cats.

[0071] Example 4 Test on duration of efficacy and antibody growth and decline

[0072] A total of 64 cats were tested, and the cats were inoculated with 3 batches of products. 28 days later, the second injection was given with the same dose and route. The control group did not receive any injection.

[0073] Results showed that serum GnRH protein antibody levels reached 1:900 to 1:1500 five weeks after the first injection, peaked seven to eight weeks after the injection, and ranged from 1:1500 to 1:3900 in the injected cats. Afterward, GnRH protein antibody levels began to decline. Thirteen months after the injection, serum GnRH protein antibody levels in the injected cats remained at least 1:1500, while those in the control group were all below 1:300. In the cohabitation experiment, 6 / 6 of the injected cats were protected from estrus, while 6 / 6 of the control cats were in estrus.

[0074] According to the results of GnRH protein antibody test and cage-to-cage test, the product was inoculated into healthy cats aged 4 to 24 months at 0.5 ml per cat. A second injection was given 28 days later at the same dose and route. The immune production period was 5 weeks after the first injection, and the immune duration was 13 months (Figure 4).

[0075] Example 5 Safety Study

[0076] 1. Safety test of overdose vaccination in mice

[0077] Test animals: 40 mice, 10 mice in each group, 3 test groups and 1 control group.

[0078] The results showed that when three batches of the product were inoculated at an overdose (600 μg / mouse) into healthy 6-week-old Kunming mice, they were observed for 14 consecutive days after injection. The mice showed no significant changes in their drinking, feeding, or mental state, no systemic adverse reactions, and no local adverse reactions such as redness, swelling, or lumps at the injection site. Autopsy showed no pathological changes at the injection site, liver, or kidneys, which were no significant differences from the control group.

[0079] 2. Local irritation test

[0080] Test animals: 10 cats, 5 cats in each group, 1 test group and 1 control group.

[0081] The results showed that a single dose of 200 μg / batch of the product did not cause significant inflammation at the injection site, nor did it cause any local adverse reactions such as redness, swelling, or lumps. Microscopic examination of local tissue samples revealed no pathological changes at the injection site.

[0082] 3. Systemic allergy test

[0083] Test animals: 20 cats, 5 cats in each group, 3 test groups and 1 control group.

[0084] The results showed that the second vaccination was conducted 28 days after the inoculation of 3 batches of products (200 μg / cat). After 14 consecutive days of observation, none of the cats in each injection group showed allergic symptoms such as systemic shock, skin allergies, respiratory tract abnormalities, and digestive tract abnormalities, and there was no significant difference from the control group.

[0085] 4. Single-dose (200 μg / animal) safety study

[0086] Test animals: 10 cats, 5 cats in each group, 1 test group and 1 control group.

[0087] The results showed that after a single dose of the product from batch 1 was administered to the test cats, there were no significant changes in body temperature, food intake, water intake, spirits, etc. There were no systemic adverse reactions, and no local adverse reactions such as redness, swelling, and lumps at the injection site.

[0088] 5. Safety of repeated single doses (200 μg / animal)

[0089] Test animals: 10 cats, 5 cats in each group, 1 test group and 1 control group.

[0090] The results showed that when the second inoculation was carried out 28 days after the first batch of products, there were no significant changes in the cats' body temperature, food intake, water intake, and spirits, no systemic adverse reactions, no local adverse reactions such as redness, swelling, and lumps at the injection site, and no lesions in any organs after autopsy.

[0091] 6. Overdose (600 μg / animal) safety test

[0092] Test animals: 20 cats, 5 cats in each group, 3 test groups and 1 control group.

[0093] The results showed that after overdose inoculation with three batches of products, the test cats were observed for 14 consecutive days. There were no obvious changes in body temperature, food intake, water intake, spirit, etc., no systemic adverse reactions, no local adverse reactions such as redness, swelling, and lumps at the injection site, and no lesions were found at the injection site and in various organs after autopsy.

[0094] 7. Safety test on cats of non-user age

[0095] Test animals: 20 cats, 5 cats in each group, 3 test groups and 1 control group.

[0096] The results showed that when three batches of products were inoculated into healthy cats aged 2 to 3 months at an overdose (600 μg / cat) and observed for 14 consecutive days, there were no obvious changes in the body temperature, food intake, water intake, spirit, etc. of the test cats, no systemic adverse reactions, no local adverse reactions such as redness, swelling, and lumps at the injection site, and no lesions at the injection site and various organs.

[0097] 8. Safety test on pregnant cats

[0098] Test animals: 10 cats, 5 cats in each group, 1 test group and 1 control group.

[0099] Results showed that healthy female cats 3 to 6 weeks pregnant were inoculated with an overdose of one batch of the product (600 μg / cat) and observed for 14 consecutive days. The cats showed no significant changes in body temperature, food intake, water intake, or spirits, no systemic adverse reactions, and no local adverse reactions such as redness, swelling, or lumps at the injection site. There were stillbirths in the injected cats, while none of the control cats experienced miscarriage, stillbirth, or fetal malformation. These results suggest that this product may cause miscarriage or stillbirth when administered to pregnant cats. Therefore, this product is contraindicated in pregnant cats.

[0100] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for castrating animals, which comprises administering a GnRH-I-AP205 virus-like particle subunit vaccine.

2. The method according to claim 1, wherein, the administration method is injection.

3. The method according to claim 2, wherein, the injection is intramuscular injection, intravenous injection, subcutaneous injection or intradermal injection.

4. The method according to claim 3, wherein, the subcutaneous injection is subcutaneous injection behind the ear and at the neck.

5. The method according to any one of claims 1 to 4, wherein, the injection dose is 0.25 mL / animal to 1.0 mL / animal.

6. The method according to claim 5, wherein, the injection procedure is to inject 0.25 mL / animal to 1.0 mL / animal for the first time, and then inject 0.25 mL / animal to 1.0 mL / animal again 4 weeks later.

7. The method according to claim 6, wherein, the immune generation period of castration is 5 weeks after the first injection, and the immune persistence period is 12 months.

8. The method according to any one of claims 1 to 7, wherein, the animals are felines, canines or rodents.

9. The method according to claim 8, wherein, the animals are non-pregnant animals.

10. The method according to claim 8 or 9, wherein, the animals are cats, dogs or mice.

11. The method according to claim 10, wherein, the cats are cats at 4 to 24 months old.

12. The method according to claim 11, wherein, the weight of the cats is 2 to 6 kg.

13. The method according to any one of claims 1 to 12, wherein, in the GnRH-I-AP205 virus-like particle subunit vaccine, the content of aluminum hydroxide adjuvant is 400 μg / ml, and the mass ratio of GnRH-I-AP205 to the adjuvant is 1:(0.1 - 10).

14. Use of the method according to any one of claims 1 to 13 in castrating male animals.

15. The use according to claim 14, wherein, the castration includes: increasing the titer of GnRH protein antibody, decreasing the testosterone level, reducing the testicular volume or testicular mass, decreasing sperm motility and / or inhibiting mounting behavior.

16. Use of the method according to any one of claims 1 to 13 in castrating female animals.

17. The use according to claim 14, wherein, the castration includes: increasing the titer of GnRH protein antibody, decreasing the estradiol level and / or avoiding pregnancy.

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