A novel method for inducing superovulation for reproduction

Abstract

To provide an improved superovulation inducing method, and an animal propagation method using the same.SOLUTION: The present invention discloses a method for inducing superovulation in nonhuman mammals, the method including administering an anti-inhibin antibody to a female nonhuman mammal. During and after the administration of the anti-inhibin antibody, no treatment is performed with equine chorionic gonadotropin (eCG) or human chorionic gonadotrophin (hCG).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for inducing superovulation using an anti-inhibin antibody, and a method for reproducing non-human mammals using said method. [Background technology]

[0002] Research into improving reproductive capacity has long been conducted, primarily in livestock, and through repeated breeding and selection, breeds with high reproductive capacity and good growth have become mainstream. However, an increase in the number of fetuses inevitably increases the burden on the mother, resulting in poorer growth of the offspring, poorer postnatal survival rates, and a lower weaning rate. It has been reported that the administration of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), which are standard superovulation treatments, increases the number of ovulations, but reduces the pregnancy rate in treated individuals and does not lead to an increase in the number of offspring (e.g., Non-Patent Document 1).

[0003] The number of ovulations and litter size for each animal species and strain are regulated by the secretion of endogenous follicle-stimulating hormone (FSH). Endogenous FSH secretion is feedback-regulated by inhibin, and a method for inducing superovulation using antisera to inhibin (AIS) is known (Patent Document 1, and Non-Patent Documents 2-4). It has also been reported that good results can be obtained by co-administering AIS with eCG (Patent Document 2 and Non-Patent Document 5). However, co-administration of AIS and eCG has problems such as significant strain-specific differences in effectiveness and lack of effectiveness at older ages.

[0004] The inventors have reported that by performing AIS treatment and hCG treatment on mice with synchronized sexual cycles, more normal eggs can be obtained in many strains than with the conventional method using eCG / hCG, and that more than twice as many eggs can be obtained even in elderly mice (Non-Patent Document 6). However, the use of antisera has limitations, such as problems with uniformity and the inability to apply it to SPF mice. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 8-229624 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-155793 [Non-patent literature]

[0006] [Non-Patent Document 1] Ito et al., Relationships between numbers of ovulated ova and implantation sites in mice following superovulation treatment. Jap J Anim Reprod 1974; 19: 153-159. [Non-patent document 2] Wang et al., Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum. Reproduction 2001; 122:809-816. [Non-patent document 3] Hasegawa et al. Efficient production of offspring from Japanese wild-derived-strains of mice (Mus musculus molossinus) by improved assisted reproductive technologies. Biol Reprod 2012; 86:167. [Non-patent document 4] Mochida et al., Devising assisted reproductive technologies for wild-derived strains of mice: 37 strains from five subspecies of Mus musculus. PLoSOne 2014; 9:e114305. [Non-Patent Document 5] Takeo and Nakagata, Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL / 6 female mice. PLoSOne 2015; 10:e0128330. [Non-patent document 6] Hasegawa et al., Ogura A. High-yield superovulation in adult mice by anti-inhibin serum treatment combined with estrus cycle synchronization. Biol Reprod 2016; 94:1-8 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an improved method for inducing superovulation and an animal breeding method utilizing the same. [Means for solving the problem]

[0008] The inventors produced anti-inhibin antibodies and administered them to mice with synchronized estrous cycles to examine the effects on pregnancy. They found that inducing ovulation without administering hCG after administration of anti-inhibin antibodies prevents developmental inhibition before and after implantation, improving the chances of normal pregnancy and birth. In particular, they found that using anti-inhibin antibodies, particularly anti-inhibin monoclonal antibodies, with an appropriate range of superovulation effects can achieve higher pregnancy rates and litter size.

[0009] The present invention is based on the above findings and relates to the following [1] to

[16] . [1] A method for producing an anti-inhibin monoclonal antibody, comprising: immunizing a non-human mammal with a peptide containing inhibin or a fragment thereof to obtain a hybridoma; administering a sample containing the monoclonal antibody produced by the hybridoma to a female mouse to perform a superovulation test; and selecting an anti-inhibin monoclonal antibody that induces superovulation of 15 to 40 eggs per mouse as the desired antibody. [2] A method for producing an anti-inhibin monoclonal antibody described in [1], wherein the peptide is a peptide containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 14. [3] The method according to [1] or [2], wherein the antibody is administered to a female non-human mammal to induce superovulation. [4] A breeding reagent for non-human mammals, comprising an anti-inhibin monoclonal antibody, which induces 15 to 40 superovulation eggs per mouse in a superovulation test. [5] The reagent described in [4], wherein the anti-inhibin monoclonal antibody specifically binds to a peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 14. [6] The reagent according to [4] or [5], wherein the anti-inhibin monoclonal antibody is an antibody produced by a hybridoma assigned the accession number NITE P-03503. [7] The reagent according to any one of [4] to [6], which is to be administered to a female non-human mammal. [8] The reagent according to [7], wherein the female non-human mammal is a female non-human animal whose sexual cycle is synchronized. [9] The reagent according to [8], wherein the synchronization of the sexual cycle is carried out by treatment with any one or more selected from the group consisting of progesterone, prostaglandin F2α, and LH-RH compounds, and derivatives thereof. [Effects of the Invention]

[0010] According to the present invention, a large number of normal oocytes are obtained, and the number of implantations and offspring is improved compared to superovulation induction methods using anti-inhibin serum (AIS) or eCG / hCG. Furthermore, anti-inhibin monoclonal antibodies are highly homogeneous, providing stable effects, eliminating the need for individual animals, and being free of microbiological issues, they can also be used in SPF animals. The method of the present invention is effective in elderly animals and is expected to be effective in many strains, making it more versatile than conventional methods. [Brief explanation of the drawings]

[0011] [Figure 1-1] 1 is an alignment of the amino acid sequences of porcine, bovine, human and rat inhibin α subunits. [Figure 1-2] Alignment of the amino acid sequences of porcine, bovine, human and rat inhibin βA subunits. [Figure 1-3] Alignment of the amino acid sequences of porcine, bovine, human and rat inhibin βB subunits. [Figure 2-1] This graph shows the results of a screening test using a superovulation test after AIMA injection. The gray bars indicate the number of eggs (number / individual) collected from 1-2 females per clone. The black bars indicate the results of the standard superovulation method (eCG). [Figure 2-2]This shows the results of a superovulation test using AIMA from clones #14 and #19. This is a graph showing the number of eggs collected from females injected with each AIMA. After synchronizing the estrous cycle with progesterone injection, AIMA from clones #14 and #29 were injected into two females each. [Figure 3] This is an experimental scheme for superovulation treatment in mice. The diagram shows a schematic of superovulation treatment using eCG and hCG or GnRH (A), and treatment with AIS or AIMA injection after synchronization of the estrous cycle with progesterone (P4) injection (B). Treated females were mated with males on day 3 (A) and day 4 (B), respectively, and the presence of a vaginal plug was observed on day 4 (A) and days 5 to 8 (B). Note that some of the AIS-treated groups were also administered GnRH on day 6 (*). [Figure 4] Graph showing the distribution of mating rates after successive matings with males. Females were injected with progesterone (P4) and administered 1 / 4 AIS / GnRH (A), 1 / 4 AIS (B), 1 / 6 AIS (C), or AIMA (D). Values ​​in the bars represent the number of females with vaginal plugs out of the total number of females mated. [Figure 5-1] (A, B) Graphs showing the number of implantations and the number of resuscitated pups (*P<0.05; **P<0.01 (Mann-Whitey U test)). [Figure 5-2] (C) Graph showing the mean weight of pups at cesarean section after treatment and after excess embryo transfer (Mann-Whitey U test). (D) Graph showing the number of implantations and cumulative number of pups. Asterisks indicate significantly higher ratios of implantation sites to total (Fisher's exact test). (E) Graph showing the percentage of resuscitated pups by birth weight range. (F) Graph showing the distribution of birth weights of all pups after each treatment. [Figure 6] Graph showing total number of pups resuscitated per treated female (average number per animal) after several treatments. [Figure 7] 1 is a graph showing the number of implantations and live births in aged mice treated with AIMA. [Figure 8] 1 is a graph showing the number of live offspring in ICR mice treated with AIMA. [Figure 9] 1 is a graph showing the mean number of live pups after i-GONAD treatment in Syrian hamsters treated with AIMA, AIS, and untreated mice. The asterisk in the figure indicates a P<0.05 result in the Mann-White U test for the number of pups in the control group and the AIMA-treated group. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Inhibin Inhibin is a gonadal hormone secreted by the female ovaries and the male testes. While its secretion is stimulated by the action of follicle-stimulating hormone (FSH), it acts as an activin antagonist in the anterior pituitary gland, suppressing FSH secretion. Inhibin consists of an α-chain and a β-chain, which are covalently linked by disulfide bonds to form subunits. There are two types of β-subunits, the βA subunit and the βB subunit, which combine with the α-subunit to form inhibin A and inhibin B. The structure of inhibin is known to show little species-specific variation, i.e., high sequence identity between species. Figure 1-1 shows an alignment of the amino acid sequences of the inhibin α-subunits of pig, bovine, human, and rat. SEQ ID NO: 2 shows the amino acid sequence of porcine inhibin α subunit (NCBI reference number: NP_999354.1), SEQ ID NO: 3 shows the amino acid sequence of bovine inhibin α subunit (NCBI reference number: NP_776519.2), SEQ ID NO: 4 shows the amino acid sequence of human inhibin α subunit (NCBI reference number: NP_002182.1), and SEQ ID NO: 5 shows the amino acid sequence of rat inhibin α subunit (NCBI reference number: NP_036722.1). Figure 1-2 shows an alignment of the amino acid sequences of inhibin βA subunits. SEQ ID NO: 6 shows the amino acid sequence of porcine inhibin βA subunit (NCBI reference number: NP_999193.1), SEQ ID NO: 7 shows the amino acid sequence of bovine inhibin βA subunit (NCBI reference number: NP_776788.1), SEQ ID NO: 8 shows the amino acid sequence of human inhibin βA subunit (NCBI reference number: NP_002182.1), and SEQ ID NO: 9 shows the amino acid sequence of rat inhibin βA subunit (NCBI reference number: NP_058824.1). Figures 1-3 show an alignment of the amino acid sequences of inhibin βB subunits. SEQ ID NO: 10 shows the amino acid sequence of porcine inhibin βB subunit (NCBI reference number: NP_001158314.1), SEQ ID NO: 11 shows the amino acid sequence of bovine inhibin βB subunit (NCBI reference number: NP_789822.2), SEQ ID NO: 12 shows the amino acid sequence of human inhibin βB subunit (NCBI reference number: NP_002184.2), and SEQ ID NO: 13 shows the amino acid sequence of rat inhibin βB subunit (NCBI reference number: NP_542949.1).Table 1 shows the sequence identity of each subunit of porcine inhibin to the corresponding amino acid sequences of other species. As shown in Table 1, the β subunit of inhibin has a very high sequence identity between species, and the α subunit also has a high sequence identity of over 80%.

[0013] [Table 1]

[0014] As used herein, "inhibin" refers to a protein that has an α subunit having an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 2, and a βA subunit having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 6 or a βB subunit having 90% or more identical to the amino acid sequence of SEQ ID NO: 10, and that functions as an activin antagonist.

[0015] As used herein, "sequence identity" can be determined using a protein or gene search system such as BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) or FASTA (http: / / www.genome.jp / tools / fasta / ), with or without introducing gaps (Zheng Zhang et al., 2000, J. Comput. Biol., Vol. 7, pp. 203-214; Altschul SF et al., 1990, Journal of Molecular Biology, Vol. 215, pp. 403-410; Pearson WR et al., 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 2444-2448).

[0016] 2. Method for inducing superovulation in non-human mammals The present invention provides a method for inducing superovulation in a non-human mammal, which comprises administering an anti-inhibin antibody to a female non-human mammal, and inducing superovulation under conditions that do not place excessive strain on the animal. Specific embodiments are as follows:

[0017] In a first embodiment, the method of the present invention comprises administering an anti-inhibin antibody to a female non-human mammal, and is characterized in that the method does not include treatment with either equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) during or after administration of the anti-inhibin antibody.

[0018] In conventional methods for inducing superovulation using inhibin antiserum (AIS), ovulation is induced by administering eCG simultaneously with AIS or by administering hCG after AIS administration. The present invention is characterized by not using eCG or hCG during or after the administration of anti-inhibin antibodies. If necessary, female non-human mammals administered with anti-inhibin antibodies may be housed with males to stimulate ovulation using the Whitten effect. Methods that do not use hCG are suitable for the breeding methods described below, as they can avoid developmental inhibition before and after implantation.

[0019] It is preferable to use a female non-human mammal whose sexual cycle is synchronized. The method for synchronizing the sexual cycle is not particularly limited, and can be carried out using, for example, progesterone, prostaglandin F2α, or derivatives thereof, such as LH-RH compounds (Honda et al., 2019, Scientific Report).

[0020] The timing of administration of the anti-inhibin antibody is not particularly limited, but is preferably 24 to 72 hours after the completion of the synchronization procedure, more preferably 40 to 56 hours after, for example, in the case of progesterone administration, the anti-inhibin antibody is administered approximately 48 hours after the completion of two progesterone administrations.

[0021] The anti-inhibin antibody of the present invention preferably induces superovulation of 40 or fewer eggs per mouse in a superovulation test, and more preferably induces superovulation of 15 to 40 eggs per mouse. Conventional antisera (AIS) induce ovulation of 40 or more eggs per mouse, or even more than 50 eggs per mouse. In the present invention, an antibody that does not induce too many eggs is used, which reduces the impact on pregnancy and increases the number of offspring born.

[0022] As used herein, the term "superovulation test" refers to a test performed using the following method: Randomly selected mature female mice are administered 2 mg of progesterone per mouse at 24-hour intervals for two days, followed 48 hours later by 0.5 mg of the antibody to be tested per mouse, and 48 hours later by 5 IU of hCG per mouse. 16 hours after hCG administration, eggs are collected from the oviducts to evaluate the number of ovulations. The mice used in the superovulation test are not particularly limited as long as they are Mus musculus, and any readily available species can be used. For example, ICR mice or hybrid mice can be used.

[0023] Anti-inhibin antibodies can be produced using inhibin or a fragment thereof as an immunogen by methods known in the art. As mentioned above, inhibin has high interspecies amino acid sequence identity, so the resulting anti-inhibin antibodies are likely to cross-react with inhibin derived from a species different from the antigen. Therefore, the inhibin used as the immunogen for antibody production does not necessarily have to be derived from the same animal species as the subject to which the antibody is administered. The inhibin used as the immunogen is not particularly limited, and inhibin derived from any mammal, such as mouse, rat, rabbit, cow, horse, goat, pig, sheep, or human, may be used. When an inhibin fragment is used as an immunogen, the fragment peptide preferably contains the amino acid sequence of the surface-exposed portion of the inhibin molecule. For example, a peptide containing the amino acid sequence derived from porcine inhibin shown in SEQ ID NO: 1 can be used as an immunogen (Mayo et al., PNAS 1986, Vol. 83, pp. 5849-5853).

[0024] The anti-inhibin antibody is preferably an anti-inhibin monoclonal antibody, and may be a monoclonal antibody newly prepared using the method described below in the section on methods for preparing anti-inhibin monoclonal antibodies.

[0025] More preferably, the anti-inhibin monoclonal antibody is an antibody produced by a hybridoma assigned the accession number NITE P-03503. The hybridoma assigned the accession number NITE P-03503 was internationally deposited under the Budapest Treaty on August 3, 2021, with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number NITE P-03503. The hybridoma was prepared from splenocytes obtained by immunizing a rat with a peptide having the amino acid sequence set forth in SEQ ID NO: 1, using the method described in the method for producing an anti-inhibin monoclonal antibody described below.

[0026] The anti-inhibin antibody may be an antibody that binds to either the α subunit or the β subunit (βA subunit or βB subunit) of inhibin, but is preferably an antibody that binds to a portion exposed on the surface of the α subunit. For example, it may be an antibody that binds to a partial peptide of the porcine inhibin α subunit having the amino acid sequence from positions 2 to 27 of SEQ ID NO: 1. Alternatively, it may be an antibody that binds to a partial peptide containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 14. The amino acid sequence of SEQ ID NO: 14 is the portion of the amino acid sequence of SEQ ID NO: 1 (amino acids 7 to 24) that has particularly high sequence identity with the corresponding portion of inhibin derived from other animal species.

[0027] In a second embodiment, the method of the present invention comprises administering an anti-inhibin monoclonal antibody to a female non-human mammal. Preferably, the female non-human mammal is an animal whose estrous cycle is synchronized. The estrous cycle can be synchronized using the methods described above.

[0028] In the second embodiment, it is preferable that eCG is not administered simultaneously with the administration of the anti-inhibin monoclonal antibody, and it is even more preferable that no treatment with either eCG or hCG is included during or after the administration of the anti-inhibin monoclonal antibody.

[0029] The method for inducing superovulation of the present invention can be applied to any mammal other than humans, such as mice, rats, marmosets, hamsters, rabbits, cows, horses, goats, pigs, and sheep. There are no particular limitations on the mammal, and it may be a naturally ovulating animal (such as a mouse, rat, hamster, cow, pig, goat, dog, or primate) or a mating-ovulating animal (such as a rabbit, cat, or ferret). Naturally ovulating animals are particularly preferred. Animals with an incomplete sexual cycle, such as a mouse, rat, or hamster, may also be used. Since naturally ovulating animals have a distinct sexual cycle, administering an anti-inhibin monoclonal antibody at the time of follicle development can more efficiently increase the number of ovulations. On the other hand, since mating-ovulating animals have an unclear sexual cycle, it is difficult to determine the timing of administration, making it difficult to induce superovulation with a single administration. However, superovulation can be induced by administering multiple times.

[0030] For example, in the case of mice, the target strain is not particularly limited and may be any strain, such as C57BL / 6, BALB / c, ICR, B6D2F1, A, C57BL / 10, C3H / He, DBA / 2, FVB, B6C3F1, NOD, or CBA.

[0031] For example, in the case of rats, the target strain is not particularly limited, and may be any strain such as F344, Wistar, BN, SD, LE, or THA.

[0032] For example, in the case of hamsters, the target species is not particularly limited, and may be any species such as Syrian hamsters, Chinese hamsters, and Djungarian hamsters.

[0033] 3. Reproduction methods for non-human mammals The present invention also provides a method for breeding non-human mammals using the above-mentioned method for inducing superovulation. The breeding method of the present invention comprises inducing superovulation in a non-human mammal by the above-mentioned method, and then artificially inseminating or mating the non-human mammal to obtain offspring. Preferably, the non-human mammal obtains offspring through natural mating.

[0034] In the case of artificial insemination, for example, the female is placed with a vasally ligated male 40 to 72 hours after administration of the anti-inhibin monoclonal antibody. 40 to 90 hours after administration of the anti-inhibin monoclonal antibody, sperm are directly injected into the oviduct of a female for which mating has been confirmed or will be confirmed, via transvaginal, transuterine, or abdominal surgery, to impregnate her and produce offspring.

[0035] In the case of natural mating, females administered with anti-inhibin monoclonal antibodies are placed together with males.

[0036] The method of the present invention produces a larger number of normal oocytes than superovulation treatment using hormone administration, and does not place excessive strain on the mother, resulting in a higher implantation rate and a larger number of live offspring. The offspring produced have normal body weights, a high survival rate, and good growth.

[0037] 4. Method for producing anti-inhibin monoclonal antibodies The present invention also provides a method for producing an anti-inhibin monoclonal antibody for use in the present invention. The method comprises immunizing a non-human mammal with inhibin or a peptide containing a fragment thereof to obtain a hybridoma, administering a sample containing the anti-inhibin monoclonal antibody produced by the hybridoma (e.g., hybridoma culture supernatant or ascites obtained by transplanting the hybridoma into a mouse) to female mice to perform a superovulation test, and selecting an anti-inhibin monoclonal antibody that induces superovulation of 15 to 40 eggs per mouse as the antibody of interest.

[0038] Hybridomas can be established according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497) by immunizing a non-human mammal (e.g., the above-mentioned non-human mammals, preferably rodents such as rabbits, mice, or rats, preferably rats) with inhibin or a fragment thereof as an immunogen, and fusing the resulting antibody-producing cells with myeloma cells.

[0039] The obtained hybridomas are evaluated for their ability to bind to inhibin using ELISA, and are also subjected to the above-mentioned superovulation test using mice to select those that induce superovulation at about 15 to 40 eggs per individual.

[0040] The peptide used as an immunogen is not particularly limited as long as it is a peptide containing inhibin or a fragment thereof, but when an inhibin fragment is used, it is particularly preferable that it contains the amino acid sequence of the portion exposed on the surface of the inhibin molecule. The origin of inhibin or its fragment is not particularly limited, and inhibin or its fragment from any mammal may be used. An example of such an immunogen is a peptide containing the amino acid sequence of SEQ ID NO: 1. It is a peptide having the amino acid sequence from the 2nd to the 27th amino acids of SEQ ID NO: 1, i.e., a peptide containing an amino acid sequence derived from porcine inhibin, and the amino acid sequence of SEQ ID NO: 1 is a sequence in which a cysteine ​​has been added to the N-terminus of the amino acid sequence corresponding to the 231st to the 256th amino acids of the amino acid sequence of SEQ ID NO: 2. Alternatively, the immunogen may be a peptide having the amino acid sequence of SEQ ID NO: 3. 227th from 252nd , or a peptide comprising the amino acid sequence from positions 233 to 258 of the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence from positions 234 to 259 of the amino acid sequence of SEQ ID NO: 5. Alternatively, the immunogen may be a partial peptide comprising an amino acid sequence having 90% or more, particularly 94% or more, or 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 14.

[0041] 5. Reproduction reagents (reagents for inducing superovulation) The anti-inhibin monoclonal antibody produced as described above is used as a reagent for breeding non-human mammals or a reagent for inducing superovulation in non-human mammals, and the present invention also provides such a reagent for breeding non-human mammals or a reagent for inducing superovulation (hereinafter referred to as the reagent of the present invention).

[0042] The dosage form of the reagent of the present invention is not particularly limited, but examples thereof include injections including intravenous drips, vaginal preparations, suppositories, nasal preparations, sublingual preparations, and transdermal preparations.

[0043] The dosage of the reagent of the present invention is determined appropriately depending on the target animal. For example, in the case of mice, it is usually administered at 0.25 to 1.0 mg / female.

[0044] In addition to the anti-inhibin monoclonal antibody, the reagent of the present invention may contain a pharmacologically acceptable carrier, such as a diluent, solubilizing agent, suspending agent, isotonicity adjusting agent, stabilizer, preservative, isotonicity adjusting agent, wetting agent, or pH adjuster.

[0045] Diluents include purified water, physiological saline, phosphate buffer, and the like.

[0046] Examples of solubilizing agents include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0047] Examples of suspending agents or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, and polyoxyethylene hydrogenated castor oil.

[0048] Examples of isotonic agents include sodium chloride, potassium chloride, sugars, glycerin, and urea.

[0049] Stabilizers include polyethylene glycol, dextran sulfate sodium, and other amino acids.

[0050] Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0051] Examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0052] Examples of wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

[0053] The reagent of the present invention uses a monoclonal antibody as the active ingredient, and therefore, compared to conventional antisera, it does not require the use of individual animals, is microbiologically clean, highly uniform, and can be produced inexpensively. It is also effective in elderly animals, and provides stable effects across many strains. Furthermore, it can be administered at 72-hour intervals, which is longer than the 48-hour administration interval for antisera, offering significant advantages in terms of workability.

[0054] 6. Use of the present invention By combining the present invention with genome editing technologies using ZFN, Talen, CRISPR / Cas9, or other gene modification technologies, the number of offspring obtained can be increased, enabling more efficient gene modification. Genome editing in laboratory animals, for example, in mice, involves injecting a genome editing reagent such as CRISPR / Cas9 into fertilized eggs, culturing them, and then implanting the resulting embryos into female individuals for birth. By combining this method with the present invention, the ovulation rate of female mice increases, making it possible to obtain fertilized eggs more efficiently. On the other hand, for example, Syrian hamsters have difficulty using techniques such as embryo culture and embryo transfer because fertilized eggs are fragile in experimental environments. For such animals, a method can be used in which a genome editing reagent is directly injected into the oviduct containing the fertilized eggs, electroporation is performed on the oviduct, and genome editing is performed, followed by birth (i-GONAD method), without manipulating the fertilized eggs in vitro. By combining this method with the present invention, the number of offspring born can be increased, making it possible to obtain genome-edited offspring more efficiently. In particular, the present invention is a method that places less strain on the mother, and therefore it is possible to improve the efficiency of the combined technique without affecting the effect of the combined technique. [Example]

[0055] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0056] Example 1: Induction of superovulation and increase in litter size in mice 1. Materials and Methods animal For all superovulation and mating experiments, 10-13 week-old females and 11-64 week-old males of the standard inbred mouse strain C57BL / 6JJcl (B6; CLEA Japan) were used. For embryo transfer experiments, 10-20 week-old ICR female mice and 3-12 month-old vasectomized male mice were used.

[0057] Preparation of anti-inhibin monoclonal antibody (AIMA) Adult female rats (Wistar; Nippon Clea) were immunized with a Cys-added peptide antigen (27AA (SEQ ID NO: 1)) of the porcine inhibin α-subunit (Mayo et al., PNAS 1986, Vol. 83, pp. 5849-5853, supra) and their spleen cells were fused with myeloma cells (P3U1) to generate hybridomas. Monoclonal antibodies in the supernatants of these cells were evaluated by both ELISA using the peptide antigen and superovulation tests. Finally, selected clones were expanded and cryopreserved. 5 × 10 cells dissolved in saline were used. 6 The cells were intraperitoneally administered to nude mice (BALB / cAJcl-nu, CLEA Japan) at a concentration of 1 / mL. After 1-2 months, the ascites fluid was collected and purified using a Protein G Sepharose column (Cytiva). The ascites fluid was then used in the superovulation experiment as AIMA (5.0 mg / mL).

[0058] Collection of MII eggs after superovulation treatment As a standard superovulation method, C57BL / 6JJcl mice were intraperitoneally injected with 5 IU of equine chorionic gonadotropin (eCG, PMSA; Nippon Zenyaku Kogyo Co., Ltd.) in the evening, followed 48 hours later by injection of 5 IU of human chorionic gonadotropin (hCG, gonadotropin; Asuka Pharmaceutical Co., Ltd.). In the other treatment group, females received subcutaneous injections of 2 mg (0.08 mL per female) of progesterone (P4, Progehormon; Mochida Pharmaceutical Co., Ltd.) once daily in the evening for two days (days 1 and 2) to synchronize their estrous cycles. On day 4, AIS (diluted at 1 / 1, 1 / 2, 1 / 4, and 1 / 8 concentrations, respectively), AIMA (0.5 mg / 0.1 mL per female), or saline (control) were administered instead of eCG. 48 hours after AIS or AIMA administration, 5 IU of hCG was administered per female. Sixteen to seventeen hours after hCG administration, the female oviduct was punctured with a needle using 80 μL of in vitro fertilization medium (mHTF) to collect mature MII oocytes. Morphologically normal and abnormal oocytes were counted under a stereomicroscope.

[0059] Mating test after superovulation treatment For the mating experiment, female mice in the eCG / hCG injection group received intraperitoneal injections of 5 IU eCG, 5 IU or 1 IU hCG, or 0.6 mg of a gonadotropin-releasing hormone (GnRH) agonist (buserelin acetate; Wako Pure Chemical Industries) every 48 hours, and were placed in the cage of a sexually experienced male. In the AIS and AIMA injection groups, 2 mg of progesterone was administered subcutaneously once daily in the evening for two days (Days 1 and 2) to synchronize the estrous cycle. Then, on Day 4, mice were intraperitoneally injected with AIS (1 / 4 and 1 / 6 dilutions) or AIMA (0.5 mg / 0.1 mL per female), followed by injections with or without GnRH every 48 hours. Females and males were then mated on Day 4. To determine the timing of mating, the presence or absence of a vaginal plug was observed every morning for four days until a plug was found.

[0060] As a control group, the vaginal appearance of C57BL / 6 females was examined. Females with gaps and red-pink swelling around the vagina were considered to be in proestrus. These females were paired with males overnight and observed the following morning (day 1) for the presence or absence of a vaginal plug. Females that appeared to be pregnant were subcutaneously injected with 2 mg of progesterone on the evenings of days 18 and 19 to prevent spontaneous labor. On the morning of day 20, these females were subjected to cesarean section to check for implantation and the number and presence of fetal abnormalities. Fetuses that were alive at the time of cesarean section were considered liveborn. Fetuses whose lungs turned white and whose breathing became rhythmic during the resuscitation process were considered liveborn. Pups were also weighed to assess their health before lactation.

[0061] in vitro fertilization We attempted to transfer excess embryos generated by in vitro fertilization (IVF) to confirm the maximum number of offspring. IVF was performed as follows: Female C57BL / 6 mice were superovulated using AIS / hCG injection as described above. 16–17 h after hCG injection, MII stage oocytes were collected from the female oviducts and incubated in HTF medium containing hypotaurine (0.11 mg / mL, Merck Millipore), 0.3% bovine serum albumin (BSA, Calibochem, Merck Millipore), and 1.25 mM reduced glutathione. Sperm collected from the cauda epididymis of male mice were suspended in HTF medium containing 1 mg / mL polyvinyl alcohol (PVA) as a BSA substitute and 0.4 mM methyl-β-cyclodextrin, and incubated in a 5% CO2, 37°C incubator for approximately 60 min. For insemination, pre-cultured sperm were transferred to 80 μL of fertilization solution containing oocytes at a final concentration of 200–400 sperm / μL. After 3–4 hours, oocytes were transferred to CZB medium and cultured overnight. The following day, two-cell embryos were counted and preserved by vitrification.

[0062] Embryo cryopreservation by vitrification Two-cell embryos were cryopreserved using high-osmolarity vitrification (HOV) solution as previously described. Briefly, approximately 20–30 embryos were placed on the surface of 50 μL of equilibration solution (5% dimethyl sulfoxide and 5% ethylene glycol in PB1) using a sterile glass capillary (diameter φ100–120 μm) containing a small amount of medium at room temperature. After 3 min, embryos that had sunk to the bottom were picked up and transferred to cryotubes (MS-4501; Sumitomo Bakelite Co., Ltd.) containing 50 μL of vitrification solution (PB1 supplemented with 42.5% (v / v) ethylene glycol, 17.3% (w / v) Ficoll, and 1.0 M sucrose). After 1 min, the cryotubes were placed in liquid nitrogen (LN2) at -196 °C. On the day of embryo transfer, the cryotubes were retrieved and the caps were quickly removed to allow the LN2 to evaporate. After leaving the tube at room temperature for 2 minutes, 850 μL of 0.75 M sucrose-PB1 was gently added. After 4 minutes, the solution in the tube was mixed five times by gentle pipetting. The entire solution was then transferred to an empty plastic dish. After 1–3 minutes, the embryos were collected and transferred to 0.25 M sucrose-PB1. After equilibration for 1–3 minutes, the collected embryos were further washed with 0.25 M sucrose-PB1 and transferred to CZB medium. Embryos with normal morphology (i.e., intact cell membranes and clear cytoplasm) were considered viable. Surviving embryos were cultured at 37°C in humidified air with 5% CO2 until embryo transfer (<3 hours).

[0063] Embryo transfer After mating with vasectomized ICR males, 14 or 7 frozen-thawed 2-cell embryos were transferred into each oviduct of pseudopregnant ICR females on day 1. To prevent spontaneous labor, all recipient females were injected subcutaneously with 2 mg of P4 on the evenings of days 18 and 19. On the morning of day 20, recipient females were subjected to cesarean section, and fetal number and viability were assessed.

[0064] statistical analysis Statistical analysis was performed to compare control data with the treatment groups. The number of eggs, live births, implantations, and resuscitated pups was analyzed using the Mann-Whitey U test. The proportions of pregnancy, implantation, and resuscitated pups were analyzed using the Fisher exact test. A P value of <0.05 was considered statistically significant.

[0065] 2.Results AIMA production and superovulation test Each cell supernatant sample extracted from the 40 hybridoma clones, except for sample #40, was subjected to a superovulation test (Figure 2-1). C57BL / 6JJcl mice were subcutaneously administered 2 mg / mouse (0.08 mL) of progesterone (P4, Progehormon; Mochida Pharmaceutical) once daily in the evening for two days. On the fourth day, 0.5 mg / mouse (0.1 mL) of each sample was administered. 48 hours after sample administration, 5 IU / mouse of human chorionic gonadotropin (hCG) was administered. 16–17 hours after hCG administration, the ampulla of the female oviduct was punctured with an injection needle using 80 μL of mHTF. Mature MII-stage oocytes were collected and counted under a stereomicroscope. The number of oocytes obtained for each sample is shown in Figure 2-1. The number of oocytes obtained when 7.5 IU / mouse of eCG was administered instead of the sample is also shown.

[0066] For mice that produced a high number of MII oocytes in the above test, samples from hybridoma cells #14 and #29 were diluted to 1 / 2 volume (0.05 mL), 1 volume (0.1 mL), and 2 volumes (0.2 mL) and subjected to a second superovulation test (Figure 2-2). As a result, even with a 1 / 2 volume, the sample from #29 yielded 31.3 oocytes, demonstrating stable oocyte production regardless of the dosage. Sample #29 was selected as the AIMA and used in the following experiment. Hybridoma #29 was internationally deposited with the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center and was assigned the accession number NITE P-03503 on August 3, 2021.

[0067] Results of superovulation tests using various treatments When eCG and hCG were administered as a standard superovulation method, the average number of morphologically normal ovulations per female was 17.1, and the female ovulation rate was 89% (33 / 37) (Table 2). When progesterone was administered to synchronize the estrous cycle (2P4 in the table), followed 48 hours later by the administration of diluted AIS or AIMA, and then 48 hours later by hCG, the highest number of normal oocytes (51-52) was obtained with AIS and 1 / 2 diluted AIS. AIMA yielded 24.6 normal oocytes, which was similar to the 1 / 4 diluted AIS, and almost no abnormal oocytes were observed. As a control for the cycle-synchronized group, saline administration yielded 6.2 oocytes.

[0068] [Table 2]

[0069] Results of mating tests after various treatments When females in proestrus, monitored by vaginal color change, were housed with sexually experienced males, mating was confirmed the next day in 32% of cases, resulting in a 100% pregnancy rate (Table 3). In the eCG / hCG group, mating rates were 60-63%, but pregnancy rates tended to be lower at 40-50% (Figure 3A). Furthermore, the group receiving GnRH instead of hCG had a significantly higher mating rate. After synchronizing the estrus cycle, when females were housed with males after receiving a 1 / 4 dilution of AIMA or AIS, mating rates increased to 71-100% over a total of 4 days (Figure 3B). Mating occurred between days 6 and 8, with the highest frequency occurring on day 7 in the AIS group and days 7-8 in the AIMA group (Figure 4).

[0070] Effect on size and number of resuscitated pups In the untreated control group, the litter size per female was 8.6, and the live birth rate relative to the number of implantations (97%, 62 / 64) and the resuscitation rate by cesarean section (100%) were also very high (Table 3, Figure 5). In contrast, the number of implantations did not increase in the eCG / hCG-treated group, and the number of live pups per female was low, ranging from 0.5 to 5 (Figure 5B). When GnRH was administered instead of hCG, both eCG and AIS treatments resulted in a high mating rate, but the resuscitation rate after cesarean section decreased and the number of resuscitated pups tended to be lower (Figure 5B). In the AIS group, the number of implantations was higher, but the number of live pups did not exceed that of the control group (Figure 5A, B). In contrast, the AIMA group had significantly higher numbers of implantations and live pups than the control group (16.3 and 11.9, respectively), and these pups were morphologically normal (Figure 5A, B). In an additional experiment, females were given half the dose of AIMA (0.25 mg), and the resulting efficiency was comparable, including mating rate (79%), number of implantations (13.8), and number of live offspring (11.3).

[0071] [Table 3]

[0072] Examination of maximum number of offspring produced by embryo transfer To determine the maximum number of pups that can be developed in C57BL / 6 mothers, we attempted to transfer an excess number of embryos. Two-cell embryos produced by in vitro fertilization were vitrified, retrieved, and transferred. When seven embryos were transferred per oviduct (14 per female), the birth rate was 68% and the number of live pups per female was 9.6 (Table 4). When an excess number of embryos (28 per female) was transferred per oviduct, the birth rate was 51% and the number of live pups was 13.3 (Table 4, Figure 5). This number of live pups was comparable to the 11.9 pups obtained with AIMA administration.

[0073] [Table 4]

[0074] Assessment of pup weight The birth weight of pups in all treatment groups was significantly lower than that of the control group (Figure 5C). In the AIMA-treated group, the weight of pups was equivalent to that of pups with an excess number of embryos transferred. The post-implantation development rate tended to be higher in the AIS and embryo transfer groups (Figure 5D). Furthermore, when we examined the weight of all pups born and the resuscitation failure rate, approximately two-thirds of pups weighing less than 0.8 g failed to be resuscitated (Figure 5E). When we examined the weight ratio of all pups born in each treatment group, in groups with significantly lower post-implantation development rates (eCG / hCG (1 IU), 2P4-1 / 4AIS / GnRH, 2P4-1 / 4AIS, and 2P4-1 / 6AIS), more than 20% of pups weighed less than 0.8 g (Figure 5F). When comparing the number of offspring per treated female, the AIS and AIMA groups had a higher litter size than the control group, with AIMA being 2.4 times more effective than the control group (Figure 6).

[0075] 3. Discussion We confirmed that the use of AIMA can increase the number of offspring in mice. Standard eCG / hCG treatment is known to result in low pregnancy rates, no increase in implantation numbers, and low fetal weights. In contrast, administration of AIS instead of eCG resulted in a higher number of normal oocytes, and increased mating, pregnancy, and implantation rates. However, the higher the ovulation rate, the lower the birth weight of the offspring and the lower the survival rate. In contrast, AIMA treatment not only improved mating rates, implantation numbers, and litter size, but also reduced the fetal weight, resuscitation rate, and weaning rate, suggesting that it places no excessive strain on the mother. These findings suggest that AIMA, unlike existing superovulation reagents, has minimal adverse effects on implantation and pregnancy, making it an effective breeding reagent. It is expected to be useful in inbred mouse strains and in cases where breeding is not progressing well.

[0076] Example 2: Effect on aged mice The effects of the standard eCG / hCG method and AIMA treatment were compared using 40-50 week old C57BL / 6J female mice. The results are shown in the table below and in Figure 7.

[0077] [Table 5]

[0078] As shown in Table 5 and Figure 7, AIMA showed favorable improvements in mating rate, pregnancy rate, number of implantations, and number of live offspring compared to the standard method, even in aged mice.

[0079] Example 3: Increased litter size in ICR mice The effect of AIMA on the number of offspring born to ICR mice was examined under the same conditions as in Example 1. The results are shown in Figure 8. It was confirmed that administration of AIMA significantly increased the number of offspring born compared to normal ICR mice.

[0080] Example 4: Increased litter size in A strain mice The effect of AIMA on the number of offspring in A strain mice was examined under the same conditions as in Example 1. The results are shown in Table 6. It was confirmed that administration of AIMA significantly increased the number of offspring compared to normal A strain mice.

[0081] [Table 6]

[0082] Example 5: Effect of AIMA administration on the number of offspring in F344 rats Ten female F344 / Jcl rats, aged 8 weeks or older, in metestrus or estrus were intraperitoneally administered 0.2 mL of AIMA per rat (Day 1). On Day 2, they were housed with male F344 / Jcl rats and mated. From the following day, vaginal plugs were checked every morning, and those with confirmed vaginal plugs were separated from the males. Eight rats were confirmed pregnant. From Day 19 to Day 21 after vaginal plugs were confirmed, progesterone was administered subcutaneously at 0.5 mL per rat daily. The rats were then euthanized and their offspring were confirmed by Caesarean section. The results are shown in Table 7. The control in the table indicates the number of offspring born to normal F344 rats. It was confirmed that administration of AIMA nearly doubled the number of offspring born to normal F344 rats.

[0083] [Table 7]

[0084] Example 6: Effect of AIMA administration on Syrian hamster litter size AIMA was administered intraperitoneally at a rate of 0.25 mL per animal to eight female Syrian hamsters in metestrus (day 1). Metestrus was determined by the presence or absence of mucus secretion from the vagina. On day 3, the females were housed with the same number of male Syrian hamsters and mated. After mating was confirmed on day 4, the females were separated from the males. On day 15 after mating was confirmed, the females were euthanized and the offspring were confirmed by Caesarean section. The results are shown in Table 8. The control in the table shows the results of Syrian hamsters treated in the same way except that saline was administered instead of AIMA. Comparing the AIMA-administered group with the control group, the average number of offspring tended to be higher in the AIMA-administered group.

[0085] [Table 8]

[0086] Example 7: Effect of AIMA administration on the number of offspring produced by genome-edited mice using the i-GONAD method in Syrian hamsters As in Example 6, female Syrian hamsters were administered AIMA and mated with males. For each female individual for which mating was confirmed, the oviduct was injected with CRISPR / Cas9 genome editing reagents and electroporation was performed (i-GONAD method: for detailed conditions, see Gurumurthy CB, et al., Nature Protocol, 2019, Vol. 14, pp. 2452-2482). After the above treatment, the females were allowed to give birth naturally, and the number of pups born was counted 2-3 days after birth. The results are shown in Table 9 and Figure 9. "AIS" in the table and figure indicates the results for the group administered 0.2 mL of AIS instead of AIMA, and the control group was the group not administered AIMA or AIS. The asterisk in the figure indicates a P<0.05 in the Mann-Whitey U test for the number of pups born between the control group and the AIMA-treated group. As shown in the figure, the actual P=0.04. As shown in Table 9 and Figure 9, it was confirmed that the number of offspring born after i-GONAD treatment in the AIMA-administered group was significantly higher than that in the control group. This demonstrated that the effect of AIMA in increasing the number of offspring born is useful in obtaining genome-edited experimental animals.

[0087] [Table 9]

[0088] As shown in Figure 1, Table 1, etc., inhibin has a very high degree of identity in amino acid sequence between animal species, and therefore AIMA can react with inhibin from a variety of animal species and function in a variety of animal species. In Examples 5 to 7, it was demonstrated that AIMA can increase litter size in multiple animal species. [Industrial Applicability]

[0089] According to the present invention, a larger number of normal eggs can be obtained, improving the number of offspring born. The method for inducing superovulation and the breeding method of the present invention are useful for improving the reproductive rate of laboratory animals and livestock, particularly in strains that have traditionally not thrived well.

[0090] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0091] The present invention includes the following embodiments. [1] A method for inducing superovulation in a non-human mammal, comprising administering an anti-inhibin antibody to a female non-human mammal, characterized in that the method does not include treatment with either equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) during or after administration of the anti-inhibin antibody. [2] A method for inducing superovulation in a non-human mammal, comprising administering an anti-inhibin antibody to a female non-human mammal, wherein the anti-inhibin antibody is an anti-inhibin monoclonal antibody. [3] The method described in [2], which does not include treatment with equine chorionic gonadotropin (eCG) at the time of administration of the anti-inhibin antibody. [4] The method according to [2], which does not include treatment with either equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) during or after administration of the anti-inhibin antibody. [5] The method according to [2], wherein the anti-inhibin monoclonal antibody is an antibody produced by a hybridoma assigned the accession number NITE P-03503. [6] The method according to any one of [1] to [5], wherein the female non-human mammal is a female non-human mammal whose sexual cycle is synchronized. [7] The method according to [6], wherein the synchronization of the sexual cycle is carried out by treatment with any one or more selected from the group consisting of progesterone, prostaglandin F2α, and LH-RH compounds, and derivatives thereof. [8] The method according to any one of [1] to [5], wherein the anti-inhibin antibody induces superovulation of 40 or less eggs per mouse in a superovulation test. [9] The method according to [8], wherein the anti-inhibin antibody induces superovulation of 15 to 40 eggs per mouse in a superovulation test.

[10] The method according to any one of [1] to [5], wherein the anti-inhibin antibody is produced using a peptide containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 14 as an immunogen.

[11] A method for breeding a non-human mammal, comprising inducing superovulation in a non-human mammal by the method according to any one of [1] to [5], and artificially inseminating or mating the non-human mammal to obtain offspring.

[12] A method for producing an anti-inhibin monoclonal antibody, comprising: immunizing a non-human mammal with a peptide containing inhibin or a fragment thereof to obtain a hybridoma; administering a sample containing the monoclonal antibody produced by the hybridoma to a female mouse to perform a superovulation test; and selecting an anti-inhibin monoclonal antibody that induces superovulation of 15 to 40 eggs per mouse as the desired antibody.

[13] A method for producing an anti-inhibin monoclonal antibody described in

[12] , wherein the peptide is a peptide containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 14.

[14] A non-human mammalian breeding reagent containing an anti-inhibin monoclonal antibody.

[15] The reagent described in

[14] , wherein the anti-inhibin monoclonal antibody uses a peptide containing the amino acid sequence shown in SEQ ID NO: 1 as an antigen and induces 15 to 40 superovulation eggs per individual in a superovulation test using mice.

[16] The reagent according to

[14] , wherein the anti-inhibin monoclonal antibody is an antibody produced by a hybridoma assigned the accession number NITE P-03503.

Claims

1. 1. A method for producing an anti-inhibin monoclonal antibody, comprising: Non-human mammals, excluding mice, A first peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or A second peptide consisting of an amino acid sequence from the 227th to the 252nd positions in the amino acid sequence shown in SEQ ID NO: 3, an amino acid sequence from the 233rd to the 258th positions in the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence from the 234th to the 259th positions in the amino acid sequence shown in SEQ ID NO: 5, with cysteine ​​added to the N-terminus thereof. an obtaining step of obtaining a hybridoma by immunizing the mouse with the above; a superovulation test step in which a sample containing the monoclonal antibody produced by the hybridoma is administered to a female mouse to perform a superovulation test; and a selection step of selecting an anti-inhibin monoclonal antibody that induces superovulation of 15 to 40 eggs per individual in the superovulation test step as the target antibody; The method comprising:

2. The method described in claim 1, wherein the first peptide comprises the amino acid sequence shown in SEQ ID NO:

14.

3. The method described in claim 1, wherein the first peptide consists of the amino acid sequence shown in SEQ ID NO:

1.

4. The method of claim 1, wherein the non-human mammal is a rat.

5. The method described in claim 1, wherein the anti-inhibin monoclonal antibody selected in the selection process is used to induce superovulation in a mammal selected from the group consisting of hamsters, rabbits, ferrets, cows, horses, goats, pigs, and sheep.

6. A breeding reagent comprising an anti-inhibin monoclonal antibody produced by a hybridoma assigned the accession number NITE P-03503.

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