Novel superovulation inducing method for propagation

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 utilizing said method. [Background technology]

[0002] Research into improving reproductive ability has long been conducted mainly with livestock, and by repeated breeding and selection, breeds with high reproductive ability and good growth have become mainstream. However, an increase in the number of fetuses inevitably increases the burden on the mother, which tends to result in poor growth of the offspring, poor resuscitation rate after birth, and a low weaning rate. It has been reported that the administration of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), which is a standard superovulation treatment, increases the number of ovulations, but reduces the pregnancy rate of the treated individuals and does not lead to an increase in the number of offspring (Non-Patent Document 1, etc.).

[0003] The number of ovulations and the number of offspring for each animal species and lineage are regulated by the secretion amount of endogenous follicle-stimulating hormone (FSH). The secretion of endogenous FSH is feedback-regulated by inhibin, and a method of inducing superovulation using antiserum against 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 simultaneously administering AIS with eCG (Patent Document 2 and Non-Patent Document 5). However, the simultaneous administration of AIS and eCG has problems in that the effect varies greatly depending on the lineage, and is ineffective at higher weeks of age.

[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 by the conventional method using eCG / hCG, and more than twice as many eggs can be obtained even in aged mice (Non-Patent Document 6). However, the use of antisera has limitations, such as the problem of 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] JP 2016-155793 A [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, administered them to mice with synchronized sexual cycles, and investigated the effects on pregnancy. As a result, they found that by inducing ovulation without administering hCG after administration of anti-inhibin antibodies, it is possible to prevent developmental inhibition before and after implantation and improve the probability of normal pregnancy and birth. In particular, they found that by using anti-inhibin antibodies with an appropriate range of superovulation effect, especially anti-inhibin monoclonal antibodies, it is possible to achieve higher pregnancy rates and litter sizes.

[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 individual as the desired antibody. [2] A method for producing an anti-inhibin monoclonal antibody described in [1], wherein the peptide is a peptide having 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 individual in a superovulation test using a mouse. [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 having the accession number NITE P-03503. [7] The reagent according to any one of [4] to [6], for administration to a female non-human mammal. [8] The reagent described in [7], wherein the female non-human mammal is a female non-human animal whose sexual cycle is synchronized. [9] The reagent described in [8], wherein synchronization of the sexual cycle is achieved by treatment with any one or more selected from the group consisting of progesterone, prostaglandin F2α, and LH-RH compounds, and derivatives thereof. Effect 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. In addition, anti-inhibin monoclonal antibodies are highly uniform, so stable effects can be obtained, and since there is no need to use individual animals and there are no microbiological problems, they can also be used for SPF animals. The method of the present invention is also effective in elderly animals, and is expected to be effective in many lineages, making it more versatile than conventional methods. [Brief description 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] 1 is an 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 is a graph showing the results of screening tests using superovulation tests after AIMA injection. The number of eggs (number / individual) collected from 1-2 females per clone is shown in gray bars. The black bars show the results of the standard superovulation method (eCG). [Figure 2-2]The results of the superovulation test of AIMA from clones #14 and #19 are shown. The graph shows the number of eggs collected from females injected with each AIMA. After synchronizing the estrus cycle with progesterone injection, AIMA from clones #14 and #29 were injected into two females, respectively. [Diagram 3] This is an experimental scheme for superovulation treatment of 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 sexual cycle by progesterone (P4) injection (B). Treated females were mated with males on the 3rd day (A) and 4th day (B), respectively, and the presence of a vaginal plug was observed on the 4th day (A) and from the 5th to 8th days (B). GnRH was administered to some of the AIS-administered groups on the 6th day (*). [Figure 4] Graph showing distribution of mating rates by successive matings with males. Females were injected with progesterone (P4) and administered 1 / 4AIS / GnRH (A) or 1 / 4AIS (B) or 1 / 6AIS (C) or AIMA (D). Values ​​in bars are 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 mean weight of pups at Caesarean section after treatment and after excess embryo transfer (Mann-Whitey U test). (D) Graph showing number of implantations and cumulative number of pups. Asterisks indicate significantly higher ratio of implantation sites to total (Fisher's exact test). (E) Graph showing percentage of resuscitated pups by birth weight range. (F) Graph showing distribution of birth weight of all pups after each treatment. [Figure 6] FIG. 1 is a graph showing the total number of resuscitated pups per treated female (average number per animal) after several treatments. [Figure 7] Graph showing the number of implantations and live offspring in aged mice treated with AIMA. [Figure 8] 1 is a graph showing the number of live offspring in ICR mice following AIMA treatment. [Figure 9] 1 is a graph showing the mean number of live offspring after i-GONAD treatment in AIMA-treated, AIS-treated and untreated Syrian hamster mice. The asterisk in the figure indicates P<0.05 in the Mann-Whitey U test for the number of live offspring in the control and AIMA-treated groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Inhibin Inhibin is a type of gonadal hormone secreted from the ovaries of females or the testes of males. It is known that inhibin secretion is promoted by the action of follicle-stimulating hormone (FSH), while in the anterior pituitary gland it acts as an activin antagonist and suppresses the secretion of FSH. Inhibin is composed of α-chains and β-chains, which are covalently bonded to each other by disulfide bonds as subunits. There are two types of β-subunits, βA subunits and βB subunits, which combine with α-subunits to form inhibin A and inhibin B. The structure of inhibin is known to have little species-specific difference, i.e., high sequence identity between species. Figure 1-1 shows the alignment of amino acid sequences of inhibin α-subunits from pigs, cows, humans, and rats. SEQ ID NO: 2 shows the amino acid sequence of the porcine inhibin α subunit (NCBI reference number: NP_999354.1), SEQ ID NO: 3 shows the amino acid sequence of the bovine inhibin α subunit (NCBI reference number: NP_776519.2), SEQ ID NO: 4 shows the amino acid sequence of the human inhibin α subunit (NCBI reference number: NP_002182.1), and SEQ ID NO: 5 shows the amino acid sequence of the rat inhibin α subunit (NCBI reference number: NP_036722.1). Figure 1-2 shows the alignment of the amino acid sequences of the inhibin βA subunit. SEQ ID NO: 6 shows the amino acid sequence of the porcine inhibin βA subunit (NCBI reference number: NP_999193.1), SEQ ID NO: 7 shows the amino acid sequence of the bovine inhibin βA subunit (NCBI reference number: NP_776788.1), SEQ ID NO: 8 shows the amino acid sequence of the human inhibin βA subunit (NCBI reference number: NP_002182.1), and SEQ ID NO: 9 shows the amino acid sequence of the rat inhibin βA subunit (NCBI reference number: NP_058824.1). Figures 1-3 show the alignment of the amino acid sequences of the inhibin βB subunit. 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 the amino acid sequences of each subunit of porcine inhibin with the corresponding amino acid sequences of other species. As shown in Table 1, the amino acid sequence identity of the β subunit of inhibin is very high between species, and the α subunit also shows a high sequence identity of over 80%.

[0013] [Table 1]

[0014] As used herein, "inhibin" refers to a protein having an α subunit having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, and a βA subunit having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 6 or a βB subunit having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, and which 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, p. 203-214; Altschul SF et al., 1990, Journal of Molecular Biology, Vol. 215, p. 403-410; Pearson WR et al., 1988, Proc. Natl. Acad. Sci. USA, Vol. 85, p. 2444-2448).

[0016] 2. Method for inducing superovulation in non-human mammals The present invention relates to a method for inducing superovulation in a non-human mammal, which is characterized by administering an anti-inhibin antibody to a female non-human mammal, and inducing superovulation under conditions that do not place an excessive burden 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, characterized in that the method does not comprise 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 of inducing superovulation using inhibin antiserum (AIS), ovulation is induced by administering eCG simultaneously with AIS or by administering hCG after AIS. The present invention is characterized in that eCG or hCG is not used during or after the administration of anti-inhibin antibody. If necessary, a female non-human mammal to which an anti-inhibin antibody has been administered may be cohabited with a male to promote ovulation by utilizing the Whitten effect. Methods that do not use hCG are suitable for the breeding method described below, since 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 of synchronizing the sexual cycle is not particularly limited, and can be performed using, for example, progesterone, prostaglandin F2α, and 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. Preferably, the anti-inhibin antibody is administered 24 to 72 hours after the completion of the synchronization procedure, more preferably 40 to 56 hours after the completion of the synchronization procedure, for example, in the case of administration of progesterone, the anti-inhibin antibody is administered about 48 hours after the completion of two administrations of progesterone.

[0021] The anti-inhibin antibody of the present invention is preferably one that induces superovulation of 40 or less eggs / individual in a superovulation test using mice, and more preferably one that induces superovulation of 15 to 40 eggs / individual. The number of ovulation eggs with conventional antisera (AIS) exceeds 40 or 50 eggs / individual. In the present invention, an antibody that does not induce too many ovulation eggs is used, which has little effect on pregnancy and can increase the number of offspring.

[0022] In this specification, the term "superovulation test" refers to a test carried out by the following method. That is, randomly selected mature female mice are administered 2 mg / mouse of progesterone at 24-hour intervals for two days, 48 ​​hours later, 0.5 mg / mouse of the antibody to be tested is administered, and 48 hours later, 5 IU / mouse of hCG is administered. 16 hours after hCG administration, eggs are collected from the oviduct 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 easily available species can be used. For example, ICR mice, hybrid mice, etc. can be used.

[0023] Anti-inhibin antibodies can be produced by methods known in the art using inhibin or a fragment thereof as an immunogen. As described above, inhibin has a high amino acid sequence identity between species, so the obtained anti-inhibin antibody is likely to cross-react with inhibin derived from a species different from the antigen. Therefore, the origin of inhibin as an immunogen used for antibody production does not necessarily have to be the same species of animal as the subject to which the antibody is administered. Inhibin as an immunogen is not particularly limited, and inhibin derived from any mammal, such as mouse, rat, rabbit, cow, horse, goat, pig, sheep, and human, may be used. When a fragment of inhibin is used as an immunogen, it is preferable that the fragment peptide used contains the amino acid sequence of a portion exposed on the surface 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 by the method described below in the method for preparing an anti-inhibin monoclonal antibody.

[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 at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on August 3, 2021, under the accession number NITE P-03503. The hybridoma is a hybridoma prepared from spleen cells obtained by immunizing a rat with a peptide having the amino acid sequence shown in SEQ ID NO: 1 using the method described in the method for producing an anti-inhibin monoclonal antibody described later.

[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 of 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 having 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 (7th to 24th amino acids) of the amino acid sequence of SEQ ID NO: 1 that has a 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. It is preferable to use a female non-human mammal whose sexual cycle is synchronized. The method of synchronizing the sexual cycle can be the method described above.

[0028] In the second embodiment, it is preferable that eCG is not administered simultaneously with administration of the anti-inhibin monoclonal antibody, and it is even more preferable that the administration of the anti-inhibin monoclonal antibody does not include treatment with either eCG or hCG during or after administration.

[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 is no particular limitation as long as it is a mammal, and it may be a naturally ovulating animal (mouse, rat, hamster, cow, pig, goat, dog, primate, etc.) or a mating ovulating animal (rabbit, cat, ferret, etc.). In particular, it is preferable to use a naturally ovulating animal. It may be an animal with an incomplete sexual cycle, such as a mouse, rat, or hamster. Since naturally ovulating animals have a clear sexual cycle, the number of ovulations can be increased more efficiently by administering anti-inhibin monoclonal antibodies at the timing when the follicles develop. On the other hand, in the case of mating ovulating animals, the sexual cycle is unclear, so it is difficult to determine the timing of administration and it is difficult to induce superovulation with a single administration, but it is possible to induce superovulation 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. Methods for Reproducing Non-Human Mammals The present invention also provides a method for breeding a non-human mammal using the above-mentioned method for inducing superovulation. The breeding method of the present invention includes inducing superovulation in a non-human mammal by the above-mentioned method, and artificially inseminating or mating the non-human mammal to obtain offspring. Preferably, the non-human mammal obtains offspring by natural mating.

[0034] In the case of artificial insemination, for example, 40 to 72 hours after administration of the anti-inhibin monoclonal antibody, the female is placed in the same cage with a vasectomized male. Sperm is directly injected into the oviduct of a female who has been confirmed or will be confirmed to have mated, via transvaginal, transuterine, or abdominal surgery, within 40 to 90 hours after administration of the anti-inhibin monoclonal antibody, to impregnate her and produce offspring.

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

[0036] The method of the present invention produces a large number of normal oocytes and does not place an excessive burden on the mother, compared to superovulation treatment using hormone administration, and therefore the implantation rate is high and the number of live offspring is also high. The offspring obtained have normal body weight, a high survival rate, and grow well.

[0037] 4. Method for producing anti-inhibin monoclonal antibodies The present invention also provides a method for producing an anti-inhibin monoclonal antibody used in the present invention, which comprises immunizing a non-human mammal with a peptide containing inhibin or a fragment thereof to obtain a hybridoma, administering a sample containing the anti-inhibin monoclonal antibody produced by the hybridoma (e.g., a culture supernatant of the hybridoma or ascites obtained by transplanting the hybridoma into a mouse) 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 individual 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 obtained antibody-producing cells with myeloma cells.

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

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

[0041] 5. Reproduction reagents (reagents for inducing superovulation) The anti-inhibin monoclonal antibody prepared as described above is used as a breeding reagent for non-human mammals or a reagent for inducing superovulation in non-human mammals, and the present invention also provides such a breeding reagent or reagent for inducing superovulation in non-human mammals (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 drip infusions, vaginal preparations, suppositories, nasal preparations, sublingual preparations, transdermal preparations, and the like.

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

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

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

[0046] Examples of the solubilizing agent 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, polyoxyethylene hydrogenated castor oil, and the like.

[0048] The isotonicity agent includes sodium chloride, potassium chloride, sugars, glycerin, urea and the like.

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

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

[0051] Examples of the buffer include buffer solutions of phosphates, acetates, carbonates, citrates, and the like.

[0052] Wetting agents include, for example, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

[0053] The reagent of the present invention uses monoclonal antibodies as an active ingredient, and therefore does not require the use of individual animals, is microbiologically clean, highly uniform, and can be produced at low cost compared to conventional antisera. It is also effective in elderly animals, and stable effects can be obtained in many strains. Furthermore, it can be administered at 72-hour intervals, which is longer than the administration interval of antisera (48-hour administration interval), and this has great advantages in terms of work.

[0054] 6. Use of the present invention The present invention can increase the number of offspring obtained by combining with genome editing techniques using ZFN, Talen, CRISPR / Cas9, or other gene modification techniques, enabling more efficient gene modification. For example, in the case of mice, genome editing in laboratory animals is performed by injecting a genome editing reagent such as CRISPR / Cas9 into a fertilized egg, culturing the egg, and then implanting the resulting embryo into a female individual to give birth. By combining this method with the present invention, the number of ovulations in female mice increases, making it possible to obtain fertilized eggs more efficiently. On the other hand, for example, Syrian hamsters have fragile fertilized eggs in an experimental environment, making it difficult to use techniques such as embryo culture and embryo implantation. For such animals, a method can be used in which a genome editing reagent is directly injected into the oviduct containing the fertilized egg without manipulating the fertilized egg in vitro, and genome editing is performed by electroporation of the oviduct and then the animal is allowed to give birth (i-GONAD method). By combining this method with the present invention, the number of offspring born increases, making it possible to obtain genome-edited offspring more efficiently. In particular, since the present invention is a method that places less of a burden on the mother, it is possible to improve the efficiency of the combined technique without affecting the effect of the combined technique. EXAMPLES

[0055] The present invention will be specifically described 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 Standard inbred mice, C57BL / 6JJcl (B6; CLEA Japan), females aged 10-13 weeks and males aged 11-64 weeks were used for all superovulation and mating experiments. In addition, ICR (CLEA Japan) female mice aged 10-20 weeks and vasectomized male mice aged 3-12 months were used to prepare pseudopregnant female mice for embryo transfer experiments.

[0057] Preparation of anti-inhibin monoclonal antibody (AIMA) Mature female rats (Wistar; Japan CLEA) were immunized by injection with a peptide antigen of the porcine inhibin α subunit (Mayo et al., supra, PNAS 1986, Vol. 83, pp. 5849-5853) with a Cys residue (27AA (SEQ ID NO: 1)), 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 by superovulation tests. Finally, selected clonal cells were expanded and cryopreserved. 5 × 10 6 The cells were intraperitoneally administered at 1.0 × 10 cells / mL into nude mice (BALB / cAJcl-nu, CLEA Japan). After 1 to 2 months, ascites was collected and purified using a Protein G Sepharose column (Cytiva), and then used in superovulation experiments as AIMA (5.0 mg / mL).

[0058] Collection of MII oocytes 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, progesterone (P4, Progehormon; Mochida Pharmaceutical Co., Ltd.) was subcutaneously injected at 2 mg (0.08 mL per female) once daily in the evening for 2 days (days 1 and 2) to synchronize the estrous cycle of the females. On day 4, AIS (diluted at 1 / 1, 1 / 2, 1 / 4, and 1 / 8 concentrations, respectively) or AIMA (0.5 mg / 0.1 mL per female) or saline (control) was administered instead of eCG. 48 hours after AIS or AIMA administration, 5 IU / animal of hCG was administered. Sixteen to seventeen hours after administration of hCG, the female oviduct was punctured with an injection needle using 80 μL of in vitro fertilization medium (mHTF) to collect mature MII stage oocytes. Morphologically normal and abnormal oocytes were counted under a stereomicroscope.

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

[0060] As a control group, the vaginal appearance of C57BL / 6 females was examined, and females with gaps and red-pink swelling of the tissues around the vagina were considered to be in proestrus. These females were paired with males overnight and observed the next 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 natural birth. On the morning of day 20, these females were subjected to cesarean section to check for implantation and the number and presence of abnormalities of fetuses. Fetuses that were alive at the time of cesarean section were considered live-born, and fetuses whose lungs turned white and whose breathing became rhythmic during the resuscitation process were considered to be resuscitated. The weight of the pups was also measured to evaluate their health before lactation.

[0061] in vitro fertilization We attempted to transfer excess embryos produced by 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. MII stage oocytes were collected from the female oviducts 16-17 hours after hCG injection, and HTF medium containing hypotaurine (0.11 mg / mL, Merck Millipore) and 0.3% bovine serum albumin (BSA, Calibochem, Merck Millipore) was added, and 1.25 mM reduced glutathione was added. Sperm collected from the cauda epididymis of male mice were suspended in HTF medium containing 1 mg / mL polyvinyl alcohol [PVA] and 0.4 mM methyl-β-cyclodextrin as a substitute for BSA, and cultured in an incubator at 37°C with 5% CO2 for approximately 60 minutes. For insemination, pre-cultured sperm were transferred to 80 μL of insemination solution containing oocytes at a final concentration of 200–400 sperm / μL. After 3–4 h, oocytes were transferred to CZB medium and cultured overnight. The following day, 2-cell stage 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 a cryotube (MS-4501; Sumitomo Bakelite Co., Ltd.) containing 50 μL of vitrification solution (PB1 plus 42.5% (v / v) ethylene glycol, 17.3% (w / v) Ficoll, and 1.0 M sucrose). After 1 min, the cryotube was placed in liquid nitrogen (LN2) at -196 °C. On the day of embryo transfer, the cryotube was retrieved and the cap was quickly removed to allow LN2 to evaporate. The tube was left at room temperature for 2 min, and then 850 μL of 0.75 M sucrose-PB1 at room temperature was gently added. After 4 min, the solution in the tube was mixed 5 times by gentle pipetting. The entire amount of the solution was then transferred to an empty plastic dish. After 1–3 min, the embryos were collected and transferred to 0.25 M sucrose-PB1. After equilibration for 1–3 min, 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 membrane and clear cytoplasm) were considered viable. Viable embryos were cultured at 37 °C in humidified air with 5% CO2 until embryo transfer (<3 h).

[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 parturition, 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 Caesarean section and fetal number and viability were assessed.

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

[0065] 2.Results AIMA creation and superovulation test Each sample of cell supernatant extracted from 40 hybridoma clones was subjected to a superovulation test, except for sample #40 (Figure 2-1). Progesterone (P4, Progehormon; Mochida Pharmaceutical) was administered subcutaneously to C57BL / 6JJcl mice at 2 mg / mouse (0.08 mL) once a day 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 administration of the sample, 5 IU / mouse of hCG was administered, and 16 to 17 hours after administration of hCG, the ampulla of the female oviduct was punctured with an administration needle using 80 μL of mHTF, mature MII stage eggs were collected, and the number of eggs was counted under a stereomicroscope. The number of eggs obtained for each sample is shown in Figure 2-1. The number of eggs obtained by administering 7.5 IU / mouse of eCG instead of the sample is also shown.

[0066] For mice that had a large number of MII-stage eggs in the above test, samples obtained 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 the superovulation test was performed again (Figure 2-2). As a result, 31.3 eggs were obtained from the sample obtained from #29 even at 1 / 2 volume, and eggs were obtained stably regardless of the dosage. Sample #29 was selected and used as AIMA for the following experiment. Hybridoma #29 was internationally deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center and was assigned the accession number NITE P-03503 on August 3, 2021.

[0067] Results of superovulation trials using various procedures 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 ovulation rate in females was 89% (33 / 37) (Table 2). On the other hand, when progesterone was administered to synchronize the sexual cycle (2P4 in the table), diluted AIS or AIMA was administered 48 hours later, and hCG was administered 48 hours later, the highest number of normal oocytes (51-52 oocytes) was obtained with AIS and 1 / 2 diluted AIS. AIMA yielded 24.6 normal oocytes, which was the same as 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 were housed with sexually experienced males based on the observation of the change in color of the vaginal appearance, mating was confirmed the next day in 32% of the cases, and the pregnancy rate was 100% (Table 3). In the eCG / hCG administration group, the mating rate was 60-63%, but the pregnancy rate tended to be low at 40-50% (Fig. 3A). In addition, the mating rate was significantly higher in the group administered GnRH instead of hCG. When the females were housed with males after the administration of a 1 / 4 dilution of AIMA or AIS after synchronizing the estrus cycle, the mating rate improved to 71-100% in a total of 4 days (Fig. 3B). The timing of mating was distributed from the 6th to the 8th day, with the 7th day being the most common in the AIS group and the 7th to 8th days being the most common in the AIMA group (Fig. 4).

[0070] Effect on size and number of resuscitated pups In the untreated control group, the number of pups born per female (litter size) was 8.6, and the 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). On the other hand, in the eCG / hCG-treated group, the number of implantations did not increase, and the number of live pups born per female was low at 0.5 to 5 (Figure 5B). When GnRH was administered instead of hCG, the mating rate was high in both eCG and AIS, but the resuscitation rate after cesarean section decreased and the number of resuscitated pups tended to be small (Figure 5B). In the AIS group, the number of implantations was high, but the number of surviving pups did not exceed that of the control group (Figure 5A, B). On the other hand, in the AIMA group, the number of implantations and the number of live pups were significantly higher than in 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 results including mating rate (79%), number of implantations (13.8 eggs), and number of live offspring (11.3 pups) were similarly efficient.

[0071] [Table 3]

[0072] Maximum number of offspring born through embryo transfer To determine the maximum number of pups that can develop in a C57BL / 6 mother, we attempted to transfer an excess number of embryos. Two-cell stage embryos produced by in vitro fertilization were vitrified, retrieved, and transferred. When seven embryos were transferred per oviduct (14 embryos / individual), the birth rate was 68% and the number of live pups was 9.6 per female (Table 4). When an excess number of embryos (14 embryos / individual), 14 per oviduct, was transferred, the birth rate was 51% and the number of live pups was 13.3 (Table 4, Figure 5). This number of live pups was equivalent to 11.9 when AIMA was administered.

[0073] [Table 4]

[0074] Assessment of pup weight The weight of pups at birth was significantly lower in all treatment groups than in 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 incidence of post-implantation development tended to be higher in the AIS and embryo transfer groups (Figure 5D). In addition, when the weight of all pups born and the rate of failure to resuscitate were examined, approximately two-thirds of pups weighing less than 0.8 g failed to be resuscitated (Figure 5E). When the weight ratio of all pups born in each treatment group was examined, more than 20% of pups weighed less than 0.8 g in groups with significantly lower incidence of post-implantation development (eCG / hCG (1IU), 2P4-1 / 4AIS / GnRH, 2P4-1 / 4AIS, and 2P4-1 / 6AIS) (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 It was confirmed that the use of AIMA can increase the number of mice born. It is known that standard eCG / hCG treatment results in low pregnancy rates, no increase in implantation numbers, and light fetal weights. On the other hand, when AIS was administered instead of eCG, more normal eggs were obtained, and the mating rate, pregnancy rate, and implantation rate also increased, but the higher the number of ovulations, the lower the birth weight of the pups and the lower the survival rate. In contrast, AIMA treatment not only improved the mating rate, implantation number, and number of pups born, but also suggested that there was no excessive burden on the mother, based on the fetal weight, resuscitation rate, and weaning rate. From the above, unlike existing superovulation reagents, AIMA has little adverse effect on implantation and pregnancy, and is effective as a breeding reagent, and is expected to be applied to inbred mice and cases where breeding is not progressing well.

[0076] Example 2: Effects 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 FIG. 7, even in aged mice, AIMA showed favorable improvements in mating rate, pregnancy rate, number of implantations, and number of live offspring compared to the standard method.

[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 litter size in F344 rats Ten female rats, F344 / Jcl, aged 8 weeks or older, in metestrus or estrus were intraperitoneally administered 0.2 mL / rat of AIMA (day 1). On day 2, they were housed together with male rats, F344 / Jcl, and mated. From the following day, vaginal plugs were checked every morning, and those with vaginal plugs were separated from the males. Eight rats were confirmed to be pregnant. From day 19 to day 21 after the vaginal plug was confirmed, progesterone was subcutaneously administered 0.5 mL / rat every day, and the rats were euthanized and then the offspring were confirmed by Caesarean section. The results are shown in Table 7. The control in the table shows the number of offspring of normal F344 rats. It was confirmed that the administration of AIMA increased the number of offspring by nearly twice that of normal F344 rats.

[0083] [Table 7]

[0084] Example 6: Effect of AIMA administration on Syrian hamster litter size AIMA was administered intraperitoneally at 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 animals were housed with the same number of male Syrian hamsters and mated. After mating was confirmed on day 4, the animals were separated from the males. On day 15 after mating was confirmed, the animals 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 that were 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 of genome-edited mice using i-GONAD method in Syrian hamsters In the same manner 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 reagent 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 hamsters were allowed to give birth naturally, and the number of live births was confirmed 2-3 days after birth. The results are shown in Table 9 and Figure 9. "AIS" in the table and figure indicates the results of the group administered 0.2 mL of AIS instead of AIMA, and the control group was not administered AIMA or AIS. The asterisk in the figure indicates that the Mann-whitey U test of the number of live births in the control group and the AIMA-administered group was P<0.05. In reality, as shown in the figure, P=0.04. As shown in Table 9 and Figure 9, it was confirmed that the number of offspring 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 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, so AIMA can react with inhibin of various animal species and function in various 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, and the number of offspring born can be improved. 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 not been successful in breeding in the past.

[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, the method being 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 according to [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 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 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 of [1] to [5], wherein the anti-inhibin antibody is produced using a peptide comprising 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 individual as the desired antibody.

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

[12] , wherein the peptide is a peptide having 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 comprising an anti-inhibin monoclonal antibody.

[15] The reagent described in

[14] , wherein the anti-inhibin monoclonal antibody uses a peptide including the amino acid sequence shown in SEQ ID NO:1 as an antigen and induces 15 to 40 superovulations 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 having the accession number NITE P-03503.

Claims

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

2. The method according to claim 1, wherein the peptide is a peptide comprising 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 of claim 1, wherein the antibody is used to induce superovulation by administration to a female non-human mammal.

4. Contains an anti-inhibin monoclonal antibody, The anti-inhibin monoclonal antibody induces superovulation of 15 to 40 eggs per mouse in a superovulation test. Reagents for breeding non-human mammals.

5. The reagent described in claim 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 claim 4, wherein the anti-inhibin monoclonal antibody is an antibody produced by a hybridoma assigned the accession number of NITE P-03503.

7. The method of claim 4 for administration to a female non-human mammal.

8. The method according to claim 7, wherein the female non-human mammal is a female non-human animal whose sexual cycle is synchronized.

9. The reagent according to claim 8, wherein the synchronization of the sexual cycle is achieved by treatment with any one or more selected from the group consisting of progesterone, prostaglandin F2α, and LH-RH compounds, and derivatives thereof.

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