Ruminant conception chance-improving composition

Abstract

The present invention provides: a conception chance-improving composition for normalization of EGF concentration profile and conception chance improvement for a ruminant; and a method for improving ruminant conception chance using said composition. The present invention is: a ruminant conception chance-improving composition comprising, as an active ingredient, a peptide or polypeptide including the amino acid sequence represented by SEQ ID NO: 1; a ruminant conception chance-improving composition having, as an active ingredient, bovine osteopontin protein, a mutant of bovine osteopontin protein, or a partial peptide of these proteins, in which the protein, the mutant, and the partial peptide include the amino acid sequence represented by SEQ ID NO: 1; and a method for improving ruminant conception chance in which either of the conception chance-improving compositions is administered to the fornix vaginae, or the vicinity thereof, of the vaginal portion of a ruminant on an estrous day or the period immediately before / after such.

Description

Composition for improving fertility in ruminants

[0001] The present invention relates to compositions for improving fertility in ruminants, such as cattle. This application claims priority to U.S. Patent Application No. 63 / 403,303, filed September 2, 2022, the contents of which are incorporated herein by reference.

[0002] A decline in the reproductive ability of dairy cows is directly linked to a decline in milk production, and is therefore one of the main causes of deterioration in farm management. Among cows that have difficulty conceiving (subfertile cows), cows that do not become pregnant even after repeated mating, despite showing no abnormalities in reproductive examinations and no clinical symptoms or abnormalities in the reproductive cycle, are called repeat breeder (RB) cows. RB cows experience significant delays in pregnancy due to repeated mating, which causes significant economic losses in dairy farming. The biggest obstacle to addressing RB cow infertility is the lack of clarity about the cause of infertility, and identifying the abnormalities that lead to this problem will lead to solutions.

[0003] Many RB cattle are thought to have impaired uterine function, the site of embryonic development. For example, the expression level of epidermal growth factor (EGF), a growth factor in the endometrium, is one indicator of uterine function. Approximately 70% of RB cattle exhibit abnormal endometrial EGF concentration profiles, specifically, the increase in EGF concentration observed on days 2-4 and 13-14 after estrus disappears or decreases. Cows with endometrial EGF concentrations below 4.7 ng / g tissue weight on day 3 after estrus have low conception rates (Non-Patent Documents 1 and 2). Correcting this abnormal endometrial EGF concentration profile also restores fertility (Non-Patent Document 2). Because EGF is involved in embryonic development and the mechanism by which the mother recognizes the presence of an embryo, its abnormality is thought to be directly linked to reduced fertility. Aside from measures to address bacterial infections in the uterus, which are common in cattle, correcting the abnormal endometrial EGF concentration profile is currently the only method for restoring fertility in RB cattle by targeting the uterus.

[0004] To normalize abnormal endometrial EGF concentration profiles, treatments combining hormone preparations are currently used. This approach is expected to be a new option for treating bovine infertility, as it is consistently effective. It has been empirically proven that mating bulls with RB cows that have failed to conceive despite repeated artificial insemination (natural mating) results in high conception rates. Therefore, when a protein contained in seminal plasma, a liquid component of semen, was administered to RB cows, endometrial EGF concentrations were normalized and fertility was restored (Non-Patent Document 3). This effect was attributed to osteopontin (OPN) contained in seminal plasma (Non-Patent Document 4). Furthermore, when OPN prepared from milk was administered to RB cows (Non-Patent Document 5) or when a recombinant OPN protein was administered to RB cows, the endometrial EGF concentration profile was normalized and fertility was improved, similar to that achieved with seminal plasma-derived OPN.

[0005] Katagiri and Takahashi, Theriogenology, 2004, vol.62, p.103-112.Katagiri and Takahashi, Animal Reproduction Science, 2006, vol.95, p.54-66.Badrakh et al., Journal of Reproduction and Development, 2020, vol.66(2), p.149-154.Badrakh et al., Japanese Journal of Veterinary Research, 2020, vol.68(2), p.91-103.Kyaw et al., Theriogenology, 2022, vol.184, p.26-33.

[0006] RB cows, which have abnormal endometrial EGF concentration profiles and do not become pregnant even after repeated mating, account for 5-6% of all dairy cows. Furthermore, approximately 25% of dairy cows have abnormal endometrial EGF concentration profiles similar to those of RB cows 60 days after calving, which is the start of the next breeding period after calving. Postpartum cows also include cattle other than dairy cows, such as beef cattle. In addition, even among livestock other than cattle, there are subfertile females that do not become pregnant even after repeated mating, despite showing no abnormalities in reproductive examinations and no clinical symptoms or abnormalities in the reproductive cycle. In response to these issues, if fertility could be improved by normalizing the endometrial EGF concentration profile, it is expected that economic losses could be eliminated.

[0007] The present invention aims to provide a composition for improving fertility for normalizing the endometrial EGF concentration profile and improving fertility in ruminants such as cows, and a method for improving fertility in ruminants such as cows using the composition.

[0008] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that when a peptide containing the integrin-binding domain (SVAYGLK: SEQ ID NO: 1) in bovine OPN was administered to RB cows, the endometrial EGF concentration profile was normalized and fertility improved, similar to the case of recombinant OPN protein, and that administration of the peptide containing the integrin-binding domain or the full-length bovine OPN protein also improved fertility in postpartum cows, thereby completing the present invention.

[0009] That is, the present invention provides a composition for improving fertility in ruminants, a method for improving fertility in ruminants, and an artificial insemination straw. [1] A composition for improving fertility in ruminants, comprising, as an active ingredient, a peptide or polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK). [2] The composition for improving fertility according to [1], wherein the peptide or polypeptide comprises the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK). [3] The composition for improving fertility according to [1] or [2], wherein the peptide or polypeptide is a partial peptide of bovine osteopontin. [4] A composition for improving fertility in ruminants, comprising, as an active ingredient, a bovine osteopontin protein, a variant of bovine osteopontin protein, or a partial peptide of these proteins, wherein the protein, the variant, and the partial peptide comprise the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK). [5] The composition for improving fertility according to [4] above, wherein the protein, the variant, and the partial peptide comprise the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK). [6] The composition for improving fertility according to [4] or [5] above, wherein the amino acid sequence of the variant of bovine osteopontin protein is an amino acid sequence having 90% or more sequence identity with the amino acid sequence of bovine osteopontin protein. [7] The composition for improving fertility according to [4] above, wherein an active ingredient is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2. [8] The composition for improving fertility according to any of [1] to [7] above, wherein the ruminant is a postpartum ruminant. [9] The composition for improving fertility according to any of [1] to [8] above, wherein the ruminant is a cow, goat, sheep, deer, or giraffe.

[10] The composition for improving fertility according to [8] above, wherein the ruminant is a cow 45 to 80 days postpartum.

[11] The composition for improving fertility according to any of [1] to [9] above, wherein the ruminant is a subfertile cow.

[12] The composition for improving fertility according to

[11] above, wherein the subfertile cow is a cow in which the increase in epidermal growth factor concentration in the endometrium, which is seen in normally conceived cows on days 2 to 4 from estrus, has disappeared or is reduced.

[13] The composition for improving fertility according to

[11] , wherein the low-fertility cow is a cow having an endometrial epidermal growth factor concentration per tissue weight on the third day from estrus of less than 4.7 ng / g-tissue.

[14] The composition for improving fertility according to

[11] , wherein the active ingredient is a protein that is a mutant of bovine osteopontin protein and comprises the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), a partial peptide of bovine osteopontin protein and comprises the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), or a partial peptide of a mutant of bovine osteopontin protein and comprises the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), and the low-fertility cow is a repeat breeder cow.

[15] The fertility-enhancing composition according to any one of [1] to

[14] , wherein administration of the fertility-enhancing composition normalizes the concentration profile of epidermal growth factor in the endometrium over a period corresponding to one estrus cycle from the day of administration.

[16] A method for improving the fertility of a ruminant, comprising administering the fertility-enhancing composition according to any one of [1] to

[15] to the vaginal fornix or its vicinity in the vaginal part of the ruminant on the day of estrus and for a period corresponding to the day before and after estrus.

[17] The method for improving fertility according to

[16] , wherein the fertility-enhancing composition is a solution obtained by dissolving the active ingredient in 0.1 to 20 mL of a solution that can be administered to a ruminant, and the solution that can be administered to a ruminant is a solution that can be administered to the vagina, cervix, and uterus of a ruminant, and is a solution obtained by dissolving water, a buffer solution, the semen of the ruminant, or any of these with an appropriate and pharmaceutically acceptable additive that can be administered to the vagina, cervix, and uterus of the ruminant.

[18] The method for improving fertility according to

[16] or

[17] , wherein the amount of the active ingredient contained in the fertility-enhancing composition is 40 nmol or more.

[19] The method for improving fertility according to any of

[16] to

[18] , wherein the fertility-enhancing composition is administered before, after, or simultaneously with artificial insemination.

[20] The method for improving fertility according to any one of

[16] to

[18] , wherein the ruminant is a ruminant that undergoes natural mating.

[21] The method for improving fertility according to any one of

[16] to

[18] , wherein the ruminant is a ruminant into which a fertilized egg is implanted.

[22] The method for improving fertility according to any one of

[16] to

[21] above, wherein the ruminant is a cow, goat, sheep, deer, or giraffe.

[23] An artificial insemination straw filled with the fertility improvement composition according to any one of [1] to

[15] above, wherein the fertility improvement composition is a solution obtained by dissolving the active ingredient in 0.1 to 20 mL of a solution that can be administered to a ruminant, and the solution that can be administered to a ruminant is a solution that can be administered to the vagina, cervix, and uterus of a ruminant, and is a solution obtained by dissolving water, a buffer solution, semen of the ruminant, or any of these with an appropriate pharmaceutically acceptable additive that can be administered to the vagina, cervix, and uterus of the ruminant.

[0010] According to the present invention, it is possible to normalize the endometrial EGF concentration profile in ruminants with an abnormal endometrial EGF concentration profile or in postpartum ruminants, thereby improving fertility.

[0011] 1 is a diagram showing a schematic representation of the change over time (solid line) in the endometrial EGF concentration of RB cows from after estrus. FIG. 2 is a diagram showing the schedule of OPN treatment in Reference Example 1. FIG. 3 is a diagram showing the results of measurement of the endometrial EGF concentration on the third day after treatment of RB cows that were treated with PBS, SP, and rOPN in Reference Example 1. FIG. 4 is a diagram showing a schematic representation of the structure of the partial peptide or partial protein of OPN used in Example 1. FIG. 5 is a diagram showing the results of measurement of the endometrial EGF concentration on the third day after treatment of RB cows that were treated with PBS, rOPN, peptide 1, peptide 2, peptide 3, and C-rOPN in Example 1. FIG. 6 is a diagram showing the schedule of OPN treatment in Example 4.

[0012] Hereinafter, embodiments of the present invention will be specifically described.

[0013] In the present invention and the present specification, "X １ ~X ２ (X １ and X ２ is X １ <X ２ "X" is a real number that satisfies １ More than X ２ means "below."

[0014] In the present invention and this specification, the term "polypeptide" refers to a polymer of 20 or more amino acids bonded together by peptide bonds, and the term "peptide" refers to a polymer of 2 to 19 amino acids bonded together by peptide bonds.

[0015] In the present invention and the present specification, unless otherwise specified, "osteopontin (OPN)" collectively refers to OPN derived from various tissues of ruminants. For example, "bovine osteopontin (OPN)" collectively refers to OPN derived from various bovine tissues (e.g., seminal plasma, milk, urine, blood, and bone tissue). OPN includes various isoforms obtained by alternative splicing, and also includes OPN that has undergone post-translational modifications. Examples of post-translational modifications include phosphorylation, glycosylation, methylation, acetylation, nitrosylation, and ubiquitination. OPN also includes not only natural OPN (produced in the animal's body) but also recombinant proteins.

[0016] In the present invention and this specification, the term "osteopontin protein" refers to the full-length mature osteopontin protein. For example, it refers to a protein consisting of the region from positions 17 to 278 of the amino acid sequence of bovine OPN set forth in SEQ ID NO: 4 (Uniprot ID: P31096). The N-terminal region from positions 1 to 16 of the amino acid sequence set forth in SEQ ID NO: 4 is a signal sequence and is therefore cleaved in the mature protein that is secreted.

[0017] In the present invention and this specification, unless otherwise specified, "osteopontin protein variant" or "osteopontin variant" means a protein in which one or more of the amino acids that make up the osteopontin protein have been substituted, deleted, or added.

[0018] In the present invention and this specification, "amino acid deletion in a polypeptide (protein) or peptide" means that some of the amino acid residues constituting the polypeptide or peptide are lost (removed). In the present invention and this specification, "amino acid substitution in a polypeptide (protein) or peptide" means that an amino acid residue constituting the polypeptide or peptide is replaced with a different amino acid residue. In the present invention and this specification, "amino acid addition in a polypeptide (protein) or peptide" means that a new amino acid residue is inserted into the polypeptide or peptide.

[0019] In the present invention and this specification, "estrus" of a ruminant means a physiological state in a female ruminant in which mating is possible, and typically, behaviors and signs specific to ruminants that allow mating are observed. For example, estrus in cattle occurs periodically at intervals of 18 to 24 days, with each estrus lasting from several hours to a day. In the present invention and this specification, the "day of estrus" of a ruminant means the day on which behaviors and signs specific to ruminants that are observed during the estrus period are confirmed.

[0020] In the present invention and the specification, the term "abnormal endometrial EGF concentration profile" refers to an abnormality in which the periodic increase in EGF concentration in the endometrium of a ruminant animal disappears or decreases. For example, in cattle, this refers to an abnormality in which the increase in concentration (which appears as peaks on the concentration profile) seen in normal cattle on days 2 to 4 and days 13 to 14 after estrus disappears or decreases. In normal cattle, the endometrial EGF concentrations at both peaks are generally within the range of 4.70 to 13.50 ng / g-tissue. In the present invention and the specification, the endometrial EGF concentration was measured on day 3 after estrus or artificial insemination, and cattle with a concentration of less than 4.70 ng / g-tissue were determined to have an abnormal endometrial EGF concentration profile.

[0021] In the present invention and this specification, "normalization of the endometrial EGF concentration profile" means that in a ruminant that had an abnormal endometrial EGF concentration profile, a periodic increase in EGF concentration in the endometrium, as seen in normal ruminants, is confirmed. In the case of cattle, this means that in a cattle that had an abnormal endometrial EGF concentration profile, a periodic increase in EGF concentration, as seen in normal cattle, on days 2 to 4 and days 13 to 14 after estrus, is confirmed. When the endometrial EGF concentration on day 3 after estrus or the day of artificial insemination of a cattle that had an abnormal endometrial EGF concentration profile is 4.70 ng / g-tissue or higher, it can be confirmed that the endometrial EGF concentration profile of the cattle has been normalized.

[0022] The endometrial EGF concentration profile of a ruminant can be determined by collecting endometrial samples from the ruminant over time during the estrous cycle and measuring the amount of EGF in the tissue. Endometrial samples can be collected using a commonly used biopsy instrument, as described in the Examples below. The amount of EGF in endometrial tissue can be measured using an ELISA method or the like, as described in Non-Patent Documents 1 to 4.

[0023] In the present invention and the specification of this application, "subfertile cows" means "female cows that have difficulty becoming pregnant, although no abnormalities have been detected in their reproductive organs or estrous cycle," and "RB cows" means "female cows that have no abnormalities in their reproductive organs or estrous cycle, but have not become pregnant even after three or more inseminations." These conditions are usually diagnosed after about 150 days after calving.

[0024] <Composition for Improving Fertility> The composition for improving fertility of this embodiment contains, as an active ingredient, a peptide or polypeptide including the SVAYGLK motif (SEQ ID NO: 1), which is an integrin-binding motif. As shown in the Examples below, the SVAYGLK motif is a domain present in OPN and is involved in the resolution of abnormal endometrial EGF concentration profiles by OPN.

[0025] A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (a peptide consisting only of the SVAYGLK motif) can be used as an active ingredient in the composition for improving fertility of this embodiment. By administering this peptide to the vagina of a ruminant, the effect of normalizing the endometrial EGF concentration profile and improving the conception rate can be obtained. In other words, the SVAYGLK motif peptide has the ability to normalize the endometrial EGF concentration profile and improve the conception rate.

[0026] The active ingredient of the fertility-enhancing composition of this embodiment is not particularly limited, as long as it has an SVAYGLK motif and is capable of normalizing the endometrial EGF concentration profile and improving the conception rate. Whether a substance has the ability to normalize the endometrial EGF concentration profile and improve the conception rate can be determined by intravaginally administering the substance to a ruminant confirmed to have an abnormal endometrial EGF concentration profile on the day of estrus (including one day before and after the day of estrus) or at the time of artificial insemination, and measuring the endometrial EGF concentration or examining the conception rate at a time corresponding to the period in which normal animals show a cyclic increase in endometrial EGF (in the case of cattle, from the third day after the day of estrus or artificial insemination). If the endometrial EGF concentration on the third day after the day of estrus or artificial insemination is 4.70 ng / g-tissue or higher, the substance is determined to have the ability to normalize the endometrial EGF concentration profile.

[0027] The active ingredient of the fertility improving composition of this embodiment is not particularly limited as long as it contains the amino acid sequence represented by SEQ ID NO: 1, and may be a peptide or a polypeptide (protein).

[0028] An example of an active ingredient of the composition for improving fertility of this embodiment is a partial protein (which may be a partial peptide) of OPN that contains the SVAYGLK motif. Specifically, this includes a peptide or polypeptide consisting of a partial region containing the SVAYGLK motif in the amino acid sequence represented by SEQ ID NO: 4 (the region from amino acids 155 to 161 of the amino acid sequence of SEQ ID NO: 4).

[0029] As an active ingredient of the fertility-enhancing composition of this embodiment, a peptide or polypeptide consisting of a partial region containing both the RGD motif (the region from positions 152 to 154 of the amino acid sequence of SEQ ID NO: 4) and the SVAYGLK motif in OPN is particularly preferred. By having both of the two integrin-binding motifs in OPN, it is possible to have a more excellent ability to normalize the endometrial EGF concentration profile and improve fertility rates than peptides containing only the SVAYGLK motif. Examples of such peptides include peptides or polypeptides containing the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK).

[0030] The active ingredient of the fertility-enhancing composition of this embodiment may be a partial protein (or a partial peptide) of an OPN variant, which contains the SVAYGLK motif and has the ability to normalize the endometrial EGF concentration profile and improve the conception rate. The OPN variant is not particularly limited, as long as it does not impair the ability of the SVAYGLK motif to normalize the endometrial EGF concentration profile and improve the conception rate. Examples of OPN variants include proteins consisting of an amino acid sequence that shares 80% or more, preferably 90% or more but less than 100%, more preferably 95% or more but less than 100%, and even more preferably 98% or more but less than 100% sequence identity with the full-length amino acid sequence of OPN. The active ingredient of the fertility-enhancing composition of this embodiment is preferably a partial protein (which may be a partial peptide) of a protein consisting of an amino acid sequence that has 80% or more, preferably 90% or more but less than 100%, more preferably 95% or more but less than 100%, and even more preferably 98% or more but less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 4, which contains the SVAYGLK motif and has the ability to normalize the endometrial EGF concentration profile and improve the conception rate.The active ingredient of the fertility-enhancing composition of this embodiment is particularly preferably a partial protein (which may be a partial peptide) of a protein consisting of an amino acid sequence that has 80% or more, preferably 90% or more but less than 100%, more preferably 95% or more but less than 100%, and even more preferably 98% or more but less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 4, which contains the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK) and has the ability to normalize the endometrial EGF concentration profile and improve the conception rate.

[0031] The active ingredient of the fertility-enhancing composition of this embodiment is preferably a partial protein (which may be a partial peptide) of a protein consisting of an amino acid sequence that has 80% or more, preferably 90% or more but less than 100%, more preferably 95% or more but less than 100%, and even more preferably 98% or more but less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 4, which contains the SVAYGLK motif and has at least one of the ability to normalize the endometrial EGF concentration profile and the ability to improve the conception rate.The active ingredient of the fertility-enhancing composition of this embodiment is particularly preferably a partial protein (which may be a partial peptide) of a protein consisting of an amino acid sequence that has 80% or more, preferably 90% or more but less than 100%, more preferably 95% or more but less than 100%, and even more preferably 98% or more but less than 100% sequence identity with the amino acid sequence represented by SEQ ID NO: 4, which contains the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK) and has at least one of the ability to normalize the endometrial EGF concentration profile and the ability to improve the conception rate.

[0032] The active ingredient of the fertility-enhancing composition of this embodiment may be an OPN protein, or a mutant of the OPN protein that contains an SVAYGLK motif, preferably the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK), and has the ability to normalize the endometrial EGF concentration profile and improve the conception rate.

[0033] The sequence identity (homology) between amino acid sequences is determined by aligning two amino acid sequences with gaps at the insertion and deletion sites so that the most corresponding amino acid residues are identical, and calculating the percentage of identical amino acid residues relative to the entire amino acid sequence excluding gaps in the resulting alignment. The sequence identity between amino acid sequences can be determined using various homology search software known in the art. For example, the sequence identity value of amino acid sequences can be calculated based on the alignment obtained using the known homology search software BLASTP.

[0034] The peptide or polypeptide serving as an active ingredient of the composition for improving fertility of this embodiment may have other amino acid sequence portions in addition to a portion derived from a partial region of OPN, such as a peptide consisting of an SVAYGLK motif or the amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK). The other amino acid sequence portion may be added to the N-terminal side or the C-terminal side of the portion derived from the partial region of OPN.

[0035] Examples of such other amino acid sequence portions include various signal peptides and various tag peptides. These signal peptides and tag peptides can be appropriately selected from various signal peptides and tag peptides commonly used in the production of recombinant proteins. Examples of such tag peptides include His tags, HA (hemagglutinin) tags, Myc tags, and Flag tags.

[0036] Generally, short-chain synthetic peptides have problems such as being easily decomposed and having low stability. Furthermore, the addition of charges to the N-terminus or C-terminus during the production process of the synthetic peptide may affect the function of the peptide itself. To solve these problems, when the active ingredient of the fertility-enhancing composition of this embodiment is a fragment peptide of OPN (e.g., a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2), various measures known in the art, such as cyclization, modification of amino acid residues, and substitution with mirror (optical) isomers, can be taken.

[0037] Cyclization of peptides can mimic natural peptides, increase their stability in vivo, and increase their resistance to hydrolysis.

[0038] Modification of amino acid residues at the N-terminus or C-terminus is also preferred. By removing the charge at the N-terminus or C-terminus of a synthetic peptide, it is possible to impart properties equivalent to those of a natural peptide. Therefore, when the active ingredient of the fertility-enhancing composition of this embodiment is a synthetic peptide, the terminal charge can be removed by acetylating the N-terminus or amidating the C-terminus. Furthermore, these modifications make the peptide less susceptible to degradation by peptidases and improve stability.

[0039] All amino acids contained in natural peptides are L-isomers, but by replacing them with their mirror isomers (D-isomers), the function of the peptide can be modified. Even when the active ingredient of the fertility-enhancing composition of this embodiment is a synthetic peptide, improving stability can be expected by replacing some or all of the amino acids with D-amino acids. In addition, some or all of the constituent amino acids of the synthetic peptide as the active ingredient can also be replaced with artificial amino acids.

[0040] When the active ingredient of the fertility-enhancing composition of this embodiment is a peptide, it can be easily synthesized using commonly used peptide synthesis techniques, and is economically efficient. When the active ingredient of the fertility-enhancing composition of this embodiment is a protein, it can be easily produced using a commonly used expression system such as Escherichia coli.

[0041] The fertility-enhancing composition of this embodiment may be a composition consisting solely of the active ingredient, or may be a formulation containing the active ingredient and various additives. Examples of such additives include commonly used excipients, binders, lubricants, disintegrants, fluidizers, solvents, solubilizers, buffers, suspending agents, emulsifiers, isotonicity agents, stabilizers, preservatives, antioxidants, flavoring agents, and colorants. The formulation of the fertility-enhancing composition of this embodiment is not particularly limited, and may be a solid formulation such as powder, granules, or tablets, or a liquid formulation. Such liquid formulations also include frozen formulations.

[0042] The fertility-enhancing composition of this embodiment may contain, in addition to the active ingredient or the active ingredient and an additive, a semen component of the ruminant to which the fertility-enhancing composition is to be administered. The ruminant semen component may be semen itself or a fraction such as seminal plasma. For example, a mixture of semen used for artificial insemination with the active ingredient is also included in the fertility-enhancing composition of this embodiment.

[0043] When the fertility-enhancing composition of this embodiment is administered to a ruminant animal with an abnormal endometrial EGF concentration profile, the endometrial EGF concentration profile is normalized over the course of one estrus cycle from the time of administration. In cattle, the period of one estrus cycle varies depending on the breed of cattle, but is approximately 20 to 30 days, and most often 21 days.

[0044] <Method for Improving Fertility in Ruminants> The method for improving fertility in ruminants of this embodiment is a method for administering the fertility-improving composition of this embodiment to the vaginal fornix or its vicinity in the vaginal part of a ruminant on the day of estrus and the period before and after. By directly administering the fertility-improving composition of this embodiment to the vaginal fornix or its vicinity in the vaginal part of a ruminant, it can efficiently exert its effect on the endometrium.

[0045] The fertility-enhancing composition of this embodiment is administered to ruminants on the day of estrus and the period before and after estrus. By administering the fertility-enhancing composition of this embodiment before and after estrus, the effects of normalizing abnormal endometrial EGF concentration profiles and improving conception rates are more fully exerted. The fertility-enhancing composition of this embodiment is administered, for example, within a period from 5 days, preferably 4 days, more preferably 3 days, and even more preferably 2 days before the day of estrus to 5 days, preferably 4 days, more preferably 3 days, and even more preferably 2 days after the day of estrus. It is particularly preferred that the fertility-enhancing composition of this embodiment be administered within a period from 1 day before the day of estrus to 1 day after the day of estrus.

[0046] The ruminant to which the fertility-improving composition of this embodiment is administered may be a ruminant in which estrus or ovulation has been artificially induced by hormone administration, or may be a ruminant in natural estrus. By administering the composition to a ruminant in natural estrus, the conception rate of natural mating is also improved.

[0047] The fertility-improving composition of this embodiment is preferably administered to ruminants to be artificially inseminated. When artificial insemination is performed, the fertility-improving composition of this embodiment may be administered before, after, or simultaneously with artificial insemination. The fertility-improving composition of this embodiment is administered, for example, within a period from 5 days, preferably 4 days, more preferably 3 days, even more preferably 2 days, and even more preferably 1 day before artificial insemination to 5 days, preferably 4 days, more preferably 3 days, even more preferably 2 days, and even more preferably 1 day after artificial insemination. The fertility-improving composition of this embodiment may be administered immediately before artificial insemination, immediately after artificial insemination, or at the time of artificial insemination. For example, by mixing the fertility-improving composition of this embodiment with semen for artificial insemination, the fertility-improving composition can be administered simultaneously with artificial insemination.

[0048] The ruminant to which the fertility-enhancing composition of this embodiment is administered may be a ruminant that undergoes natural mating. Even in the case of natural mating, the conception rate can be improved by administering the composition on the day of estrus and one day before and one day after the estrus.

[0049] The ruminant to which the fertility-enhancing composition of this embodiment is administered may be a ruminant to which a fertilized egg is implanted. The fertility-enhancing composition of this embodiment can improve the conception rate of the implanted fertilized egg. By administering the fertility-enhancing composition of this embodiment during the scheduled estrus day before implantation and the period before and after it (for example, the period from five days before the estrus day to five days after the estrus day), the conception rate of the implanted fertilized egg can be improved.

[0050] By administering the fertility-improving composition of this embodiment to a ruminant having an abnormal endometrial EGF concentration profile, the abnormality can be eliminated or alleviated, thereby improving the conception rate. Therefore, the fertility-improving composition of this embodiment can be widely administered to ruminants in need of fertility improvement, and is particularly preferably administered to ruminants having an abnormal endometrial EGF concentration profile.

[0051] The ruminant to which the fertility-improving composition of this embodiment is administered is not particularly limited, and may be any ruminant such as cattle, goats, sheep, deer, giraffes, etc. The ruminant to which the fertility-improving composition of this embodiment is administered is preferably cattle, goats, or sheep, and particularly preferably cattle. The fertility-improving composition of this embodiment is preferably used for dairy cows, for which fertility improvement is particularly desired.

[0052] The fertility-improving composition of this embodiment is preferably used for, for example, subfertile cows, and is particularly preferably administered to RB cows. In particular, the fertility-improving composition of this embodiment is preferably administered to cows in which the increase in endometrial epidermal growth factor concentration seen in normally conceived cows on days 2 to 4 from estrus has disappeared or decreased, i.e., cows in which an abnormal endometrial EGF concentration profile has occurred, and more preferably to cows in which the endometrial epidermal growth factor concentration on day 3 from estrus is less than 4.7 ng / g-tissue.

[0053] In postpartum ruminants, conception may be difficult due to the influence of the recovery state of the uterus. Furthermore, due to significant differences in the metabolic and physiological states related to nutrition, they may not respond to hormone treatments used in standard reproductive care. Since the fertility-enhancing composition of this embodiment can normalize the EGF concentration profile in the endometrium over a period equivalent to one estrus cycle after administration, administering the fertility-enhancing composition of this embodiment to postpartum ruminants is expected to improve conception rates. When administered to postpartum ruminants, the fertility-enhancing composition of this embodiment is preferably administered around the time of the first insemination after parturition. Specifically, the period after calving is preferably within the period from the number of days corresponding to two estrous cycles to the number of days corresponding to five estrous cycles, more preferably within the period from the number of days corresponding to two estrous cycles to the number of days corresponding to four estrous cycles, even more preferably within the period from the number of days corresponding to 2.5 estrous cycles to the number of days corresponding to four estrous cycles, and even more preferably within the period from the number of days corresponding to 2.5 estrous cycles to the number of days corresponding to 3.5 estrous cycles. For example, in the case of cattle, the fertility-enhancing composition of this embodiment is preferably administered within the period from 40 to 100 days after calving, more preferably within the period from 40 to 90 days, even more preferably within the period from 40 to 80 days, and even more preferably within the period from 45 to 80 days. In particular, the fertility enhancing composition of this embodiment is preferably administered to cows within the period of 50 to 80 days after calving, preferably within the period of 60 to 80 days, and more preferably within the period of 60 to 70 days.

[0054] The fertility-enhancing composition of this embodiment can be administered to the vaginal vault or its vicinity in a manner similar to that used for administering semen during artificial insemination. For example, the fertility-enhancing composition of this embodiment can be dissolved or diluted in a solution suitable for administration to ruminants to prepare a solution, which can then be injected into the vagina using a catheter syringe or the like for artificial insemination. Examples of solutions suitable for administration to ruminants include solutions suitable for administration to the vagina, cervix, and uterus of ruminants, such as water, buffer solutions, semen of the ruminant, or solutions prepared by dissolving appropriate pharmaceutically acceptable additives suitable for administration to the vagina, cervix, and uterus of the ruminant. The buffer solution is not particularly limited as long as it can be safely administered to the vagina, etc. of ruminants, and examples include phosphate-buffered saline (PBS) and physiological saline. The additives can be the same as those listed above, and additives that are well tolerated by the vagina and endometrium are preferred.

[0055] The solution of the fertility-enhancing composition of this embodiment to be injected into the vagina of a ruminant is preferably 0.1 to 20 mL, more preferably 2 to 20 mL, even more preferably 5 to 20 mL, even more preferably 10 to 20 mL, and particularly preferably 10 to 15 mL, per treatment. Amounts within these ranges allow administration deep into the vagina to stimulate a wide range of receptors with a small amount of peptide, resulting in effective results. In particular, since the natural ejaculation volume of cattle is 2 to 10 mL, administration within these ranges is particularly preferred for cattle.

[0056] Furthermore, the amount of the active ingredient of the fertility-enhancing composition of this embodiment in the solution need only be sufficient to achieve the effects of normalizing the endometrial EGF concentration profile and improving the conception rate, and for example, the concentration of the peptide or polypeptide as the active ingredient per treatment solution can be 40 nmol or more, preferably 50 nmol or more, more preferably 100 nmol or more, even more preferably 200 nmol or more, and even more preferably 320 nmol or more. The upper limit of the concentration of the peptide or polypeptide as the active ingredient per treatment solution is not particularly limited, and can be, for example, 2400 nmol or less, preferably 2000 nmol or less, and more preferably 1600 nmol or less.

[0057] By using an artificial insemination straw filled with the fertility-enhancing composition of this embodiment, the fertility-enhancing composition can be more easily administered to the vagina of a ruminant. The fertility-enhancing composition of this embodiment filled into the artificial insemination straw is preferably a solution in which the active ingredient, a peptide or polypeptide, is dissolved in 0.1 to 20 mL of a solution suitable for administration to ruminants. The solution suitable for administration to ruminants can be the same as those listed above, and the amount of the solution in which the active ingredient is dissolved is preferably 2 to 20 mL, more preferably 5 to 20 mL, even more preferably 10 to 20 mL, and particularly preferably 10 to 15 mL. By using artificial insemination semen as this solution or as part thereof, an artificial insemination straw can be obtained that can be used for both administering the fertility-enhancing composition of this embodiment and artificial insemination.

[0058] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0059] All cows used in the following experiments were lactating Holstein cows (parity: 2-6, age: 3-9 years, milk yield: >9500 kg / 305 days of fat-corrected milk) raised on commercial farms in the Hokkaido region of Japan. Cows with problems requiring veterinary consultations three or more times during the calving and postpartum period were excluded from the study.

[0060] <Measurement of bovine endometrial EGF concentration> Measurement of endometrial EGF concentration was carried out according to the method described in Non-Patent Document 1. Specifically, the measurement was carried out as follows.

[0061] (1) Collection of endometrial samples: After administering epidural anesthesia to the cows, the external genitalia were washed and a biopsy instrument (Fujihira Industry Co., Ltd.) was inserted into the vagina to collect endometrial tissue samples. The collected endometrial tissue samples were weighed, frozen in liquid nitrogen within 10 minutes of collection, and stored at −80°C until EGF assay.

[0062] (2) Preparation of EGF Assay Measurement Samples (Endometrial Tissue Extracts) First, the tissue samples were minced and homogenized in ice-cold 1 M acetic acid (5 mL / g tissue), and the homogenate was centrifuged. The supernatant after centrifugation was collected and stored, and the remaining pellet was homogenized again and centrifuged to collect the supernatant. The first and second supernatants were combined and concentrated. The resulting concentrated sample was applied to a Sephadex G-50 column (Amersham Biosciences) and eluted with 10 mM PBS (pH 7.2) for size fractionation. Fractions containing proteins greater than 10 kJ / ml were combined into one sample, lyophilized, and stored at -80°C. The frozen samples were reconstituted by dissolving in PBS on the day of the EGF assay.

[0063] (3) EGF Assay Measurement The EGF concentration in endometrial tissue extracts was measured by ELISA using a 96-well plate. All assays were performed in triplicate. Anti-human EGF mouse monoclonal antibody (clone: ​​MAB636, R&D Systems) was used as the solid-phase antibody, and anti-human EGF rabbit antiserum (product number: 5022-100, Biogenesis Ltd.) was used as the detection antibody. The sensitivity of the assay was 10 pg / well. The intra- and inter-assay coefficients of variation at 50 pg / well were 4.2% and 6.8%, respectively.

[0064] Figure 1 shows a schematic diagram of the time course of endometrial EGF concentrations in RB cows (solid line) after estrus. In normal cows, peaks of increased EGF concentrations are observed on days 2-4 and 13-14 after estrus (dotted lines in the figure). In contrast, in the majority of RB cows, endometrial EGF concentrations on days 2-4 and 13-14 after estrus are 4.7 ng / g-tissue or less, and the concentration peaks disappear or decrease.

[0065] In the subsequent studies, endometrial EGF concentrations were measured on day 3 (the day of estrus or artificial insemination (AI) was defined as day 0). If the endometrial EGF concentration was within the normal range on day 3 (4.70 to 13.50 ng / g-tissue), the endometrial EGF concentration profile was considered normal.

[0066] <Ovulation synchronization> In RB cows, ovulation is induced by the Ovsynch protocol, E ２ Ovsynch protocol with (estradiol) treatment, or P ４ The cows were synchronized by the Ovsynch protocol with progesterone supplementation. In the Ovsynch protocol, cows were first treated with gonadotropin-releasing hormone (GnRH) (100 μg intramuscular fertirelin acetate, Concerall® Injection, MSD Animal Health) and then 7 days later with PGF ２α (25 mg intramuscular dinoprost tromethamine) was administered, followed by PGF ２α Approximately 55±2 hours after treatment, a second dose of GnRH (100 mg fetirelin acetate intramuscularly) was administered. In the Ovsynch protocol with E2 treatment, in addition to the Ovsynch protocol steps, PGF ２α One day after the treatment, the animals were treated with estradiol benzoate (1 mg intramuscular estradiol injection, Kyoritsu Pharmaceutical Co., Ltd.). ４ In the Ovsynch protocol with supplementation, on the day of the first GnRH treatment, in addition to the Ovsynch procedure, 1.9 g of P ４ Intravaginal placement type P ４ A vaginal infusion device (CIDR) (product number: CIDR 1900, manufactured by Zoetis Japan) was inserted. The CIDR was removed 7 days after insertion.２ The Ovsynch protocol with treatment was initiated between days 5 and 10 or between days 18 and 20 of the estrous cycle. ４ The Ovsynch protocol with supplementation was initiated on days 13 and 17. When cows were ovulated, the day after the second GnRH treatment was considered as day 0.

[0067] Artificial insemination (AI) Artificial insemination was performed by a professional AI technician using commercially available frozen and thawed Holstein semen.

[0068] <Injection of treatment solutions into the vagina> All treatment solutions (samples) were prepared to a volume of 10 mL using PBS. The samples were loaded into a 10 mL syringe and injected into the vagina. A disposable plastic catheter for AI was attached to the syringe loaded with the sample to be injected, and the tip of the plastic catheter was introduced into the vagina. The tip of the catheter was guided deep into the vagina adjacent to the external opening of the cervix using the AI ​​method. Next, the plunger of the syringe was pressed to inject the sample. The sample was injected into the vagina immediately after AI.

[0069] [Reference Example 1] OPN is well known as a protein with many post-translational modifications, which exert various functions through these modifications. Therefore, we first examined the effects of bovine full-length recombinant OPN (rOPN), which is produced using E. coli and lacks post-translational modifications, on the normalization of EGF concentration profiles and fertility in RB cows.

[0070] <Test Cows> Lactating Holstein RB cows were selected as test cows (n = 216, postpartum days: 120-180 days) based on the following criteria: estrous cycle length, clinical signs, no detectable abnormalities in the reproductive tract, and no pregnancy after three or more artificial inseminations. Prior to the study, all RB cows were confirmed to have normal uterine conditions by transrectal ultrasound and uterine cytology using a cytobrush.

[0071] <Preparation of rOPN> rOPN was prepared using an Escherichia coli expression system to produce OPN without post-translational modifications. Specifically, a gene consisting of a nucleotide sequence encoding the predicted mature protein of bovine OPN (a protein consisting of the region from amino acids 17 to 278 of the amino acid sequence of SEQ ID NO: 4) was introduced into Escherichia coli to obtain a transformant.

[0072] The gene encoding bovine OPN (Uniprot ID: P31096) was synthesized using "GeneArt® Gene Synthesis" (provided by Thermo Fischer Scientific) with codon optimization for expression in E. coli. DNA encoding the predicted mature protein region of OPN (the region from leucine at position 17 to asparagine at position 278 in the amino acid sequence of SEQ ID NO: 4) was amplified by PCR using the primers listed in Table 1 and DNA polymerase "KOD Plus Neo®, manufactured by Toyobo Co., Ltd." In the base sequences in Table 1, the underlined portion in the forward primer (sense strand) indicates the NdeI site, and the underlined portion in the reverse primer (antisense strand) indicates the HindIII site.

[0073]

[0074] The PCR product was purified using a QIAEX II Gel Extraction Kit (Qiagen) and then inserted into the NdeI-HindIII site of plasmid pET-28a (Merck Millipore) using SLiCE (Seamless Ligation Cloning Extract). The resulting plasmid vector was named SPP1 / pET-28a. This vector produces a recombinant protein consisting of an amino acid sequence in which a histidine tag-containing plasmid vector-derived amino acid sequence (MGSSHHHHHHSSGLVPRGSHM: SEQ ID NO: 7) is added to the N-terminal side of the predicted mature protein region of OPN (region from positions 17 to 278 of SEQ ID NO: 4).

[0075] Escherichia coli Rosetta (DE3) (Merck Millipore) carrying SPP1 / pET-28a was cultured overnight at 37°C on an LB agar plate containing 50 μg / mL kanamycin and 30 μg / mL chloramphenicol. The resulting single colony was inoculated into 150 mL of LB medium containing 50 μg / mL kanamycin and 30 μg / mL chloramphenicol and grown overnight with shaking at 37°C to prepare a seed culture. The seed culture was inoculated into 3,000 mL of LB medium containing 50 μg / mL kanamycin and grown at 37°C. The OD of the medium was ６００ When the RI reached 0.4-0.8, the medium was cooled in ice for 10 minutes and 0.1 mM IPTG (isopropyl β-D-thiogalactopyranoside) was added to induce protein expression. The medium was then cultured at 30°C for 20-22 hours. The resulting culture was centrifuged (10,000 × g, 25°C, 5 minutes) to recover E. coli cells, which were then frozen at -80°C until rOPN purification.

[0076] rOPN was purified as follows: E. coli was resuspended in buffer A (20 mM sodium phosphate buffer, pH 7.0, 0.3 M NaCl) at 4°C, disrupted by sonication, and then centrifuged (4°C, 6,000 × g, 20 minutes) to obtain a cell-free lysate. 2+The rOPN was applied to a 1000-mM HCl-charged Chelating Sepharose Fast Flow column (Cytiva). The column was washed with Buffer A containing 20 mM imidazole, and then rOPN was eluted with a linear gradient of 20 to 500 mM imidazole in Buffer A. The purified rOPN was dialyzed against PBS (Takara Bio) using a cellulose membrane (cellulose tubing (30 / 32), Viskase Companies). Purification and dialysis of rOPN were carried out at 4°C. 1% (w / v) trehalose was added to the dialyzed protein solution containing rOPN as a cryoprotectant, followed by filtration (filter pore size: 0.22 μm). A solution containing 0.3 mg of rOPN was dispensed into 10 mL vials and lyophilized. The lyophilized rOPN was stored at 4°C until injection into cattle. The rOPN concentration in the dialyzed protein solution was calculated by calculating the crude protein concentration by ultraviolet spectrophotometry and correcting the concentration by the purity evaluated by polyacrylamide gel electrophoresis. 0.3 mg of rOPN contains 16 nmol of rOPN as determined by amino acid analysis.

[0077] <Treatment with OPN> OPN treatment was performed by injecting a solution (rOPN treatment solution) prepared by dissolving 0.3 mg of rOPN in 10 mL of PBS into the vagina. As a positive control, a solution (SP treatment solution) prepared by diluting 0.5 mL of seminal plasma with PBS to a total volume of 10 mL was used. PBS was used as a negative control.

[0078] The OPN treatment schedule is shown in Figure 2. First, all cows were screened to confirm that they were RB cows, and then estrus was synchronized. Next, endometrial biopsies were performed on the third day after estrus. Cows that showed abnormal endometrial EGF concentration profiles based on the endometrial biopsies were selected as test cows. Next, GnRH was administered on the seventh to nineth day after estrus, and prostaglandin F2α (PGF2α) was administered seven days later to synchronize estrus. Estrus was confirmed two to four days after PGF2α administration. OPN treatment was performed with artificial insemination (AI) within four to 12 hours after estrus confirmation. 74 cows were treated with PBS, 37 with SP, and 105 with rOPN.

[0079] The day of artificial insemination and OPN treatment was defined as the day of estrus, and endometrial EGF concentrations were measured again three days later. Furthermore, pregnancy tests were performed 30 to 35 days after estrus, and conception rates were calculated and compared using Fisher's exact test. Pregnancy tests were performed using transrectal ultrasound.

[0080]

[0081] The results of EGF concentration measurements and conception rates (%) on day 3 after treatment for each treatment group are shown in Figure 3 and Table 2. As shown in Figure 3 and Table 1, rOPN treatment improved EGF concentrations on day 3 after estrus to a level similar to that of SP treatment, confirming that rOPN normalizes abnormal endometrial EGF concentration profiles. Furthermore, in both groups, the conception rate was higher in cows with normalized EGF concentration profiles than in cows without normalized EGF concentration profiles. As a result, the conception rate was significantly higher in the rOPN-treated group than in the PBS-treated group, confirming that rOPN treatment improves fertility.

[0082] [Example 1] OPN is a protein that has multiple physiologically active sites and regulates a variety of biological phenomena. Therefore, we attempted to identify a partial region of OPN that is involved in resolving abnormalities in the endometrial EGF concentration profile.

[0083] <Test Cows> As in Reference Example 1, Holstein RB cows in which an abnormal endometrial EGF concentration profile was confirmed were used as test cows (n=294).

[0084] <Preparation of Partial Peptides and Partial Proteins of OPN> OPN has a thrombin cleavage site (serine at position 162 in the amino acid sequence of SEQ ID NO: 4), and cleavage by thrombin generates an N-terminal fragment (amino acid region at positions 17 to 161 in the amino acid sequence of SEQ ID NO: 4) and a C-terminal fragment (amino acid region at positions 162 to 278 in the amino acid sequence of SEQ ID NO: 4). The N-terminal fragment has an integrin-binding domain. The integrin-binding domain consists of two integrin-binding motifs: an RGD motif on the N-terminus (region at positions 152 to 154 in the amino acid sequence of SEQ ID NO: 4) and an SVAYGLK motif on the C-terminus (SEQ ID NO: 1: region at positions 155 to 161 in the amino acid sequence of SEQ ID NO: 4). These two integrin-binding motifs bind to different types of integrins. Furthermore, the C-terminal fragment targets CD44, but the binding domain is unknown.

[0085] Based on the domain structure of OPN, peptides and polypeptides consisting of the amino acid sequences shown in Figure 4 were synthesized. Peptide 1 (SEQ ID NO: 2: GRGDSVAYGLK) is a peptide in the region from positions 151 to 161 of the amino acid sequence of SEQ ID NO: 4, peptide 2 (SEQ ID NO: 3: GRGDS) is a peptide in the region from positions 151 to 155 of the amino acid sequence of SEQ ID NO: 4, and peptide 3 (SEQ ID NO: 1: SVAYGLK) is a peptide in the region from positions 155 to 161 of the amino acid sequence of SEQ ID NO: 4.

[0086] Peptide 1 and peptide 3 were synthesized in the form of trifluoroacetate salts (Peptide Institute). Peptide 2 was a fibronectin active fragment (GRGDS) in the form of acetate salts (Peptide Institute). Each peptide provided as a lyophilized peptide was dissolved in sterile ultrapure water and filtered (filter pore size: 0.22 μm). The filtered peptide-containing solution was used as a peptide sample, dispensed into vials at 32 nmol, 320 nmol, and 1600 nmol, and lyophilized. The lyophilized peptides were stored at 4°C until injection into cattle. The amount of peptide was quantified based on amino acid analysis.

[0087] The rOPN used was the same as that used in Reference Example 1. C-rOPN (the region from amino acids 162 to 278 of the amino acid sequence of SEQ ID NO: 4) was a recombinant protein synthesized in Escherichia coli in the same manner as rOPN. Each purified recombinant protein was lyophilized and stored at 4°C until use. The amount of protein was quantified based on amino acid analysis.

[0088] <OPN Treatment> For the OPN treatment, a solution of 0.3 mg of rOPN dissolved in 10 mL of PBS was used as the treatment solution for the rOPN treatment, which served as a positive control. For the C-rOPN treatment, a solution of 28 nmol or 46 nmol of C-rOPN dissolved in 10 mL of PBS was used as the treatment solution. For the peptide 1 treatment, a solution of 32 nmol, 320 nmol, or 1600 nmol of peptide 1 dissolved in 10 mL of PBS was used as the treatment solution. For the peptide 2 treatment, a solution of 320 nmol or 1600 nmol of peptide 2 dissolved in 10 mL of PBS was used as the treatment solution. For the peptide 3 treatment, a solution of 320 nmol or 1600 nmol of peptide 3 dissolved in 10 mL of PBS was used as the treatment solution. PBS was used as a negative control.

[0089] The OPN treatment schedule was the same as in Reference Example 1 (Figure 2), except that estrus synchronization for artificial insemination and OPN treatment was changed to ovulation synchronization, and artificial insemination was performed 16 to 20 hours after the second GnRH treatment in the ovulation synchronization treatment. The results of measuring EGF concentrations on day 3 after treatment for each treatment group are shown in Figure 5, and the conception rates (%) are shown in Table 3. In Figure 5, the numbers at the top of each column represent the normalization rate of the EGF concentration profile ([number of cows with EGF concentrations of 4.70 ng / g-tissue or higher] / [number of treated cows] x 100%).

[0090]

[0091] As shown in Figure 5, the 32-1600 nmol Peptide 1 treatment group and the 1600 mol Peptide 3 treatment group showed improvement in endometrial EGF concentrations on day 3 after estrus, similar to the rOPN treatment group. Furthermore, the 320-1600 nmol Peptide 1 treatment group and the 1600 mol Peptide 3 treatment group showed improvement in the EGF concentration profile on day 3 after estrus, similar to the rOPN treatment group. On the other hand, the normalization rates of endometrial EGF concentrations and EGF concentration profiles for the other samples were lower than those for the rOPN group and comparable to those for the PBS group. Furthermore, as shown in Table 2, in all groups, cows with normal EGF concentration profiles showed higher conception rates than those with abnormal EGF concentration profiles. Furthermore, the conception rates for each group were higher in the 320-1600 nmol Peptide 1 treatment group and the 1600 nmol Peptide 3 treatment group than the PBS control group, similar to those for the rOPN treatment group. On the other hand, the overall conception rate in the 1600 nmol peptide 2 treatment group and the 320 nmol peptide 3 treatment group was intermediate between those in the PBS treatment group and the rOPN treatment group. The 320 nmol peptide 2 treatment group and the C-rOPN treatment group had conception rates similar to those in the PBS treatment group. These results indicated that the integrin-binding domain, and in particular the SVAYGLK motif, plays an important role in the normalization of the endometrial EGF concentration profile and the improvement of fertility by OPN.

[0092] Example 2 The effect of the integrin-binding domain of OPN on normalizing the endometrial EGF concentration profile and restoring fertility in RB cows was investigated.

[0093] <Test Cows> As in Reference Example 1, Holstein RB cows in which an abnormal endometrial EGF concentration profile was confirmed were used as test cows (n=586).

[0094] <Preparation of partial peptides and partial proteins of OPN> rOPN and peptides 1 to 3 prepared in Example 1 were used. Peptide 4 was a peptide (SEQ ID NO: 8) in which the fourth D (aspartic acid) of peptide 1 was substituted with E (glutamic acid). This amino acid substitution mutation converts the RGD motif into an RGE sequence that does not have integrin-binding activity. Peptide 4 was synthesized in the form of a trifluoroacetate salt (manufactured by Peptide Institute), and a peptide sample was prepared in the same manner as for peptide 1, etc.

[0095] OPN treatment was performed as in Example 1. rOPN (0.3 mg, n = 111), peptide 1 (GRGDSVAYGLK) (32 nmol, 320 nmol, or 1600 nmol, n = 25, 128, and 50, respectively), peptide 2 (GRGDS) (320 nmol or 1600 nmol, n = 20 each), peptide 3 (SVAYGLK) (320 nmol or 1600 nmol, n = 25 and 55, respectively), peptide 4 (GRGESVAYGLK) (320 nmol or 1600 nmol, n = 50 and 26, respectively), and PBS (n = 77) were administered at the time of artificial insemination.

[0096]

[0097] Table 4 shows the results of measuring the endometrial EGF concentration and the normalization rate of the EGF concentration profile in each administration group. As shown in Table 4, the normalization rate was higher in the groups administered with peptide 1 or peptide 3 than in the PBS-administered group. In particular, the groups treated with 320 to 1600 nmol of peptide 1 and the group treated with 1600 nmol of peptide 3 showed significantly higher normalization rates of the endometrial EGF concentration and EGF concentration profile than the control PBS-administered group, similar to the rOPN-administered group. On the other hand, no improvement in endometrial EGF concentration or normalization of the EGF concentration profile was observed in the group treated with peptide 2. These results confirmed that administration of peptide 1 or peptide 3 can normalize the endometrial EGF concentration profile, similar to administration of rOPN, that this effect is exerted at lower concentrations with peptide 1 than with peptide 3, and that peptide 3 (i.e., the SVAYGLK motif) is the minimum functional unit of OPN's ability to normalize the endometrial EGF concentration profile.

[0098] On the other hand, in the group administered with peptide 4, in which the integrin-binding activity of the RGD motif was inactivated by introducing an amino acid substitution mutation into peptide 1, the improvement in endometrial EGF concentration and normalization of the EGF concentration profile observed with peptide 3 were not confirmed. This result suggests that the RGD motif is also involved in the normalization of endometrial EGF concentration by the SVAYGLK motif.

[0099]

[0100] The effects on fertility of the administration groups in which an improvement in the normalization rate of the endometrial EGF concentration profile was confirmed were investigated, and the results are shown in Table 5. As a result, fertility was improved to the same extent as in the rOPN administration group in all peptide and dosage groups.

[0101] Example 3 The effects of rOPN on endometrial EGF concentration profiles and fertility in cows at the first insemination after parturition were investigated.

[0102] <Cows to be tested> Cows to be tested were Holstein cows at the time of their first insemination after calving, and cows that showed no detectable abnormalities in estrous cycle, clinical signs, uterine cytology, vaginal examination, rectal examination, and reproductive examination by ultrasound before inclusion in the study (n = 106).

[0103] <OPN treatment> OPN treatment was performed at the time of first insemination 60-70 days after calving. Of the test cows, 52 were artificially inseminated only (untreated group), and 54 were administered rOPN (16 nmol) along with artificial insemination (rOPN-administered group). Endometrial EGF concentrations were measured 3 days after the artificial insemination date. Pregnancy was diagnosed by transrectal ultrasound between 30 and 35 days after the artificial insemination date.

[0104]

[0105] The measurement results of the endometrial EGF concentration, normalization rate, and conception rate for each administration group are shown in Table 6. As a result, the rOPN administration group had a higher endometrial EGF concentration, an improved normalization rate, and an improved conception rate compared to the untreated group.

[0106] Example 4 The effects of rOPN and peptide 1 on endometrial EGF concentration profiles and fertility in cows at the first insemination after parturition were investigated.

[0107] <Test cows> As in Example 3, test cows were Holstein cows that were to undergo artificial insemination for the first time after calving and that had no detectable abnormalities in estrous cycle, clinical signs, uterine cytology, vaginal examination, rectal examination, and reproductive examination by ultrasound before inclusion in the study (n=220).

[0108] The OPN treatment schedule is shown in Figure 6. Biopsies to measure endometrial EGF concentrations on day 3 of the estrous cycle were performed on day 51 after calving. Next, the cows' ovulation was synchronized. Cows were artificially inseminated on day 62 after calving, and simultaneously, rOPN (16 nmol, n = 41) or peptide 1 (GRGDSVAYGLK, 320 mol, n = 97) was injected into the vagina. In the untreated group (n = 82), only artificial insemination was performed. A second endometrial EGF concentration measurement was performed on day 3 after artificial insemination (day 65 after calving). Pregnancy diagnosis was performed by transrectal ultrasound approximately 38 days after artificial insemination (approximately 100 days after calving).

[0109]

[0110] The measurement results of endometrial EGF concentration, normalization rate, and conception rate for each administration group are shown in Table 7. As a result, when rOPN and peptide 1 were administered to cows that had an abnormal endometrial EGF concentration profile, the abnormal endometrial EGF expression was corrected and the conception rate also improved. In cows that originally had a normal endometrial EGF concentration profile, no change in conception rate was observed with administration of rOPN or peptide 1. As a result, the conception rate was improved in the rOPN administration group and peptide 1 administration group compared to the untreated group.

Claims

1. A composition for improving the fertility of ruminants, comprising a peptide or polypeptide as an active ingredient, which contains the amino acid sequence represented by Sequence ID No. 1 (SVAYGLK).

2. The fertility-enhancing composition according to claim 1, wherein the peptide or polypeptide comprises an amino acid sequence represented by SEQ ID NO: 2 (GRGDSVAYGLK).

3. The fertility-enhancing composition according to claim 1, wherein the peptide or polypeptide is a partial peptide of uciosteopontin.

4. The active ingredient is the protein of uciosteopontin, a variant of the protein of uciosteopontin, or a partial peptide of these proteins. The protein, the mutant, and the partial peptide include the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), A composition for improving the fertility of ruminants.

5. The fertility-enhancing composition according to claim 4, wherein the protein, the variant, and the partial peptide comprise the amino acid sequence represented by Sequence ID No. 2 (GRGDSVAYGLK).

6. The fertility-enhancing composition according to claim 4, wherein the amino acid sequence of the mutant of the uciosteopontin protein is an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the uciosteopontin protein.

7. The fertility-enhancing composition according to claim 4, comprising a peptide consisting of an amino acid sequence represented by Sequence ID No. 1 or 2 as an active ingredient.

8. The fertility-enhancing composition according to any one of claims 1 to 7, wherein the ruminant is a ruminant that has given birth.

9. The fertility-enhancing composition according to any one of claims 1 to 7, wherein the ruminant animal is a cattle, goat, sheep, deer, or giraffe.

10. The fertility-enhancing composition according to claim 8, wherein the ruminant is a cattle that is 45 to 80 days postpartum.

11. The fertility-enhancing composition according to any one of claims 1 to 7, wherein the ruminant is a low-fertility cow.

12. The low-fertility cow is a cow in which the increase in epidermal growth factor concentration in the endometrium, which is observed in normally conceiving cows 2 to 4 days after estrus, has disappeared or decreased, according to claim 11.

13. The fertility-enhancing composition according to claim 11, wherein the low-fertility cow is a cow in which the concentration of epithelial growth factor in the endometrium on the third day from estrus is less than 4.7 ng / g-tissue per unit tissue weight.

14. The aforementioned active ingredient, A variant of the Ushiosteopontin protein, comprising the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), A partial peptide of the Ushiosteopontin protein, comprising the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK), or A partial peptide of a variant of the Ushiosteopontin protein, comprising the amino acid sequence represented by SEQ ID NO: 1 (SVAYGLK). And, The fertility-enhancing composition according to claim 11, wherein the low-fertility cow is a repeat breeder cow.

15. The fertility-enhancing composition according to any one of claims 1 to 7, wherein administration of the fertility-enhancing composition normalizes the epidermal growth factor concentration profile in the endometrium over a period of one estrous cycle from the time of administration.

16. A method for improving the fertility of a ruminant, comprising administering the fertility-enhancing composition described in any one of claims 1 to 7 to the vaginal fornix or its vicinity in the vaginal area of ​​the ruminant during the estrus day and the period immediately before and after.

17. The fertility-enhancing composition is a solution obtained by dissolving the active ingredient in 0.1 to 20 mL of a solution that can be administered to ruminants. The method for improving fertility according to claim 16, wherein the solution that can be administered to the ruminant is a solution that can be administered to the vagina, cervix, and uterus of the ruminant, and is a solution obtained by dissolving water, a buffer, the semen of the ruminant, or an appropriate and pharmaceutically acceptable additive that can be administered to the vagina, cervix, and uterus of the ruminant in the same.

18. The method for improving fertility according to claim 16, wherein the amount of the active ingredient contained in the fertility-enhancing composition is 40 nmol or more.

19. The method for improving fertility according to claim 16, wherein the fertility-enhancing composition is administered before, after, or simultaneously with artificial insemination.

20. The method for improving fertility according to claim 16, wherein the ruminant is a ruminant that mates naturally.

21. The method for improving fertility according to claim 16, wherein the ruminant is a ruminant to which a fertilized egg is implanted.

22. The method for improving fertility according to claim 16, wherein the ruminant is a cow, a goat, a sheep, a deer, or a giraffe.

23. An artificial insemination straw filled with the fertility-enhancing composition described in any one of claims 1 to 7, The fertility-enhancing composition is a solution obtained by dissolving the active ingredient in 0.1 to 20 mL of a solution that can be administered to ruminants. An artificial insemination straw wherein the solution administerable to the ruminant is a solution administerable to the vagina, cervix, and uterus of the ruminant, and is a solution obtained by dissolving water, a buffer, the semen of the ruminant, or a suitable and pharmaceutically acceptable additive that can be administered to the vagina, cervix, and uterus of the ruminant in the same.