Method for promoting estrus regression in animals and estrus regression promoter for animals

Abstract

To provide a method for promoting the return of estrus of an animal and an agent for promoting the return of estrus of an animal.SOLUTION: The method for promoting estrus regression of an animal includes a step of administering mesenchymal stem cells or a composition containing the mesenchymal stem cells to the animal. The mesenchymal stem cells are preferably adipose-derived stem cells, and the animal is preferably a domestic animal, a dog or a cat.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for promoting the return of estrus to an animal and an agent for promoting the return of estrus to an animal. [Background technology]

[0002] From a management perspective, improving animal reproductive efficiency is an important issue for ranches and breeders that handle livestock and pets. Measures to improve reproductive efficiency include improving the health of offspring and parents, avoiding stillbirths and deaths after birth, and shortening the birth interval.

[0003] Meanwhile, the concept of animal welfare has become more widespread around the world in recent years. The World Organization for Animal Health (WOAH), an international organization whose goal is to improve animal health worldwide, defines animal welfare as "the physical and mental state of animals in relation to the conditions under which they live and die." Japan's Ministry of Agriculture, Forestry and Fisheries believes that raising livestock in a comfortable environment is important for reducing stress and disease in livestock, which ultimately leads to improved productivity and the production of safe livestock products, and is working to promote livestock rearing management that takes animal welfare into account.

[0004] Given the above circumstances, there is a need to improve reproductive efficiency in a way that places as little strain on animals as possible. Research into improving reproductive efficiency has traditionally focused on shortening the number of days until estrus returns, which is the process by which animals can quickly return to a breeding state after giving birth.

[0005] For example, Non-Patent Document 1 discloses a study of factors affecting the return of estrus after parturition, particularly the relationship between feeding and blood components.

[0006] Furthermore, Non-Patent Document 2 investigated the effectiveness of supplementing energy intake by feeding medium-chain triglycerides (MCT) and administering gonadotropin-containing hormones as a measure to delay the return of estrus in breeding sows in the summer, and reported that MCT had no effect on shortening the number of days until the return of estrus, while administration of hormones was effective.

[0007] However, there are limits to how much a delay in the return of estrus can be avoided by changing or improving feed, and although the administration of hormones is expected to be effective, there are countries in which the administration of hormones is prohibited. For these reasons, there is still room for improvement in methods for shortening the number of days (hours) until the return of estrus. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Tohoku Livestock Clinical Research Association Journal, Vol. 16, No. 1 [Non-patent document 2] Aichi Agricultural Research Institute Report 47:151-154(2015) Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, a new method for shortening the number of days until animals return to estrus is desired. Therefore, an object of the present invention is to provide a method for promoting the return of estrus to animals and an agent for promoting the return of estrus to animals. [Means for solving the problem]

[0010] As a result of extensive research to solve the above problems, the present inventors discovered that administration of mesenchymal stem cells to animals can promote the return of estrus, leading to the completion of the present invention.

[0011] That is, the present invention includes the following [1] to [9]. [1] A method for promoting reversion to estrus in an animal, comprising the step of administering to the animal mesenchymal stem cells or a composition containing mesenchymal stem cells. [2] The method for promoting reversion to estrus in an animal according to [1], wherein the mesenchymal stem cells are adipose-derived stem cells. [3] The method for promoting the return of estrus to an animal according to [1], wherein the animal is a livestock, a dog, or a cat. [4] The method for promoting the return of estrus to animals according to [3], wherein the livestock is a cow, a horse, or a pig. [5] The method for promoting return of estrus to an animal according to any one of [1] to [4], wherein the animal is a postpartum animal. [6] The method for promoting the return of estrus to animals according to [1], which can shorten the number of days required for the return of estrus. [7] A method for promoting recovery of reproductive function in an animal, comprising the step of administering mesenchymal stem cells or a composition containing mesenchymal stem cells to a postpartum animal. [8] A method for promoting the return of estrus, characterized by containing animal mesenchymal stem cells. [9] An agent for promoting the recovery of reproductive function in animals, characterized by containing animal mesenchymal stem cells. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a method for promoting the return of estrus to an animal and an agent for promoting the return of estrus to an animal. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a graph showing the results of an example. [Figure 2] FIG. 1 is a graph showing the results of an example. DETAILED DESCRIPTION OF THE INVENTION

[0014] <Method for promoting the return of estrus in animals> The method for promoting return to estrus in an animal of the present invention is characterized by comprising the step of administering mesenchymal stem cells or a composition containing mesenchymal stem cells to the animal. In the present invention, promoting the return of estrus in an animal means promoting the return of estrus in an animal, preferably hastening the return of estrus in an animal, and more preferably shortening the number of days until the return of estrus in an animal. By promoting the return of estrus, the time until the animal is ready to become pregnant after parturition can be shortened. Furthermore, promoting the return of estrus can suppress a delay in the return of estrus in an animal. In other words, the method for promoting the return of estrus in an animal of the present invention preferably includes a method for suppressing a delay in the return of estrus in an animal.

[0015] [Target of administration] In the present invention, there are no particular limitations on the animals (excluding humans) to which mesenchymal stem cells or compositions containing mesenchymal stem cells are administered. Female animals are preferred. Suitable animals include livestock, dogs, and cats, with preferred livestock being cows, horses, pigs, goats, and sheep.

[0016] There are no particular limitations on the age of the animal to be administered, and the animal is preferably of an age at which mesenchymal stem cells can be administered, or at which pregnancy and childbirth are possible.

[0017] [Mesenchymal stem cells] Mesenchymal stem cells (MSCs) are somatic stem cells found in mammalian organisms. They can be easily cultured outside the body to expand their cell numbers, and because they have properties such as angiogenic, immunosuppressive, and anti-inflammatory properties, clinical research and trials are being conducted on them as immunosuppressants and therapeutic agents for intractable autoimmune diseases, and they are attracting attention as an important field in cell therapy.

[0018] Mesenchymal stem cells are a type of adult stem cell that have been reported to be found in bone marrow, fat, umbilical cord, synovium (tissue surrounding joints), etc. The mesenchymal stem cells used in the present invention preferably have the following characteristics: (1) they are positive for the cell surface antigens CD44, CD73, CD90, and CD105, and negative for CD14, CD19, CD34, CD45, and MHC Class II; (2) they adhere to plastic under standard culture conditions and proliferate with a fibroblast-like morphology; and (3) they have the ability to differentiate into adipocytes, osteoblasts, and chondroblasts. The above description of the cell surface antigens in (1) is one preferred example, and known surface antigens can be selected for each animal species. For example, in dogs, it is preferable that CD44 and CD90 are positive and CD45 and MHC class II are negative. Furthermore, it is preferable that any one or more of CD29, CD73, and CD105 are positive. In cats, it is preferable that CD29 and CD44 are positive and CD34 and MHC class II are negative. Furthermore, it is preferable that any one or more of CD73, CD90, and CD166 are positive. In cats, it is preferable that any one or more of CD29, CD44, CD73, CD90, CD105, and CD166 are positive and any one or more of CD11b, CD14, CD31, CD34, CD45, and CD117 are negative. In horses, it is preferable that one or more of CD29, CD44, CD73, CD90, CD105, and CD166 are positive, and one or more of CD11b, CD14, CD31, CD34, CD45, and MHC class II are negative. In goats, it is preferable that one or more of CD29, CD44, CD73, CD90, and CD105 are positive, and one or more of CD11b, CD14, CD31, CD34, and CD45 are negative. The above examples can be modified as appropriate, and surface antigens can be changed and selected according to publicly known or commonly known information.

[0019] Mesenchymal stem cells can be collected from bone marrow, but they can also be collected from adipose tissue. Subcutaneous fat has the following advantages over bone marrow: (1) tissue collection is relatively easy, and (2) the amount of mesenchymal stem cells contained per unit volume of tissue is several hundred times higher than that of bone marrow. Because it is easy to secure the number of cells, subcutaneous fat is a preferred resource for mesenchymal stem cells. Therefore, in the present invention, it is preferable to use adipose-derived stem cells as mesenchymal stem cells. Adipose-derived stem cells may contain cells other than mesenchymal stem cells.

[0020] [Adipose-derived stem cells] Adipose-derived stem cells (ASCs) are somatic stem cells contained in adipose tissue. For example, they can be collected from subcutaneous adipose tissue. In the present invention, adipose-derived stem cells from animals, particularly mammals, are preferably used. Either autologous or allogeneic ASCs may be used, but allogeneic ASCs are preferred considering the cost and effort involved in regular administration. When using autologous ASCs, stem cells are isolated from the adipose tissue of the animal to be administered, cultured, and then administered to the animal. When using allogeneic ASCs, adipose-derived stem cells prepared in advance can be used. In the case of allogeneic ASCs, mesenchymal stem cells or adipose-derived stem cells derived from the same species of animal as the animal to be administered are preferred.

[0021] Adipose-derived stem cells can be prepared by harvesting and culturing them using known methods. For example, in the case of allogeneic transplantation, subcutaneous fat is harvested from a healthy animal under anesthesia using surgical instruments, and the wound is sutured closed. The harvesting site is the abdomen. Adipose-derived stem cells are then recovered from the harvested subcutaneous fat by enzyme treatment, cultured for several days, for example, about 7 days, and cryopreserved to obtain adipose-derived stem cells. They are thawed appropriately before use.

[0022] For adipose-derived stem cells, surface antigen markers can include those similar to those of the mesenchymal stem cells described above. In the case of dogs, adipose-derived stem cells are preferably CD90 ≥ 85% and / or CD44 ≥ 85%. Furthermore, CD45 ≤ 3% and MHCII ≤ 3% are preferred. In the case of cats, adipose-derived stem cells are preferably CD29 ≥ 50% and / or CD44 ≥ 80%. Furthermore, CD34 ≤ 3% and MHCII ≤ 3% are preferred. Here, "~%" refers to the positive rate of a specific marker in cells contained in the cell population, and can be measured by known methods. If these markers are confirmed, it is not necessary to confirm the above-mentioned preferred mesenchymal stem cell markers.

[0023] When administering allogeneic adipose-derived stem cells, immunosuppressants can be used in combination, but they can also be administered without the use of immunosuppressants. When not using immunosuppressants, it is preferable to check for the presence or absence of markers that affect immune responses, such as MHC class II. For example, if the MHC class II positivity rate of the adipose-derived stem cells to be administered exceeds 3%, it is highly likely that they will cause an immune rejection reaction, so it is preferable to avoid administering them to animals.

[0024] [Preparation of adipose-derived stem cells] An example of a procedure for preparing adipose-derived stem cells (procedures such as culturing, selection, recovery, freezing, and thawing) is described below. Specifically, one embodiment of a process for isolating and obtaining adipose-derived stem cells from adipose tissue contained in a dog, as an example of an animal, is shown. However, the present invention should not be construed as being limited thereto. Similar procedures can also be adopted for animals other than dogs, and may be modified as appropriate depending on the animal species.

[0025] (1) Preparation of cell populations from adipose tissue Adipose tissue is harvested by excision or other means under anesthesia. The harvested adipose tissue is briefly exposed to 70% ethanol to clean and sterilize any bacteria or viruses adhering to the tissue, then immersed in a buffer solution or culture medium and subjected to the following enzyme treatment. Sterilization treatments also include methods using known disinfectants such as 10% iodine solution.

[0026] The enzymatic treatment involves enzymatically digesting the adipose tissue with a collagenase Type I enzyme solution (0.1-5 mg / mL) at 37°C for 30-120 minutes to obtain a cell population (solution) containing adipose-derived stem cells. The enzymes used to decompose the adipose tissue include trypsin, dispase, and other commercially available digestive enzymes for adipose tissue, as used in known methods.

[0027] To separate the cell population containing adipose-derived stem cells, centrifugation is performed. The enzyme-treated solution is dispensed into a 50 mL centrifuge tube and centrifuged at 750 to 1500 G (1.0 G = 9.80665 m / s 2) and collect a cell population containing adipose-derived stem cells from the sediment fraction at the bottom of the tube. The centrifugal acceleration may be changed depending on the amount of adipose tissue. The sediment fraction is added to a new centrifuge tube containing D-PBS and washed to remove remaining oil and fat components and other impurities. The solution containing adipose-derived stem cells is filtered through a cell strainer with a pore size of 70-100 μm to remove ECM and undegraded adipose tissue from the adipose tissue. The buffers, filtration filters, and devices used in this section may also include other known buffers, filtration membranes, and devices.

[0028] (2) Selective culture of adherent fibroblast-like cells and collection of cells (P0) The solution containing the cells is centrifuged at 750 to 1500 G, and the precipitated fraction at the bottom of the tube is collected as a cell population containing adipose-derived stem cells. An appropriate medium is added to this and the cells are suspended, and then the cells are centrifuged at 225 cm 2 The suspension is transferred to a culture flask and cultured for 7 to 10 days, with repeated washing and medium changes every 3 to 4 days. The culture environment in the incubator is 37°C with a carbon dioxide concentration of 5%. Conventional animal cell culture media can be used. Examples include Dulbecco's Modified Eagle's Medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, etc.), α-MEM (Fujifilm Wako Pure Chemical Corporation, etc.), DMED:Ham's F12 mixed medium (1:1) (Fujifilm Wako Pure Chemical Corporation, etc.), and Ham's F12 Medium (Fujifilm Wako Pure Chemical Corporation, etc.). Serum derived from fetal bovine serum (FBS), human serum, sheep serum, etc. can be used as the medium. Serum or serum substitutes can be added to the medium in an amount ranging from 5% (v / v) to 30% (v / v), for example.

[0029] To recover the proliferated cells, the recovery procedure follows the standard method for detaching cells adhered to the bottom of the flask, for example, by detaching the cells after enzymatic treatment (trypsin or dispase treatment). The detached cells are suspended in a medium containing fetal bovine serum to inhibit trypsin activity, and then centrifuged at 500-1500G to remove medium components. The cell population is then washed again with D-PBS and used as a passage 0 (P0) adipose-derived stem cell suspension.

[0030] (3) Selective culture of adipose-derived stem cells and collection of cells (P1) 3–5 × 10 adipose-derived stem cells from P0 4 cells / cm 2 The cells were prepared at a concentration of 225 cm. 2 The suspension is transferred to a culture flask and cultured for 7-10 days, with repeated washing and medium changes every 3-4 days. The culture environment in the incubator is 37°C with a carbon dioxide concentration of 5%. Regular animal cell culture media can be used for the culture media, as with P0 cell culture.

[0031] The proliferated cells (P1) can be harvested using standard methods for detaching cells adhered to the flask bottom, such as by enzymatic treatment (trypsin or dispase). The detached cells are suspended in a medium containing FBS to inhibit trypsin activity, then washed with D-PBS and centrifuged at 500-1500 G to wash away any remaining medium components. The cell population is then washed again with D-PBS and used as the mesenchymal stem cell suspension for passage 1 (P1).

[0032] (4) Cell cryopreservation method P0 and P1 cells were frozen at 1 × 10 6 ~1×10 7The cells are suspended in a cryopreservation solution at a concentration of 1000 cells / mL, dispensed into cryotubes, and slowly frozen in a slow freezer at -80°C. A cryopreservation solution typically used for freezing animal cells can be used. Commercially available cell freezing solutions containing or not containing dimethyl sulfoxide, such as CELL BANKER I (TAKARA), COS BANKER (Cosmo Bio Co., Ltd.), and Banbanker (Nihon Genetics), can also be used. The dimethyl sulfoxide (DMSO) contained in the freezing solution is, for example, 0% (v / v) to 10% (v / v).

[0033] (5) Cell thawing method The frozen P0 and P1 cells are thawed according to standard cell thawing methods. The thawed cell suspension is centrifuged at 500-1500 G, the cell freezing solution is removed, and then a buffer solution such as D-PBS is added to the cell mass, followed by repeated washing and centrifugation. Methods for thawing frozen tubes include rapid thawing using an automated thawing device, the Thawstar cell freeze-thaw station (Biocision).

[0034] [Composition containing mesenchymal stem cells] The composition containing mesenchymal stem cells may contain, in addition to mesenchymal stem cells, a solvent, auxiliary components, etc. A cell suspension is preferred. The preferred composition of the composition containing mesenchymal stem cells is the same as that of the agent for promoting return to estrus, which will be described later.

[0035] [Administration method] The method of administering mesenchymal stem cells is not particularly limited and any known method can be used. From the viewpoint of reducing the burden on animals, intravenous drip infusion, intravenous injection, subcutaneous injection, or intramuscular injection is preferred. Subcutaneous injection or intramuscular injection is more preferred because it allows for a shorter administration time than intravenous drip infusion.

[0036] [Dosage] The preferred dose of mesenchymal stem cells is 1 x 10 per administration. 4 pieces / kg(weight)~1×10 8 1×10 5 pieces / kg~1×107 Pieces / kg (body weight).

[0037] [Administration schedule] In the method of the present invention, mesenchymal stem cells are administered once or multiple times. For example, if administered once before pregnancy or after delivery, it is expected that the disease preventive effect will be exerted for a long period thereafter. Periodic administration is also preferable. Periodic administration is expected to continuously promote the return of estrus thereafter. For example, administration schedules include administration once a year or once every few months. Regarding the timing of administration, administration after delivery is preferable, and administration once after delivery is more preferable. Administration once every delivery is also preferable.

[0038] <Method for promoting reproductive function recovery> The method for promoting recovery of reproductive function in an animal of the present invention is characterized by comprising the step of administering mesenchymal stem cells or a composition containing mesenchymal stem cells to the animal. In the present invention, the method for promoting recovery of reproductive function in an animal means promoting recovery of reproductive function so that the animal becomes able to reproduce again after giving birth, and preferably includes recovery of the mother's physical strength and promotion of her health after giving birth. The mesenchymal stem cells, the subjects to be administered, the administration method, etc. are the same as those in the above-mentioned method for promoting return to estrus.

[0039] <Estrus return promoter> The agent for promoting the return of estrus of the present invention is characterized by containing mesenchymal stem cells and is used to promote the return of estrus in animals. That is, the agent for promoting the return of estrus of the present invention is an agent or composition for use in promoting the return of estrus in animals.

[0040] The agent for promoting the return of estrus of the present invention contains mesenchymal stem cells, preferably adipose-derived stem cells, as an active ingredient. The agent is preferably a cell suspension. The concentration of mesenchymal stem cells in the agent for promoting the return of estrus is preferably 1×10 4 cells / mL or more, and more preferably 1×10 5 cells / mL or more, more preferably 4 x 10 6The agent for promoting return to estrus of the present invention may contain, in addition to the active ingredient, a solvent such as water, a buffer solution, or other accessory ingredients.

[0041] Examples of the buffer solution include Ringer's solution, L-sodium lactate Ringer's solution, 5% glucose-lactated Ringer's solution, acetated Ringer's solution, 5% glucose-acetated Ringer's solution, Dulbecco's phosphate buffered saline (D-PBS), saline, Good's buffer, Hank's balanced salt solution, phosphate buffer (PBS), imidazole buffer, triethanolamine hydrochloride buffer (TEA), or a combination thereof.

[0042] The agent for promoting the return of estrus of the present invention may also contain nutrients and chelating agents. Furthermore, it is preferable that the agent for promoting the return of estrus of the present invention is substantially free of medium components for animal cells, organic solvents, and dextran.

[0043] The agent for promoting the return to estrus of the present invention comprises animal mesenchymal stem cells, preferably adipose-derived stem cells, suspended in a buffer solution, and the concentration of mesenchymal stem cells in the agent for promoting the return to estrus is 1×10 5 Preferably, the concentration of ascorbic acid in the buffer solution is 1 mmol / L to 150 mmol / L, and the concentration of ethylenediaminetetraacetic acid or citric acid in the buffer solution is 1 mmol / L to 100 mmol / L, and the concentration of ethylenediaminetetraacetic acid or citric acid in the buffer solution is 1 mmol / L to 100 mmol / L.

[0044] The agent for promoting return to estrus of the present invention may be stored frozen and kept at 0 to 10°C.

[0045] [Dosage form] The dosage form of the agent for promoting reversion to estrus of the present invention is not particularly limited, and may be any known dosage form. Examples include a cell suspension, an injection solution, an intravenous drip solution, a capsule, etc. A cell suspension in which cells are suspended in a solvent is preferred, and an injection solution or an intravenous drip solution made of a cell suspension is more preferred.

[0046] The subjects to which the agent for promoting reversion to estrus of the present invention is administered are the same as those in the method for promoting reversion to estrus described above.

[0047] <Reproductive function recovery promoter> The reproductive function promoter of the present invention is characterized by containing mesenchymal stem cells and is used to promote the recovery of reproductive function in animals. That is, the reproductive function recovery promoter of the present invention is an agent or composition for use in promoting the recovery of reproductive function after birth in animals. The constituent components, administration target, etc. are the same as those of the above-mentioned estrus return promoter. [Example]

[0048] The abbreviations and trade names used in the examples have the following meanings. PBS: D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium ascorbate: L(+)-sodium ascorbate (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0049] <Preparation of canine adipose-derived stem cells> (1) Preparation of cell populations from adipose tissue Adipose tissue was collected from the subcutaneous fat of healthy dogs. The collected adipose tissue was briefly sterilized with 70% ethanol and then enzymatically digested with collagenase Type I enzyme solution (0.1-5 mg / mL) at 37°C for 60 minutes to obtain a cell population (solution) containing adipose-derived stem cells.

[0050] To fractionate the cell population containing adipose-derived stem cells, the above-described enzyme-treated solution was dispensed into a 50 mL centrifuge tube and centrifuged at 750-1500 G. The precipitated fraction at the bottom of the tube was collected as the cell population. The precipitated fraction was then transferred to a new centrifuge tube containing PBS and rinsed to remove any remaining oils and other impurities. The extracellular matrix and undegraded adipose tissue in the adipose tissue were then removed by filtration through a 70 μm pore cell strainer, yielding a cell-containing solution.

[0051] (2) Cultivation and collection of adipose-derived stem cells The solution containing the cells was centrifuged at 1500 G, and the precipitated fraction at the bottom of the tube was collected as a cell population containing adipose-derived stem cells. 2 The suspension was transferred to a culture flask and cultured for 7–10 days, with repeated washing and medium changes every 3–4 days. Culture was carried out at 37°C and 5% carbon dioxide. The culture medium used was the Mesenchymal Stem Cell Growth Medium Bullet Kit™ (MSCGM, Lonza, Inc.).

[0052] To recover the proliferated cells, the recovery procedure followed the standard method for detaching cells adhered to the bottom of the flask. The cells were recovered by reacting with 0.05% (v / v) trypsin at 37°C for 5 minutes. The detached cells were suspended in medium containing fetal bovine serum (FBS, Fujifilm Wako Pure Chemical Corporation) and then centrifuged at 500-1500G to remove the trypsin. The cells were then suspended in medium to obtain the P0 adipose-derived stem cell suspension.

[0053] (3) Cultivation and collection of adipose-derived stem cells P0 mesenchymal stem cells were cultured at 225cm 2The suspension was transferred to a culture flask and cultured for 7-10 days, with repeated washing and medium changes every 3-4 days. Culture was carried out at 37°C and a carbon dioxide concentration of 5%. The culture medium used was D-MEM (Fujifilm Wako Pure Chemical Corporation) or similar, supplemented with FBS. After reaction with trypsin in the same manner as for the collection of P0 above, centrifugation was carried out to obtain the P1 adipose-derived stem cell suspension.

[0054] (4) Cell cryopreservation P1 cells were frozen at 1 × 10 6 ~5×10 6 The cells were suspended in a cryopreservation solution at a concentration of 1000 cells / mL, dispensed into cryotubes, and slowly frozen in a slow freezer at -80°C. A commercially available cell freezing solution containing dimethyl sulfoxide (DMSO) was used as the cryopreservation solution. The cells were then stored in an ultra-low temperature freezer (-80°C to -150°C) or in liquid nitrogen (-196°C).

[0055] (5) Thawing of cells Frozen P1 cells were thawed using an automated cell freeze-thaw station, Thawstar (Biocision), following standard cell thawing procedures. The cells were centrifuged at 2000 G to remove the frozen cell solution. The cell population was suspended in saline and centrifuged, after which the cells were suspended in PBS containing 25 mM sodium ascorbate.

[0056] <Administration of adipose-derived stem cells to dogs> The suspension of canine adipose-derived stem cells prepared above was administered once by intramuscular injection to 17 female dogs (including various breeds) immediately after giving birth (on average, 42.6 days after giving birth). The dose was approximately 1 x 10 6 The number was 1 / kg (body weight). The number of days to estrus was investigated before and after administration. The number of days to estrus was determined by visual inspection by the inventors (actual measurements) or by checking the breeder's rearing records (breeder interviews), which were the number of days from birth to the next estrus. The results are shown in Figures 1 and 2. Figure 1 shows the number of days to estrus measured by the inventors plus the breeder's interviews (average of the actual measurements and the breeder's interviews), with (A) showing the average value for all individuals and (B) showing the distribution. Figure 2 shows the number of days to estrus measured by the inventors, with (A) showing the average value for all individuals and (B) showing the distribution.

[0057] Figures 1 and 2 show that the number of days to estrus return was 55 days shorter on average after administration (actual measurements and results from interviews with breeders; the actual measurement was 67 days). Furthermore, a t-test confirmed that the difference between the administration group and the non-administration group was significant. This demonstrates that administering mesenchymal stem cells can promote the return of estrus in animals. One possible reason for this effect is that the administration of mesenchymal stem cells quickly repairs damage to the mother after birth, but this is not the only possible explanation. Based on this speculated mechanism, it is understandable that similar effects would be achieved in animals other than dogs.

Claims

1. A method for promoting reversion to estrus in an animal, comprising the step of administering to the animal mesenchymal stem cells or a composition containing mesenchymal stem cells.

2. The method for promoting reversion to estrus in an animal according to claim 1, wherein the mesenchymal stem cells are adipose-derived stem cells.

3. 2. The method for promoting the return of estrus to an animal according to claim 1, wherein the animal is a livestock, a dog, or a cat.

4. 4. The method for promoting the return of estrus to animals according to claim 3, wherein the livestock is a cow, a horse or a pig.

5. The method for promoting reversion to estrus in an animal according to any one of claims 1 to 4, wherein the animal is a postpartum animal.

6. 2. The method for promoting the return of estrus to animals according to claim 1, which can shorten the number of days until the return of estrus to animals.

7. A method for promoting recovery of reproductive function in an animal, comprising the step of administering mesenchymal stem cells or a composition containing mesenchymal stem cells to an animal after giving birth.

8. An agent for promoting the return of estrus, characterized by containing animal mesenchymal stem cells.

9. An agent for promoting the recovery of reproductive function in animals, characterized by containing animal mesenchymal stem cells.

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