Medium for culturing fertilized eggs of mammals, method for culturing fertilized eggs, and medium composition for culturing fertilized eggs

Abstract

To achieve a technique for efficiently acquiring high-quality fertilized eggs.SOLUTION: A medium for culturing fertilized eggs of mammals is a non-essential amino acid-containing medium including a group of non-essential amino acids, where the L-serine content is 200 μM or more and 1500 μM or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a medium for culturing mammalian fertilized eggs, a method for culturing fertilized eggs, and a medium composition for culturing fertilized eggs. [Background technology]

[0002] Although artificial insemination and embryo transfer are used almost 100% in domestic cattle production, a decline in reproductive performance significantly delays the next calving, leading to an extension of the dry period in dairy cows and a decrease in the number of calves produced in beef cows, all of which have a negative economic impact. Therefore, ensuring the conception rate is extremely important for the economic stability of farmers.

[0003] In IVF / Egg Transfer, the quality of the fertilized eggs is directly related to the conception outcome. In general, IVF / Egg Transfer is considered to have a lower conception rate than artificial insemination, which is a major issue in the spread of IVF / Egg Transfer. Therefore, there is a demand for the development of technology to produce high-quality fertilized eggs with high efficiency.

[0004] As a technique for producing fertilized eggs, Patent Document 1 describes the addition of non-essential amino acids to a culture medium. In addition, Non-Patent Document 1 describes a technique for selecting animal embryos with a high conception rate from those obtained by in vitro culture of fertilized eggs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 3-503841 [Non-patent literature]

[0006] [Non-Patent Document 1] Selection criteria and methods for highly fertile bovine in vitro fertilized embryos, Japanese Journal of Embryo Transfer, 2015, Vol.36, No.1, 37-41 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in the technology described in Patent Document 1, the quality of the cultured fertilized eggs is not sufficiently high, and it is not easy to obtain high-quality fertilized eggs with a high conception rate. In addition, in the technology described in Non-Patent Document 1, it is necessary to select high-quality fertilized eggs with a high conception rate from the cultured fertilized eggs, and it is not easy to efficiently obtain high-quality fertilized eggs with a high conception rate.

[0008] One aspect of the present invention is to solve the above-mentioned problems, and an object of the present invention is to realize a technique for efficiently obtaining high-quality fertilized eggs. [Means for solving the problem]

[0009] In order to solve the above problems, a medium for culturing mammalian fertilized eggs according to one embodiment of the present invention is a non-essential amino acid-containing medium that includes a group of non-essential amino acids, and has an L-serine content of 200 μM or more and 1500 μM or less.

[0010] Furthermore, a method for culturing a mammalian fertilized egg according to one embodiment of the present invention includes a culture step of culturing a mammalian fertilized egg in a non-essential amino acid-containing medium containing a group of non-essential amino acids and having an L-serine content of 200 μM or more and 1500 μM or less.

[0011] Furthermore, the medium composition for culturing mammalian fertilized eggs according to one embodiment of the present invention contains a group of non-essential amino acids, and has an L-serine content of 200 μM or more and 1500 μM or less when added to the medium. Effect of the Invention

[0012] According to one aspect of the present invention, a technique for efficiently obtaining high-quality fertilized eggs can be realized. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of an example. [Diagram 2] FIG. 1 shows the results of an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Although artificial insemination and embryo transfer are used almost 100% in domestic cattle production, a decline in reproductive performance significantly delays the next calving, leading to an extension of the dry period in dairy cows and a decrease in the number of calves produced in beef cows, all of which have a negative economic impact. Therefore, ensuring the conception rate is extremely important for the economic stability of farmers.

[0015] In addition, with the widespread use of sex-sorted semen, which ensures that dairy cows produce females with a 90% or higher probability, it has become possible to secure successor cows while also increasing income by producing crossbreeds with Japanese Black cattle, which are more expensive than dairy bulls, or by producing purebred Japanese Black cattle through embryo transfer.

[0016] However, the conception rate of cattle continues to decline year by year, and in particular that of dairy cows has fallen from 60% to 40% over the past 30 years. In IVF transfer, the quality of the fertilized eggs is directly linked to conception success, and it is generally believed that IVF transfer has a lower conception rate than artificial insemination, which has been a major obstacle to the widespread use of IVF transfer.

[0017] In the oviduct fluid, where fertilized eggs develop in the body, the concentrations of non-essential amino acids vary greatly depending on the type. However, in conventional fertilized egg culture media such as those described in Patent Document 1, all non-essential amino acids are added at a uniform concentration (e.g., 100 μM), which differs from the concentration in the oviduct fluid. The present inventors have focused on the effect of the concentration of non-essential amino acids in the medium on the quality of fertilized eggs and conducted extensive research, discovering an appropriate concentration of non-essential amino acids for producing high-quality fertilized eggs, and have completed the present invention.

[0018] According to one aspect of the present invention, by using a medium for culturing mammalian fertilized eggs, which is a non-essential amino acid-containing medium containing a group of non-essential amino acids and has an L-serine content of 200 μM or more and 1500 μM or less, high-quality fertilized eggs can be efficiently obtained, and as a result, fertilized eggs with a high conception rate can be obtained.

[0019] [Medium for fertilized egg culture] A medium for culturing fertilized eggs according to one embodiment of the present invention is a non-essential amino acid-containing medium that contains a group of non-essential amino acids, and has an L-serine content of 200 μM or more and 1500 μM or less.

[0020] The non-essential amino acids added to the medium for fertilized egg culture consist of L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, L-alanine, and L-serine. The non-essential amino acids may be contained in the medium for fertilized egg culture in the form of free amino acids or pharmacologically acceptable salts. The non-essential amino acids contained in the medium for fertilized egg culture may be those that can be hydrolyzed and converted into free amino acids.

[0021] The medium for culturing fertilized eggs contains electrolytes as basic components, and may contain other compounds such as carbohydrates, essential amino acids, vitamins, hormones, cell growth factors, lipids or their components, carrier proteins, extracellular matrix components (adhesion factors), reducing substances, and even trace metal elements in addition to non-essential amino acids.

[0022] Examples of electrolytes include sodium chloride, sodium acetate, sodium citrate, potassium chloride, calcium chloride, calcium gluconate, magnesium chloride, magnesium sulfate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium bicarbonate, sodium pyruvate, sodium lactate, calcium lactate, etc. Examples of carbohydrates include glucose, maltose, fructose, xylitol, sorbitol, trehalose, etc. Examples of vitamins include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, biotin, folic acid, etc. Examples of trace metal elements include zinc, iron, manganese, copper, iodine, selenium, cobalt, etc.

[0023] Examples of hormones include insulin, hydrocortisone, dexamethasone, and triiodothyronine. Examples of cell growth factors include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, and growth hormone. Examples of lipids or their components include essential unsaturated fatty acids such as oleic acid, linoleic acid, and linolenic acid, cholesterol, ethanolamine, and choline. Examples of carrier proteins include serum albumin and transferrin. Examples of extracellular matrix components (adhesive factors) include phygronectin, collagen, and gelatin. Examples of reducing substances include 2-mercaptoethanol, dithiothreitol, and reduced glutathione. The medium for culturing fertilized eggs is usually composed of a combination of some compounds appropriately selected from the compounds described above.

[0024] Furthermore, antibiotics such as penicillin, streptomycin, kanamycin, gentamicin, and erythromycin, and antifungal agents such as amphotericin B and nystatin may be appropriately added to the culture medium for fertilized eggs.

[0025] Furthermore, the medium for fertilized egg culture may be used by mixing with known balanced salt solutions or media consisting of the above-mentioned compounds that may constitute the medium composition. Examples of balanced salt solutions include Tyrode's solution, Krebs-Ringer bicarbonate solution, Earle's solution, Hank's solution, Dulbecco's phosphate buffer, or modified solutions thereof. Examples of modified solutions include Whitten medium, m-KRB medium, Kane & Foote medium, BWW medium, Brinster medium, m-Tyrode's medium, Hoppes & Pitts medium, BO medium, KSOM medium, CR1 medium, SOF medium, HTF medium, T6 medium, etc. Examples of media suitable for tissue culture containing more complex components include 199 medium, BME medium, CMRL1066 medium, MEM medium, McCoy-5A medium, Weymouth medium, Trowill T-8 medium, Ham's medium, Leibovitz L-15 medium, NCTC medium, Williams-E medium, MCDB104 medium, or modified media thereof.

[0026] The medium for culturing fertilized eggs can be produced by mixing the components by a conventional method, and is usually a liquid medium. The medium for culturing fertilized eggs may contain additives such as a pH adjuster, a stabilizer, and an excipient, as necessary. Examples of pH adjusters include hydrochloric acid, acetic acid, and sodium hydroxide. Examples of stabilizers include HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), sodium sulfite, sodium hydrogen sulfite, and sodium pyrosulfite. The medium for culturing fertilized eggs may also contain a pH indicator such as phenol red.

[0027] Of the non-essential amino acids contained in the medium for culturing fertilized eggs, the contents of L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, and L-proline are, for example, 50 μM or more and 150 μM or less, respectively, and may be 100 μM.

[0028] The content of L-serine contained in the medium for fertilized egg culture is 200 μM or more and 1500 μM or less. This allows high-quality fertilized eggs to be produced with high efficiency. Here, the quality of the fertilized eggs can be evaluated by the following method. Specifically, a fertilized egg that has divided into two cells at 27 hours from the time of insemination during in vitro fertilization, has not divided into three or more cells at 31 hours, and does not have cell fragments, and has divided into six or more cells at 55 hours, and is a blastocyst at the time of 8 days after insemination while satisfying all of these indicators, can be evaluated as a high-quality fertilized egg (see Non-Patent Document 1). In addition, a blastocyst at the time of 8 days after insemination that does not satisfy any one or more indicators at 27 hours, 31 hours, or 55 hours from the time of insemination, or a high-quality fertilized egg, can be evaluated as a transplantable fertilized egg (see Non-Patent Document 1). High-quality fertilized eggs evaluated in this way have a high conception rate.

[0029] Furthermore, the content of L-serine in the medium for culturing fertilized eggs is preferably 800 μM or more and 1000 μM or less, and more preferably 1000 μM, which allows high-quality fertilized eggs to be produced more efficiently.

[0030] The medium for culturing fertilized eggs may not contain L-alanine or may have an L-alanine content of 50 μM or less. That is, the medium for culturing fertilized eggs may not contain any L-alanine, or if it does contain L-alanine, it is preferable that it contains it at a low concentration of 50 μM or less. This allows high-quality fertilized eggs to be produced more efficiently. The content of L-alanine in the medium for culturing fertilized eggs is more preferably 25 μM or less, even more preferably 10 μM or less, and most preferably 0 μM.

[0031] The embryo culture medium can be used for culturing the eggs or fertilized eggs of any mammal, but is particularly suitable for culturing bovine fertilized eggs, and can produce bovine fertilized eggs of high quality and with a high conception rate with high efficiency.

[0032] The fertilized eggs cultured in the medium for fertilized egg culture are fertilized mammalian eggs fertilized outside the mammalian body. The medium for fertilized egg culture can be used to develop the fertilized eggs into blastocysts and produce blastocyst-stage embryos. The medium for fertilized egg culture can produce blastocyst-stage embryos of high quality fertilized eggs evaluated as described above with high efficiency.

[0033] [fertilized egg culture method] A method for culturing a mammalian fertilized egg according to one embodiment of the present invention includes a culture step of culturing a mammalian fertilized egg in a non-essential amino acid-containing medium containing a group of non-essential amino acids and having an L-serine content of 200 μM or more and 1500 μM or less.

[0034] In the culture step, a mammalian fertilized egg is cultured in a medium containing a non-essential amino acid with an L-serine content of 200 μM or more and 1500 μM or less. The culture conditions and culture period in the culture step can be the conventionally known culture conditions and culture period for fertilized eggs. As an example, in the culture step, the fertilized egg is cultured until the development rate of blastocyst stage embryos exceeds a predetermined ratio, or the ratio of the fertilized egg divided into a predetermined number of cells exceeds a predetermined ratio.

[0035] In the method for culturing a mammalian fertilized egg according to one aspect of the present invention, the non-essential amino acid-containing medium used for culturing the mammalian fertilized egg is one aspect of the non-essential amino acid-containing medium of the medium for culturing a fertilized egg according to the aspect of the present invention described above. Therefore, for details of the non-essential amino acid-containing medium in the method for culturing a fertilized egg according to one aspect of the present invention, the explanation of the non-essential amino acid-containing medium in the medium for culturing a fertilized egg according to the aspect of the present invention described above is incorporated herein by reference.

[0036] [Additives for fertilized egg culture medium] A medium composition for culturing mammalian fertilized eggs according to one embodiment of the present invention contains a group of non-essential amino acids, and has an L-serine content of 200 μM or more and 1500 μM or less when added to the medium.

[0037] The medium composition for mammalian fertilized egg culture can be added to a conventionally known basal medium for embryo development to produce a medium for fertilized egg culture. Examples of conventionally known basal medium for embryo development can be the above-mentioned balanced salt solutions and media. The medium for fertilized egg culture produced in this manner can be used to culture mammalian fertilized eggs and develop embryos. The medium composition for mammalian fertilized egg culture contains a group of non-essential amino acids, and when added to a medium such as a basal medium for embryo development, the content of L-serine is 200 μM or more and 1500 μM or less. The medium composition for mammalian fertilized egg culture contains L-serine in an amount such that the content of L-serine is 200 μM or more and 1500 μM or less when added to the medium.

[0038] The medium composition for mammalian fertilized egg culture may contain components other than the non-essential amino acids, an example of which is as exemplified in the description of the medium for fertilized egg culture according to one embodiment of the present invention above. Therefore, for details of the components contained in the medium composition for mammalian fertilized egg culture according to one embodiment of the present invention, the description of the components contained in the medium for fertilized egg culture according to one embodiment of the present invention is incorporated herein by reference.

[0039] The above-mentioned configuration leads to the maintenance and development of the livestock industry. Such an effect also contributes to the achievement of Goal 15 of the Sustainable Development Goals (SDGs) advocated by the United Nations, "Protect and sustain the life on land."

[0040] [Additional Note 1] The present invention can be expressed as follows.

[0041] The medium for culturing mammalian fertilized eggs according to embodiment 1 of the present invention is a non-essential amino acid-containing medium that contains a group of non-essential amino acids and has an L-serine content of 200 μM or more and 1500 μM or less.

[0042] The medium for culturing mammalian fertilized eggs according to aspect 2 of the present invention, in accordance with aspect 1 above, preferably has an L-serine content of 800 μM or more and 1000 μM or less.

[0043] The medium for culturing mammalian fertilized eggs according to aspect 3 of the present invention may be the same as that according to aspect 1 or 2 above, and may contain no L-alanine or may have an L-alanine content of 50 μM or less.

[0044] A fourth aspect of the present invention relates to a medium for culturing mammalian fertilized eggs, and is a medium for culturing mammalian fertilized eggs according to any one of the first to third aspects, the medium being for culturing bovine fertilized eggs as described in claim 1 or 2.

[0045] The method for culturing a mammalian fertilized egg according to aspect 5 of the present invention includes a culture step of culturing a mammalian fertilized egg in a non-essential amino acid-containing medium containing a group of non-essential amino acids and having an L-serine content of 200 μM or more and 1500 μM or less.

[0046] A medium composition for culturing a mammalian fertilized egg according to a sixth aspect of the present invention contains a group of non-essential amino acids, and has an L-serine content of 200 μM or more and 1500 μM or less when added to the medium.

[0047] [Additional Note 2] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0048] Example 1 Cumulus cell-oocyte complexes were aspirated from bovine ovarian antral follicles and cultured in vitro for maturation in 199 medium supplemented with 10% FCS for 22 hours. 6 The embryos were inseminated for 6 hours using sperm adjusted to a concentration of 1 / mL. After insemination, the denuded embryos were cultured in microdroplets of serum-free modified CR1 (containing 0.3% BSA and 2% BME Essential amino acid (Sigma-Aldrich: M6766)).

[0049] Fertilized egg culture media were prepared by adding non-essential amino acids other than L-alanine to MEM medium. The L-serine concentrations were 100 μM, 500 μM, and 1000 μM, respectively, and the amounts of non-essential amino acids other than L-serine and L-alanine were 100 μM.

[0050] 137 fertilized eggs were cultured in each medium for fertilized egg culture, and each fertilized egg was evaluated according to the rate of embryo development. Embryos that reached 2 cells at 27 hours after insemination, had not divided into 3 or more cells at 31 hours, had no fragments, divided into 6 or more cells at 55 hours, and were blastocysts on the 8th day after insemination were evaluated as high-quality fertilized eggs. In addition, blastocysts on the 8th day after insemination that did not meet any or more of the criteria at 27, 31, or 55 hours after insemination, and high-quality fertilized eggs were also evaluated as transferable fertilized eggs.

[0051] The fertilized eggs were evaluated according to the above-mentioned indices, the number of high-quality fertilized eggs and transferable fertilized eggs were measured, and their ratio to the total fertilized eggs was calculated. The results are shown in Table 1.

[0052] [Table 1]

[0053] As shown in Table 1, when the L-serine concentration was 500 μm and 1000 μm, the ratio of high-quality fertilized eggs and the ratio of transferable fertilized eggs were improved compared to when the L-serine concentration was 100 μm.

[0054] Example 2 The incidence of normal blastocysts was examined in the first and second half of the culture when L-alanine was not added and L-serine concentrations were 100 μM and 1000 μM. Details of the medium, culture conditions, etc. were the same as those in Example 1 described above. The evaluation was performed on high-quality fertilized eggs, which were 2 cells at 27 hours after insemination, had not divided into 3 cells or more at 31 hours, had no fragments, and divided into 6 cells or more at 55 hours, and were blastocysts on the 8th day after insemination. In addition, blastocysts on the 8th day after insemination that did not meet any or multiple indicators at 27 hours, 31 hours, and 55 hours after insemination, and high-quality fertilized eggs were also evaluated as implantable fertilized eggs.

[0055] The results are shown in Figure 1. Figure 1A shows the rate of embryos that cleared the indicators up to 55 hours after IVF, and Figure 1B shows the development rate after 55 hours after IVF. As shown in Figure 1A, there was little difference due to the L-serine concentration in the first half of the culture. On the other hand, when the blastocyst formation rate was calculated using the number of embryos that cleared the indicators up to 55 hours after IVF as the denominator, the development rate increased significantly in the category where the L-serine concentration was 1000 μM, as shown in Figure 1B.

[0056] Example 3 The effect of L-alanine concentration on fertilized eggs was examined by setting the L-serine concentration to 1000 μM and the L-alanine concentrations to 0 μM and 100 μM. Except for setting the L-serine concentration to 1000 μM and the L-alanine concentrations to 0 μM and 100 μM, the eggs were cultured and evaluated under the same conditions as in Example 1. The results are shown in Table 2. In Table 2, "initial culture" indicates the division rate into two cells at 27 hours after insemination.

[0057] [Table 2]

[0058] As shown in Table 2, in the fertilized egg culture medium containing L-alanine, the two-cell division rate at 27 hours after insemination was low, and the ratios of high-quality fertilized eggs and transferable fertilized eggs were also low.

[0059] Example 4 The effect of high concentrations of L-serine on fertilized eggs was examined by not adding L-alanine and setting the L-serine concentrations to 1000 μM, 2000 μM, and 3000 μM. Culture and evaluation were performed under the same conditions as in Example 1, except that the L-serine concentrations were 1000 μM, 2000 μM, and 3000 μM and L-alanine was not added. The results are shown in Table 3. Details of the medium, culture conditions, evaluation index, etc. were the same as in Example 1 described above.

[0060] [Table 3]

[0061] As shown in Table 3, when the L-serine concentration was high at 2000 μM and 3000 μM, the ratios of high-quality fertilized eggs and transferable fertilized eggs were lower than when the L-serine concentration was 1000 μM.

[0062] Example 5 The effect of L-alanine concentration on cell division was examined by setting the L-alanine concentration to 0 μM, 10 μM, 25 μM, and 50 μM, and the L-serine concentration to 1000 μM. Except for setting the L-alanine concentration to 0 μM, 10 μM, 25 μM, and 50 μM, and the L-serine concentration to 1000 μM, the culture was performed under the same conditions as in Example 1. The fertilized eggs were evaluated by the two-cell division rate 27 hours after fertilization. The results are shown in FIG. 2. As shown in FIG. 2, when the L-alanine concentration was 0 μM, 10 μM, 25 μM, and 50 μM, there was no effect on the two-cell division rate of the fertilized eggs. [Industrial Applicability]

[0063] The present invention can be used in agriculture and animal husbandry.

Claims

1. A non-essential amino acid-containing medium comprising a group of non-essential amino acids, A medium for culturing mammalian fertilized eggs, the medium having an L-serine content of 200 μM or more and 1500 μM or less.

2. 2. The medium for culturing mammalian fertilized eggs according to claim 1, wherein the L-serine content is 800 μM or more and 1000 μM or less.

3. 3. The medium for culturing mammalian fertilized eggs according to claim 1, which does not contain L-alanine or has an L-alanine content of 50 μM or less.

4. 3. The medium for culturing mammalian fertilized eggs according to claim 1 or 2, for culturing bovine fertilized eggs.

5. A method for culturing a mammalian fertilized egg, comprising a culturing step of culturing the mammalian fertilized egg in a non-essential amino acid-containing medium containing a group of non-essential amino acids and having an L-serine content of 200 μM or more and 1500 μM or less.

6. A medium composition for culturing mammalian fertilized eggs, which contains a group of non-essential amino acids and has an L-serine content of 200 μM or more and 1500 μM or less when added to the medium.

Images