Culture medium for mammalian fertilized eggs, method for culturing fertilized eggs, and composition of culture medium for 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 Art

[0002] Artificial insemination and fertilized egg transplantation are almost 100% used in domestic cattle production, but the decline in reproductive performance greatly delays the next calving, leading to an extended dry period in dairy cows and a reduction in the number of calves produced in beef cattle, which exerts an adverse economic impact. Therefore, ensuring the conception rate is extremely critical to the economic stability of farmers.

[0003] In in vitro fertilized egg transplantation, the quality of fertilized eggs is directly linked to conception performance. In general, the conception rate of in vitro fertilized egg transplantation is considered to be lower than that of artificial insemination, which has become a major challenge in the popularization of in vitro fertilized egg transplantation. Accordingly, there is a demand for the development of a technique for efficiently producing high-quality fertilized eggs.

[0004] As a technique for producing fertilized eggs, Patent Document 1 describes adding non-essential amino acids to a culture solution. In addition, Non-Patent Document 1 describes a technique for selecting animal embryos obtained by in vitro culture of fertilized eggs that have a high conception rate.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of Invention

[0007] However, the technology described in Patent Document 1 does not produce sufficiently high-quality fertilized eggs after culture, making it difficult to obtain high-quality fertilized eggs with a high conception rate. Furthermore, the technology described in Non-Patent Document 1 requires selecting high-quality fertilized eggs with a high conception rate from the cultured fertilized eggs, making it difficult to efficiently obtain high-quality fertilized eggs with a high conception rate.

[0008] One aspect of the present invention solves the above-mentioned problems, and its objective is to realize a technology for efficiently obtaining high-quality fertilized eggs. [Means for solving the problem]

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

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

[0011] Furthermore, the culture medium composition for mammalian fertilized eggs according to one aspect of the present invention contains non-essential amino acids, and the L-serine content when added to the culture medium is 200 μM or more and 1500 μM or less. [Effects of the Invention]

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

[0013] [Figure 1] This figure shows the results of the example. [Figure 2] This figure shows the results of the example. [Modes for carrying out the invention]

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

[0015] Furthermore, the widespread use of sex-sorted semen, which results in over 90% of female births in dairy cows, has made it possible to increase income by securing replacement heifers while producing crossbreeds with Japanese Black cattle, which are more expensive than male dairy calves, or by producing purebred Japanese Black cattle through embryo transfer.

[0016] However, the conception rate in cattle continues to decline year after year, and in particular, the conception rate in dairy cows has fallen from 60% to 40% in the last 30 years. In in vitro fertilization (IVF) transplantation, the quality of the fertilized egg directly impacts the conception rate, and IVF is generally considered to have a lower conception rate than artificial insemination, which is a major challenge in the widespread adoption of IVF.

[0017] In the fallopian tube fluid, where fertilized eggs develop in the body, the concentration of non-essential amino acids varies greatly depending on the type. However, in conventional culture media for fertilized eggs, 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 fallopian tube fluid. The inventors focused on the fact that the concentration of non-essential amino acids in the culture medium affects the quality of fertilized eggs and conducted extensive research. As a result, they found an appropriate concentration of non-essential amino acids for producing high-quality fertilized eggs, thus completing 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 comprising 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. As a result, fertilized eggs with a high conception rate can be obtained.

[0019] [Medium for culturing fertilized eggs] The medium for culturing fertilized eggs according to one aspect of the present invention is a non-essential amino acid-containing medium comprising 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 group of non-essential amino acids added to the medium for culturing fertilized eggs consists of L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, L-alanine, and L-serine. The group of non-essential amino acids may be contained in the medium for culturing fertilized eggs in a free form or a pharmacologically acceptable salt form. Further, the group of non-essential amino acids contained in the medium for culturing fertilized eggs may be those that can be hydrolyzed and converted into free amino acids.

[0021] The medium for culturing fertilized eggs contains electrolytes as basic constituents, and may contain, as other compounds other than the group of non-essential amino acids, carbohydrates, a group of essential amino acids, vitamins, hormones, cell growth factors, lipids or constituents thereof, carrier proteins, extracellular matrix components (adhesion factors), reducing substances, and even trace metal elements and the like.

[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, and calcium lactate. Examples of carbohydrates include glucose, maltose, fructose, xylitol, sorbitol, and trehalose. Examples of vitamins include vitamin A, B vitamins, vitamin C, D vitamins, vitamin E, nicotinic acid, biotin, and folic acid. Examples of trace metal elements include zinc, iron, manganese, copper, iodine, selenium, and cobalt.

[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 (adhesion factors) include figronectin, collagen, and gelatin. Examples of reducing substances include 2-mercaptoethanol, dithiothreitol, and reduced glutathione. Culture media for fertilized eggs are 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, as well as antifungal agents such as amphotericin B and nystatin, may be added to the culture medium for fertilized eggs as appropriate.

[0025] Furthermore, culture media for fertilized egg culture can also be used in combination with known equilibrium salt solutions or media consisting of the above-mentioned compounds that can constitute the medium composition. Examples of equilibrium salt solutions include Tyrode's solution, Krebs-Ringer's bicarbonate solution, Earl's solution, Hanks' solution, Dulbecco's phosphate buffer, or modified versions thereof. Examples of modified versions 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, and T6 medium. Examples of media suitable for tissue cultures with more complex components include 199 medium, BME medium, CMRL1066 medium, MEM medium, McCoy-5A medium, Weymouth medium, Trowill T-8 medium, Hamm medium, Leibowitz L-15 medium, NCTC medium, Williams-E medium, MCDB104 medium, or modified versions thereof.

[0026] Culture media for fertilized eggs can be manufactured by conventional methods by combining their components, and are usually liquid media. Furthermore, culture media for fertilized eggs may contain additives such as pH adjusters, stabilizers, and excipients as needed. 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 bisulfite, and sodium pyrosulfite. Additionally, pH indicators such as phenol red may be added to the culture media for fertilized eggs.

[0027] The content of L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, and L-proline in the culture medium for fertilized eggs, among the non-essential amino acids, is, for example, 50 μM or more and 150 μM or less, respectively, but may also be 100 μM.

[0028] The L-serine content in the culture medium for fertilized eggs is between 200 μM and 1500 μM. This allows for the efficient production of high-quality fertilized eggs. The quality of fertilized eggs can be evaluated by the following method. Specifically, a fertilized egg that has divided into 2 cells at 27 hours after insemination in in vitro fertilization, has not divided into 3 or more cells at 31 hours, and has no cell fragments, and has divided into 6 or more cells at 55 hours, and is a blastocyst at 8 days after insemination, while satisfying all of these indicators, can be evaluated as a high-quality fertilized egg (see Non-Patent Literature 1). In addition, fertilized eggs that are blastocysts at 8 days after insemination but do not satisfy one or more of the indicators at 27 hours, 31 hours, or 55 hours after insemination, along with high-quality fertilized eggs, can be evaluated as transferable fertilized eggs (see Non-Patent Literature 1). High-quality fertilized eggs evaluated in this way have a high conception rate.

[0029] Furthermore, the L-serine content in the culture medium for fertilized eggs is preferably 800 μM or more and 1000 μM or less, and more preferably 1000 μM. This allows for the more efficient production of high-quality fertilized eggs.

[0030] The culture medium for fertilized eggs may be L-alanine-free or contain L-alanine at a concentration of 50 μM or less. In other words, it is preferable that the culture medium for fertilized eggs contains no L-alanine at all, or, if L-alanine is present, at a low concentration of 50 μM or less. This allows for the more efficient production of high-quality fertilized eggs. The L-alanine content in the culture medium for fertilized eggs is more preferably 25 μM or less, even more preferably 10 μM or less, and most preferably 0 μM.

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

[0032] The fertilized eggs cultured in the fertilized egg culture medium are mammalian fertilized eggs that have been fertilized outside the mammalian body. The fertilized egg culture medium can be used to develop fertilized eggs into blastocysts and produce blastocyst-stage embryos. Using the fertilized egg culture medium, it is possible to produce blastocyst-stage embryos of high-quality fertilized eggs, as evaluated above, with high efficiency.

[0033] [fertilized egg culture method] A method for culturing mammalian fertilized eggs according to one aspect of the present invention comprises a culture step of culturing mammalian fertilized eggs in 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.

[0034] In the culture process, mammalian fertilized eggs are cultured in a non-essential amino acid-containing medium having an L-serine content of 200 μM or more and 1500 μM or less. Conventional known culture conditions and durations for fertilized eggs can be used for the culture conditions and duration in the culture process. For example, in the culture process, fertilized eggs are cultured until the development rate of blastocyst-stage embryos exceeds a predetermined ratio, or until the ratio of fertilized eggs divided into a predetermined number of cells exceeds a predetermined ratio.

[0035] In a method for culturing mammalian fertilized eggs according to one aspect of the present invention, the non-essential amino acid-containing medium used for culturing mammalian fertilized eggs is one aspect of the non-essential amino acid-containing medium for fertilized egg culture according to one aspect of the present invention described above. Therefore, for details regarding the non-essential amino acid-containing medium in the method for culturing mammalian fertilized eggs according to one aspect of the present invention, refer to the description of the non-essential amino acid-containing medium in the fertilized egg culture medium according to one aspect of the present invention described above.

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

[0037] Mammalian embryo culture medium compositions can be used to produce embryo culture medium by adding them to conventionally known basic culture media for embryo development. Examples of conventionally known basic culture media for embryo development include the equilibrium salt solutions and culture media described above. The embryo culture medium thus produced can be used to culture mammalian embryos and induce embryo development. Mammalian embryo culture medium compositions contain non-essential amino acids and have an L-serine content of 200 μM or more and 1500 μM or less when added to a culture medium such as a basic culture medium for embryo development. Mammalian embryo culture medium compositions contain an amount of L-serine such that the L-serine content when added to a culture medium is 200 μM or more and 1500 μM or less.

[0038] The culture medium composition for mammalian fertilized eggs may contain components other than non-essential amino acids, an example of which is illustrated in the description of the culture medium for fertilized eggs according to one aspect of the present invention described above. Therefore, for details of the components contained in the culture medium composition for mammalian fertilized eggs according to one aspect of the present invention, refer to the description of the components contained in the culture medium for fertilized eggs according to one aspect of the present invention described above.

[0039] According to the above configuration, it will contribute to the maintenance and development of the livestock industry. Such effects will also contribute to achieving goals such as Goal 15 of the United Nations' Sustainable Development Goals (SDGs), "Protect and restore life on land."

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

[0041] The culture medium for mammalian fertilized eggs according to Embodiment 1 of the present invention is a non-essential amino acid-containing medium that includes a group of non-essential amino acids, wherein the L-serine content is 200 μM or more and 1500 μM or less.

[0042] In the culture medium for mammalian fertilized eggs according to aspect 2 of the present invention, it is preferable that the L-serine content is 800 μM or more and 1000 μM or less, as in aspect 1 above.

[0043] The culture medium for mammalian fertilized eggs according to embodiment 3 of the present invention may, in embodiment 1 or 2 above, not contain L-alanine, or may contain L-alanine in a quantity of 50 μM or less.

[0044] A mammalian fertilized egg culture medium according to aspect 4 of the present invention is a mammalian fertilized egg culture medium according to claim 1 or 2, for culturing bovine fertilized eggs in any of the above aspects 1 to 3.

[0045] A method for culturing mammalian fertilized eggs according to aspect 5 of the present invention comprises a culture step of culturing mammalian fertilized eggs in 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.

[0046] The mammalian fertilized egg culture medium composition according to aspect 6 of the present invention contains non-essential amino acids, and the L-serine content when added to the medium is 200 μM or more and 1500 μM or less.

[0047] [Additional Note 2] The present invention is not limited to the embodiments described above, 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 oophorus cell-oocyte complexes were aspirated from antral follicles of bovine ovaries and cultured in vitro for 22 hours in 199 medium supplemented with 10% FCS. In vitro fertilization was performed with a final concentration of 5 × 10⁶. 6 Sperm adjusted to 1 / mL was used for 6-hour insemination. After insemination, the denatured embryos were cultured in microdroplets of serum-free modified CR1 (containing 0.3% BSA and 2% BME essential amino acid (Sigma-Aldrich: M6766)).

[0049] A culture medium for fertilized eggs was prepared by adding non-essential amino acids other than L-alanine to MEM medium. Culture media for fertilized eggs were prepared with L-serine concentrations of 100 μM, 500 μM, and 1000 μM, respectively, and the amount of other non-essential amino acids other than L-serine and L-alanine was set to 100 μM.

[0050] 137 fertilized eggs were cultured in various embryo culture media, and each fertilized egg was evaluated based on its embryonic development rate. A high-quality fertilized egg was defined as one that reached 2 cells at 27 hours after insemination, had not divided into 3 or more cells at 31 hours, was free of fragments, had divided into 6 or more cells at 55 hours, and was a blastocyst on day 8 after insemination. Additionally, fertilized eggs that were blastocysts on day 8 after insemination but did not meet one or more of the indicators at 27, 31, or 55 hours after insemination, along with high-quality fertilized eggs, were collectively evaluated as transferable fertilized eggs.

[0051] The fertilized eggs were evaluated using the indicators described above, and the number of high-quality and transferable fertilized eggs was measured. Their ratio to the total number of fertilized eggs was then calculated. The results are shown in Table 1.

[0052] [Table 1]

[0053] As shown in Table 1, when the L-serine concentration was set to 500 μm and 1000 μm, the proportion of high-quality embryos and the proportion of embryos suitable for transfer improved compared to when the concentration was 100 μm.

[0054] [Example 2] The development rate of normal blastocysts was investigated in the first and second halves of culture, with L-alanine omitted and L-serine concentrations of 100 μM and 1000 μM. Details of the culture medium and conditions were the same as in Example 1 described above. High-quality embryos were defined as those that reached 2 cells at 27 hours after insemination, had not divided into 3 or more cells at 31 hours, were free of fragments, had divided into 6 or more cells at 55 hours, and were blastocysts on day 8 after insemination. In addition, embryos that were blastocysts on day 8 after insemination but did not meet one or more of the indicators at 27, 31, or 55 hours after insemination, along with high-quality embryos, were evaluated as embryos suitable for transfer.

[0055] The results are shown in Figure 1. Figure 1A shows the percentage of embryos that met the criteria up to 55 hours after in vitro fertilization, and Figure 1B shows the developmental rate after 55 hours after in vitro fertilization. As shown in Figure 1A, there was little difference in L-serine concentration during the first half of the culture period. On the other hand, when the blastocyst formation rate was calculated using embryos that met the criteria up to 55 hours after in vitro fertilization as the denominator, as shown in Figure 1B, the developmental rate significantly increased in the group with an L-serine concentration of 1000 μM.

[0056] [Example 3] The effect of L-alanine concentration on fertilized eggs was investigated by setting the L-serine concentration to 1000 μM and the L-alanine concentrations to 0 μM and 100 μM. Culture and evaluation were performed under the same conditions as in Example 1, except that the L-serine concentration was 1000 μM and the L-alanine concentrations were 0 μM and 100 μM. The results are shown in Table 2. In Table 2, "Initial Culture Stage" indicates the cell division rate into two cells at 27 hours after insemination.

[0057] [Table 2]

[0058] As shown in Table 2, in embryo culture media containing L-alanine, the two-cell cleavage rate at 27 hours after insemination was low, and the proportion of high-quality embryos and embryos suitable for transfer was also low.

[0059] [Example 4] The effects of high concentrations of L-serine on fertilized eggs were investigated by setting L-serine concentrations to 1000 μM, 2000 μM, and 3000 μM, without the addition of L-alanine. Culturing and evaluation were performed under the same conditions as in Example 1, except for the L-serine concentrations of 1000 μM, 2000 μM, and 3000 μM and the absence of L-alanine. The results are shown in Table 3. Details such as culture medium, culture conditions, and evaluation indicators were the same as in Example 1 described above.

[0060] [Table 3]

[0061] As shown in Table 3, the proportion of high-quality embryos and embryos suitable for transfer was lower when the L-serine concentration was high at 2000 μM and 3000 μM compared to when it was 1000 μM.

[0062] [Example 5] The effect of L-alanine concentration on cell division was investigated by setting L-alanine concentrations to 0 μM, 10 μM, 25 μM, and 50 μM, and L-serine concentration to 1000 μM. The cells were cultured under the same conditions as in Example 1, except that the L-alanine concentrations were 0 μM, 10 μM, 25 μM, and 50 μM, and the L-serine concentration was 1000 μM. Fertilized eggs were evaluated by the two-cell division rate 27 hours after fertilization. The results are shown in Figure 2. As shown in Figure 2, L-alanine concentrations of 0 μM, 10 μM, 25 μM, and 50 μM did not affect the two-cell division rate of fertilized eggs. [Industrial applicability]

[0063] This invention can be used in agriculture and livestock farming.

Claims

1. A culture medium containing non-essential amino acids, including a group of non-essential amino acids, The L-serine content is 500 μM or more and 1000 μM or less. The aforementioned non-essential amino acid group consists of L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine. It does not contain L-alanine, or its L-alanine content is 50 μM or less. Culture medium for bovine fertilized eggs.

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

3. The method includes a culture step of culturing bovine fertilized eggs in a non-essential amino acid-containing medium that contains non-essential amino acids and has an L-serine content of 500 μM or more and 1000 μM or less. The aforementioned non-essential amino acid group consists of L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine. The aforementioned non-essential amino acid-containing medium does not contain L-alanine, or its L-alanine content is 50 μM or less. A method for culturing bovine fertilized eggs.

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