JP2010187629A - Culture medium composition for artificial insemination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new culture medium composition insusceptible to thermal stress in a culturing process of a bovine embryo and its storage process. <P>SOLUTION: There is provided a culture medium composition for artificial insemination (external fertilization), containing xanthophyll such as astaxanthin and a method for culturing and storing bovine embryo after the artificial insemination of the embryo, using the composition. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a medium composition for artificial fertilization, in particular, a medium composition for in vitro fertilization used when culturing or storing bovine embryos, and a method for culturing and storing bovine embryos after in vitro fertilization using the composition.

  Cattle are usually limited to a maximum of about 10 offspring in their lifetimes that can be left by one female for parturition. In order to produce a good calf efficiently, ovulation is induced to a good cow with an ovulation inducer, and after in vivo artificial insemination using a good bull's sperm, an embryo is collected from the uterus. An in-vivo artificial insemination method that preserves and transplants embryos into the fallopian tubes or uterus of other cows is widely used. Currently, 270,000 embryos from fresh embryos and 280,000 embryos from cryopreserved embryos are transplanted worldwide. It has been implemented (Non-patent Document 1).

  In order to produce better calves more efficiently, immature eggs are collected from the ovary of beef cattle in the meat market, fertilized with eggs and sperm in vitro in a culture medium, and then embryos are developed. In vitro fertilization methods that preserve and transplant embryos into the fallopian tubes or uterus of dairy cows are widely used. In vitro fertilization is generally known as a more suitable method for mass production than in-vivo artificial insemination because about 100 immature eggs are collected from 10 ovaries that are usually discarded and about 10 calves can be produced. Then, about 2000 calves are born each year (Non-patent Document 1). Furthermore, in vitro fertilization using an egg collected from a live cow using an ultrasonic diagnostic apparatus has entered the practical application stage (Non-patent Document 1).

  In vitro fertilization, the culture and storage of the embryo in the culture medium greatly affects the production efficiency. Therefore, a composition comprising 23 kinds of amino acids as a medium for in vitro fertilization (Patent Document 1), a medium containing a metalloproteinase inhibitor (Patent Document 2), a medium containing casein phosphopeptide (Patent Document 3), a medium containing cycloheximide (Patent Document 4), and a medium substantially free of glutamine (Patent Document 5) are known. There is no known medium that imparts heat stress tolerance during culture and storage.

  Xanthophyll refers to a carotenoid composed of carbon, hydrogen, and oxygen. Astaxanthin is a red pigment found in shrimps, crabs and the like, and it has been clarified that xanthophylls containing astaxanthin have excellent physiological activity including antioxidation. Moreover, although it is used for feed since it has an effect of producing semen from livestock (Patent Document 6, Patent Document 7), the effect on fertilized embryos is not known, and it is not known as a medium additive.

Japanese Patent No. 3660026 Japanese Patent Publication No. 7-34698 JP-A-6-197665 JP 2001-89302 A JP 2001-17160 A Japanese Patent No. 3897978 Japanese Patent No. 3620858

Akira Iritani, Introduction to the latest developmental engineering, p28-62, 2001

  The direct heat stress caused by high temperature inhibits the development of bovine early embryos, and it has been a problem to reduce the embryo development rate and conception rate in artificial fertilization. For this reason, the development of a novel medium composition that is not easily affected by heat stress in the culture process and preservation process of bovine embryos is desired.

  The inventors have intensively studied and found that, in the bovine embryo culturing process, by adding xanthophyll to the culture solution, a medium that is resistant to heat stress and useful in improving the conception rate is provided.

  The present invention includes (1) a medium composition for artificial fertilization characterized by containing xanthophyll, (2) the medium composition according to (1) above, wherein xanthophyll is astaxanthin, and (3) artificial (1) to (1), wherein the fertilization is in vitro fertilization, and the medium composition according to (1) or (2) above, and (4) the bovine embryo cultured after artificial fertilization The medium composition according to any one of 3).

  The present invention also provides (5) a method for culturing an embryo characterized by using a medium containing xanthophyll in the culture of an embryo after artificial fertilization, (6) the xanthophyll is astaxanthin (5) (7) The embryo culture method according to (5) or (6) above, wherein the artificial fertilization is in vitro fertilization, and (8) The embryo is an early bovine embryo The embryo culturing method according to any one of (5) to (7) above,

  The medium composition of the present invention has an effect that an embryo becomes less susceptible to thermal stress by adding xanthophyll to a culture solution in the culture and storage process of bovine embryos. Therefore, the medium composition of the present invention containing such xanthophyll has an advantageous effect on improving the conception rate and embryo development rate in artificial fertilization.

  In the present invention, xanthophylls include lutein, zeaxanthin, canthaxanthin, astaxanthin and the like, and it is particularly preferable to use astaxanthin. Examples of astaxanthin include astaxanthin obtained by chemical synthesis, astaxanthin extracted from marine products such as krill, astaxanthin extracted from algae, astaxanthin extracted from yeasts such as phafiarodozyma, and the like. The concentration of xanthophyll in the medium composition is 2.5 ppm to 1000 ppm, preferably 2.5 ppm to 100 ppm, particularly preferably 25 ppm to 100 ppm.

  The artificial fertilization medium refers to a medium that can culture egg cells, a medium that can be used for fertilization of egg cells outside the body, a medium that can culture embryos collected by in vitro artificial insemination or produced by in vitro fertilization, and the like. Specifically, 199 medium, BME medium, CMRL 1066 medium, MEM medium, McCoy-5A medium, Weymouth medium, Trowell T-8 medium, Ham medium, Leibovitz L-15 medium, NCTC medium, Williams-E medium, Kane -Kane and Foote medium, MCDB104 medium, Brinster medium, m-Tyrode medium, BWW medium, Whitten medium, TYH medium, Hoppes & Pitts medium M-KRB medium, BO medium, T6 medium, HTF medium, GPM medium, synthetic fallopian tube fluid (SOF), Charles Ronsenkrans medium, or a modified medium thereof.

  In the medium, amino acids, carbohydrates, electrolytes, vitamins, trace metal elements, hormones, cell growth factors, lipids or constituents thereof, carrier proteins, extracellular matrix components (adhesion factors), reducing substances, etc., as necessary May be added.

  As amino acids, L-phenylalanine, L-tryptophan, L-lysine, L-threonine, L-valine, L-methionine, L-isoleucine, L-leucine, L-proline, glycine, L-alanine, L-tyrosine, Examples include L-histidine, L-arginine, taurine, L-aspartic acid, L-serine, L-glutamic acid, L-cystine, L-glutamine, L-hydroxyproline, L-asparagine and the like.

  Glucose, glucose, maltose, fructose, xylitol, sorbitol, trehalose, etc., and electrolytes include sodium chloride, sodium acetate, sodium citrate, potassium chloride, calcium chloride, calcium gluconate, magnesium chloride, magnesium sulfate, phosphorus Examples include sodium dihydrogen acid, dipotassium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, and sodium lactate.

  Vitamins include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, nicotinic acid, biotin, folic acid, etc., and trace metal elements include zinc, iron, manganese, copper, iodine, selenium, cobalt Etc.

  Hormones include insulin, hydrocortisone, dexamethasone, triiodothyronine, and cell growth factors include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, growth hormone, etc. .

  Lipids or constituents thereof include essential unsaturated fatty acids such as oleic acid, linoleic acid or linolenic acid, cholesterol, ethanolamine, choline, etc., and carrier proteins include serum albumin, transferrin, etc. Factors include fibronectin, collagen, gelatin, and the like, and examples of the reducing substance include 2-mercaptoethanol (ME), dithiothreitol, reduced glutathione, and the like.

  In addition, fertilizers such as antibiotics such as penicillin, streptomycin, kanamycin, gentamicin and erythromycin, antifungal agents such as amphotericin B and nystatin, metalloprotease inhibitors, cyclophosphamide and casein peptides are appropriately added to the medium. May be.

  The medium composition of the present invention can be used for embryo culture or preservation in artificial fertilization. Embryo culture or storage in artificial fertilization refers to the culture of animal egg cells in in vitro fertilization, culture of embryos during or after fertilization, or preservation after culture, and in in vivo artificial insemination, the uterus of the generated embryo, etc. It refers to the culture or preservation after being collected from.

  The embryo may be any animal such as a human or a cow as long as it is an animal embryo, but is preferably a mammal, more preferably a livestock animal such as a cow, pig or sheep, and particularly preferably a cow.

  In the culture of the egg cell and the embryo at the time of fertilization in the present invention, when the artificial fertilization is in vitro fertilization, the medium of the present invention containing xanthophyll is added to immature eggs to culture the egg cells. Next, in general, 37-39 ° C, preferably 38.5 ° C, 5% carbon dioxide gas / 95% air, or 5% carbon dioxide gas / 5% oxygen / 90% nitrogen, in a humidified environment, fertilize by adding sperm. And culture for about 1 day in the medium of the present invention.

  In the culture of the embryo after artificial fertilization in the present invention, the embryo culture method characterized by using a medium containing xanthophyll is performed as follows. A fertilized egg obtained by artificial fertilization is naked, that is, granulosa cells are removed, and the naked fertilized egg is cultured in this medium. Replace the medium every 2-3 days. The above culture is usually performed at 37 ° C. to 39 ° C., preferably 38.5 ° C., in a humidified environment in 5% carbon dioxide gas / 5% oxygen / 90% nitrogen. Usually after 7-8 days of culture, the fertilized egg reaches the blastocyst.

  The developed embryo can be stored in liquid nitrogen. In in vivo artificial insemination, embryos collected from the uterus can be added to the medium of the present invention and stored in liquid nitrogen.

  The definitions of xanthophyll, embryo, etc. are as defined above.

  In this specification, “fertilization” refers to a biological phenomenon in which male and female germ cells are joined, and “insemination” refers to artificially causing fertilization. Also, in this specification, “artificial insemination” refers to a technique for insemination that artificially injects semen into the genital organs, and “in vitro fertilization” refers to insemination that fuses sperm and eggs outside the body. The term “artificial insemination” refers to a technique for insemination including artificial insemination and in vitro fertilization.

  Next, the present invention will be described more specifically with reference to examples.

Composition of TCM199 (Kurihara T, Kurome M, Wako N, Ochiai T, Mizuno K, Fujimura T, Takahagi Y, Murakami H, Kano K, Miyagawa S, Shirakura R, Nagashima H. Developmental competence of in vitro matured porcine oocytes after With reference to electrical activation. J Reprod Dev. 2002; 48: 271-279), a medium with the following composition (Composition 1) is produced:
CaCl _{2 (anhydrous) 200.00mg / L, Fe (NO} 3) 3 · 9H 2 O 0.72mg / L, KCl 400.00mg / L, MgSO 4 ( anhydrous) 97.67mg / L, NaCl 6800.00mg / L, NaH ₂ PO ₄ .H ₂ O 140.00 mg / L, adenosine sulfate 10.00 mg / L, ATP (2Na salt) 1.00 mg / L, adenylic acid 0.20 mg / L, cholesterol 0.20 mg / L, Deoxyribose 0.50 mg / L, D-glucose 1000.00 mg / L, Glutathione (GSH) 0.05 mg / L, Guanine / HCl 0.30 mg / L, Hypoxanthine (Na salt) 0.354 mg / L, Phenol red 20.00 mg / L, ribose 0.50 mg / L, sodium acetate 50.00 mg / L, chi 0.30 mg / L, Tween 80 (registered trademark) 20.00 mg / L, uracil 0.30 mg / L, xanthine (Na salt) 0.344 mg / L, DL-alanine 50.00 mg / L, L-arginine · HCl 70.00 mg / L, DL-aspartic acid 60.00 mg / L, L-cysteine · HCl · H ₂ O 0.11 mg / L, L-cystine · 2HCl 26.00 mg / L, DL-glutamic acid · H ₂ O 150 .00 mg / L, L-glutamine 100.00 mg / L, glycine 50.00 mg / L, L-histidine · HCl · H ₂ O 21.88 mg / L, L-hydroxyproline 10.00 mg / L, DL-isoleucine 40 .00 mg / L, DL-leucine 120.00 mg / L, L-lysine · HCl 70.00 mg / DL-methionine 30.00 mg / L, DL-phenylalanine 50.00 mg / L, L-proline 40.00 mg / L, DL-serine 50.00 mg / L, DL-threonine 60.00 mg / L, DL-tryptophan 20 0.000 mg / L, L-tyrosine (2Na salt) 57.88 mg / L, DL-valine 50.00 mg / L, Ascorbic acid 0.05 mg / L, α-tocopherol phosphate (2Na salt) 0.01 mg / L, d -Biotin 0.01 mg / L, Calciferol 0.10 mg / L, Calcium p-pantothenate 0.01 mg / L, Choline chloride 0.50 mg / L, Folic acid 0.01 mg / L, i-Inositol 0.05 mg / L , Menadione 0.01 mg / L, niacin 0.025 mg / L, niacinamide 0.025 mg / L, p-aminobenzoic acid 0.05 mg / L, pyridoxal · HCl 0.025 mg / L, pyridoxine · HCl 0.025 mg / L, riboflavin 0.01 mg / L, thiamine · HCl 0.01 mg / L Vitamin A (acetate) 0.14 mg / L, Astaxanthin (Fuji Chemical Industries) 25 mg / L.

  A medium (composition 2) having the same composition is produced except that the concentration of astaxanthin in composition 1 is changed to 50 mg / L.

  A medium (composition 3) having the same composition except that the concentration of astaxanthin in the composition 1 is changed to 100 mg / L is produced.

  A medium (composition 4) in which 20% NBS (newborn calf serum) is added to the composition 1 is produced.

  A medium (composition 5) is produced in the same manner except that 0.1 mM β-ME is added to the composition 4.

Preparation of basal medium: modified synthetic oviduct fluid (Takahashi Y, First NL. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992; 37: 963-978) With reference to the composition, a medium (basic medium) having the following composition was prepared:
NaCl 6294.00 mg / L, KCl 534.00 mg / L, KH ₂ PO ₄ 162.00 mg / L, MgCl ₂ (hexahydrate) 99.60 mg / L, CaCl ₂ (dihydrate) 251.40 mg / L, NaHCO ₃ 2106.00 mg / L, L-arginine · HCl 21.00 mg / L, L-cystine 12.00 mg / L, L-histidine 8.00 mg / L, L-isoleucine 26.00 mg / L, L- 3. Leucine 26.00 mg / L, L-lysine · HCl 36.98 mg / L, L-methionine 7.50 mg / L, L-phenylalanine 16.50 mg / L, L-threonine 24.00 mg / L, L-tryptophan 00 mg / L, L-tyrosine 18.00 mg / L, L-valine 23.50 mg / L, L-glutamine 1 6.00 mg / L, L-alanine 8.90 mg / L, L-asparagine (monohydrate) 15.00 mg / L, L-aspartic acid 13.30 mg / L, L-glutamic acid 14.70 mg / L, glycine 7.50 mg / L, L-proline 11.50 mg / L, L-serine 10.50 mg / L, sodium bibrate 55.00 mg / L, sodium lactate 370.00 mg / L, bovine serum albumin 3000.00 mg / L, Penicillin 10,0000.00 units / L, streptomycin 100 mg / L, amphotericin B 0.25 mg / L.

  Recovery of immature eggs and mature culture: Bovine ovaries were recovered from cattle dismantled in the meat market, transferred to a Dewar bottle containing physiological saline, and transported to the laboratory. Within 6 hours after disassembly, the follicle contents were aspirated from a follicle having a diameter of 2 to 8 mm on the surface of bovine ovary using a 10 ml syringe with a 21G injection needle, and granulosa cell-attached ova were recovered from the contents. Medium with the same composition (egg) except that bovine serum albumin is not added to the basal medium but D-glucose 1000.00 mg / L, follicle stimulating hormone 200 IU / L and fetal bovine serum 10% (V / V) are added. A maturation medium) was prepared, and 50 μl of a drop was prepared on a 60 × 15 mm Petri dish and covered with liquid paraffin. Ten collected granule membrane cell-attached eggs were transferred per drop and cultured in an incubator of 38.5 ° C., saturated humidity, 5% carbon dioxide, 5% oxygen, 90% nitrogen for 22 hours.

In vitro fertilization of mature eggs: Prepare a medium with the same composition except adding 20 mg / L of heparin in the basal medium, prepare a 45 μl drop on a 60 × 15 mm Petri dish, cover with liquid paraffin, and drop for waiting for the egg It was. Japanese black frozen semen (0.5 ml) was thawed in 37 ° C. warm water and placed on a 2 ml of 90% percoll and 2 ml of 45% percoll prepared in a 15 ml conical tube. Centrifugation was performed at 25 ° C. and 700 g for 30 minutes, and the supernatant was removed by suction. The remaining sperm precipitate was suspended in 10 ml of basal medium, centrifuged for 10 minutes, and the supernatant was removed by aspiration. This was used as a washed sperm, the sperm concentration was measured with a hemocytometer, and diluted with a basal medium so that the sperm concentration was 2 × 10 ⁶ sperm / ml. The granulosa cell-attached eggs after matured culture are transferred to the drop for waiting for ovum 10 pieces per drop, and 50 μl of the sperm dilution solution is poured into the drop for waiting for ovum containing granule-cell-attached ovum at 38.5 ° C., The cells were cultured for 20 hours in an incubator of saturated humidity, 5% carbon dioxide, 5% oxygen, and 90% nitrogen.

  Culture of in vitro fertilized embryo: A medium (Composition 6) having the same composition was prepared except that 270.00 mg / L of D-glucose and 0.025% (v / v) of dimethyl sulfoxide were added to the basal medium. A medium (composition 7) having the same composition was prepared except that 2.5 ppm of astaxanthin (Marine Chemical Laboratory) was added to the composition 6. A medium (composition 8) having the same composition was prepared except that the concentration of astaxanthin in composition 7 was changed to 25 ppm. At 20 hours after the start of in vitro fertilization, granulosa cells were removed from the granulosa cell-attached ovum to obtain a 1-cell stage embryo. The obtained 1-cell stage embryo is cultured at a temperature of 38.5 ° C. in the normal treatment, and in the heat treatment, the temperature is increased to 40.5 ° C. twice for 0-10 hours and 24-34 hours after the start of the culture The cells were cultured in a set incubator. All incubators were set to saturation humidity, 5% carbon dioxide, 5% oxygen, and 90% nitrogen. As the medium under each temperature condition, Composition 6, Composition 7, or Composition 8 was put in a 4-well plate at 500 μl per well. That is, normal treatment and no astaxanthin added (usually-no addition), normal treatment and astaxanthin added 2.5 ppm or 25 ppm (usually -Ax2.5 or normal -Ax25), heat treatment and no astaxanthin added (hot-no addition) )), Heat treatment, and astaxanthin 2.5 ppm or 25 ppm added (hot heat-Ax 2.5 ward or hot heat-Ax 25 ward). The start date of in vitro fertilization was defined as day 0, and the medium was replaced with a medium having the same composition except that 270.00 mg / L of D-glucose was added to the basal medium on the third day. Furthermore, on the fifth day, the medium was replaced with a medium having the same composition except that D-glucose 270.00 mg / L, β-ME 0.1 mM and fetal calf serum 10% (v / v) were added in the basal medium. The medium after the third day was used in a state where 50 μl drops were prepared on 60 × 15 mm Petri dishes and covered with liquid paraffin. The division rate on the third day, the incidence to the 5-8 cell stage, and the blastocyst incidence on the eighth day were measured. The division rate and the incidence rate were subjected to multiple comparisons using the Sidak method after a one-way analysis of variance. As a result, compared with the normal-no additive group, the division rate, the 5-8 cell stage, and the incidence of blastocysts were significantly reduced in the hot-additive group. On the other hand, in the heat-Ax2.5 or the heat-Ax25, the embryo development rate recovered depending on the concentration of astaxanthin. It was. From the above results, it was shown that the development of bovine early embryos was directly inhibited by physiological heat conditions, and that astaxanthin could relieve the inhibition of the development (Table 1).

Claims (8)

  A medium composition for artificial fertilization, comprising xanthophyll.   The medium composition according to claim 1, wherein the xanthophyll is astaxanthin.   The medium composition according to claim 1 or 2, wherein the artificial fertilization is in vitro fertilization.   The medium composition according to any one of claims 1 to 3, which is used for culturing bovine embryos after artificial insemination.   A method for culturing an embryo, wherein a medium containing xanthophyll is used for culturing an embryo after artificial insemination.   6. The embryo culture method according to claim 5, wherein the xanthophyll is astaxanthin.   The method for culturing an embryo according to claim 5 or 6, wherein the artificial fertilization is in vitro fertilization.   The embryo culture method according to any one of claims 5 to 7, wherein the embryo is an early bovine embryo.