JP2002153267A - Method for culturing animal ovum and early embryo with deep water - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a method for culturing an animal ovum and early embryo with ocean deep water at a low cost and at a high maturation rate. SOLUTION: This method for culturing the animal ovum, characterized by adding ocean deed water to a serum-containing or serum-free in vitro maturation culture medium preferably in an amount of 5 to 25% based on the culture medium to maturate the ovum, and the method for culturing the early embryo from the ovum.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for in vitro culture of animal ova and early embryos using deep ocean water.

[0002]

2. Description of the Related Art In vitro culture techniques for egg and early embryos of animals such as cattle have recently become increasingly important due to the development and development of in vitro fertilization and cloning techniques. It has been reported that miscarriages and oversized children are more likely to occur than in vivo-derived embryos. It is considered that the cause is the generation of active oxygen due to an incomplete in vitro culture system and the excess or deficiency of vitamins, minerals, trace elements, and the like.

[0003] Deep ocean water is rich in inorganic nutrients compared to surface water (Table 1). Further, the deep ocean water of Toyama Bay is called "Cold water peculiar to the Sea of Japan", and it is assumed that marine organisms and physiologically active substances having completely different characteristics from the deep ocean water on the Pacific Ocean side exist. Temperature of cold water specific to the Sea of Japan in Toyama Bay (2
° C or lower; Table 2) is considerably lower than the deep water in the North Pacific Ocean (approximately 9 ° C; Table 3) facing the open sea at Cape Muroto, Kochi Prefecture, and has a higher dissolved oxygen content and stable salinity (34.0-34.1psu). The cold water unique to the Sea of Japan in Toyama Bay is young as deep water. Comparing the respective deep waters, Toyama Bay has more inorganic nutrients such as phosphate phosphorus and nitrate nitrogen. Thus, the present inventors have used the deep water of Toyama Bay (cold water peculiar to the Sea of Japan), which is low in temperature and rich in minerals and trace elements, as an in vitro culture method for animal eggs and early embryos. This led to the finding of a high maturation rate.

[0004]

[Table 1]

[0005]

[Table 2]

[0006]

[Table 3]

[0007]

Accordingly, an object of the present invention is to develop a method for culturing animal eggs and early embryos which is inexpensive and has a high maturation rate.

[0008]

According to the first aspect of the present invention, deep-sea water is added to an extracorporeal maturation medium, and an animal egg is cultured. With this configuration, an animal egg can be cultured at a high maturation rate. Specifically, the in vitro maturation medium includes TCM199 medium and the like, and the animals include cow, pig, sheep, mouse and the like. Examples of deep ocean water include cold water specific to the Sea of Japan in Toyama Bay. According to the second aspect of the present invention, instead of the medium containing serum, deep sea water is added to a serum-free medium containing PVP, and the eggs of the animal are cultured and matured. With such a configuration, an egg of an animal can be cultured without serum, and an animal egg can be cultured at a low cost and a high maturation rate without using expensive serum. According to the invention described in claim 3, deep sea water is
Add 5-25% of serum-containing or serum-free in vitro maturation medium,
Culture the egg of the animal. With this configuration, the maturation rate of the animal egg can be more effectively increased. Furthermore, according to the fourth aspect of the present invention, the early embryo is cultured using an egg cultured in an extracorporeal maturation medium to which deep water has been added.
With this configuration, the division rate and the blastocyst rate can be increased, and the embryo can be transplanted to an oocyte to be fertilized.

Next, a method for culturing eggs will be described. The deep water to be added is prepared by filtering and sterilizing the deep sea water of Toyama Bay (cold water unique to the Sea of Japan) with a filter and adjusting the osmotic pressure. The immature ova are collected by suction from the follicles on the surface of the ovary of the animal,
Among them, the cumulus cells adhere to several layers or more and the cytoplasm of the egg is normal. Then, deep water is added to an in vitro maturation medium such as a TCM199 medium, and maturation culture is performed. Cumulus cell-egg complex matured and cultured in a deep water-supplemented medium is sperm suspended in a sperm suspension that has acquired fertilization by the caffeine-heparin method, and after insemination, a medium based on CR1aa or the like. Development culture of the early embryo is performed in the inside. In addition, the mature cultured animal ova are enucleated and used as recipient eggs for nuclear transplantation.They are fused with nucleated cells of animal somatic cells, activated and cultured for development. Embryo transfer and conception can also be performed.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. The present invention is not limited to these examples.

Example 1 First, deep water pumped up in the Toyama Prefectural Fisheries Experimental Station was used for 0.1 minute.
The solution was sterilized by filtration through a 22 μm filter and stored at 4 ° C. TCM19
9 Deep water to be added to the culture medium is ultrapure water (18.2 MΩ) before use.
The osmotic pressure was adjusted to 292m0sm with. The osmotically adjusted deep water was added to an in vitro maturation medium based on TCM199 medium by 20% and used for in vitro maturation of immature eggs. Next, the immature ova were sucked and collected from a follicle having a diameter of 2 to 5 mm on the surface of a bovine ovary from a slaughterhouse. Among the collected immature eggs, cumulus cells adhered to several layers or more, and those with a normal cytoplasmic state of the eggs were subjected to in vitro maturation.

In vitro maturation in a medium supplemented with deep water was 38
The maturation rate was determined from the observation result of the nuclear phase by 1% acetoorcein staining at 19 ° C. In vitro fertilization is 20
The culture was performed by culturing for 22 hours in a medium supplemented with 5% deep water. The cumulus cell-egg complex was placed in a sperm suspension adjusted to a concentration of 5,000,000 / mL, which had acquired fertilization by the caffeine-heparin method, and was inseminated for 4 hours. After completion of insemination, embryos are cultured for development at 39 ° C and 5% CO ₂ in a medium based on CR1aa medium. The cell rate was investigated.

A bovine egg matured and cultured in a medium supplemented with 20% deep water was enucleated and used as a recipient egg for nuclear transfer. Bovine somatic cells were used as nucleated cells, and the cells were fused with recipient eggs and activated. After nuclear transfer, developmental culture was performed, and embryos that had developed into a transferable state were transferred to embryo-bearing cows to examine fertility.

The maturation rate, the division rate after in vitro fertilization, and the blastocyst rate of bovine ovum matured and cultured in a TCM199 medium supplemented with 20% deep water were examined. The maturation rate was high, with significant differences. Also, the split ratio,
The blastocyst rate was higher for bovine eggs matured and cultured in a medium supplemented with deep water.

[0015]

[Table 4]

Further, the produced embryos were transplanted into oviposited cows, and the fertility of the nuclear transfer embryos produced using deep water was examined. As a result, 5 out of 9 (56%) fertilized.

Example 2 In a mature culture, a CS medium (TCM199 + 5% CS + FSH.E2 (CS))
And PVP medium (TCM199 + 0.3% PVP + FSH.E2 (PVP)), and add 10 or 20% of deep water to the whole medium, respectively. The maturation rate was examined using this. As a result, there was no significant difference in the maturation rate when the deep water was added to the CS medium containing serum, but in the PVP medium added as a macromolecular substance to remove the effect of serum,
The addition of deep water significantly increased the maturation rate (FIG. 1).
Therefore, the effect of increasing the maturation rate by adding deep sea water under serum-free conditions was recognized.

[0018]

According to the present invention, the maturation medium of animal eggs can be matured only by adding the deep sea water of Toyama Bay, and the eggs can be fertilized to generate embryos. The rate and blastocyst rate can be increased for conception, and it can be produced at extremely low cost and in large quantities as compared with the case where a commercially available hormone or growth factor is added. In addition, deep water may contain hormones added during maturation culture, nutrients or bioactive substances that cannot be supplemented with commercially available TCM199 medium, and in vitro fertilization technology that requires in vitro culture, improvement of nuclear transfer technology, It can be applied to various cell cultures and the like.

[Brief description of the drawings]

FIG. 1 is a diagram showing a maturation rate when deep water is added to a serum-free medium.

Claims (4)

[Claims] 1. A method for culturing animal ovum, comprising adding deep-sea water to an in vitro maturation medium for animal ovum. 2. The method for culturing an animal egg according to claim 1, wherein the in vitro maturation medium is a serum-free medium. 3. The method for culturing an animal egg according to claim 1, wherein 5 to 25% of the extracorporeal maturation medium is added to deep ocean water. 4. A method for culturing an early embryo using an egg matured and cultured by the culture method according to claim 1.