JPH0651601B2 - Frozen fertilized egg of pig and method for cryopreservation - Google Patents

Description

TECHNICAL FIELD The present invention relates to a frozen fertilized egg of pig and a method of cryopreserving the fertilized egg.

[Conventional method]

Generally, in the pig farming industry, it is desired to produce many genetically excellent pigs in order to increase production efficiency.

Conventionally, breeding of pigs has been carried out by mating boars with the breeding pigs to cause pregnancy and delivery. According to this method, it takes about 114 days from mating to delivery so that the number of deliveries within the industrially possible range of a single-breeding sow is up to 10 or more.

Thus, there is a limit to the number of piglets that one breeding sow can pass to the next generation, and fertilized egg transplantation has been used as a method to overcome this. However, that method required that fertilized eggs collected from the pigs that provided the fertilized eggs be transplanted into the hungry pig within at least a few days.

By the way, cryopreservation technology for fertilized eggs is used for mice, rabbits, goats,
It has been developed for sheep, cows, etc., and morula-stage embryos or early blastocysts are generally used for cryopreservation. On the other hand, the technology for preserving fertilized eggs of pigs has not yet been developed.

[Problems to be Solved by the Invention]

Fertilized egg transplantation is an important technique for breeding and breeding livestock, but since the fertilized egg cannot be stored for a long period of time, its effect could not be fully exerted. If the fertilized eggs can be cryopreserved, this problem can be solved, but the successful case with pigs is not known yet.

[Means for Solving the Problems]

The present inventor has conducted various studies to develop a method for preserving fertilized porcine eggs for a long period of time. The present invention has been completed by confirming that those that have grown to the absence of the perivitelline are still alive.

That is, the present invention relates to a fertilized egg of a pig that has grown to the absence of the perivitelline space or has been developed at a later development period and has been stored at minus 35 degrees Celsius or less, and a method for storing the same.

In the fertilized egg, the 1-cell stage embryo divides to become the 2-cell stage embryo, and further divides to repeat the 4-cell stage embryo, 8-cell stage embryo, morula stage embryo,
It grows with early blastocyst, diastolic blastocyst, zona pellucida escaped blastocyst and implants in the uterus. On the other hand, when the blastocyst enters the diastole, the perivitelline space, which is the space between the fertilized egg and the zona pellucida, disappears. In the present invention, this diastolic blastocyst or zona pellucida escaped blastocyst is used. 5 to 8 days after fertilization, which is 5
Those on the 7th day are preferable. Growth until a post-fertilization diffusion stage blastocyst or a zona pellucida escaped blastocyst may be performed in the body of a pig that provides a fertilized egg, but may also be performed by artificial culture. A known culture medium for culturing mammalian cells may be used, and examples thereof include TCM-199, Ham's F-10 and Brins.
Ter solution (BMOC-3), Menezo's B-2, SOF-modified PBS, etc. can be used by adding fetal bovine serum and the like if necessary. The culture conditions may be the same as those for fertilized bovine eggs.

Such fertilized eggs are placed in a culture medium containing a suitable cryoprotective agent used for cell freezing and allowed to stand at 20-40 ° C for a certain period of time to equilibrate. As the culture solution, for example, the above-mentioned one can be used. Cryoprotectants include glycerin, dimethylsulfoxide, ethylene glycol, polyvinylpyrrolidone,
Sucrose or the like may be used. It is advisable to divide the cryoprotective agent into several concentrations and immerse the fertilized egg in a liquid with a higher concentration one after another. The final concentration of glycerin is preferably about 1 to 2 molar, and for example, a five-stage solution of 0.2, 0.35, 0.65, 0.85, 1.0 can be used. The immersion time of each solution may be about 5 to 10 minutes, and the final concentration solution is immersed for about 15 minutes to 1 hour. The final concentration of DMSO is also preferably about 1 to 2 molar, and it is preferable to use a solution divided into stepwise concentrations.

Fertilized eggs that have been equilibrated with a cryoprotectant are placed in a cryopreservation container and gradually cooled to about -5 to -10 ° C, and then transplanted. A general container used for cryopreservation of fertilized eggs may be used as the container, and for example, a small test tube, a small ampoule, a frozen semen straw tube, etc. can be used. Cooling is preferably performed at a constant rate using a program freezer or the like. The planting of ice may be carried out by introducing small ice pieces, bringing a metal piece cooled with liquid nitrogen into contact with the outside of the container, blowing cold air, giving a light vibration, or the like.

After the ice is frozen, the fertilized eggs are frozen by further cooling to about -30 to -40 ° C. This cooling is also preferably performed at a constant rate using a program freezer or the like.

The frozen fertilized egg thus obtained is stored in a freezer or liquid nitrogen.

The thawing when using a fertilized egg may be rapid thawing or slow thawing.

It does not matter what kind of pig the present invention can be applied to.

〔Example〕

The semen of a Duroc male was artificially fertilized to a female of a hybrid of Landrace and Large York or a Hampshire, and the fertilized egg was surgically recovered 5 to 6 days after fertilization.

The collected fertilized eggs were immersed in a solution containing 20% fetal bovine serum in phosphate buffer and glycerin as a cryoprotectant. The glycerin concentration was set to 5 steps, and the glycerin was immersed in a liquid having a high concentration in order from a low concentration (glycerin equilibrium). After equilibrating with glycerin, the fertilized eggs were suction-sealed in a plastic straw having a volume of 0.25 ml. The enclosed straws were placed in a program freezer, cooled from 35 degrees Celsius to minus 6.8 degrees Celsius for 1 minute at a rate of 1 degree Celsius, and iced at minus 6.8 degrees Celsius and held for 10 minutes. After that minus
It was cooled to 35 ° C. at a rate of 0.3 ° C. for 1 minute, further cooled to −36 ° C. at a rate of 0.1 ° C. for 1 minute, and put into liquid nitrogen (−196 ° C.) for storage. The fertilized eggs thus cryopreserved were put directly into hot water of 37 degrees Celsius to dissolve them, and glycerin was removed by the procedure reverse to the glycerin equilibrium. Fertilized eggs made in this way are 37 degrees Celsius.
The culture was carried out in an incubator with a carbon dioxide concentration of 5%.

As a result, 28 frozen embryos that were younger than the early blastocysts (fertilized eggs in which the blastocyst cavity was soon formed and had a perivitelline cavity) were frozen but survived after freezing. There was only one, but in fertilized eggs after the diastolic blastocyst, 134 frozen and 49 survived.

〔The invention's effect〕

Freezing fertilized swine eggs has enabled long-term storage. Although conventional fertilized egg transplants for pigs could not preserve fertilized eggs, they were not useful for gene preservation.
The present invention enables preservation. For example, even after the sow that has produced the fertilized egg has died, the offspring can be obtained, and the breeding improvement of the pig and the great effect on breeding can be brought about.

Claims (2)

[Claims] 1. A fertilized egg of a pig that has grown to the absence of the perivitelline space or has been developed after that and has been stored at a temperature of −35 ° C. or lower. 2. A method for preserving fertilized eggs of pigs, characterized in that fertilized eggs of pigs that have grown to the absence of the perivitelline space or are in the subsequent stage of development are stored at −35 ° C. or lower.