JPH0685792B2 - Cryopreservation method of fertilized pig egg - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for storing fertilized porcine eggs. The present invention also relates to a method for efficiently carrying out transplantation of fertilized eggs by using the fertilized eggs of pigs thus stored.

More specifically, the present invention is a method of storing a fertilized egg of a pig at a low temperature of 15 ° C. or lower without impairing its viability, and transplanting a fertilized egg using a fertilized egg of a pig thus preserved efficiently. It relates to a method to carry out.

[Conventional technology]

As a breeding method for large mammals including humans, such as cows, horses, pigs, sheep, goats and small mammals, for example, rabbits, mice, rats, etc., the fertilized eggs fertilized in the body or in vitro are returned to the body. The conception has already been performed experimentally and many successful cases have been reported. In order to make this method easier and to make fertilized eggs collected at remote locations available, many attempts have been made to cryopreserve or cryopreserve fertilized eggs. And in the fertilized eggs of the above-mentioned mammals other than pigs, such attempts have been successful, and by establishing a low temperature or cryopreservation technique for such fertilized eggs, it becomes possible to transport the fertilized eggs to a remote place, and each animal species This has helped to improve the breed of cultivar and diversify the means of reproduction.

[Problems to be solved by the invention]

Similar low-temperature or cryopreservation attempts have been made for fertilized eggs of pigs, but fertilized eggs of pigs die only when exposed to low temperatures of 15 ° C or lower (for example, by C. Polge et al. C
According to ryobiology, Vol. 11, p. 560 (1974)), such an attempt was unsuccessful, and therefore cryopreservation of fertilized swine eggs was considered completely impossible.

In view of such a situation, the present inventors have completed the present invention by studying the conditions under which fertilized porcine eggs can be stored at a low temperature of 15 ° C or lower without impairing their viability.

[Means for solving problems]

Attempts at cryopreservation or cryopreservation of the above-mentioned mammalian fertilized eggs have hitherto been made to fertilized eggs up to the 2-cell stage, 4-cell stage, 8-cell stage, morula stage and blastocyst stage. Since these fertilized eggs at the developmental stage are wrapped in the egg membrane called the zona pellucida and are under the protective action of such a membrane, generally, in order to prevent the fertilized eggs from being damaged by low temperature, the fertilized eggs at the developmental stage should be handled. Was expected to bring good results. In fact, this prediction was correct for fertilized eggs of mammals other than pigs, but not as expected for fertilized eggs of pigs, as described above.

The present inventors have surprisingly found that fertilized eggs of pigs at the stage of the unshelled blastocyst stage, which appeared by breaking the zona pellucida, are not damaged by low temperature in the present invention. It was completed.

That is, the present invention relates to a method for preserving a fertilized egg of a pig, which comprises cooling the fertilized egg of a pig at the stage of the unshelling stage blastocyst to a low temperature.

Furthermore, the present invention also relates to a method for breeding pigs, characterized in that fertilized eggs of pigs in the stage of unshelling stage blastocysts are cooled and stored at a low temperature, and the fertilized eggs thus preserved are transplanted. .

In the present invention, the fertilized egg of the pig at the stage of the molting stage blastocyst (hatched blastocyst) that can be stored by cooling to low temperature means a fertilized egg that has undergone molting, that is, escape from the zona pellucida (hatching), Typically, it may be obtained by culturing the expansion stage blastocyst in an appropriate culture medium and visually observing the completion of dehulling by visual means or other means, for example, a photoelectric monitoring means. Needless to say, it does not have to be obtained through the means of culturing, and if such an unshelled blastocyst can be obtained by some other means, it can be used as such.

However, as a reliable means for obtaining the unshelled blastocysts, it is possible to develop them by means of artificial culture of the expanded blastocysts and wait for the end of unshelling.

The culture in this case can be performed by various known means. Various kinds of culture liquid can be selected, for example, Whittingham's liquid (M-16; 1971), Whittingham's modified PBS (PBI, 1971), Whitten's liquid (1957), Brinster liquid (BMOC-1). , 1963), Brinster solution (BMOC-2,1965), Brinst
er solution (BMOC-3,1971), Whitten solution (1968), Biggers solution (1971), TYH solution (1971), Bavister solution (1969) (the above Krebs-Ringer bicarbonate solution is improved) and the like. The one appropriately modified can be used for culture.

Expanding blastocysts dehull in several hours to 30 hours when cultured in these cultures at temperatures around 37 ° C.

The unhulled blastocysts obtained by unhulling in this manner are added to an appropriate preservation solution, for example, Darbetuco's phosphate buffer at 10 to 20%.
Calf serum, fetal calf serum, swine serum, etc., or 1 mg to 16 mg / ml bovine serum albumin or 10 to 20% fetal calf serum, fetal calf serum, swine serum, etc. was added to the culture solution. It is cooled and stored using a thing.

After storage for a certain period of time, the sample was transferred to a 37 ° C. incubator and heated for 30 minutes, the embryo was recovered after heating, and the viability was determined by culture or transplantation. As a result, it was confirmed that the embryos survive even when cooled to 11 ° C, 6 ° C and -20 ° C.

The unhulled blastocyst used in the method of the present invention may be one that has just undergone unhulling or one that has become an expanded unhulled blastocyst, which is the next stage of development.

In fertilized eggs of pigs, unlike fertilized eggs of other mammals,
It is not clear why unshelled blastocysts are resistant to low temperatures, but fertilized eggs of pigs have more fat in the cell membrane than those of other mammals, and this fat is metabolized as energy during unshelling. Since the fat mass decreases after shelling, it is presumed that the fluctuation of the fat mass is related to the low temperature viability of the fertilized egg. In the present invention, the fertilized egg at the time when the low temperature tolerance is improved is stored at low temperature. However, the reason for the improvement of the low temperature tolerance of the fertilized egg of a pig described above is based on an inference, and therefore the reason for such inference does not affect the establishment of the present invention.

The present invention will be described in detail below with reference to examples.

〔Example〕

LW breeding sows are mated with Deyurott males, and laparotomy is performed 5 to 6 days after the end of estrus, and the blastocysts of the expansive stage (expandin
g blastocyst) or shelling stage blastocyst (hatching blast)
oyst or hatched blastocyst) was collected. These embryos were treated with Whittingham (1971) M-16 medium and Dulbecco.
The mixture was cultured for about 24 hours in a mixed culture solution of a solution prepared by adding fetal bovine serum (16%) to the phosphate buffer solution (PBS) of.

By this culture treatment, the stages of embryo development proceeded as follows.

1) Expanding blastocyst → hatched blastocyst …… 2) Unsealed blastocyst or h
atching blastocyst) → expanding blastocyst
hatched blastocyst) …… The names of these embryos are in vitro molting blastocyst, in
It was used as an in vitro expanded unshelled blastocyst.

In this way, “a blastocyst whose developmental stage has been advanced by culture”
Was subjected to the following low temperature storage.

Cryopreservation method Embryos were aspirated into a 0.25 ml plastic straw with PBS containing 16% fetal bovine serum, and encapsulated with gelatin powder.
This sample was cooled at 1 ° C / min to 5 ° C / min at 11 ° C,
It was cooled to 6 ° C or -20 ° C. The holding time of these three kinds at the final temperature was as follows. Final temperature holding time 11 ° C. 3 minutes 6 ° C. 1 hour 6 ° C. 16 hours −20 ° C. 10 minutes After cooling, the sample was transferred to a 37 ° C. thermostat and held for 30 minutes to heat. After heating, the embryos were collected from straws and cultured using the above-mentioned culture medium to examine the viability of the embryos.

Some embryos were transferred to embryo recipient females (recipients) and examined for their ability to develop into fetuses.

The results of cryopreservation of fruit embryos are shown in the table below.

Claims (4)

[Claims] 1. A method for preserving a fertilized egg of a pig, which comprises cooling the fertilized egg of a pig at the stage of the unshelling stage blastocyst to a low temperature. 2. The method according to claim 1, wherein the unshelled blastocyst is obtained by culturing an expanded blastocyst of a fertilized egg of a pig and allowing it to escape from the zona pellucida. 3. A method for breeding pigs, characterized in that fertilized eggs of a porcine blastocyst stage are cooled and stored at a low temperature, and the fertilized eggs thus preserved are transplanted. 4. The method according to claim 3, wherein the unhulled blastocyst is obtained by culturing an expanded blastocyst of a fertilized egg of a pig and allowing it to escape from the zona pellucida.