Culture and transportation of bovine embryos

Details Culture and transportation of bovine embryos Culture and transportation of bovine embryos

1993-02-17

2001-05-29

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of storing cells in a viable state

C435S325000, C435S383000

Reexamination Certificate

active

06238920

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of culturing or transporting effectively bovine embryos for transplanting.
BACKGROUND OF THE INVENTION
So far bovine embryos obtained by in vitro fertilization and/or in vivo fertilization have been cultured for several days in in vivo (oviducts of rabbits and sheep) or in vitro (co-culture in the presence of oviductal or cumulus cells) for their development.
When bovine embroys obtained by in vitro culture of in vitro fertilized oocytes were transported to the distant place where the embryo transfer of them was carried out, they were cryopreserved using glycerol and sucrose as cryoprotectans, stored and transported in liquid nitrogen. However, this method of transportation of embryos is nothing but a trial.
It is well known that bovine embryos do not develop beyond 8 cell stage (8 cell block) in in vitro culture. To overcome this 8 cell block embryos have been cultured in vivo or in vitro (co-culture with a monolayer of cells).
However, it is time and effort consuming to culture embryos in vivo, because embryos must be transferred to recipient animals and collected from them surgically. In addition, as for culturing embryos with supporting (oviduotal or cumulus) cells in vitro, establishment of those cells is also time and effort consuming. Furthermore, sometimes the supporting cells cover the surface of bovine embryos resulting in distortion of the embryos. Therefore, it is necessary to develop more efficient in vitro culture system for bovine embryos.
When bovine embryos are transported to the distant place after cryopreservation, the viability of them decreases sharply during freezing and thawing of them. In addition, before transfer of cryopreserved embryos, a special facility for treatment of the cryopreserved embryos and examination of the viability of the embryos after thawing are needed. Therefore, it is necessary to develope a handy and reliable method for the transportation of bovine embryos.
While we have been investigating the suitable culture and transportation methods for bovine embryos, we found out that the reduced state of embryos during culture and transportation are important for them to develop. And we concluded that low molecular thiol compounds are suitable to make intracellular conditions of embryos reduced state and completed a new method of culture or transportation of bovine embryos.
SUMMARY OF THE INVENTION
This invention provides methods for culture and transportation of bovine embryos. In particular, the present invention relates a method for culture or transportation of bovine embryos which comprises adding thiol compound, especially low molecular thiol compound to a medium for culture or transportation of them.
DETAILED DESCRIPTION OF THE INVENTION
In this invention, low molecular thiol compounds are low molecular compounds that have thiol, such as &bgr;-mercaptoethanol (hereinunder it may be abbreviated as &bgr;-ME, molecular weight (m.w.):78.13) , &bgr;-mercaptoethylamine (hereinunder it may be abbreviated as cysteamine, m.w.:77.14), glutathione (m.w.:307.3), dithiothreitol (m.w.:154.24), &agr;-thioglycerol (m.w.:108.16) etc.
To culture bovine embryos (in vitro fertilized etc.) up to blastocyst stage low molecular thiol compounds mentioned above must be added to the medium. The effective concentration of the compounds in the medium is within the range of 10-100 &mgr;M preferably 10-50 &mgr;M .
For the medium, Tissue Culture Medium (TCM)-199, DMEM, MEM, CMRL 1066 and NCTC 109 etc. are used and those media are supplemented with 10% (V/V) fetal calf serum (FCS), calf serum (CS), bovine serum albumin (BSA), polyvinylpyrrolidone (PVP) and polyvinylalchohol (PVA) etc.
To transport bovine embryos without decreasing their viability the addition of low molecular thiol compounds at concentrations of 10-150 &mgr;M , preferably 100-150 &mgr;M to the medium of transportation is necessary.
For the medium of utilizing transportation, modified TCM-199 etc. supplemented with 20% calf serum are used.
According to the present invention, it is possible to culture bovine embryos up to blastocyst stage efficiently in the medium containing low molecular thiol compound without in vivo culture or coculture using feeder cells.
Also when low molecular thiol compound is added to the transportation medium for bovine embryos, almost 100% embryos are still viable after long distant transportation. This transportation method makes it possible to transfer embryos to recipient cows as soon as they are transported to the place where embryo transfer is carried out. With this method, there is no need for a facility for thawing of frozen embryos or judgment of viability of frozen-thawed embryos.


REFERENCES:
Takahashi et al., Thereogenology, 39(1), p. 326, Jan. 1993.*
Ealy et al., Cell Biology International Reports, vol. 16, No. 2, pp. 125-131, Feb. 1992.*
Hasler et al, J. of Animal Science, vol. 49, Supplement I, pp 135-136, Feb. 1979.*
Bannai, Hum. Cell, 5 (3), pp. 292-297, Sep. 1992 (Biosis Abstrat).

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