Prolonged hypothermic storage of oocytes of the European common frog Rana temporaria in a gas mixture of oxygen and carbon monoxide

Abstract

The maximum hypothermic storage time of amphibian oocytes is several hours, which is due to the peculiarities of the structure of the cell envelope. The authors of this paper have already demonstrated the possibility of increasing the storage period of unfertilized oocytes of the common frog (Rana temporaria) up to 5–7 days. The aim of the current study was to determine the possibility of using a 6.5 atm gaseous mixture of carbon monoxide and oxygen, for prolonged hypothermic preservation of unfertilized oocytes for 4 to 12 days. After four days, oocytes stored under CO+O2 conditions exhibited fertilization and hatching rates that were 1.6 and 2.2-fold higher than control, respectively. While no oocytes in the control group survived to day twelve, oocytes held under CO +O2 gas exhibited a 39±14% (38 out of 99 oocytes in total) fertilization rate, however only 1±2% (1/99) of those hatched. This approach is promising for the storage of genetic material from female amphibians, particularly in respect to managing and restoring endangered species, but may also be applicable to oocytes of other classes of vertebrates.

Ecological Civilization COP 15 and Amphibian Sustainability through Reproduction Technologies, Biobanking, and Conservation Breeding Programs (RBCs)

“…Dry storage of cool-temperate anuran oocytes at 4°C in Eppendorf tubes, to prevent dehydration and to maintain ionic stasis, over five days maintained 70% fertility and 40% hatch rates, with some fertility and development extended to eight days [312]. Gagarinskiy et al [313] showed that a 6.5 atm gaseous mixture of carbon monoxide and oxygen doubled the refrigerated storage viability of the unfertilised oocytes of a cool temperate anuran, compared with controls when fertilised over eight days. However, from four to eight days of storage the hatch rate declined to a low level like that of controls.…”

“…However, from four to eight days of storage the hatch rate declined to a low level like that of controls. Therefore, the causes of oocyte loss of viability are ions or proteins diffusing from the gel that are needed for sperm motility or oocyte metabolism [314][315][316], or damage to the cell envelope [313].…”