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

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.

Time Limiting Boundaries of Reversible Clinical Death in Rats Subjected to Ultra-Deep Hypothermia.

BACKGROUND: It is well known that body temperature maintenance between 20 and 35°C prevents hypoxic damage. However, data regarding the ideal duration and permissible temperature boundaries for ultra-deep hypothermia below 20°C are rather fragmentary. The aim of the present study was to determine the time limits of reversible clinical death in rats subjected to ultra-deep hypothermia at 1-8°C. RESULTS: Rat survival rates were directly dependent on the duration of clinical death. If clinical death did not exceed 35 min, animal viability could be restored. Extending the duration of clinical death longer than 45 min led to rat death, and cardiac functioning in these animals was not recovered. The rewarming rate and the lowest temperature of hypothermia experienced did not directly influence survival rates. CONCLUSIONS: In a rat model, reversible ultra-deep hypothermia as low as 1-8°C could be achieved without the application of hypercapnia or pharmacological support. The survival of animals was dependent on the duration of clinical death, which should not exceed 35 min.

The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days.

There is a catastrophic decrease in the biodiversity of amphibians coupled with the loss of genetic variation. The perpetuation of amphibian biodiversity demands a multifaceted approach, including the use of reproduction technologies (RTs), to enable efficient reproduction in captivity and to prevent the loss of genetic variation. Reproduction technologies for the storage of amphibian sperm for days to weeks, when refrigerated at 4°C, or for millennia when cryopreserved have recently undergone rapid development. Sperm from amphibians may be obtained through excision and maceration of testes; however, this is sometimes not possible with rare or endangered species. Alternate methods of obtaining sperm are through hormonal induction, or as spermatozoa from the carcasses of recently dead amphibians. The use of sperm from carcasses of recently dead amphibians is particularly valuable when sampled from genetically important founders in conservation breeding programs, or where catastrophic mortality is occurring in natural population. Sperm harvested over a period of 7 days from the testes of European common frog (Rana temporaria) carcasses stored in a refrigerator were assessed for percentage and progressive motility, cell membrane integrity, nuclear DNA fragmentation, and fertilizing ability. In addition, the survival of resulting embryos to hatch was recorded. Results indicated that some sperm of R. temporaria remain motile and fertile when harvested from frog carcasses refrigerated up to 7 days post-mortem, and resulting embryos can develop to hatch.