EFFECT OF STORAGE TIME ON FROZEN BEEF CATTLE SEMEN
Biological cells like bovine sperm and embryos are frozen by exposing them to cryoprotectants then slow cooling the samples at specific rates to allow the exodus of intracellular water molecules prior to being plunged into liquid nitrogen for long term storage. Slow cooling rates along with cryoprotectants allow the formation of very small ice crystals in the extracellular solutions during freezing. The primary goal during cell freezing is to remove intracellular water, which minimizes the formation of intracellular ice that is created when intracellular water molecules crystallize at sub-freezing temperatures. Intracellular ice damages cell membranes, cellular organelles, and even chromosomes therefore intracellular water must be removed before reaching crystallizing temperatures. Bovine embryos are typically exposed to either glycerol or ethylene glycol for several minutes at room temperature and then ramped to a temperature of about – 35° Celsius (C) before being plunged into liquid nitrogen ( – 196° C). It is important to note for both sperm and embryos that once they are cooled to a point below – 130°C, the glass transition temperature of water, they cannot be raised above that temperature and then be re-exposed to below – 130° C or cell damage can occur8. The damage to the cells is caused by a reorganization or transformation of very small ice crystals in the extracellular fluids into much larger crystals during the temperature changes from below - 130° C to above – 130° C, and back to below – 130° C. This biological effect is called recrystallization. Damage occurs when the transformed large crystals physically invade the cell membranes and cellular organelles of either sperm or embryos. The severity of damage to cells is dependent upon two factors; one, how high the temperature gets above – 130° C, and two, the duration of exposure above – 130°C. Rapatz8 reported that although some cell damage can occur at - 130° C ice is relatively stable at - 100° C, but becomes more vulnerable at - 80° C. Since the temperature in the necks of most standard ranch Dewars ranges from to – 75° C all the way to room temperature, it is very common for frozen semen and embryos to be exposed and damaged, or even destroyed, during routine handling by those involved in daily Dewar management. The purpose of this paper is to review the damaging effects of recrystallization, and to point out common mistakes made by those who routinely handle frozen semen and embryos on the farm or ranch. Also, the economics of damaged semen and embryos due to mishandling will be discussed.