Effects of Cooling and Warming Rate to and from −70°C, and Effect of Further Cooling from −70 to −196°C on the Motility of Mouse Spermatozoa1

Abstract

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.

Optimization of a Protocol for Cryopreservation of Mouse Spermatozoa Using Cryotubes

“…By varying the position of the cryotube relative to the surface of the LN 2 and the volume of sperm suspension, we found that the survival of spermatozoa in cryotubes was improved to a level similar to that observed in spermatozoa frozen in plastic straws. Previous studies on the cryopreservation of mouse spermatozoa have reported optimal cooling rates of 27-130 [29] or 39-114 C/min [30]. These data are consistent with ours because the cooling rate in our experiments was -143.4 C/ min in cryotubes, although there was a shoulder at around -10 C (Fig.…”

Vitrification Technique - New Possibilities for Male Gamete Low-Temperature Storage

“…To date, publications dedicated to this topic are rare (Nawroth, et al, 2002;Koshimoto & Mazur, 2002;Isachenko et al, 2004aIsachenko et al, , b, 2008. Recent work has reversed this situation in that favorable results have been obtained in human spermatozoa after excluding permeable cryoprotectants from cryopreservation solutions, increasing the cooling rate and using carbohydrates, proteins and other extracellular agents, to increase the viscosity of the surrounding medium of cells and prevent the formation of any intra-and big extracellular crystals (Isachenko et al, 2004a, b).…”

“…In general, the inclusion of osmotically active, non-permeating compounds into the vitrification solution leads to additional rehydration of cells and, as a result, to decreasing toxic effects of the permeable cryoprotectants on intracellular structures. The non-permeable cryoprotectant sugars possess a unique property: stabilization of a cell membrane (Nakagata & Takeshima, 1992Koshimoto et al, 2000;Koshimoto & Mazur, 2002).…”

“…It has been suggested that raffinose plays the role of a membrane stabilizing and dehydrating agent. Comparative investigation of three different sugars, monosaccharide glucose, disaccharide sucrose and trisaccharide raffinose, showed that protection against freezing/thawing injuries is independent of the kind of sugar itself, but depends more on the sugar's concentration (Koshimoto & Mazur, 2002). Based on this evidence, we have decided to investigate the different concentrations of sucrose on the viability of cryopreserved spermatozoa.…”

Optimization of Ethylene Glycol Concentrations, Freezing Rates and Holding Times in Liquid Nitrogen Vapor for Cryopreservation of Rhesus Macaque (Macaca mulatta) Sperm

“…The synergistic effect of the CPA and freezing medium on nonhuman primate sperm cryopreservation may also explain the low efficiency of cryopreservation of nonhuman primate sperm. Studies have indicated that the highest survival rate can be obtained by freezing sperm at their optimal freezing rate [9,22]. In the present study, four freezing rates determined by the distances between cryostraws with sperm and the LN 2 surface were chosen to determine the optimal freezing rate for rhesus macaque sperm cryopreservation.…”