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Title: COMPARISON OF GLYCEROL, OTHER POLYOLS, TREHALOSE AND RAFFINOSE TO PROVIDE A DEFINED CRYOPROTECTANT MEDIUM FOR MOUSE SPERM CRYOPRESERVATION

Author
item STOREY, B - UNIV OF PENN MED CENTER
item Noiles, Esther
item THOMPSON, K - UNIV OF PA MED CENTER

Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Mouse sperm in the past have proved more resistant to the cryopreservation process as compared to sperm of other mammalian species. A major incentive to optimize procedures for the cryopreservation of mouse sperm stems from the need to maintain lines of transgenic mice, which have proved to be powerful models for the study of the regulation and functional consequences sof gene expression. Different combinations of sugars and polyols were compared as cryoprotectant agents with mouse sperm. In addition, addition and removal of cryoprotectants were studied. It was determined that 6% glycerol/7.5 % trehalose combination provides a defined cryoprotectant, which, when used with addition by dialysis prior to freezing and direct insemination after thawing, yields a satisfactory yield of fertilized eggs in a mouse in vitro fertilization system. This study provided a defined cryoprotectant medium, lacking egg yolk, that would allow in vitro fertilization with cryopreserved mouse sperm to attain a percentage of egg fertilized equal to or greater than half that obtained with fresh sperm.

Technical Abstract: Most procedures for mouse sperm cryopreservation have utilized raffinose to control cell dehydration prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane, and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and d20% sugar showed that 6% glycerol and 7.5% sugar consistently gave maximal protection. Comparsion of raffinose and trehalose at this concentration showed trehalose to give better (P<.01) recovery of intact cells (48.6+6% for trehalose, 36+9% for raffinose, mean+SEM, N=5). Less hydrophilic polyols should prove more permeant to the membrane than glycerol and possibly inhibit lethal intracellular ice formation more effectively than glycerol alone. The polyols tested as supplements to 6% glycerol were: propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydrolymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% sugar, the two diols and THME gave less cryoprotection than glycerol alone, and EHMP reduced post-thaw membrane integrity to nil, thus invalidating the hypothesis. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; dialysis addition and direct insemination without cryoprotectant removal. The last proved superior (P<.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal & 32% for serial addition and dilution. The glycerol/trehalose combination provides a defined cryoptotectant, when used with addition by dialysis pre- freeze and direct insemination post-thaw, yields a satisfactory yield of fertilized eggs in an in mouse vitro fertilization system.