[J] Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent. Three of the sugars--glucose, arabinose, and rhamnose--protected the sperm during equilibration and freezing. Xylose was less effective, but its addition resulted in slightly better sperm survival than glycerol alone. It was found also that the methylene-blue reduction time (metabolic test for semen quality) was faster in samples to which the sugars had been added--after glycerolization, after equilibration, and after freezing the samples. This is confirming evidence for the presence of more living and actively metabolizing sperm in the portions to which sugars had been added. Table 13.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm Motility During the Freezing Procedures[K] (Average of 10 ejaculates) ====================================================================== Sperm motility (percent) ------------------------------------------------- Stage of Glycerol Glycerol Glycerol Glycerol Glycerol observation only and and and and glucose arabinose xylose rhamnose ---------------------------------------------------------------------- Fresh diluted semen 63 63 63 63 63 After glycerolization 54 55 54 57 60 After 18 hours equilibration 39 43 44 39 46 After freezing to -79 deg. C. and immediate thawing 28 34 34 29 24 After 4 days at -79 deg. C. 23 26 26 25 27 ----------------------------------------------------------------------- [K] Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent. =Substitutes for glycerol.= Since glycerol was so effective in protecting sperm during freezing, many have assumed that related compounds might be even better. Several compounds, some related to glycerol and some not, have been tried as substitutes for glycerol in the freezing procedure. They include ethylene glycol, propylene glycol, trimethylene glycol, mannitol, sorbitol, dextrans, and seminal-plasma proteins. None of these materials has been as effective as glycerol in protecting sperm during freezing. In fact, several of the materials proved to be injurious to sperm prior to attempts to freeze the samples. While the work in our laboratory with these substances as glycerol substitutes was by no means finally conclusive, because of the many possible interactions of experimental conditions, sufficient data were gathered to lead us to abandon further study until greater promise of success might be evident. FREEZING RATE