The Influence of Extraneous Factors 65 



and electrolytes from the colloidal particles. This damage can be 

 substantially reduced if freezing is carried out in the presence of 

 certain organic substances such as sucrose, glucose, fructose, 

 glycerol, ethylene glycol, gelatin, albumin and various gums, all of 

 which have been used extensively in the past in freezing and freeze- 

 drying experiments on bacteria, yeasts, protozoa and various other 

 cells and tissues. The application of these substances, however, to 

 sperm is comparatively recent. In 1938, Luyet and Hodapp ob- 

 served that frog spermatozoa fail to survive the temperature of 

 liquid air, but if the cooling is carried out in the presence of 40% 

 sucrose at least 20% of them revive. Shaffner, Henderson and Card 

 (1941) were able to keep alive 30% of fowl spermatozoa by freezing 

 them to -79°, after treatment with fructose. An attempt to use 

 glycerol in connection with survival experiments on frog sperma- 

 tozoa was made by Rostand (1946) but it was not until 1949 when 

 the remarkable properties of glycerol were brought into prominence 

 thanks to Polge, Smith and Parkes (1949) as a result of their studies 

 on the low temperature resistance of glycerol-treated fowl semen. In 

 fowl semen diluted with an equal volume of Ringer solution and 

 vitrified at -79° for 20 min. and then rapidly thawed, there was no 

 significant revival of spermatozoa. On the other hand, when the 

 dilution was carried out with Ringer solution containing 40% 

 glycerol, the spermatozoa resumed full motility on thawing. This 

 observation was soon extended to the semen of other animals, 

 including the bull (Smith and Polge, 1950). A large number of cows 

 have been inseminated by Polge and Rowson (1952) with glycerol- 

 treated bull semen which had been stored at -79° for periods of 

 many months, and the excellent fertilizing capacity of such 'deep- 

 frozen' semen was proved when pregnancy occurred in 66% of the 

 inseminated animals. Glycerol-frozen and thawed human semen has 

 been reported to contain motile and fertile spermatozoa (Sherman 

 and Bunge, 1953; Bunge and Sherman, 1953). 



As yet, there is no adequate explanation for the effect of glycerol 

 on semen. The suggestion that it acts by supporting some sort of 

 residual metabolism in the frozen spermatozoa is difficult to reconcile 

 with our own observation that glycerol, unlike sugars and fatty acids, 

 is not oxidized by bull or ram spermatozoa. More probably, glycerol 

 exerts a protective influence on spermatozoa, preventing denaturation 



