The immobilization of spermatozoa by freezing and thawing and the protective action of glycerol.

It was discovered by Polge, Smith & Parkes (1949) that spermatozoa could be frozen and thawed without loss of motility if glycerol was included in their suspending medium. Following this discovery the spermatozoa of the domestic fowl (Polge, 1951), ofthe bull (Polge & Rowson, 1952) and ofthe herring (Blaxter, 1953) have been preserved without loss of function for long periods in the frozen state. The importance of the practical applications of this method has been discussed (Polge & Parkes, 1952). The investigation of the protective action of glycerol was facilitated by the observation that red blood cells also were protected against the adverse effects of freezing and thawing by means of glycerol (Smith, 1950), and the nature of the damage occurring to red blood cells when they are frozen and thawed, and of the protection given by glycerol was then described (Lovelock, 1953a, b). Briefly, haemolysis by freezing and thawing is due to the destructive action of the concentrated salt solution to which the cells are exposedwhen water is removed as ice. In the presence of glycerol the electrolyte concentration at temperatures below the freezing point is sufficiently reduced to explain in full the protection afforded by glycerol. The apparent simplicity of the processes causing the haemolysis of red blood cells by freezing and thawing, and of the protective action of glycerol, made it ofsome interest to observe the behaviour of other living cells on freezing and thawing. It seemed that this provisional explanation of the effects of freezing could best be tested using living cells with a wide range of sensitivity to hypertonic salt solutions. It also seemed desirable to use a criterion of damage more delicate than haemolysis. Both of these requirements are met by the spermatozoa of different animals. Those of the rabbit are particularly sensitive to hypertonic salt solutions (Emmens, 1948), while those of the herring clearly will tolerate salt solutions at least as strong as sea water. In addition, the observation of the motility of spermatozoa, even if somewhat lacking in precision, provides a different and sensitive criterion for the assessment of freezing damage. This paper records the effects of freezing and thawing upon the motility ofthe spermatozoa of the rabbit, the bull, the fowl and the herring. The modification of these effects by glycerol is also described.