The Influence of Extraneous Factors 79 



confirms the adverse effect of dilution is, that although the addition 

 of fructose to an isotonic salt diluent prolongs the metabolic activity 

 of bull sperm suspensions, excessive dilution with 'Ringer-fructose' 

 solution leads to a decline in the rate of fructolysis (Mann and 

 Lutwak-Mann, 1948). Up to a point, the changes due to the 'dilution 

 effect' resemble those produced by extensive washing of spermatozoa. 

 Thus for example, whereas one careful washing of centrifuged ram 

 sperm with several volumes of Ringer solution causes but negligible 

 damage, repeated washing results in a progressive decline in motility 

 and metabolism (Mann, 1945^; White, 1953). 



The use of artificial diluents in the storage of semen 



It has been noticed repeatedly that the deleterious effect of salt 

 diluents on spermatozoa can be at least partly overcome by the 

 inclusion of certain organic substances, mainly of colloidal nature. 

 These observations stimulated the development of the so-called 

 'media', 'pabula', 'dilutors' and 'extenders', for use in the storage 

 of semen for artificial insemination. 



The use of diluents in the storage of semen has its origin in 

 certain experiments made by Donne, de Quatrefages, and Koelliker, 

 who examined the effect of blood plasma, milk, various proteins 

 and sugars, on the spermatozoa. Early in this century, Hirokawa 

 (1909), Champy (1913), and others, experimented with blood plasma 

 and serum. Particularly illuminating results were obtained by 

 Grodzinski and Marchlewski (1938) who stored cock semen diluted 

 ten times with chicken serum at 2° for periods up to eight days, 

 and found, on increasing the temperature to 37°, that the sperma- 

 tozoa were motile. In addition, however, these authors also found, 

 in agreement with Bernstein and Lazarev (1933), that it is advisable 

 to use blood serum which has been pre-treated at 55°, as otherwise 

 the spermatozoa tend to agglutinate. An agglutinating and spermi- 

 cidal factor, present in fresh serum but inactivated by a few days' 

 storage at 4°, or 10 minutes' heating at 56°, was found by Chang 

 (1947a) in human, bovine, rabbit, guinea pig, and rat serum. It is 

 mainly because of its agglutinating properties that blood serum has 

 not found a wider application as a semen diluent. On the other 

 hand, as a direct result of immunological studies on the reactions 

 between blood serum and spermatozoa, rapid progress has been 



