78 The Biochemistry of Semen 



CO2. In some experiments this is supplemented with (i) 0-5 g. 

 fructose/ 100 ml. ('Ringer-fructose'), (ii) 20 ml. 0-25 M-phosphate 

 buffer pH 7-4, the latter made up by mixing 19 ml. of an aqueous 

 solution containing 0-71 g. Na2HP04 (or 1-79 g. NasHPO^.UHaO) 

 with 1 ml. N-HCl ('Ringer-phosphate') or (iii) both fructose and 

 phosphate ('Ringer-fructose-phosphate'). Glass-distilled water and 

 analytical grade reagents are used throughout (Mann, 1946^). 



If the dilution of mammalian sperm with saline is excessive it 

 leads after a short spell of increased activity to a permanent loss 

 of motility, metabolic activity and fertilizing capacity. According 

 to Milovanov (1934), the resistance (R) of sperm to the immobiliz- 

 ing effect of dilution with large volumes of 1% NaCl, varies in 

 different animals; he believes that specimens of semen capable of 

 high resistance possess at the same time high fertilizing capacity. 



V 

 The formula R=— used by Milovanov in the 'resistance test', is cal- 



V 



culated from the volume of 1% NaCl (V) required to arrest in a 

 given volume of semen (v) all progressive movement of sperm-heads. 

 In bull semen the test is carried out with 002 ml. semen, at 17-24°, 

 and sperm motility is assessed by microscopic examination after the 

 addition of successive lots of 10 ml. 1% NaCl solution (Nagornyi 

 and Smimov, 1939). The R values given by Milovanov are: bull 

 300-20,000, ram 100-5000, stallion 100-1500, boar 60-1000, dog 

 200-600. Some doubts however, have arisen as to the significance of 

 Milovanov's test in the assessment of male fertility, as well as his 

 statement that the immobilizing action is due to the toxicity of 

 sodium chloride as such (Emmens and Swyer, 1947, 1948; Cheng, 

 Casida and Barrett, 1949). But there is general agreement that 

 excessive dilution is invariably harmful to spermatozoa. This is 

 reflected, among others, in Chang's (1946) finding that a constant 

 number of rabbit spermatozoa has a greater fertilizing capacity 

 in a small, as opposed to a large, volume of salt diluent. Chang insem- 

 inated superovulated does with saline suspensions of spermatozoa, 

 removed the ova 38^2 hr. later, and counted the cleaved ova. 

 Results showed that the insemination of a total of 30-40 thousand 

 spermatozoa in 1 ml. 0-9^o NaCl was followed by the cleavage of 

 0-6% of ova, whereas the same number of cells suspended in 0- 1 ml. 

 produced cleavage in 17-42% of ova. Another observation which 



