===================================================================== Microliters of oxygen Semen sample tested utilized per 10^{ 8} sperm -------------------------- First hour Second hour --------------------------------------------------------------------- Fresh diluted semen 10.3 8.1 Fresh diluted semen glycerol tipped in at end of first hour 9.7[L] 12.9[L] Aged 20 to 24 hours at 5° C. 11.2 8.3 Aged 20 to 24 hours at 5° C. glycerol tipped in at end of first hour 11.8[L] 12.9[L] After 20 hours equilibration with glycerol 11.7[L] 7.8[L] After freezing and thawing 9.7 6.3 --------------------------------------------------------------------
=Effect of freezing procedures on oxygen utilization by sperm.= Limited data have been obtained on the effects of some of the freezing procedures on the oxygen utilization of bull sperm. The results obtained in these experiments confirmed the earlier findings that tipping glycerol directly into the diluted semen at 37° C. caused an increase in oxygen consumption (Table 16). All other steps in the freezing procedure had little effect on oxygen consumption by the sperm. Except where glycerol was added during the determination, the rate of oxygen utilization was lower the second hour than during the first. The oxygen uptake of semen that had been frozen and thawed seemed to drop faster than that of unfrozen samples.
=Effect of freezing procedures on methylene-blue reduction time.= The methylene-blue reduction test has been used as a means of measuring semen quality and is dependent on the metabolic activity of the sperm. The effects of various freezing procedures on the ability of samples to decolorize methylene blue were determined with 10 semen samples. Sperm numbers were standardized to 300 × 10^{ 6} cells per milliliter and the time required for these cells to reduce a 1:40,000 solution of methylene blue was determined on freshly diluted semen, after the addition of glycerol, after equilibration, and after freezing and thawing. Portions of each diluted sample were tested at these stages of the procedure with glycerol alone added and with glycerol and various sugars added.
A marked increase in the time required for the sperm to reduce methylene blue occurred when the glycerol was added (Table 17). This increase was greatest in the portions with glycerol alone and with glycerol and glucose. The time increase was less pronounced in the presence of the three pentose sugars used. Following equilibration, the samples regained the ability to reduce methylene blue at a rate only slightly slower than when they were fresh. Freezing and storage of semen resulted in slower reduction of the methylene blue than was shown after equilibration with glycerol. Since freezing usually kills some of the sperm, a slowing of the reduction time after freezing would be expected.