=Allowing sperm to equilibrate with the glycerol.= Allowing sperm to stand in the presence of glycerol is considered by some to be necessary in order that the glycerol penetrate the sperm heads before freezing. From the first successful attempts at freezing bull sperm came the practice of allowing 12 to 20 hours for this process of equilibration. A long equilibration time results in aging the sperm. Data from a number of sources indicate that a drop of approximately 5 percent in fertility in the field occurs with each 24 hours of aging in the test tube. Thus it would seem desirable to reduce the equilibration time to a minimum commensurate with good freezability in order to reduce the effects of aging (at 5 deg. C.). Results of attempting to reduce equilibration time are given in Table 11. At 4.5 deg. C., little variation in motility following freezing and thawing was found after equilibration times of 2, 6, and 18 hours. At the higher temperatures of 10 deg. and 15.5 deg. C., the shortest equilibration time--2 hours--was slightly more detrimental with the differences significant at the 5-percent level at 15.5 deg. C. For all temperatures combined, 6 hours was significantly better than 2 or 18 hours. =Sugar additions and equilibration time.= Early in their experiences in freezing semen, the Australian workers found a short equilibration time--30 minutes--to be satisfactory if sugars were added to the diluent.[5] This protective action of sugars during the equilibration period was confirmed in our investigations. The results of one phase of this study are shown in Table 12. From these data it can be seen that the presence of glucose or rhamnose at a level of 1.25 percent improved sperm survival during the period of equilibration. In another trial these sugars and two others, arabinose and xylose, were tested for their protective action in freezing semen. The percentages of surviving sperm remaining after the various steps in the freezing procedure with and without the presence of these sugars are shown in Table 13. Table 12.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm Motility During Equilibration With Glycerol[J] ============================================================ Sperm motility (percent) ------------------------------------- Stage when observed Glycerol Glycerol Glycerol only and glucose and rhamnose ------------------------------------------------------------ Fresh diluted semen 56 56 56 After glycerolization 54 54 54 After equilibration 2 hours 51 53 53 6 hours 48 52 53 12 hours 46 50 51 18 hours 40 46 46 ------------------------------------------------------------