Sperm cells centrifuged three times and washed twice in 0.9 percent sodium chloride solution withstood freezing well when finally resuspended and frozen in yolk-citrate diluent. The percentage of survival in three samples subjected to this treatment was 60 percent. Thus it appears that the seminal plasma itself is not essential for ejaculated sperm to survive the rigors of freezing. This is not surprising, since it had already been found that epididymal sperm, which also are free of accessory gland secretions, can withstand freezing and thawing. EXTENDERS FOR FREEZING BULL SEMEN Both of the extenders that are widely used in routine storage of bull semen at 5 deg. C. are used for freezing semen. These are the egg yolk-sodium citrate and whole or skimmilk extenders. Most of the research with extenders for freezing bull semen in this laboratory has been done with the yolk-citrate diluents. =Proportion of egg yolk in the final diluent.= Some early experiences with a diluent consisting of one part yolk and one part 2.9 percent sodium citrate dihydrate in distilled water showed poor sperm survival following freezing. The final mixture with this diluent consisted of about 45 percent yolk. In other attempts at adding glycerol in order to freeze semen, the final proportion of yolk was diminished and better sperm survival was obtained. Several experiments were carried out to test the effect of varying levels of egg yolk. In the first efforts to find the optimum level of egg yolk, the level of yolk in the final frozen mixture was varied from about 6 to 46 percent. These levels were obtained by varying the proportion of yolk to 2.9 percent citrate solution in the original extending media and also in the media added in glycerolating the samples. Split portions of 20 semen samples were frozen in each of the extender combinations indicated in Table 4. The mean percentages of motile sperm found before and after freezing and thawing are shown also. The highest percentages were found with extenders containing 23 and 24 percent yolk. The highest percentage of yolk, resulting when a 1:1 (yolk to citrate) extender was used for both extending and glycerolating, proved to be most detrimental to sperm survival during freezing. The lowest percentage of yolk used (6 percent) was not as effective in protecting sperm during freezing as the intermediate levels tested (Fig. 2). Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen (Average of 20 semen samples)