Freezability of several consecutive ejaculates. The fact that second ejaculates sometimes withstood freezing better than first ejaculates suggested that the maturity of the sperm might be a factor affecting freezability. An opportunity to check this idea came when 20 consecutive ejaculates were collected from each of 6 bulls within a 4-hour period. The sperm in samples collected in this manner might be expected to be less mature with each additional collection.
The results obtained in freezing several consecutive ejaculates are shown in [Figure 1] as averages for the ejaculates from 6 bulls. In same instances, there was an insufficient quantity of semen available to test the freezability. (Procedure: Diluted to 30 × 106 sperm per ml. with 1:1 yolk-citrate, then cooled and glycerolated with an equal volume of 14 percent glycerol (percent by volume) in 2.9 percent sodium citrate. Final sperm concentration, 15 × 106. Equilibration time, 15 hours. Freezing rate, 2° C. per minute from +5° to -19° C. then 4° C. per minute from -19° to -79° C. Held frozen for 5 or more hours then thawed in water at 5° C. and checked for motility.)
| Percent of motile sperm before and after freezing consecutive ejaculates collectedwithin a 4-hour period | |
| from each of 6 bulls | (Fig. 1) |
In general, the motility before freezing improved slightly from the first to the fourth to sixth ejaculate and then declined until about the 12th or 14th ejaculate, at which point the prefreezing motility seemed to level off through the 20th ejaculate ([Fig. 1]). The percentage of motile sperm found after freezing and thawing followed the same trend at an average level 10 to 15 percent lower than the prefreezing level. As is readily seen from the trend lines in [Fig. 1], the difference between the prefreezing motility and the post-thawing motility increased gradually after about the fifth ejaculate. Although the absolute difference did not increase greatly, the percentage of survival after freezing dropped from 81 percent on the first 5 ejaculates to 26.5 percent on the last 5 ([Table 2]).
Table 2.—Comparison of the Freezability of 20 Consecutive
Ejaculates Collected Within a 4-Hour Period
(Weighted averages for 6 bulls)
| Ejaculate | Number of ejaculates | Prefreezing motility (percent) | Post-thawing motility (percent) | Survival (Percent) |
| 1st to 5th | 29 | 53.3 | 43.2 | 81.0 |
| 6th to 10th | 26 | 43.8 | 30.2 | 69.0 |
| 11th to 15th | 23 | 28.6 | 14.5 | 50.7 |
| 16th to 20th | 19 | 18.1 | 4.8 | 26.5 |
Freezability of epididymal sperm. Since the freezability of bull semen seemed to be better in second than in first ejaculates and some improvement in freezability was evident through the first 4 to 6 ejaculates taken consecutively, the question of whether epididymal sperm would withstand freezing seemed to be important. Although when 20 collections were made, the later ejaculates no doubt contained fewer mature sperm, the lowered freezability could have been due to accessory gland secretions rather than changes in the sperm themselves. Removing sperm directly from the epididymis would eliminate any effect that the accessory gland secretions could be exerting. Further, if epididymal sperm could be frozen, obtaining and using semen from a bull shortly after his death should be possible.