| Glycerol level (percent) | Sperm motility | |||||||||||||||||||
| Post- thawing | After storage at 5° C. | Average | ||||||||||||||||||
| 1 day | 3 days | 7 days | ||||||||||||||||||
| percent | rate | percent | rate | percent | rate | percent | rate | percent | rate | |||||||||||
| Control[I] | 56 | 2.5 | 55 | 1.9 | 46 | 1.8 | 38 | 1.4 | 48 | 1.90 | ||||||||||
| 0 | 54 | 2.4 | 44 | 1.9 | 46 | 1.8 | 36 | 1.4 | 45 | 1.87 | ||||||||||
| 5 | 52 | 2.2 | 50 | 1.9 | 46 | 1.7 | 32 | 1.4 | 45 | 1.80 | ||||||||||
| 1 | 0 | 52 | 2.3 | 46 | 1.8 | 42 | 1.7 | 28 | 1.6 | 42 | 1.85 | |||||||||
| 2 | 0 | 52 | 2.1 | 50 | 1.7 | 44 | 1.6 | 38 | 1.1 | 46 | 1.62 | |||||||||
| 3 | 0 | 50 | 0.7 | 44 | 0.5 | 42 | 0.4 | 30 | 0.4 | 42 | 0.51 | |||||||||
| Average | 53 | 2.0 | 3 | 47 | 1.6 | 2 | 44 | 1.5 | 0 | 34 | 1.2 | 2 | .. | .... | ||||||
[I] The control differed from the 0-glycerol treatment in that no additional citrate or glycerol solution was added.
A similar trial was carried out with seven samples; one portion was diluted 1:9; the other was extended at the outset to 15 million sperm per milliliter. Results for both tests are summarized in [Table 7].
From [Table 7] it can be seen that refreezing following an initial freezing further reduced the number of surviving sperm. The second freezing was more detrimental to the portion of the samples extended to 15 million sperm per milliliter than to the portion that was refrozen at a higher sperm concentration. The percentage of motile sperm remained fairly high in the portions that were diluted to 15 million sperm and stored at 5° C. However, in all cases, survival was best in the samples at the lower dilution levels.
GLYCEROL ADDITIONS
When the British procedure for freezing bull semen was first tried in this country, many of the refinements of the technique still had not been defined. It was known that glycerol worked well in protecting sperm during freezing. The effects of glycerol on sperm at 5° C., the appropriate levels to use in freezing, and the manner of adding it were not well established. Therefore, a number of trials were conducted in an attempt to establish the best procedures.
Effect of glycerol on sperm survival at 5° C. Since early work indicated the need for adding glycerol to diluted semen in order to protect the sperm during freezing, it was considered important to determine the levels of glycerol that sperm would tolerate at 5° C. Ten semen samples were extended 1:9 (semen to diluent) in a 1:1 yolk-citrate diluent (yolk to 2.9 percent sodium citrate dihydrate). Each sample was then split into 6 portions and an equal volume of citrate solution containing glycerol was added slowly to each to bring the glycerol in the final mixture to 0, 5, 10, 20, or 30 percent (by volume). These samples were stored at 5° C. and examined for motile sperm after 1, 3, and 7 days. The effects of glycerol levels on the percentage of sperm surviving and the rate (or speed) of their forward motion (0 = no forward motion; 4 = extremely rapid progressive motility) are presented in [Table 8].
The percentage of motile sperm decreased slightly at the higher levels of glycerol. The most noticeable effect of the increase in glycerol level was the reduction in the rate of forward motion of the sperm. At the 30-percent level, the sperm moved slowly and could be seen to rotate as they moved forward. Some samples were checked after slowly bringing the diluent up to a level of 40 percent glycerol; the sperm seemed to be immobilized completely in this solution.
Glycerol levels for freezing semen. The British procedure called for the use of 10 percent glycerol in the final mixture of semen and extender prior to freezing. Yet, as shown in [Table 6], in our laboratory 5 percent glycerol resulted in the survival of a higher percentage of sperm than did 10 or 15 percent. In order to define more clearly the optimum glycerol level, several ejaculates of semen were subsampled and portions were frozen after the addition of yolk-citrate extender and glycerol in varying quantities. From [Table 9] it can be seen that glycerol levels of 6 and 8 percent in the final mixture resulted in maximum sperm survival during freezing. These results were confirmed in tests on the survival of sperm at 5° C. storage for 3 days following freezing and thawing with varying glycerol levels (see [Table 10]).