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)
| Diluent | Medium | Yolk in final mixture[D] (percent) | Pre- freezing motility (percent) | Post- thawing motility (percent) | Survival (percent) | ||||||
| Extending | Glycerolating[C] | ||||||||||
| yolk | : | citrate | yolk | : | citrate | ||||||
| 1 | 1 | : | 1 | 1 | : | 1 | 45.7 | 65 | 5 | 8 | |
| 2 | 1 | : | 1 | 1 | : | 3 | 34.9 | 64 | 27 | 42 | |
| 3 | 1 | : | 3 | 1 | : | 1 | 33.6 | 63 | 33 | 52 | |
| 4 | 1 | : | 1 | 0 | : | 1 | 24.2 | 64 | 39 | 61 | |
| 5 | 1 | : | 3 | 1 | : | 3 | 22.8 | 63 | 37 | 59 | |
| 6 | 1 | : | 3 | 0 | : | 1 | 12.1 | 59 | 33 | 56 | |
| 7 | 1 | : | 7 | 1 | : | 7 | 11.4 | 56 | 35 | 62 | |
| 8 | 1 | : | 7 | 0 | : | 1 | 6.0 | 52 | 26 | 50 | |
| 9 | 1 | : | 15 | 1 | : | 15 | 5.7 | 49 | 25 | 51 | |
[C] This mixture included 14 percent glycerol.
[D] The average initial sperm concentration was 900 × 106/ml. Sufficient extender was added to give 30 × 106/ml. at the first extension. Thus the final concentration was 15 × 106 sperm/ml. after glycerolization.
Since rather large changes in the percentages of yolk were used in this experiment, two further trials were conducted in which 16, 24, and 32 percent yolk in the final mixture were compared, with the final citrate percentages held constant. In these tests, 16 and 24 percent yolk maintained sperm better at all citrate levels tried than 32 percent yolk. The 16 percent level was slightly better at most of the levels of citrate tested ([Fig. 3]).
Citrate level in the final diluent. The early work of the British indicated that a final citrate level near 2 percent in the diluent was satisfactory for freezing bull sperm. Later, in a personal communication, Polge of the British group suggested that a citrate level of about 2.35 percent might be best with a final glycerol concentration of 7 percent. Some of the first attempts in this laboratory at establishing the optimum yolk-to-citrate ratios are shown in [Fig. 3]. In these experiments, the optimum levels of citrate appeared to be lower than anticipated from the British work. Thus a more complex experiment was set up to test a wider range of citrate levels using 16 and 24 percent egg yolk in the final freezing mixture. The average percentages of motile sperm found after freezing 10 semen samples at each of the citrate and yolk levels in this experiment are shown also in [Fig. 3]. Little difference in freezability was found between citrate percentages of 1.55 and 1.95. When the rate of sperm motility following freezing and thawing was considered along with the percent of motile sperm, a slight advantage was found with 16 percent yolk and a citrate concentration of 1.55 percent.
