| Percent of motile sperm after freezing and thawing semen in diluents containingvarious levels of egg | |
| yolk | (Fig. 2) |
From the results of these experiments, and from several reports in the literature, [5],] [6],] [7],] [9],] [10] it appears that a diluting medium resulting in a final concentration of 16 to 25 percent yolk and 1.55 to 2.2 percent sodium citrate dihydrate is highly satisfactory for freezing.
Storing and freezing diluent. In some instances it would be advantageous to have prepared diluent on hand for use at any time. The suitability of stored diluent was tested with a yolk-citrate (equal parts yolk and citrate without antibiotics added) diluent prepared and stored at 5° C. for 0, 2, 5, 7, and 9 days. Seven semen samples were diluted and frozen in these diluents. No difference was noted in the survival of sperm that could be attributed to the age of the diluent.
| Percent of motile sperm after freezing and thawing semen in diluents containing various levels of egg | |
| yolk and various percentages of sodium citrate | (Fig. 3) |
In another trial, a similar diluent (1:1 yolk to citrate with 1000 units of penicillin and 5000 units of streptomycin) was prepared and stored in the freezer compartment of a refrigerator at -15° C. Upon thawing, it was whitish in color and more viscous than freshly prepared diluent. Except for the fact that the viscosity seemed to reduce the rate of sperm motility, this frozen diluent stored for 65 days compared favorably with freshly prepared diluent for freezing semen.
Other diluents. Without the protective action of egg yolk or milk, few bull sperm will survive freezing. Several diluents were compared on a limited scale for freezing bull sperm. The results of these trials are compiled in [Table 5]. In this trial the yolk-citrate extender served best in maintaining sperm motility during freezing. Yolk-phosphate and homogenized whole milk were slightly less protective and yolk-saline seemed to furnish the least protection to sperm during freezing.
A number of investigations in other laboratories have now proven that milk can be used as effectively as the yolk-citrate diluent for freezing bull sperm.[6],] [7]