=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] Table 5.--Comparison of the Freezability of 4 Semen Samples in Different Extenders ====================================================================== Dilution Pre- Post- Motility Extender rate freezing thawing Survival after (semen: motility motility (percent) storage[E] extender) (percent) (percent) (percent) ---------------------------------------------------------------------- Yolk-citrate 1:1 60 49 82 46 1:10 53 45 85 36 Yolk-saline 1:1 57 29 51 28 1:10 60 31 52 24 Yolk-phosphate 1:1 55 33 64 25 1:10 60 43 72 25 Whole milk 1:1 60 40 67 35 1:10 60 35 58 16 ---------------------------------------------------------------------- [E] Stored at 5 deg. C. for 7 hours after thawing. DILUTION RATES The first trials by the British at freezing bull semen were made with samples containing many millions of sperm cells. In routine artificial breeding, it is common to add extenders to semen so that one milliliter of diluted semen may contain only 10 million living sperm cells. (This number still insures optimal fertility.) Frequently the addition of 100 or more parts of the yolk extender to each part of the original semen sample is possible without reducing the sperm numbers below 10 million per milliliter. No one knew if this process of dilution would affect the resistance of bull sperm to freezing. The effect of various rates of dilution on the freezability of bull sperm was tested with 10 semen samples. The results, presented in Table 6, show that the numbers of sperm between 10 and 90 million per milliliter did not influence the percentage of sperm that survived freezing.