Table 5.—Comparison of the Freezability of 4 Semen Samples
in Different Extenders
| Extender | Dilution rate (semen: extender) | Pre- freezing motility (percent) | Post- thawing motility (percent) | Survival (percent) | Motility after storage[E] (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° 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.
In a later trial it was found that sperm survival was slightly better at lower dilution rates than in the same samples frozen following dilution to 15 million sperm per milliliter. However, field trials with frozen semen carried out by others, using sperm numbers as low as 15 million per milliliter of semen inseminated or even lower, have been highly satisfactory.[11],] [12]
During the early studies in the Illinois laboratory, the effects of glycerol level were also tested.[13] These effects are discussed in the section on [glycerol additions] beginning on [page 17].
Effect of further dilution and refreezing after the initial freezing. Under some circumstances it might be advantageous to freeze semen with a high concentration of sperm cells and then extend it further after thawing. With such a procedure less storage space is needed than when dilution is carried to the maximum before freezing. Two experiments were conducted to test the effects of dilution and storage at 5° C. and dilution and refreezing following an initial freezing of concentrated samples.