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 deg. C. and dilution and refreezing following an initial freezing of concentrated samples. Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on Freezability of Bull Sperm at -79 deg. C. (Average of 10 ejaculates) ================================================================ Post-thawing motility (percent)[F] --------------------------------------------- Glycerol level Number of sperm (millions/ml.) (percent) -------------------------------- 90 30 10 Average ---------------------------------------------------------------- 5 36.0 34.0 36.0 35.0 10 22.0 24.0 23.0 23.0 15 3.2 0.9 0.2 1.4 Average 20.3 19.8 19.9 20.0 ---------------------------------------------------------------- [F] Mean initial motility of sperm before freezing was 55 percent. Four semen samples were split and extended at rates of 1:1 (semen to extender) and 1:10. These were frozen, then thawed and halved. One half was further extended to a level of 15 million sperm per milliliter; the sperm numbers in the other remained unchanged. Each of these halves was split again, and one portion of each was stored at 5 deg. C. for 3 to 7 hours. The other two portions were refrozen. Table 7.--Effect of Further Dilution and Refreezing on Sperm Motility After the Initial Freezing of Bull Semen