Cryo-protection of some amide solutes on spermatozoa of domestic animals:I. Effects of formamide, acetamide and lactamide on the motility of bovine spermatozoa in pellet freezing
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The effect of some amides on the motility of bull spermatozoa was investigated before and after pellet freezing of semen.<BR>Each aliquot of collected semen was diluted ten times with egg yolk-citrate diluent containing one of amides (form-, lact-, acet-, propion-, malo-, butyl-, valer-), ethylene glycol, DMSO, or glycerol (control) at a final level of 0.31, 0.93, 1.55, 2.17 and 3.1 M after dilution.<BR>Sperm motility was estimated with television-video record equipment connected with phase-contrast microscope.<BR>When diluted semen was incubated at 20°C for 30 min., the best sperm motility was observed in lactamide diluent as well as in glycerol diluent (Fig. 1).<BR>Other amides were inferior to these two diluents, but acetamide maintained rather good mo-tility which was followed by propionamide, formamide and malomide. Effect of malonamide over 1.55 M level on sperm motility was unable to investigate owing to its low solubility. In butylamide sperm motility dropped near to zero at 1.55 M level and in valeramide sperm died immediately after dilution even at 0.31 M level. When diluted semen was cooled to 5°C and frozen on a dry ice block in 0.1 m<I>l</I> pellet form after equilibration for 36 hrs and thawed on a metal plate at 40°C, all amides, except butylamide and valeramide, showed some protective effect for sperm survi-val against freezing injury (Fig. 2).<BR>Among them, the best protective effect was observed in lactamide diluent, then in acetamide and formamide diluent, when semen was thawed without thawing media. Propionamide and ma-lonamide gave low protective effect.<BR>Striking and contrasted changes of post-thawed sperm motility were observed by redilution of thawed semen with thawing media (buffered milk-isotonic to raw semen)<SUP>6)</SUP> in the samples diluted with formamide and lactamide diluents.<BR>In the former, the motility was greatly improved at 0.93 M level, but in the latter the moti-lity was injured significantly by redilution.<BR>These phenomena is inferred to be related with the permeability of the amides into the cells and also with the degree of injury of living sperm after freezing and thawing.<BR>From these findings, formamide and lactamide will can be used as new cryoprotective agents for spermatozoa.<BR>Fertility triale are undergoing with these amides in cattles, swine and rabbit.<BR>Parts of the paper had been presented at the 13th annual meeting of The Japan Frozen Semen Research Society on Sept. 2 3, 1971 and at the antumn meeting of The Japanese Soc. Anim. Reprod. at Kyoto on Oct. 27, 1971.
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