New methods for cryopreservation of chicken semen using Hiroshima solution (trehalose solution)

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Since an initial report by Polge et al. who found a protective action of glycerol in chicken spermatozoa deep-frozen to -79ﾟC, there have been many investigations of freeze-storage of chicken spermatozoa. However, fertility by freeze-thaw semen is markedly below that obtained with fresh semen. The purpose of the present study is to evaluate an effectiveness of trehalose and to improve pelleted methods for cryopreservation of chicken semen. The semen was diluted with an extender containing different levels of trehalose and glycerol, and was frozen in pellets. After thawing, the spem motility and lactate dehydrogenase(LDH)activity of extracellular medium of freeze-thaw semen were measured. Along with the increased levels of trehalose, the motility of postthaw sperm increased and LDH activity in freeze-thaw semen decreased. The minimum difference between LDH leakage before and after freeze-thawing was shown to be at 0.26 M trehalose. The 9% glycerol level gave the best postthaw recovery in the sperm motility and the lowest LDH activity. The effects of PH and osmotic pressure of the extenders containg trehalose on the sperm motility and LDH leakage after freeze-thawing were investigated. The optimum pH value and osmotic pressure for the extender used to freeze semen was 7.2 and 370 mOsm, respectively. Furthermore, the effects of sperm concentration on the sperm motility and LDH leakage after freeze-thawing were estimated. The lowest sperm concentration at freezing gave the best postthaw recovery in the sperm motility and the lowest LDH activity in the extracellular medium of freeze-thaw semen. From those results, it was concluded that cryopreservation of semen diluted with 0.26 M trehalose solution (PH=7.2 ; osmotic pressure, 370 mOsm) containing 9% glycerol was the method feasible for poultry breeding programs.