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This study is the continuation of our work to determine the causes of morphological freezing injuries.Semen had been collected from 10 fowls by the massage technique and was pooled. This semen was diluted with various diluents adjusted to different osmotic pressures (161, 323, 646, 1615 and 3230mOsm/kg) and was stored at 5°C for 24hr. These stored spermatozoa were examined under light microscope and scanning electron microscope (SEM).The results obtained are as follows.1) Many neck-bending spermatozoa were observed when the semen was diluted and stored with hypo, iso and hyper tonic glucose solutions, hypo, iso and hyper tonic glycerol solutions or hypo and iso tonic salt solutions.2) When osmotic pressures of the diluents were adjusted with salt, circular spermatozoa were observed at over 2 times isotonic concentration (over 646 mOsm), a swelling of sperm head was observed at 5 times isotonic concentration (1615 mOsm) and almost all spermatozoa lost the sperm head at 10 times isotonic concentration (3230 mOsm).3) Abnormality such as dislodged mitochondria was seen at the middle piece in the semen stored with hypotonic glucose solution, hypotonic salt solution or hypo, iso and hyper tonic glycerol solutions.4) Many isolated acrosome were observed under SEM when the semen was diluted and stored with glucose solutions of over 5 times isotonic concentration (over 1615 mOsm), salt solution of 5 times isotonic concentration (1615 mOsm) and glycerol solutions of over 2 times isotonic concentration (over 646 mOsm).5) Under SEM, a swelling at the end of the sperm head was observed in the semen stored with glucose solutions of over 2 times isotonic concentration (over 646 mOsm) and salt solutions of 2 and 5 times isotonic concentration (646 and 1615 mOsm).6) From these results, the isolation of acrosome and the swelling at the end of the sperm head seemed to be caused by high osmotic pressure of diluents during freezing and thawing.
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