There was little or no loss of DNA or phospholipid from ram spermatozoa after washing three times with an equal volume of isotonic tris buffer. There was substantial loss of non-dialysable orcinoland biuret-reactive substances and also acid soluble phosphorus and phospholipids after cold-shocking and freezing ram spermatozoa. These components were also rendered more extractable with 10% NaCl after cold treatment. DNA was lost from cold-shocked and frozen ram spermatozoa after incubation at 37° C for 6 hr and the rate of degradation of DNA into dialysable components was initially higher in coldshocked or frozen spermatozoa.
Electron microscopy showed that cold shock and freezing caused profound changes in the appearance of the corrugated and terminal parts of the acrosome in the majority of spermatozoa but there were no apparent changes in the smooth region. There was considerable swelling of the acrosome with reduction in the density of the material contained within the acrosomal membranes. The effects were more pronounced in frozen than in cold-shocked spermatozoa.
In the middle pieces, changes occurred in the matrix of the mitochondria making up the mitochondrial sheath. The matrix in cold-shocked and frozen spermatozoa appeared lighter than in the controls and loss of protein from the middle piece was confirmed histochemically.
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