A new preparation protocol for measurement of testicular sperm production

In studies of male reproductive toxicity, measuring daily sperm production is a quite important criterion. However, the accuracy of the values measured by the basic protocol is still controversial. In order to enhance the homogeneity of countable testicular sperm/spermatid heads, this report introduces a new enzymatic method with a subsequent detergent treatment. The testis of rat was firstly homogenized in phosphate-buffered saline. The homogenate (buffer mix) was then treated with collagenase and trypsin, and then sodium dodecyl sulfate (SDS) was added to produce detergent-resistant sperm/spermatid heads (detergent mix). After examination by hemocytometer, the coefficient of variation (CV) of the number of sperm/spermatid heads was compared with that obtained from the buffer mix. In addition, a MicroCell chamber was applied to the examination, and the CV was compared with other cases. In both examinations, homogeneity was improved by the detergent mix preparation. Counting with the hemocytometer showed an increased number of sperm/spermatid heads compared with that of the buffer mix (P<0.001), and the CV was decreased (P<0.05). In addition, when the MicroCell chamber was applied, the numbers increased about 3-hold compared with that of the buffer mix (P<0.001). The CV of the detergent mix was 23.7%, while that of the buffer mix was 38.9%. These results clearly demonstrate that the new preparation protocol generated in this study can provide more actual and accurate values when measuring daily sperm production.