If there is cell cycle delay caused by the test agent, the second harvest time would be expected to yield variable ratios of first and second generation metaphase cells. A single harvest is recommended after multiple dose treatment. It is sufficient to harvest both negative and positive controls at only one time following treatment. Immediately after sacrifice, testicular samples should be obtained. Spermatogonial cells are exposed to hypotonic solution and fixed.
Some methods use acid or enzyme steps to increase the yield of spermatogonial cells for analysis.
The cells should then be spread on slides and stained. Chromosome preparations should be made following standard procedures. Good scientific practice suggests that every experiment should be verified in an independent repeat assay. However, assuming that the procedures described above have been properly conducted, there is no absolute requirement for a repeat assay. In the case of a marginal or equivocal response, or of a positive response at a single dose, a repeat assay should be required.