AN IMPROVED PROCEDURE FOR THE BIOLOGICAL ASSAY OF ANDROGENS BY DIRECT APPLICATION TO THE COMBS OF BABY CHICKS1

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Abstract
Experiments on improvement of the biological assay method for androgens by direct application to the combs of baby chicks led to five successive modifications in procedure, each of which was put into practice and evaluated for precision, minimum effective dose (m.e.d.), and convenience. A total of 162 assays and experiments using approximately 20,000 baby chicks was involved. The response metameter, the readily computed log [image], was selected after testing several other transformations of the data. It yielded a linear log dose-response curve, minimized the variance, and was homoscedastic. In other experiments it was shown (1) that when the volume of solution applied to the comb daily was reduced from 0.05 ml. to 0.01 ml., the m.e.d. was lowered markedly, (2) that absolute ethanol was substantially as effective (at a volume of 0.01 ml.) as well as more convenient than ether as the vehicle for application of androgen, (3) that 7 daily applications produced more precise results than 5 applications, and (4) that there was a hump at the lower end of the log dose-response curve that could be avoided, thus improving the precision of the assay. The procedure for the assay currently in use in our laboratory (the fifth modification) is described in detail. The results of 29 assays in this series were evaluated statistically and compared with the results of the previous modifications. The m.e.d. was less than 0.05 ug. androsterone per chick per day. The mean index of precision ([lambda]) was 0.34, with a standard deviation of 0.056. With 40 chicks on the standard and 20 on each unknown the expected minimum % standard error of a potency estimate was 24%. There was much less interassay error than in the first three modifications of the assay method.
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