6 FEMALE BITTERLING I 



Others are put in clean water. After 24 hours the ovipositors 

 have shortened to a length of from 2 to 4 A.U., and are most 

 ready to react. 



When it is desired to test a given preparation, it is advis- 

 able to get a number of the fishes, for control purposes, to 

 react to a standard preparation, say progesterone (5-8 y per 

 750 ml water). In this way it is possible to compare the 

 results of different days' tests, as the sensitivity of the fishes 

 fluctuates. 



The fishes may be used several times in succession, 

 providing their ovipositors have an initial length of between 

 2 and 4 A.U. for each test. Insufficient reaction to the 

 standard preparation gives warning that the fishes are to be 

 considered as finished with, and to be replaced by fresh ones. 



The preparations need not be injected; they should simply 

 be added to the water. Anterior-pituitary preparations, how- 

 ever, do not permeate the epithelium of the gills, and must 

 therefore be injected. 



It is usually sufficient for exact determination to obtain 

 an average reaction of 60 fishes. The activity of the pre- 

 parations is expressed in the curve of the (average) growth 

 of the ovipositors. The fishes should be properly fed, on a 

 diet for omnivorous fishes, since a great deal is expected of 

 them physiologically. 



It is essential that the aquaria should be properly and 

 constantly lighted, since pituitary activity is dependent on 

 light. The reaction of the ovipositor is favourably affected by 

 permanent electric light (20 — 25 % higher results). 



§ 2. THE SPECIFICITY OF THE OVIPOSITOR TEST 



We have mentioned that the female bitterling reacts to the 

 steroids of the estrane, pregnane, and androstane range. 

 Although the fishes are most sensitive to these, they also 

 react to various other substances of quite a different chemical 

 constitution. The specificity of the ovipositor test, therefore, 

 is such that one should always take into account what other 



