Further Experiences with the Male-toad Pregnancy Test Using European Species

Abstract

The satisfactory reports on the Galli Mainini male toad test for pregnancy which have been published from nearly every part of the world (Cowie, 1948 ; Bhaduri, 1951) show that the test can be performed on species of toads or frogs indigenous to the district where the laboratory is situated. In England and Europe the toads Bufo bufo (syn. B. vulgaris) and B. calamita and the frog Rana esculenta are used for the test. Favourable results with these species have been reported by Hinglais and Hinglais (1948), Haines and Ferreira (1949), Klopper and Frank (1949), Frazer andWohlzogen (1949), andBach, Szmuk, Robert, andKlinger (1949). In a later paper Frazer and Wohlzogen (1951) described a method for estimating urinary gonadotrophin using B. bufo. Modifications of the Earlier TechniqueThe principle and the technique of the test which have already been described for a South American species (Haines, 1948) are readily adapted to European species and the technical details of modifications in procedure which we found necessary for the new species have been described by Law (1949).The toads available weighed only 15-25 g. and the volume of urine injected was reduced to 2 ml., i.e. 1 ml. per 10 g. body weight. Because there was an injection mortality of up to 40% a modified purification method was tried (Scott, 1940) Reading of the TestThe end-point is taken as the appearance of spermatozoa in a specimen of toad's urine usually collected from the cloaca. Spermatozoa may be found in less than one hour after injection. Our tests were read at the end of three hours-or four if negative at three. Under circumstances where a weak positive response was to be expected (see below) readings were taken at five and six hours. A weak positive is denoted by a delayed response of the test animal and by the presence of relatively few sperms per low-power field. Increasing numbers of sperms may be found at the end of the fourth and fifth hours when only two or three per field were found at the end of the third hour. In our experience few sperms per field, in the absence of a delayed response, indicate exhaustion of the test animal. The urine of positive reactors was found clear of spermatozoa within two days of the injection. All animals used in the series were tested (and found negative) before a test was begun.