Holding water steroid hormones in the African cichlid fish Pseudocrenilabrus multicolor victoriae

Measuring hormone levels multiple times on the same individual across different life stages or treatments can facilitate our understanding of hormonal regulation of physiological and behavioral events. The conventional method of hormone measurement requires blood sampling, which is potentially lethal to small individuals. In fishes, there is an alternative non-invasive method of hormone measurement using the release of hormones across gill membranes from blood into holding water. Validation of this method is required to evaluate its application value to different species. In the present study we used the maternal mouth-brooding African cichlid fish, Pseudocrenilabrus multicolor victoriae to (i) investigate whether handling involved in using the holding water technique is a stressor by measuring excreted cortisol in male and female P. multicolor handled one or multiple times, (ii) validate use of this technique by quantifying the relationship between plasma and holding water measures of sex hormones in male P. multicolor, and (iii) demonstrate the biological relevance of this technique using excreted levels of sex hormones in female P. multicolor across different reproductive stages. Excreted cortisol and estradiol levels did not differ between fish handled one or more times, suggesting that the repeated sampling approach over the breeding cycle that we propose to use does not affect the excreted level of the hormone of interest. Measurements from plasma and holding water samples were positively related for both testosterone and estradiol, indicating that the holding water technique is a reliable index of plasma hormone levels. Excreted sex hormone levels varied with reproductive state, suggesting that the technique is a useful, non-invasive measure of sex hormone levels in P. multicolor.