Establishment of a non-invasive method for stress evaluation in farmed salmon based on direct fecal corticoid metabolites measurement.

Fish welfare is an important issue for growth of the aquaculture industry. Stress responses represent animal's natural reactions to challenging conditions and could be used as a welfare indicator. Cortisol level is relevant to fish welfare condition, and is a readily measured component of the primary stress response system. Generally, cortisol is measured by blood sampling. However, fish blood cortisol level could be instantly influenced by handling-stress at sampling. Fecal corticoid metabolites (FCM) are a mixture of several different metabolites with a wide range of polarities. Thus, feces could be promising alternative less handling-sensitive and non-invasive biological matrices for cortisol evaluation in Atlantic salmon. In this study we developed non-invasive method for determination of fecal corticoid metabolites in farmed Atlantic salmon (Salmo salar L.) using enzyme-linked immunosorbent assay (ELISA). It was demonstrated that salmon FCM extracted from salmon feces is insoluble in non-polar solvents like diethyl ether, but well soluble in polar solvents like methanol. The proper extraction ratio could be one ml 100% methanol for 100 μL of the liquid part of salmon feces or 100 mg of the solid part. The FCM directly detected in unextracted liquid part of feces correlated well with the FCM extracted from both liquid and solid part of the corresponding samples, without significant difference. Thus, it is feasible to measure FCM directly in the liquid part of salmon feces without any extraction procedure. Then, we applied this assay for FCM analysis in the group of salmon that experienced salmon pancreas disease (PD) and amoebic gill disease (AGD). We demonstrated 1) both plasma cortisol and FCM increased significantly during the outbreak of inflammatory disease (P < 0.01). Plasma cortisol level was elevated from 28 ± 40 ng/ml to 164.4 ± 62.5 ng/ml, FCM from 14.4 ± 13.2 ng/ml to 170.7 ± 89.7 ng/ml 2) Growth and starvation has no significant impact on either cortisol or FCM level. 3) FCM correlated well with plasma cortisol level (P < 0.01). Furthermore, there seems more individual variation in plasma cortisol levels than in FCM levels. These results suggest FCM could be directly analyzed in liquid part of salmon feces without extraction. This directly detected FCM level could represent the total fecal FCM level and plasma cortisol level. This simple and non-invasive method makes FCM a proper indicator for salmon welfare.