WISH analysis of the opioid nature of the β-casomorphins. (A) WT zebrafish embryos were pre-treated with 10 μM naloxone for 2 h to block the μ-opioid receptors and exposed to naloxone and 50 µg/ml of bBCM-5, bBCM-7, hBCM-5 and hBCM-7 every 24 h from 3–6 dpf. (Black arrowheads represent the insulin domain of expression). (B) Statistical analysis of the insulin domain of expression using Student's t-test. Fold change (compared to untreated control zebrafish embryos) shows insulin domain of expression significantly decreased following bBCM-5, -7 exposure and significantly increased following hBCM-5, -7 exposure. WT embryos treated with µ-opioid receptor antagonist, naloxone and bBCM-5, -7 and hBCM-5, -7 showed no changes in the insulin domain of expression compared to untreated controls. Data represent mean±s.e.m.; *P<0.05, **P<0.005; Student's t-test. (n=20).