Evaluation of dorsal aorta cannulation for immunological studies of grouper (Epinephelus malabaricus).

Blood is often withdrawn to study the immune responses of fish. However, netting, handling and anaesthetising the experimental fish, and drawing blood samples cause severe stress that may alter the effects of immune study protocols and treatments. We evaluated the effect of aorta cannulation, for use in immune studies, on grouper (Epinephelus malabaricus) plasma cortisol, total red and white blood cell counts and phagocytosis. Plasma cortisol increased from 30 to 88 ng/ml 1 day after insertion of the cannula, to a maximum of 951 ng/ml 3 to 5 days after surgery, indicating the groupers were stressed by cannulation and post-cannulation inflammation. Total RBC count decreased, and total WBC count increased after surgery. Following cannulation, the phagocytic index of peripheral blood leukocytes decreased from 100% to 46%. The adverse effects of cannulation were mitigated by continuously immersing groupers in oxytetracycline (OTC), which decreased the recovery period for treated fish. In contrast, OTC-treatment did not markedly improve the recovery of groupers subjected to caudal vessel puncture. Cortisol levels in OTC-treated grouper with caudal vessel puncture were significantly higher than in OTC-treated, cannulated grouper, and remained at a high level until day 13 of the experiment. From day 7 to 13, total RBC and WBC counts in OTC-treated, cannulated groupers were significantly different from those in OTC-treated groupers with caudal vessel puncture. OTC treatment improved the phagocytic index of groupers subjected to caudal vessel puncture, but the phagocytic index was lower than that of groupers subjected to cannulation. Cannulation minimises visual and handling disturbances, and facilitates standardisation of experimental conditions and quick and easy sampling via the dorsal aorta cannula. Therefore, dorsal aorta cannulation minimises the stress of blood sampling and should prove useful for immune studies in fish.