Shree Dhawale

Development of a method for extracting macrophages from zebrafish, Danio rerio and their use to assess stress

Macrophages are phagocytic cells that play an important role in both specific and non-specific immune responses. These cells are capable of killing a wide range of pathogens by engulfing them and producing reactive superoxide species (ROS) (Secombes 1996, Babior 2000). These functions are indicators of the non-specific immune response and the general health of the organism, and can be measured by the visible presence of engulfed bacteria and respiratory burst activity. Assays for the measurement of these macrophage functions are widely used for evaluating immune responses in many vertebrates (Pick and Mizel 1981, Ciapetti et al. 1998, Voie et al. 1998) as well as in fish (Secombes 1990, Secombes and Fletcher 1992). In such studies macrophage cells in fish are obtained and isolated from the head kidney (Mustafa et al. 2000, Brubacher and Bols 2001, Regala et al. 2001, Reynaud et al. 2001), maintained in cell culture, and then checked for the amount of ROS produced during the respiratory burst activity and the number of phagocytized bacteria. Zebrafish, Danio rerio (Hamilton, 1822), are small cyprinid fish that are widely used as genetic models for studying vertebrate development (Haffter and NussleinVolhard 1996, Dooley and Zon 2000). These fish grow optimally between 25oC and 31oC, and exhibit exceptional fecundity (Sanders et al. 2003). Although, zebrafish are very hardy and tolerant to a wide range of temperatures, there are claims that they do not do well at the upper end of the temperature range mentioned above. They also undergo stress due to frequent handling during production, grading, and distribution, as well as due to other environmental stressors, and they may become vulnerable ACTA ICHTHYOLOGICA ET PISCATORIA (2008) 38 (1): 73–77 DOI: 10.3750/AIP2008.38.1.11

Effect of Phosphatidylcholine and Beta-Carotene Supplementation on Growth and Immune Response of Nile Tilapia, Oreochromis niloticus, in Cool Water

The effects of nutraceuticals in improving growth and immune response of Nile tilapia in cool water were investigated. Fish were reared in two different environments: warm water (28°C) and cool water (16°C). Fish reared in warm water (control) were provided with a basal commercial diet, while fish reared in cool water were provided with either the basal commercial diet (cool water control) or diets supplemented with nutraceuticals (either phosphatidylcholine or β-carotene). Experiments were conducted over an 8-week period. Fish held in warm water had significantly higher growth (p < 0.05), but condition factor, blood hematocrit, plasma glucose, and phagocytic capacity of macrophage cells were similar to fish held in cool water. Within the cool water groups (basal vs. supplemented diet), fish did not show any significant difference in condition factor, blood hematocrit, and phagocytic capacity of macrophage cells. Furthermore, there were no significant differences in glucose levels until the eighth week. Collectively these data indicate that a nutraceutical supplementation to the basal diet was not significantly beneficial. Despite slower growth, fish reared in cool water remained generally healthy. The observation that phagocytic capacity of macrophage cells was not significantly different suggests that disease resistance of tilapia grown in cool water may be comparable to those reared in warm water. Thus, our experiments suggest the feasibility of stocking or rearing tilapia in water temperatures as low as 16°C during cool weather, without need for supplementation to basal commercial feed.

The Validation of Effect of Neutraceuticals on Growth and Immune Response of Nile Tilapia in Cool Water Using a Cluster-Based Approach

This interdisciplinary study investigated computational analytic methods used for biological hypothesis testing and applied the methods for the validation of the effects of nutraceuticals on growth and immune response of Nile tilapia in cool water. Farmers in cooler regions face problems with cultivating tilapia, one of the most popular cultivated fish species, due to poor survival rates at suboptimal temperatures. We hypothesized that two nutraceuticals, phosphatidylcholine and β-carotene, help tilapia adapt to cooler water temperatures and benefit tilapias growth and immune response. Fish were reared in two different environments: warm water (28°C) and cool water (16°C). Fish reared in warm water (control) were provided with a basal commercial diet, while fish reared in cool water were provided with either the basal commercial diet (cool water control) or diets supplemented with nutraceuticals (either phosphatidylcholine or β-carotene). Experiments were conducted over an eight-week period. The effects of the nutraceuticals were tested using an unsupervised learning technique in data mining and statistics called cluster analysis. An external index used for cluster validation was adopted for testing our hypothesis by formulating the level of agreement between two different partitions of samples: experimental groups and clusters based on the similarity of their features. Contrary to the findings of previous studies, which showed the beneficial effects of phosphatidylcholine and β-carotene supplementation in a range of fish including tilapias, our test results show no significant difference among the fish reared in cool water and fed with either the basal diet or diets supplemented with the nutraceutical. Despite slower growth compared with fish held in warm waters, our study suggests the feasibility of stocking or rearing tilapia in water temperatures as low as 16°C during cool weather, without need for supplementation to basal commercial feed.