Ahmed Mustafa

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.

Primary Stress Responses of Common Carp, Cyprinus carpio exposed to copper toxicity

A wide range of chemicals has been shown to cause endocrine disturbances in living organisms. Under an environmental stress, organisms are known to exhibit primary response by way of changes in hormone levels, which in turn are known to stimulate the secondary responses including physiological and biochemical changes. The homeostasis of an animal is disturbed as a result of the actions of intrinsic and/or extrinsic stimuli or stressors (Wendelaar Bonga 1997). Among stressors, heavy metals are of greater importance because of their long biological half-lives, which pose a real threat to aquatic organisms, especially fish. In sub-acute concentrations, heavy metals gradually accumulate in various organisms, as they reach higher trophic levels of the food chain but at high concentrations heavy metal are lethal to aquatic organisms.Among the heavy metals, copper has been regarded as strongly toxic to fish and other aquatic animals (Sloman 2003). Copper, at high concentrations, causes disturbances in sodiumand chloride homeostasis and alters nitrogenous metabolites in aquatic animals (Bury et al. 2003, Sloman 2004). However, little is known about the effects of copper on stress modulation. ACTA ICHTHYOLOGICA ET PISCATORIA (2007) 37 (2): 81–85 DOI: 10.3750/AIP2007.37.2.02

Effects of Puerarin on the Reduction of Glucose and Promotion of Overall Health in Acutely Stressed Chinook Salmon, Oncorhynchus Tshawytscha (Actinopterygii: Salmoniformes: Salmonidae)

Background. Farmed fish reared in aquaculture systems are exposed to physiological stressors from biological, chemical, and physical sources. The consequences of these stressors affect the productivity of farming outcomes by reducing immune response, growth, and development within fish. A 24-h study was performed to determine the effects of supplementing puerarin—the most biologically active ingredient present in the roots of kudzu (Pueraria lobata )—within farmed fish on acute handling stress. Materials and methods. Fingerlings of Chinook salmon, Oncorhynchus tshawytscha , were maintained at 16‐18oC in 10 US gallon (37.8 L) glass tanks, supplied with recirculated and aerated dechlorinated water. Puerarin was given to fish through supplementation to commercial fish feed pellets. Acute handling stress was induced through transferring the fish from their housing tanks into 8 designated tanks at 0000 h. Subsequently, at 5 h post-transfer all the fish were fed to satiation, following which the stressed groups were chased with nets. Results. The results indicated that administration of puerarin through feed supplementation reduces plasma glu cose, hematocrit, plasma protein, and improves spleen somatic indices after 24 h, and thereby counteracting the negative consequences of acute stress induced through handling . Conclusion. The results suggest that supplementation of puerarin to commercial fish feed improved overall fish health through enhancing systemic circulation while attenuating plasma glucose. Being one of the first studies to study the effects of puerarin on fish, our results are in agreement with prior research with puerarin on other ani mal models .

Effects of omega-3 fatty acid supplementation on aquaponics system-raised tilapia (Oreochromis niloticus X Oreochromis aureus) growth, physiology, and immunology

ABSTRACT This study evaluates the potential health effects of omega-3 fatty acid supplementation on tilapia physiology and immunology. Tilapia were fed diets supplemented with linseed oil (LO; high in omega-3s), sunflower oil (SO; low in omega-3s) or no extra oil (control) for 12 weeks. Blood glucose was significantly reduced in LO and SO over time and significantly lower than the control. Packed cell volume and spleen somatic index were significantly reduced in LO and SO over time but not different from the control. Plasma protein was significantly higher than the control for both LO and SO. Phagocytosis was increased only in LO and was significantly higher than SO. Higher dietary lipid levels confer some health benefits, and omega-3 supplementation enhances phagocytic activity.

Puerarin Promotes Overall Health Through Blood Flow and Immune Response within Chinook Salmon (Oncorhynchus tshawytscha) Fingerlings

An 8-week study was performed to determine the effects of supplementing puerarin, the most biologically active ingredient present in the roots of kudzu (Puerarin lobata), in diets of farmed chinook salmon (Oncorhynchus tshawytscha) fingerlings. Puerarin was given to study fish through supplementation to commercial fish feed. Administration of puerarin reduced cortisol, proteins, and hematocrit levels while improving spleen somatic indices and respiratory burst response compared to fish fed a control diet without affecting growth, and thereby counteracts the negative consequences of stress induced through handling and experienced by fish reared in aquaculture.

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.