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Background. To assess stress levels in fishes, plasma cortisol levels are measured by radioimmuno assay and phagocytic activity is assessed using macrophages. However, the small size of some fishes makes it difficult to measure stress using these physiological and immunological indicators. In this study, we investigated the possibility of obtaining macrophages from zebrafish via whole body extractions by assessing the respiratory burst activity and phagocytic capacity of extracted cells and we studied the effects of temperature stress on zebrafish using the extracted macrophages. Materials and Methods. One hundred and fifty genetically pure zebrafish, Danio rerio (Hamilton, 1822), were randomly divided into three groups and placed in three different environments: optimal (28°C), warm (32°C), and cool (23°C). Using the newly developed extraction method described in this article macrophages were extracted from whole fish bodies and the phagocytic activity of these cells were assessed. Results. The method yielded enough macrophages to examine their respiratory burst activity and phagocytic capacity. Values obtained for experimental replicates were similar and the assessment measures were sensitive enough to detect differences in these parameters among fish maintained at three different temperatures for 2, 4, 6, and 8 weeks. These results suggest that macrophages can be successfully extracted using the whole body method and the extracted macrophages are useful for studying stress. Conclusion. The method of macrophage extraction described in this article is simple and rapid, and will enable researchers to study the effects of any stressors, environmental or pathogenic, on the non-specific immune response of fish.
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 glucose, 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 animal models.
Background. Arctic charr, Salvelinus alpinus (L.), are known to harbour significantly more sea lice, Caligus elongatus von Nordmann, 1832, than do Atlantic salmon, Salmo salar L. This research investigated whether this is due to differences in initial infection or to resettlement/loss of mobile adults. Skin mucus protein profiles and epidermal histology were also studied as two characteristics that might help explain interspecific differences in host susceptibility. Materials and Methods. Atlantic salmon and Arctic charr were sampled from the sea-pen after 12 weeks of exposure to natural infection. Fishes were examined for sea lice counts and mucus was analysed for protein profiles using standard methods of poly-acrylamide gel electrophoresis. Epidermal tissue of fishes around chalimus attachment sites was also examined by transmission electron microscopy. Results. Interspecific differences in host susceptibility were determined not to be due to differences in initial infection rate, as charr and salmon had similar numbers of attached chalimi. Adult parasites were significantly more numerous on charr as a result of resettlement of adults lost by other fishes in the area. Gel electrophoresis indicated that salmon had a greater abundance of skin mucus proteins than charr, particularly in the 30–42 and 67–94 kDa range. Ultrastructural analysis of epidermal tissue indicated that charr had more mucous cells and showed evidence of possible osmotic stress. Interspecific differences also existed in intercellular adhesion characteristics. Salmon and charr showed a similar absence of inflammation around chalimus attachment sites. Conclusion. Artic charr had a more intense infection with Caligus elongatus than did salmon corresponding to fewer potentially antagonistic proteins in the mucus. Perhaps also, the epidermis of charr provides more easily accessible food for the sea lice. Higher levels of stress in charr in sea water may also have predisposed them to higher sea lice infections.
Background. Copper is a heavy metal, and an aquatic pollutant, known for its bio-accumulative and non-biodegradable properties. In the aquatic ecosystems, acute and sublethal concentrations of copper may be linked to a variety of effects. Recently, hormones, particularly those regulating vital functions, such as osmoregulation, energy metabolism, and reproduction, may be used as potential biomarkers for sublethal toxicity studies. In the present study, the potential effect of a heavy metal—copper on hormonal changes (cortisol and prolactin) in an economically important fresh-water fish—common carp, Cyprinus carpio, was examined. Materials and Methods. The experimental fish were subjected to two experimental regimes (backed by controls). In the first treatment they were exposed to the acute concentration of copper sulphate, amounting to 0.7 ppm. The second treatment featured copper sulphate concentration of 0.07 ppm, constituting 10% of LC50 (24 h). The acute-toxicity trials were carried out in two, 20-L, circular plastic tubs. Twenty fish from the tank were selected randomly and introduced to each tub. Control was maintained in 2 similar plastic tubs with 20 fish per tub. After 24 h, fish from control- and copper-exposure tubs were taken for analyses. To observe the sublethal toxicity four, 125-L, glass aquaria, filled with clean water were used. 200 fish were randomly selected from the stock and 100 of them were added to two aquaria, 50 fish in each, as experimental fish and 100 in two other aquaria, 50 in each, for as control fish. By the end of the stipulated period, 20 fish from control and 20 fish from experimental group were used for the hormone assay. Results. In both acute- and sublethal treatments, both cortisol- and prolactin levels increased. In sublethal treatment, however, plasma prolactin level decreased after 28-day exposure, showing a minimum percentage point decrease of 3.84 by the end of 35-day trial. Conclusion. The increase of the plasma cortisol was probably caused by release of cortisol from the interrenal tissue, as a mechanism of coping with stress. Significantly lower content of prolactin levels in sublethal treatment could be an indicative of a possible restored hydromineral balance or atrophy of the pituitary prolactin cells leading to inhibited prolactin secretion of the fish. These alterations of the above hormonal changes may be used as stress biomarkers in fish.
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