STRESS-INDUCED RESPONSES IN NILE TILAPIA, OREOCHROMIS NILOTICUS AND AFRICAN CATFISH, CLARIAS GARIEPINUS FED AQUEOUS EXTRACT OF MORINGA OLEIFERA LEAF SUPPLEMENTED DIETS

Abstract

This study assessed the effects of aqueous extract from Moringa oleifera leaf on stress- induced physiological responses in Nile tilapia, Oreochromis niloticus and African catfish, Clarias gariepinus fingerlings with a view towards determining the appropriate dosage required during stressful periods. The leaves of Moringa oleifera were collected from a farm settlement at Ijare, Ondo State, Nigeria. Preliminary qualitative and quantitative screening of the major bioactive components present in the aqueous extracts of M. oleifera was carried out using Gas Chromatography – Mass Spectrometry (GC–MS) and analytical High Performance Liquid Chromatography (HPLC) respectively. Six isonitrogenous and isocaloric diets were formulated to meet the requirements of 30% crude protein for O. niloticus and 40% crude protein for C. gariepinus fingerlings using feed formulation software (WinFeed soft 2.0, USA). The graded levels of M. oleifera leaf extract were 0.00g (control), 0.05g, 0.10g, 0.15g, 0.20g, 0.25g per 100g for each diet denoted as MLST0, MLST5, MLST10, MLST15, MLST20, MLST25 for O. niloticus and MLSC0, MLSC5, MLSC10, MLSC15, MLSC20, MLSC25 for C. gariepinus in treatments 1, 2,3,4,5 and 6 respectively. Fish were graded by size and groups of 15 fish of 6.00 ± 0.02g per replicate for O. niloticus and 10.00 ± 0.10g for C. gariepinus and then stocked into glass tanks of 60cm ×45cm×45cm dimension in triplicate in a flow-through system. After six weeks of the feeding trial, fish previously fed with each experimental diet were exposed to pathogenic strain of Aeromonas hydrophila. After bath exposure, fish from each dietary treatment was placed into the glass tank. They were fed with their respective diets at 5% body weight twice daily, and mortality was monitored for the remaining 4 weeks of the feeding trial. At the end of the feeding trial, fish previously fed each experimental diet were kept in plastic tanks for a 2-hour journey. Two fish specimens from each tank were removed for blood analyses. Differential white blood cell count was performed to quantify the circulatory levels of lymphocytes, granulocytes and monocytes. Hepatocellular stress activities were determined by Aspartate transaminase (AST), Alanine transaminase (ALT), Lactate dehydrogenase (LDH) and Malate dehydrogenase (MDH) tests according to standard procedure. After terminal anaesthesia of the fish, tissue samples from the liver and intestines were removed by dissection for histological study. Tissue sections were compared after examination under the microscope for differences in the morphology of the tissues. Molecular expression (abundance and diversity) of stress protein genes (heat shock protein, HSP70) in experimental fish was assessed after hyperthermia- induced stress. This experiment was designed with a completely randomised design (CRD) to test for significant differences in the mean of treatments. Differences between mean of treatments were considered significant at P ≤ 0.05 by one way analysis of variance (ANOVA) using Statistica software. Follow–up procedures were performed where significant differences occurred in the means using Tukey test. Result from experiment 1 focused on stress – induced physiological responses in Nile tilapia fingerlings fed Moringa leaf supplemented diets. Statistically, there were significant differences (P< 0.05) in the growth performance and nutrient utilisation of O. niloticus. The best weight gain and FCR were recorded in fish fed MLST10 and poorest value obtained in the control. Mortality was recorded in all the dietary treatments. However, survival of fish significantly higher (P< 0.05) in fish fed MLST10 and MLST15 diets. Haematological analyses of O. niloticus fed the experimental diets showed that the packed cell volume and haemoglobin concentration significantly increased with increasing Moringa supplementation. The inclusion of 1.00g/kg dietary Moringa leaf supplementation led to a significant reduction in the content of physiological stress biomarkers like AST, ALT, LDH, MDH, cortisol and glucose (P< 0.05). Histological examination of the intestine and liver of fish in this study showed several histopathological alterations in fish fed the MLST0 (control) diets compared with those fed the Moringa leaf supplemented diets. Molecular analyses of O. niloticus liver with the aid of TGGE gel showed similar banding patterns in fish fed the Moringa leaf supplemented diets in MLST10, MLST15, and MLST20 treatments. Dietary effects were observed in the protective expression of the HSP70 genes, with M .Oleifera based diets showing more bands on the banding profile on the gel compared with the control. Results from experiment II focused on stress – induced physiological responses in African catfish fingerlings fed Moringa leaf supplemented diets. Growth performance and nutrient utilisation were significantly higher (P< 0.05) in C. gariepinus with increasing M. oleifera supplementation. Higher survival of fish was found in fish fed MLSC10 and MLSC15 diets as 98.00 and 95.60 % respectively. Haematological analyses showed that the PCV and haemoglobin concentration significantly increased with increasing Moringa supplementation (P< 0.05). Physiological stress biomarkers like glucose and cortisol significantly reduced with increasing Moringa supplementation. The histology of the intestine of fish fed MLSC10 and MLSC15 diets showed apparently normal intestinal villi and enterocytes. Histology of the liver of C. gariepinus fed the control diet had disorganised sinusoids and highly vacuolated cytoplasm. Molecular analyses of the HSP 70 genes showed marked similarity and upregulation of the protective expression of the HSP70 in the banding patterns of fish fed the Moringa leaf supplemented diets. In conclusion, the results of this study suggest that farmed fish species especially O. niloticus and C. gariepinus can be cultured on diets containing M. oleifera leaf as a potent dietary supplement during stressful periods. The present study also showed that the leaf of M. oleifera could be used as feed additive to reduce or ameliorate the effects of stress and improve the health of O. niloticus and C. gariepinus. Fish fed the control diet in this study developed clinical signs and negative physiological responses which affected their growth performance with induced stressors. Furthermore, the current study also revealed that M. oleifera leaf as dietary supplements helped to up-regulate the expression of the stress gene, Heat Shock Protein (HSP 70) in O. niloticus and C. gariepinus. Based on the result of this study, a dose of 1.00g/kg dietary Moringa leaf supplementation per kg diet with a corresponding content of 0.21 mg /g and 0.14 mg /g Quercetin (Q) and Kaempferol (K) were sufficient to prevent reduction in growth, mortality and up-regulate the expression of HSP genes in O. niloticus and C. gariepinus