The Effects Of Long-term Exposure To Antibiotics In Feed And Water Environment On The Physiological Function Of Zebrafish

For the reason that antibiotic residues in water environment,and fish farmers use antibiotics in diet for long time,aquatic organisms face long-term risk of exposure to antibiotics in diet and water environment.The response of aquatic animals to these two different forms of antibiotics exposure has not been sufficiently considered.Sulfamethoxazole(SMZ)and tetracycline(OTC)are permitted by the ministry of agriculture for the use of antibiotics in feed,the detection rate of these two kinds of antibiotics is also very high in the water environment.According to the provisions of the ministry of agriculture on the use of fishery drugs and the average concentration of these two kinds of antibiotics in China’s water environment,zebrafish were exposed to SMZ and OTC added in diets or in water.The effects of long-term exposure of antibiotics in feed and water environment on the physiological function of zebrafish were explored by using the aspects of growth and metabolism,non-specific immunity,antioxidant properties,and intestinal flora structure.After 6 weeks of exposure,compare of the control group,SMZ-diet group,SMZ-bath group,OTC-diet group,OTC-bath group of zebrafish growth and metabolism,immunity and anti-oxidation performance,intestinal microflora.we find that the weight gain rates of zebrafish in SMZ-diet and OTC-diet groups were significantly increased,and the feed conversion ratio were significantly decreased,indicating that the long-term exposure of antibiotics in the diet promoted the growth of zebrafish.Furthermore the ratio of intestinal weight/body weight(IBW)in the antibiotics exposure groups decreased at varying levels.Intestinal digestive enzymes such as amylase,lipase activity were significantly increased in zebrafish expose to antibiotics.The expression of intestinal nutrient transporter genes such as glucose transporter 2(GLUT2)and ATPase were up-regulated in the antibiotics treated zebrafish.These results indicate that the long-term exposure of antibiotics in diet promotes the growth of zebrafish,which is achieved by reducing intestinal weight,improving digestive enzyme activity,and upregulating the expression of intestinal nutrient transporter genes.Genreally,the oxygen consumption rate of zebrafish exposed to antibiotics increased than the control group.Zebrafish exposed to SMZ-bath and OTC-bath groups had higher oxygen consumption rate than those fed on SMZ-diet and OTC-diet.These results show that zebrafish increased the metabolic rate to meet the additional energy requirement for long-term exposure to antibiotics in the diet and water environment.However,the long-term exposure to antibiotics in the water environment induces more changes in energy distribution of zebrafish than antibiotics in diets.Thus,zebrafish in the bath treatments allocates more energy to the stress of antibiotics which relatively reduces the energy allocated to growth,thus masking the growth promoting effect in SMZ-Bath,OTC-bath group.The results on body composition showed that the exposure of antibiotics had no significant effect on the content of crude protein and glycogen in whole fish.However,the crude fat content was significantly decreased in OTC-bath group.In liver and muscle tissues,the content of triglyceride(TG),total β-oxidation rate and lipid metabolism-related genes(PPARα,CPT-1,ACOX1)were decreased at different levels.These results indicate that the long-term exposure to antibiotics in feed and water environment affect the lipid metabolism of zebrafish,and the specific mechanism needs further study.After 6 weeks of exposure,zebrafish were infected with Aeromonas hydrophila via immersion bath.The results of bacteria challenge test showed that the mortality of zebrafish was significantly increased in the antibiotic exposure group,which indicated that the long-term exposure of antibiotics in feed and water environment destroyed the immune system function of zebrafish and reduced the resistance to disease.Moreover,the results showed that alkline phosphatase(AKP),acid phosphatase(ACP)activities were decreased in intestinal and liver tissues.While the expression of tumour necrosises factor(TNF-a)and interleukin-1(IL-1)genes as indicators of inflammation were increased in intestinal.Similary,the number of intestinal goblet cells were decreased in bath groups.The malondialdehyde(MDA)content was increased whereas glutathione(GSH)content,and superoxide dismutase(SOD),peroxidase(POD)activities decreased in intestinal and muscle tissues in zebrafish exposed to antibiotics.These results suggest that long-term exposure to antibiotics in the diet and water environment led to intestinal inflammation and oxidative damage in zebrafish intestinal and muscle.The results of high throughput sequencing showed that the bacteria abundances were decreased in antibiotics groups.which indicated that the long-term exposure of antibiotics reduced the abundance of intestinal flora.The results of principal component analysis(PCA)and Heatmap showed that OTC-diet,OTC-bath were clearly distinguished from the Control group,while SMZ-diet,SMZ-bath and Control group did not distinguish well.This suggests that antibiotic OTC treatment alters the structure of the gut microbiota more seriously in zebrafish than SMZ treatment.The results of this study showed that SMZ and OTC were added to feed to prevent disease,although it promoted the growth of zebrafish,but it destroyed the immune function,antioxidation function and intestinal microecological balance.Similarly,long term exposure to antibiotics in the aquatic environment also disrupts the immune and antioxidant functions of the zebrafish,as well as the intestinal microecological balance.Therefore,antibiotics should not be used as a drug to prevent the occurrence of disease,and can not be ignored the negative effect of antibiotics in the water environment at very low concentration on aquatic animals physiological function.