饲料和水环境中抗生素的长期暴露对斑马鱼生理功能的影响

发布时间：2018-05-01 20:18

本文选题：斑马鱼 + 抗生素生长代谢　；参考：《华东师范大学》2017年硕士论文

【摘要】：由于水环境中的抗生素残留,以及渔民把抗生素添加到饲料中作为预防疾病发生的药物而长期使用,导致了水生生物饱受着来自饲料和水环境中抗生素长期暴露的风险。水生动物如何应对这两种不同形式的抗生素暴露以及长期的抗生素暴露对水生生物有何影响,目前尚不明确。磺胺甲恶唑(Sulfamethoxazole,SMZ)和氧四环素(Oxytetracycline,OTC)是我国农业部允许的在饲料中添加使用的水产用抗生素,这两种抗生素在中国水环境中也普遍存在,根据农业部有关渔药使用的规定以及这两种抗生素在我国水环境中的平均浓度,本实验选取SMZ和OTC,通过药浴和饲料添加两种方式处理斑马鱼,从斑马鱼的生长和代谢、非特异性免疫与抗氧化性能、肠道菌群结构这几个方面探讨了饲料和水环境中抗生素的长期暴露对斑马鱼生理功能的影响。6周养殖结束后,和Control组相比,SMZ-diet组和OTC-diet组的斑马鱼的增重率(SGR)显著性升高、饲料系数(FCR)显著性降低,这意味着饲料中抗生素的长期暴露有助于斑马鱼的生长。进一步的研究显示,抗生素暴露组的肠道/体重的比值(IBW)出现了不同程度的下降,肠道淀粉酶(Amylase)、脂肪酶(Lipase)的活性相比于Control组得到了显著性的提升,肠道营养物质转运载体基因例如葡萄糖转运蛋白2(GLUT2)、三磷酸腺苷酶(ATPase)表达出现不同程度的上调,以上结果表明饲料中抗生素的长期暴露促进了斑马鱼的生长,这种促生长效应可能是通过以下途径来达到的:1)降低肠道重量。2)促进肠道淀粉酶以及肠道脂肪酶的活性。3)提高肠道营养物质转运载体基因例如葡萄糖转运蛋白2(GLUT2)、三磷酸腺苷酶(ATPase)基因的表达。斑马鱼全鱼耗氧率测定结果显示,和Control组相比,抗生素暴露组全鱼耗氧率出现不同程度的增加,其中抗生素药浴组(SMZ-bath、OTC-bath)全鱼的耗氧率要高于抗生素饲料组(SMZ-diet、OTC-diet),这表明斑马鱼通过提高新陈代谢速率来满足机体应对饲料和水环境中抗生素长期暴露下额外增加的能量需要。基于抗生素药浴组耗氧率高于抗生素饲料组,而抗生素药浴组生长性能低于抗生素饲料组,推测其可能的原因是:相比于饲料中抗生素的长期暴露,水环境中抗生素的长期暴露更加明显地改变了斑马鱼的能量分配方式,将更多的能量分配于应对抗生素的胁迫,相对地减少了分配于生长的能量,从而掩盖抗生素药浴处理组的促生长效应。体成分测定结果显示,抗生素的暴露对全鱼粗蛋白、糖原的含量无显著性的影响,仅OTC-bath组粗脂肪含量显著性下降。在肝脏组织中,和对照组相比,甘油三酯(TG)含量、总脂肪酸β氧化速率、PPARα、CPT-1、ACOX1)都有不同程度的下降,肌肉组织也表现出和肝脏类似的趋势。以上结果表明饲料和水环境中抗生素的长期暴露对斑马鱼脂代谢造成了影响,具体机制还有待进一步的研究。6周养殖结束后,利用嗜水气单胞菌浸浴感染斑马鱼,结果显示抗生素暴露组斑马鱼的死亡率显著性升高,这表明饲料和水环境中抗生素的长期暴露破坏了斑马鱼的免疫系统功能,降低机体对疾病的抵抗能力。在斑马鱼肠道和肝脏组织中,抗生素暴露组碱性磷酸酶(AKP)、酸性磷酸酶(ACP)活性出现了不同程度的下降。在肠道组织中,和Control组相比,抗生素暴露组中与炎症反应相关的基因例如肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)的表达出现不同程度的上调。SMZ-bath、OTC-bath组肠道杯状细胞的数量显著性低于Control组。在肠道、肌肉组织中,丙二醛(MDA)的含量不同程度的升高,谷胱甘肽(GSH)的含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性不同程度的降低。以上结果表明,饲料和水环境中抗生素的长期暴露破坏了肠道的屏障功能、引发了肠道炎症、造成了氧化损伤,肠道和肌肉是抗生素暴露造成氧化损伤的主要器官。肠道微生物高通量测序结果显示,抗生素暴露组肠道菌群丰度指数出现不同程度的下降,这表明抗生素的长期暴露降低了肠道菌群的丰度,主成分分析(PCA)、菌群热图(Heatmap)的结果显示,OTC-diet、OTC-bath 组与 Control组肠道菌群明显区分开来,表明抗生素OTC改变了斑马鱼的肠道菌群结构,而SMZ-diet、SMZ-bath组与Control组并没有很好的区分开来,这表明抗生素SMZ对肠道菌结构的影响没有OTC的大。本实验研究表明,在饲料中添加SMZ和OTC作为预防疾病发生的药物长期使用,虽然促进了斑马鱼的生长,但却降低了对疾病的抵抗能力,破坏了机体的抗氧化功能以及肠道微生态平衡。水环境中极低浓度(ng·L-1)的抗生素残留同样降低机体对疾病的抵抗能力,破坏了机体的抗氧化功能以及肠道微生态平衡。因此不宜将抗生素作为预防疾病发生的药物长期使用,并且不能忽视水环境中极低浓度的抗生素给水生动物的生理功能带来了负面效应。
[Abstract]:The long-term use of antibiotics in the water environment, as well as the long-term use of antibiotics added to the feed as a drug to prevent disease, has caused aquatic organisms to suffer from the risk of long-term exposure to antibiotics from feed and water environments. How do aquatic animals respond to these two different forms of antibiotic exposure and long-term resistance to antibiotics. The effects of raw vegetal exposure on aquatic organisms are not yet clear. Sulfonamoxazole (Sulfamethoxazole, SMZ) and Oxytetracycline (OTC) are the aquatic antibiotics allowed to be added to feed by the Ministry of agriculture in China. These two antibiotics are also prevalent in China's water environment, according to the regulations of the Ministry of Agriculture on the use of fishery drugs. The average concentration of the two antibiotics in the water environment of our country was determined. SMZ and OTC were selected in the experiment. The growth and metabolism of zebrafish, the non specific immunity and antioxidant properties, and the intestinal microflora structure were discussed in this experiment. The long-term exposure of antibiotics in the feed and water environment was discussed. The effect on the physiological function of zebrafish.6 week after culture, compared with group Control, the weight gain of zebrafish (SGR) in group SMZ-diet and OTC-diet group increased significantly, and the feed coefficient (FCR) decreased significantly. This means that the long-term exposure of antibiotics in the feed is helpful to the growth of zebra fish. Further studies show that the intestine of the antibiotic exposure group The ratio of channel / weight (IBW) decreased in varying degrees. The activity of intestinal amylase (Amylase) and lipase (Lipase) was significantly enhanced compared to the Control group. The intestinal nutrient transporter gene, such as glucose transporter 2 (GLUT2), and the expression of three adenosine adenosine (ATPase), was up-regulated in varying degrees, and the above results were found. The long-term exposure to antibiotics in the feed promotes the growth of zebrafish. This growth effect may be achieved through the following pathways: 1) reducing the intestinal weight.2) and promoting the activity of intestinal amylase and intestinal lipase activity.3) to improve the intestinal nutrient transporter basis, such as glucose transporter 2 (GLUT2), adenosine triphosphate The expression of ATPase gene. The oxygen consumption rate of all fish in zebrafish fish showed that compared with the Control group, the total fish oxygen consumption rate in the antibiotic exposed group increased in varying degrees, and the oxygen consumption rate of the whole fish in the antibiotic bath group (SMZ-bath, OTC-bath) was higher than that of the antibiotic feed group (SMZ-diet, OTC-diet), which indicated that the zebrafish was improved by the new age. The growth performance of the antibiotic bath group is lower than that of the antibiotic feed group, and the possible reason for the growth performance of the antibiotic bath group is lower than the long-term exposure to antibiotics in the feed and the water environment. The long-term exposure to antibiotics significantly changed the energy distribution pattern of zebrafish, allocated more energy to the stress response to antibiotics, reduced the energy allocated to the growth, and masked the growth promoting effect of the antibiotic bath treatment group. The content of glycogen was not significantly affected, and the content of crude fat decreased significantly in the OTC-bath group. In the liver tissue, the content of triglyceride (TG), the rate of total fatty acid beta oxidation, the PPAR alpha, the CPT-1, ACOX1 in the liver tissue decreased in varying degrees, and the muscle tissue showed a similar trend to the liver. The results showed that the feed and water ring were found. The long-term exposure to antibiotics in the border has affected the lipid metabolism of zebrafish. The specific mechanism remains to be further studied. After.6 weeks, the use of Aeromonas hydrophila to infect zebra fish shows a significant increase in the mortality of zebrafish in the antibiotic exposure group, indicating the long-term exposure of antibiotics in the feed and water environment. The immune system of zebrafish is damaged and the body's resistance to disease is reduced. In the intestinal and liver tissues of zebrafish, the alkaline phosphatase (AKP), acid phosphatase (ACP) activity of the antibiotic exposure group decreases in varying degrees. In the intestinal tissue, the gene related to the inflammatory response in the antibiotic exposure group is compared with the Control group. For example, tumor necrosis factor - alpha (TNF- alpha), the expression of interleukin -1 (IL-1) in different degrees of up regulation of.SMZ-bath, the number of intestinal goblet cells in the OTC-bath group was significantly lower than that in the Control group. In the intestine, the muscle tissue, the content of malondialdehyde (MDA) in different degrees, the content of glutathione (GSH), superoxide dismutase (SOD), and superoxide dismutase (SOD). The activity of oxide enzyme (POD) decreases in varying degrees. The above results show that the long-term exposure of antibiotics in feed and water environment destroys the intestinal barrier function, causes intestinal inflammation, causes oxidative damage, and the intestine and muscles are the main organs of oxidative damage caused by antibiotic exposure. The intestinal flora abundance index of the probiotics exposed group decreased to varying degrees, which indicated that the long-term exposure of the antibiotics decreased the abundance of intestinal flora, principal component analysis (PCA), and the results of bacterial group thermogram (Heatmap) showed that OTC-diet, OTC-bath group and Control group were distinguished from the intestinal flora, indicating that the antibiotic OTC changed the zebrafish intestines. The SMZ-diet, SMZ-bath and Control groups were not well differentiated, which showed that the effect of antibiotic SMZ on the structure of intestinal bacteria was not OTC. This experimental study showed that the addition of SMZ and OTC to the long-term use of drugs to prevent disease occurred, although it promoted the growth of zebrafish, but reduced the disease to the disease. The resistance of the disease has destroyed the body's antioxidant function and the intestinal microecological balance. The very low concentration of antibiotics in the water environment (ng. L-1) also reduces the body's resistance to the disease, destroys the body's antioxidant function and the intestinal microecological balance. Therefore, antibiotics should not be used as a drug to prevent the disease from occurring for a long time. Use, and can not ignore the extremely low concentration of antibiotics in aquatic environment has negative effects on the physiological functions of aquatic animals.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

【参考文献】