本文選題：斑馬魚 + 抗生素生長代謝��； 參考：《華東師范大學(xué)》2017年碩士論文

【摘要】：由于水環(huán)境中的抗生素殘留,以及漁民把抗生素添加到飼料中作為預(yù)防疾病發(fā)生的藥物而長期使用,導(dǎo)致了水生生物飽受著來自飼料和水環(huán)境中抗生素長期暴露的風(fēng)險(xiǎn)。水生動(dòng)物如何應(yīng)對(duì)這兩種不同形式的抗生素暴露以及長期的抗生素暴露對(duì)水生生物有何影響,目前尚不明確�；前芳讗哼�(Sulfamethoxazole,SMZ)和氧四環(huán)素(Oxytetracycline,OTC)是我國農(nóng)業(yè)部允許的在飼料中添加使用的水產(chǎn)用抗生素,這兩種抗生素在中國水環(huán)境中也普遍存在,根據(jù)農(nóng)業(yè)部有關(guān)漁藥使用的規(guī)定以及這兩種抗生素在我國水環(huán)境中的平均濃度,本實(shí)驗(yàn)選取SMZ和OTC,通過藥浴和飼料添加兩種方式處理斑馬魚,從斑馬魚的生長和代謝、非特異性免疫與抗氧化性能、腸道菌群結(jié)構(gòu)這幾個(gè)方面探討了飼料和水環(huán)境中抗生素的長期暴露對(duì)斑馬魚生理功能的影響。6周養(yǎng)殖結(jié)束后,和Control組相比,SMZ-diet組和OTC-diet組的斑馬魚的增重率(SGR)顯著性升高、飼料系數(shù)(FCR)顯著性降低,這意味著飼料中抗生素的長期暴露有助于斑馬魚的生長。進(jìn)一步的研究顯示,抗生素暴露組的腸道/體重的比值(IBW)出現(xiàn)了不同程度的下降,腸道淀粉酶(Amylase)、脂肪酶(Lipase)的活性相比于Control組得到了顯著性的提升,腸道營養(yǎng)物質(zhì)轉(zhuǎn)運(yùn)載體基因例如葡萄糖轉(zhuǎn)運(yùn)蛋白2(GLUT2)、三磷酸腺苷酶(ATPase)表達(dá)出現(xiàn)不同程度的上調(diào),以上結(jié)果表明飼料中抗生素的長期暴露促進(jìn)了斑馬魚的生長,這種促生長效應(yīng)可能是通過以下途徑來達(dá)到的:1)降低腸道重量。2)促進(jìn)腸道淀粉酶以及腸道脂肪酶的活性。3)提高腸道營養(yǎng)物質(zhì)轉(zhuǎn)運(yùn)載體基因例如葡萄糖轉(zhuǎn)運(yùn)蛋白2(GLUT2)、三磷酸腺苷酶(ATPase)基因的表達(dá)。斑馬魚全魚耗氧率測(cè)定結(jié)果顯示,和Control組相比,抗生素暴露組全魚耗氧率出現(xiàn)不同程度的增加,其中抗生素藥浴組(SMZ-bath、OTC-bath)全魚的耗氧率要高于抗生素飼料組(SMZ-diet、OTC-diet),這表明斑馬魚通過提高新陳代謝速率來滿足機(jī)體應(yīng)對(duì)飼料和水環(huán)境中抗生素長期暴露下額外增加的能量需要�；诳股厮幵〗M耗氧率高于抗生素飼料組,而抗生素藥浴組生長性能低于抗生素飼料組,推測(cè)其可能的原因是:相比于飼料中抗生素的長期暴露,水環(huán)境中抗生素的長期暴露更加明顯地改變了斑馬魚的能量分配方式,將更多的能量分配于應(yīng)對(duì)抗生素的脅迫,相對(duì)地減少了分配于生長的能量,從而掩蓋抗生素藥浴處理組的促生長效應(yīng)。體成分測(cè)定結(jié)果顯示,抗生素的暴露對(duì)全魚粗蛋白、糖原的含量無顯著性的影響,僅OTC-bath組粗脂肪含量顯著性下降。在肝臟組織中,和對(duì)照組相比,甘油三酯(TG)含量、總脂肪酸β氧化速率、PPARα、CPT-1、ACOX1)都有不同程度的下降,肌肉組織也表現(xiàn)出和肝臟類似的趨勢(shì)。以上結(jié)果表明飼料和水環(huán)境中抗生素的長期暴露對(duì)斑馬魚脂代謝造成了影響,具體機(jī)制還有待進(jìn)一步的研究。6周養(yǎng)殖結(jié)束后,利用嗜水氣單胞菌浸浴感染斑馬魚,結(jié)果顯示抗生素暴露組斑馬魚的死亡率顯著性升高,這表明飼料和水環(huán)境中抗生素的長期暴露破壞了斑馬魚的免疫系統(tǒng)功能,降低機(jī)體對(duì)疾病的抵抗能力。在斑馬魚腸道和肝臟組織中,抗生素暴露組堿性磷酸酶(AKP)、酸性磷酸酶(ACP)活性出現(xiàn)了不同程度的下降。在腸道組織中,和Control組相比,抗生素暴露組中與炎癥反應(yīng)相關(guān)的基因例如腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素-1(IL-1)的表達(dá)出現(xiàn)不同程度的上調(diào)。SMZ-bath、OTC-bath組腸道杯狀細(xì)胞的數(shù)量顯著性低于Control組。在腸道、肌肉組織中,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)、過氧化物酶(POD)活性不同程度的降低。以上結(jié)果表明,飼料和水環(huán)境中抗生素的長期暴露破壞了腸道的屏障功能、引發(fā)了腸道炎癥、造成了氧化損傷,腸道和肌肉是抗生素暴露造成氧化損傷的主要器官。腸道微生物高通量測(cè)序結(jié)果顯示,抗生素暴露組腸道菌群豐度指數(shù)出現(xiàn)不同程度的下降,這表明抗生素的長期暴露降低了腸道菌群的豐度,主成分分析(PCA)、菌群熱圖(Heatmap)的結(jié)果顯示,OTC-diet、OTC-bath 組與 Control組腸道菌群明顯區(qū)分開來,表明抗生素OTC改變了斑馬魚的腸道菌群結(jié)構(gòu),而SMZ-diet、SMZ-bath組與Control組并沒有很好的區(qū)分開來,這表明抗生素SMZ對(duì)腸道菌結(jié)構(gòu)的影響沒有OTC的大。本實(shí)驗(yàn)研究表明,在飼料中添加SMZ和OTC作為預(yù)防疾病發(fā)生的藥物長期使用,雖然促進(jìn)了斑馬魚的生長,但卻降低了對(duì)疾病的抵抗能力,破壞了機(jī)體的抗氧化功能以及腸道微生態(tài)平衡。水環(huán)境中極低濃度(ng·L-1)的抗生素殘留同樣降低機(jī)體對(duì)疾病的抵抗能力,破壞了機(jī)體的抗氧化功能以及腸道微生態(tài)平衡。因此不宜將抗生素作為預(yù)防疾病發(fā)生的藥物長期使用,并且不能忽視水環(huán)境中極低濃度的抗生素給水生動(dòng)物的生理功能帶來了負(fù)面效應(yīng)。
[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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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