飼料中添加亞精胺碳量子點對日本鰻與鱸鰻生長、腸道形態、免疫和抗病力的影響

Abstract

日本鰻（Anguilla japonica）和鱸鰻（Anguilla marmorata）是臺灣主要的鰻魚養殖種類。由於鰻線資源減少、土地成本增加和環境惡化，近年來，集約化循環水系統養殖逐漸成為鰻魚養殖的重要組成部分。集約化養殖會提高養殖密度，易加速病原的傳播，病害也不易控制。鰻魚養殖最大的挑戰是愛德華氏菌 （Edwardsiella tarda）的威脅，是養鰻業者損失的主要原因之一。傳統鰻魚養殖業者常使用抗生素對抗愛德華氏菌暴發。濫用抗生素會使細菌產生耐藥性並產生水產品安全問題。亞精胺碳量子點（spermidine carbon quantum dots, CQDSpds）作為一種新研發的碳奈米材料，由亞精胺和碳量子點通過簡單一步熱裂解合成。 CQDSpds表面攜帶的正電荷，會與表面帶負電荷的細菌結合，利用離子往不同方向振動的原理將細菌撕扯破裂，具有廣泛抗菌能力，對多種致病菌有良好的抑菌活性。 CQDSpds作為飼料添加劑，可以減少傳染性魚病發生的幾率，有望成為抗生素的部分替代品。飼料添加 CQDSpds 在鰻魚養殖中的具體效果還沒有進行過研究。 本試驗探討飼料中添加不同比例CQDSpds 對日本鰻與鱸鰻之生長性能、腸道形態、免疫相關基因表現和對愛德華氏菌抗病力的影響。經過前置試驗確認CQDSpds安全劑量後，試驗採用日本鰻與鱸鰻分別進行試驗。每次試驗分為 5 組，每組 2 重複。每組的飼料有不同 CQDSpds 添加量，分別是 0 ppm、0.25 ppm、0.5 ppm、1 ppm、2 ppm。日本鰻試驗共 250 尾，時長 56 天。鱸鰻試驗共 300 尾，時長 84 天。每 14 天測量一次魚體總長和體重，進行生長性能分析。每天記録死亡數量和餵食情況。試驗結束後每缸犧牲 4 尾，採集腸道、頭腎。採集的腸道進行石蠟包埋，切片觀察腸道形態及腸道絨毛狀態。採集的頭腎進行 qPCR 分析免疫相關基因表現。攻毒試驗使用愛德華氏菌。 試驗結果顯示，餵食適量添加 CQDSpds 的飼料會促進鰻魚生長相關參數，飼料中添加 1 ppm 與 0.5 ppm CQDSpds 的日本鰻與鱸鰻的最終魚體重量、增重率、特定成長率提升最高。投餵適量 CQDSpds 可減少日本鰻與鱸鰻在循環水系統中所受的壓力。相比對照組，投餵 1 ppm CQDSpds提高了日本鰻 LZM （lysozyme）基因表達，因此提升了日本鰻的非特異性免疫。但各組間存活率沒有顯著差異。腸道形態學結果顯示，餵食含 CQDSpds 飼料的鰻魚，腸道絨毛高度和腸道形態與未添加組相比沒有顯著差異。攻毒試驗顯示，餵食含 CQDSpds 的飼料，可增加愛德華氏菌攻毒時日本鰻和鱸鰻的生存率，添加 CQDSpds的各組生存率皆比對照組高，其中日本鰻1 ppm，鱸鰻0.5 ppm的生存率最高。飼料中添加 CQDSpds 可提升日本鰻與鱸鰻對愛德華氏菌的抵抗力，在細菌性疾病爆發時增加生存率。鰻魚養殖中可以添加 CQDSpds 以提高經濟效益。

Japanese eel（Anguilla japonica）and giant mottled eel（Anguilla marmorata） are the main breeding species in Taiwan. Due to reduced elvers, the increased land cost and the deterioration of environment in recent years, intensive recirculating aquaculture system has gradually become the essential constituent of eel breeding. Intensive farming increases the density of breeding, which easily accelerates the spread of pathogens increasing the difficulty to control diseases. The biggest pathogenic challenge of eel farming is the threat of Edwardsiella tarda, which is one of the main reasons for the loss of eels. In traditional eel farming, antibiotics are often used to fight the outbreak of E.tarda. However, abuse of antibiotics can create resistant bacterial strains and even lead to the problem of aquatic product safety. Spermidine carbon quantum dots（CQDSpds）, as a newly developed carbon nanomaterial, are synthesized from the simple one-step pyrolysis of spermidine and carbon quantum dots. The positive charges on the surface of CQDSpds binds to negatively charged bacterial cell wall, and by the principle of vibrating ions in multiple directions, the shearing force will tear and break the bacteria. The compound has a wide range of application in anti-bacterial, especially against a variety of pathogenic bacteria. When feeding it as additive, CQDSpds can reduce the prevalence of infectious diseases among aquatic animals and it is expected to become a partial substitute to antibiotics. However the specific effect of adding CQDSpds in eel culture has not been studied. This experiment was conducted to investigate the effect of different proportions of CQDSpds on growth performance, intestinal morphology, immunity and oxidative stress related to gene expression and disease resistance to E.tarda. in Japanese eel and giant mottled eel. After the pre-test confirmed the safe dose of CQDSpds, the compound was tested on both eels, respectively. There were five groups for each session of the trial and each group was duplicate. Each group was fed with different proportion of CQDSpds, namely 0 ppm, 0.25 ppm, 0.5 ppm, 1 ppm and 2 ppm. A total of 250 Japanese eels were tested for a duration of 56 days. And, for giant mottled eels, a total of 300 fish were tested for 74 days. The total body length and body weight of eels were measured every 2 weeks for growth analysis. The number of deaths and feeding condition were recorded daily. At the end of trials, 4 eels were euthanized to take the intestinal tissues and head kidneys. The sampled intestines were embedded in paraffin and prepared into slides to observe their morphology and intestinal villi under microscope. Immune related gene expression of the head kidneys were analyzed by qPCR. As for the challenge experiment, E.tarda was used. The test results showed that feeding certain amount of CQDSpds could improve several growth-related parameters of Japanese eel and giant mottled eel, final body weight, weight gain and specific growth rate of Japanese eel and giant mottled eel fed with 1 ppm and 0.5 ppm CQDSpds increased the most. Adding an appropriate amount of CQDSpds can reduce the pressure on Japanese eel and giant mottled eel in the circulating water system. Compared with the control group, administration of 1 ppm CQDSpds increased the LZM（lysozyme）gene expression of Japanese eel, thus enhancing the nonspecific immunity of eel. However, there was no significant difference in survival rate among groups. The results of intestinal morphology showed that there was no significant difference in intestinal villus height and intestinal morphology between the eels fed with CQDSpds and the control group. The challenge test showed that the survival rate of Japanese eel and giant mottled eel improved after E. tarda challenge when they were fed with CQDSpds. The survival rate of each group supplemented with CQDSpds was higher than that of the control group, among which the highest survival rates were found in groups of Japanese eel 1 ppm and giant mottled eel 0.5 ppm. The addition of CQDSpds in the diet can improve the resistance of Japanese eel and giant mottled eel to E.tarda, and increase the survival rate in the outbreak of bacterial diseases. As proven by this experiment, the additive of CQDSpds could improve eel culture and increase economic benefit.