黑水虻處理雞糞對微生物相及含蟲油之飼糧對蛋雞產蛋性能、腸道型態與菌相之影響

Abstract

家禽飼養過程中常使用抗菌藥物以治療疾病，部分藥物可能以未代謝或仍具活性的形式排出體外，導致雞糞中抗生素殘留，並促進抗藥性菌株的增長，進而成為抗生素抗藥性基因（antibiotic resistance genes, ARGs）累積與擴散的重要來源。ARGs可經由垂直或水平傳播進入環境，並可能藉由食物鏈影響人類健康。黑水虻（Hermetia illucens）為一種腐食性昆蟲，能有效分解各種有機廢棄物，並將其轉化為高蛋白與高脂質的生物質。其中，黑水虻蟲油（black soldier fly oil, BSFO）富含中鏈脂肪酸（medium-chain fatty acids, MCFAs），以月桂酸（lauric acid, C12:0）含量最為豐富。研究顯示，月桂酸具有良好的抑菌效果，其可以透過調控TLR4/NF-κB訊號傳導路徑，增加腸道屏障功能，對腸道健康具有潛在益處。腸道微生物群落是個複雜而多樣的系統，對於家禽的健康和生理功能扮演重要的角色，且易受動物年齡和飼糧組成等許多因素影響。然而，關於BSFO作為飼料油脂來源對蛋雞腸道免疫功能與健康的影響仍缺乏深入探討。 本研究分為兩個部分，試驗一以雞糞飼養黑水虻，於處理第0、6與12天，採集糞便與蟲體樣本，透過即時定量聚合酶連鎖反應分析8種雞糞中常見的ARGs，包含Sul1、tetA、tetB、tetG、blaTEM、blaCTX、blaSHV及qnrB；並結合16S rRNA定序，探討黑水虻處理對基質菌群結構的影響。同時，利用塗佈平板法測定大腸桿菌、乳酸菌及腸桿菌的菌落數。結果顯示，黑水虻處理可顯著減少基質中大腸桿菌菌落數；然而，16S rRNA定序仍偵測到如Pseudomonas、Erysipelothrix等潛在病原菌。此外，ARGs分析結果顯示，黑水虻處理沒有顯著減少基質中blaSHV、tetB和Sul1表現，且蟲體中仍可檢測到抗藥性基因殘留，顯示以廢棄物飼養黑水虻仍存在安全風險。試驗二評估不同比例BSFO取代飼糧中大豆油對蛋雞生產性能與腸道健康之影響。本次試驗採用42隻57週齡羅曼褐殼蛋雞，並將其隨機分配至對照組（100%大豆油）、20% BSFO組（替代飼糧中20%大豆油）以及40% BSFO組（替代飼糧中40%大豆油），試驗為期8週。於試驗過程中記錄體重、產蛋率與蛋重，於第8週採集血液、腸道組織及盲腸內容物，並利用16S rRNA定序分析菌相。結果顯示，BSFO不影響蛋雞體重、產蛋率或蛋重。此外，蛋黃顏色、蛋白高度和豪氏單位組間無顯著差異，但蛋殼強度隨BSFO比例增加而降低（p < 0.05），推測可能因飽和脂肪酸容易與鈣形成皂化物，降低鈣吸收。而血液生化值是評估動物健康生理狀況的重要因素，結果顯示白蛋白、球蛋白、天門冬胺酸轉胺酶、膽固醇及三酸甘油酯在各組間無顯著差異（p > 0.05），顯示BSFO不影響蛋雞健康狀態及血脂代謝。在腸道結構分析中，20% BSFO組十二指腸絨毛長度減少（p < 0.05）；同時，20%和40% BSFO組十二指腸與迴腸IL-1β表現量升高（p < 0.05），且20%與40% BSFO組空腸CLDN基因表現量顯著減少（p < 0.05），顯示BSFO可能誘導局部發炎反應並削弱腸道屏障功能，推測是因為BSFO的替代增加飼糧中飽和脂肪酸並減少Omega-6含量，刺激TLR4/NF-κB路徑，增加促炎因子釋放，導致腸道屏障功能受損。此外，菌相分析結果表示，各組菌群α多樣性沒有顯著差異，優勢菌門皆以厚壁菌門（Firmicutes）和擬桿菌門（Bacteroidetes）為主。然而，β多樣性分析顯示各處理組之間菌群組成有顯著差異（p < 0.05），BSFO組如Syngergistes及Flavonifractor菌屬相對豐度增加，相關代謝途徑包含泛酸、輔酶A合成及支鏈胺基酸代謝，可能促進短鏈脂肪酸生成，進而影響腸道免疫與屏障功能。綜上所述， BSFO不影響蛋雞生產性能及血液生化值，但高比例替代可能降低蛋殼強度，並透過改變脂肪酸組成誘導腸道局部炎症，同時影響菌群結構與代謝功能。黑水虻飼養基質對於蟲體應用價值及品質至關重要，未來可透過優化基質，調整黑水虻蟲油脂肪酸比例，進一步提升其於家禽腸道健康之應用。

In poultry farming, antimicrobial drugs are commonly used for disease prevention and treatment. However, some drugs may be excreted in unmetabolized or active forms, leading to the presence of antibiotic residues and bacterial strains in manure. Consequently, manure serves as a significant reservoir for the accumulation and dissemination of antibiotic resistance genes (ARGs), which can be transmitted vertically or horizontally into the environment and may indirectly affect human health through the food chain. The black soldier fly (Hermetia illucens) is a saprophagous insect capable of decomposing various organic wastes and converting them into protein and lipid. Black soldier fly oil (BSFO), extracted from the larvae, is rich in medium－chain fatty acids (MCFAs), particularly lauric acid (C12:0), which can comprise 40–50% of the total fatty acids. Previous studies have demonstrated that lauric acid exhibits antimicrobial activity and enhance intestinal barrier function by modulating the TLR4/NF-κB signaling pathway, thereby potentially benefiting gut health. The gut microbiota constitutes a complex and diverse ecosystem that plays a crucial role in physiological functions of poultry. It is affected by multiple factors, including the animal’s age and diets. However, the effects of BSFO as a lipid source on intestinal health and immune function in laying hens remain inadequately explored. This study consists of two parts. In trial one, black soldier fly were reared on manure, and samples were collected on days 0, 6, and 12. Quantitative PCR was used to detect eight common ARGs in manure: Sul1、tetA、tetB、tetG、blaTEM、blaCTX、blaSHV and qnrB. Traditional plating methods were used to quantify Escherichia coli, Enterobacteriaceae, and Lactobacillus spp. Results indicated that manure decomposed by the black soldier fly significantly reduced the counts of E. coli colony. However, 16S rRNA sequencing revealed the continued presence of potential pathogens such as Pseudomonas and Erysipelothrix. Also, ARGs such as blaSHV, tetB, or Sul1 remained detectable in both manure and larvae, suggesting biosafety concerns when rearing larvae on waste. In trial two, evaluated the effects of replacing soybean oil with varying proportions of BSFO on production performance and gut health in laying hens. Forty-two 57-week-old Roman brown hens were randomly distributed to 3 groups: control (100% soybean oil), 20% BSFO replacement, and 40% BSFO replacement. The trial lasted 8 weeks, during which body weight, egg production rate, and egg weight were recorded. At week 8, blood, intestinal tissues, and cecal contents were collected for 16S rRNA sequencing to analyze gut microbiota. Results indicated the production performance and egg quality were unaffected (p > 0.05), except for a decrease in eggshell strength at higher BSFO levels (p < 0.05), possibly due to reduced calcium absorption from saturated fatty acids. Blood biochemical parameters, including albumin, globulin, aspartate aminotransferase (AST), cholesterol, and triglycerides, also showed no significant different. However, intestinal analysis revealed that the villus height in the duodenum reduced in the 20% BSFO group (p < 0.05). Moreover, IL-1β expression in the duodenum and ileum was elevated in both the 20% and 40% BSFO groups, while claudin (CLDN) gene expression in the jejunum was significantly reduced (p < 0.05). These effects may be linked to increased saturated fat and reduced omega-6 content in BSFO, potentially activating the TLR4/NF-κB pathway. Microbiota analysis showed no significant differences in alpha diversity across groups, with Firmicutes and Bacteroidetes dominating the microbial communities. However, beta diversity analysis revealed significant differences in microbial composition between treatments (p < 0.05). The relative abundance of genera include Synergistes and Flavonifractor increased in BSFO groups. These bacteria are involved in metabolic pathways related to pantothenic acid, coenzyme A biosynthesis, and branched-chain amino acid metabolism, which may enhance short-chain fatty acid production and thereby influence gut immunity and barrier function. In conclusion, BSFO substitution did not affect laying hen production performance or blood biochemistry, but higher replacement may reduce eggshell strength and induce intestinal inflammation by altering fatty acid composition. This leads to weakened barrier function in microbial structure and metabolic pathway. The choice of substrate for rearing black soldier flies critically impacts the safety and quality of insect-derived products. Future research should focus on optimizing rearing substrates and modulating the fatty acid profile of BSFO to enhance its potential benefits for poultry gut health.