仔豬體外消化與發酵平台之建立及其在抗生素替代物評估之應用

Abstract

仔豬離乳最初幾週會因營養、環境等因素受到疾病之風險造成嚴重損失，隨著國際趨勢禁用抗生素作為生長促進劑的同時，抗生素替代物的研發與應用日益重要。然而，各種替代物之實際有效用量及效果可能受飼料組成、動物日齡等因素影響，致使其實際飼養上的使用效果評估仍缺乏有效測試。動物試驗雖可獲得真實的生理反應與代謝資料，但成本高且效率低。體外消化與發酵平台即是一套可大量操作，減少動物使用且低成本與快速的篩選方法。但離乳仔豬因消化能力與生長或肥育豬有相當大之差異，需建立適用於離乳仔豬之體外消化條件才能正確進行評估工作。本研究旨在建立一套適用於離乳仔豬消化條件之體外消化與發酵系統，評估抗生素替代物之效果及用量，並藉由動物試驗驗證其準確性。 試驗第一階段建立與優化離乳仔豬體外消化與發酵系統，依照離乳仔豬腸道生理特性之研究，分別調整為生長豬消化道酵素活性之50%，並分別設定胃與小腸乾物質比例30%與12%條件下，以pH 4或pH 5模擬胃部pH值消化1小時，接續以pH 6.5模擬小腸消化8或16小時。試驗透過不同粗蛋白含量之離乳仔豬飼糧評估不同消化條件對於體外消化情形、臭味物質，並與實際動物體內消化率、糞便臭味物質及生長表現間進行比對，以建立具代表性之模擬系統。進一步運用透析法評估模擬小腸吸收之時間設定，優化現有體外消化平台。體外模擬結果顯示，三種粗蛋白含量飼糧在模擬胃pH 4或5與小腸消化8或16小時的組合條件下，對乾物質消化率並無顯著差異，因此設定8小時作為模擬小腸消化時間。在胃部pH 4條件下，中蛋白與高蛋白飼糧之體外粗蛋白消化率顯著高於低蛋白組，與動物試驗結果趨勢一致。生長表現方面，中蛋白組與高蛋白組仔豬之平均日增重以及飼料效率皆優於低蛋白組。體外發酵臭味物質與糞便分析結果也具有一致趨勢。因此，依離乳仔豬腸道乾物質比例，並採生長豬酵素活性之半量進行模擬，結果顯示以胃pH 4消化1小時，接續小腸pH 6.5消化8小時，可有效模擬體內消化情形。搭配12小時透析可移除約90%游離胺基酸與還原糖，模擬腸吸收。進一步以不同粗蛋白與能量水平仔豬飼糧進行雙重驗證，結果顯示體外模擬與實際動物試驗在消化與發酵產物上皆具有相關性，進一步證實體外模擬平台與活體動物消化情形具有良好對應關係。 第二部分之研究利用第一階段建立之離乳仔豬體外消化與發酵平台，評估中鏈脂肪酸混合物（含12碳單酸甘油酯與14碳之脂肪酸）作為抗生素替代物之效用與添加量，並搭配動物試驗進行體內外添加效果之雙向驗證。結果顯示，添加0.2%混合物時，體外全腸消化率與對照組並無顯著差異，且與動物實際消化結果一致，但營養物釋放速率較0.1%為佳。體外發酵之微生物分析顯示，添加0.2%亦可提升乳酸菌比例、抑制大腸桿菌與沙門氏菌生長，且與動物實驗中之糞便菌相變化趨勢一致。進一步將透析模擬吸收作用納入評估，比對動物試驗結果，顯示模擬消化後之透析操作可有效改變發酵基質組成，反映出更貼近體內後腸發酵之產氣動力學與臭味物質分佈。 綜合上述，本研究成功建立一套涵蓋消化—吸收—發酵三階段之離乳仔豬體外消化與發酵系統，能準確反應實際飼糧對仔豬消化性能與腸道環境之影響。透過體內外雙向驗證，證實該系統可作為抗生素替代物添加之篩選之工具，有效降低動物試驗需求，並提升抗生素替代物之精準利用。

The weaner period is a critical phase in piglet development, because nutritional deficits, underdeveloped digestive function, and environmental stressors significantly increase the risk of disease and growth retardation. With the global trend of banning the use of antibiotic growth promoters, the development and application of antibiotic alternatives have become increasingly important. However, the efficacy and optimal dosages of these alternatives vary depending on factors such as feed composition and age. Therefore, there is still a lack of efficient evaluation methods on the effectiveness and dosage of their practical application. While animal experiments provide accurate physiological and metabolic data, they are costly, labor-intensive, and low efficiency. As such, an in vitro digestion and fermentation system offers a high efficiency, low-cost, and animal-sparing alternative for evaluating the efficacy and dosage of these alternatives. However, due to substantial differences in digestive capacity between weaning piglets and growing or finishing pigs, it is necessary to establish in vitro digestion conditions specifically suitable for weaning piglets to ensure accurate evaluation. The research aimed to establish an in vitro digestion and fermentation system of weaning piglets, to evaluate the effectiveness and dosage of antibiotic alternatives, and to validate the system’s accuracy through animal experiments. The first phase of the experiment focused on the development and optimization of the in vitro system. Based on studies of the gastrointestinal physiology of weaning piglets, the system was adjusted to simulate piglet digestion using 50% of the enzymatic activity of growing pigs and dry matter ratios in the stomach and small intestine of 30% and 12%, respectively. Digestion was simulated in the stomach at pH 4 or pH 5 for one hour, followed by digestion in the small intestine at a pH of 6.5 for either 8 or 16 hours. Using diets with varying crude protein (CP) levels, the study evaluated in vitro digestibility, odor compounds, and the correlation between in vitro and in vivo digestive performance, fecal odor compounds, and growth performance. Furthermore, the dialysis process was applied to evaluate and optimize simulated intestinal absorption. The in vitro experiment results showed no significant differences in dry matter digestibility across the various protein-level diets under different small intestinal digestion times or gastric pH conditions, which supports using 8 hours is suitable for small intestinal digestion. At gastric pH 4, medium- and high-CP diets exhibited significantly higher in vitro protein digestibility than the low-CP diet, consistent with in vivo results. Piglets fed medium- or high-CP diets also showed better average daily gain and feed efficiency. Additionally, odor compounds produced from in vitro fermentation and fecal samples showed similar trends. It suggested that the effectiveness of simulating gastric digestion at pH 4 for 1 hour and intestinal digestion at pH 6.5 for 8 hours under the specified dry matter ratios and enzyme activity levels. The 12-hour dialysis duration removed approximately 90% of free amino acids and reducing sugars, successfully simulating nutrient absorption. Bidirectional evaluation using diets with varying crude protein and energy levels confirmed a strong correlation between in vitro and in vivo digestive and fermentation outcomes, reinforcing the system’s reliability. In the second experiment, the in vitro digestion and fermentation platform for weaning piglets established in the first experiment was applied to evaluate the efficacy and dosage of medium-chain fatty acids (MCFA) mixture (including C12 monoglyceride and C14 fatty acids) as antibiotic alternatives with bidirectional verification. The results showed that 0.2% MCFA mixture supplementation had no significant effect on total tract digestibility compared to the control group. The nutrient release was more effective at 0.2% than at 0.1%. Furthermore, microbial analysis from in vitro fermentation indicated that 0.2% MCFA mixture increased the proportion of lactic acid bacteria while suppressing the growth of E. coli and Salmonella. This finding aligns with the changes in fecal microbiota observed in vivo. Moreover, incorporating dialysis to simulate nutrient absorption allowed for a more accurate evaluation by modifying the fermentation substrate composition, leading to fermentation kinetics and odor compound profiles that better reflected in vivo hindgut fermentation. In conclusion, this study successfully established a three-stage in vitro digestion-absorption-fermentation system that accurately simulates the digestive conditions of weaning piglets. The platform effectively evaluated the impact of different diets on digestion efficiency and gut microbial metabolism. Through bidirectional evaluation using in vivo and in vitro results, the system was proven to be a reliable tool for screening antibiotic alternatives. This approach effectively reduces reliance on animal experiments and enhances the precision in the utilization of antibiotic alternatives.