豬隻高通量體外模擬消化平台之建立

Abstract

現今評定飼糧價值時，多透過近似分析來進行，但化學分析所得成分與實際上的消化吸收仍有差距，因此需透過更客觀的方式評定飼糧的養分效價，以達到提高飼料效率的目的。利用直接餵飼動物取得結果的方法為動物體內法(in vivo)，可蒐集真實的消化代謝數據及其生理數值，但是此法相當耗時費力，尤其在同時比較多種飼糧時，需投入大量飼養成本與時間。利用動態體外消化法（dynamic in vitro digestion system）雖可省去飼養動物的負擔，並與體內法具有高度相似性，但是所需設備昂貴，無法同時進行多種飼糧間的比較，在實驗效率上較差。靜態體外消化法（static in vitro digestion system）簡化了吸收的過程，雖對於後腸發酵的模擬較不足，但有使用器材簡單、操作快速和同時可比較多種不同飼糧的特點，因此若能加以改良，則可有效發揮體外消化的優勢。

本研究利用三種體外消化來比較不同飼料之間的差異，分別以搖瓶法(shaking flask)、透析袋法(dialysis bag)以及搖瓶+透析袋法(shaking flask+ dialysis bag)來模擬胃部以及小腸的消化，並結合體外發酵(in vitro fermentation)過程，接種新鮮豬糞模擬豬隻後腸的發酵。試驗主要探討三種不同的消化方法之間對於消化率以及最終後腸發酵的影響，並且分析採食相對應飼糧之豬隻糞便所得的數據進行雙向驗證比較，以了解不同消化方法對於消化結果與臭味物質排放的影響，並建立較適合對應於豬隻的靜態體外消化方法。

利用不同豬場所蒐集的生長豬飼料以及肥育豬飼料進行測試，實驗結果顯示，不同的消化方法在胃以及小腸的乾物質和粗蛋白消化率之間具有顯著差異(p<0.05)，不論生長豬飼料或是肥育豬飼料試驗之趨勢皆相同。在乾物質消化率方面，使用透析袋法與搖瓶搭配透析袋法相近，單獨使用搖瓶法時較低。蛋白質消化率方面則為以透析袋法高於搖瓶搭配透析袋法，單獨使用搖瓶法時最低。後腸發酵實驗結果顯示，發酵12小時的模擬後腸乾物質以及蛋白質消化率，以搖瓶法消化殘餘物為基質時最高，透析袋法與搖瓶搭配透析袋法之消化殘餘物較低。在發酵48小時的後腸乾物質以及蛋白質消化率，也和發酵12小時的模式相似。整合胃與小腸部分消化酵素模擬以及大腸發酵之全腸道消化率來看，三種模擬胃與小腸之體外消化的方法之間均有顯著差異。在臭味物質的排放方面，不論是發酵12小時或是發酵48小時在對甲酚(p-cresol)、吲哚(indole)、糞臭素(skatole)以及氨態氮(amonia nitrogen, NH3-N)這四種臭味物質以及揮發性脂肪酸(volatile fatty acids, VFA)的排放方面皆具有顯著差異(P<0.05)。以搖瓶搭配透析袋法之消化殘餘物為基質，經過48小時發酵的臭味物質，與豬隻糞便所得的數據進行比較，可發現臭味物質中的對甲酚、吲哚以及氨態氮之濃度上，在體外評估與活體糞便分析值間具有較高之相關性。

綜上所述，透過本研究了解不同消化方法對於消化率的影響，及利用體內實驗所蒐集的豬隻臭味排放數據，有助於建立出更適用於豬隻飼糧評估的靜態體外消化方法。

Presently, the assessment of feed value is often conducted through approximate analysis, which may not fully reflect the actual digestion and absorption in animals. To enhance feed efficiency, a more objective approach is needed to evaluate the nutrient effectiveness of animal feed. In vivo methods involve direct feeding of animals to collect real digestion and metabolic data, but they are time-consuming and labor-intensive, especially when comparing multiple feed types simultaneously. Dynamic in vitro digestion systems, though similar to in vivo methods, are costly and impractical for parallel comparisons of different feeds, leading to lower experimental efficiency. Static in vitro digestion systems, on the other hand, simplify the absorption process and enable easy and rapid comparison of multiple feed types, but they may lack accuracy in simulating hindgut fermentation. However, with appropriate modifications, they can still effectively leverage the advantages of in vitro digestion.

In this study, three in vitro digestion methods were employed to compare differences among various feeds. Three digestion techniques, namely shaking flask, dialysis bag, and a combination of shaking flask with a dialysis bag, were utilized to simulate gastric and small intestinal digestion. Furthermore, an in vitro fermentation process was integrated by inoculating fresh pig feces to simulate hindgut fermentation. The experiment focused on investigating the impact of these three digestion methods on digestibility rates and final hindgut fermentation. Data obtained from pig feces corresponding to the respective feed intake were subjected to bidirectional validation and comparison to understand the effects of different digestion methods on digestion outcomes and emission of odorous compounds. The goal was to establish a more suitable static in vitro digestion method applicable to pig feeding studies.

Samples of growth pig feed and finishing pig feed from various pig farms were collected for testing. The results revealed significant differences (p<0.05) in dry matter and crude protein digestibility rates among the three digestion methods in both growth and finishing pig feed experiments. The dialysis bag method showed similar results to the shaking flask combined with dialysis bag method but performed lower when used alone. In terms of protein digestibility, the dialysis bag method outperformed the shaking flask combined with dialysis bag method, which, in turn, outperformed the shaking flask method used alone. The hindgut fermentation experiment indicated that the shaking flask digestion residues served as the best substrate for fermenting dry matter and protein after 12 hours, while the dialysis bag method and shaking flask combined with dialysis bag method exhibited lower digestion residues. The 48-hour hindgut fermentation results were similar to the 12-hour pattern.

Analyzing the overall digestive rates, including enzyme simulations for gastric and small intestinal digestion and fermentation in the large intestine, significant differences were observed among the three in vitro digestion methods. Regarding the emission of odorous compounds, both the 12-hour and 48-hour fermentation showed significant differences (P<0.05) in the release of four odorants, namely p-cresol, indole, skatole, and ammonia nitrogen (NH3-N), as well as volatile fatty acids (VFA). Comparing the 48-hour fermentation results for odorants with pig feces data, the shaking flask combined with dialysis bag method displayed a higher correlation between in vitro assessment and in vivo fecal analysis for p-cresol, indole, and ammonia nitrogen concentrations.

Overall, the study demonstrated that different static in vitro digestion methods significantly influence digestibility rates and fermentation characteristics in swine feeding. The shaking flask combined with dialysis bag method appears to be a promising approach for accurately evaluating nutrient digestibility and predicting odor emissions in pig feeding studies.