體外發酵與面罩技術於農副產物添加於乳牛飼糧降低甲烷排放評估之應用

Abstract

考量近年國內乳牛產業面臨飼料進口成本上升壓力，加上國際間推動淨零碳排之趨勢，使低成本、低碳排的農副產物開發為替代性飼糧原料之潛力愈受重視。替代性飼糧除須具備維持生產性能之能力，其對瘤胃甲烷排放之影響亦為重要評估指標。體外發酵法雖能有效比較飼糧餵飼後之甲烷生成潛力，但是實際降低排放之程度仍須經由體內法驗證。現行利用活體之甲烷偵測技術多具成本高昂與偵測效率低之特性，限制甲烷排放資料之取得與應用。因此，本研究旨在優化實驗室開發中之面罩偵測技術，並評估其應用於分析乳牛餵飼農副產物飼糧後，改變甲烷排放效果的可行性。 試驗第一階段針對夏季試驗中觀察到乳牛呼氣量異常偏高之情形，於原面罩裝置新增兩種單向閥，並透過乳牛呼氣測驗評估實際分流成效。結果顯示，自製閥設計可有效降低乳牛換氣頻率，所呈現之呼氣波形亦較未分流及壓縮袋閥設計更為平緩。甲烷產量結果亦顯示，分流設計有助於減少未分流下流量過高與甲烷濃度蓄積的情況，有利提升結果穩定性。而在流場模型結果，顯示流量計與分流出口之理論分流比為1：4，然而根據此比例回推之總呼氣量顯著高於乳牛標準呼氣量。顯示精確的分流比例仍須藉由建構能模擬乳牛實際呼吸模式之變動流場模型，或實際由裝置測試進行優化。 第二階段將改良後之面罩結合三種偵測時機（餵飼後0小時、2至3小時、6小時），應用於泌乳牛之農副產物替代飼糧的甲烷排放體內外整合評估試驗。第一部分將杏鮑菇菇包青貯以三種比例（0%、25%、40%）取代完全混合日糧中之燕麥草。體外發酵結果顯示，隨著菇包青貯添加比例上升，中洗纖維消化率顯著提升，甲烷產量亦呈上升趨勢。比較揮發性脂肪酸（volatile fatty acid, VFA）、輔酶M（coenzyme M, CoM）與甲烷產量間關聯性，顯示由VFA推估之甲烷產量與實測結果具高度相關性，可作為甲烷生成間接評估指標。乳牛餵飼試驗結果顯示，乳牛採食量與乳量在不同菇包取代率下並無顯著差異，面罩試驗資料亦顯示飼糧間甲烷產量無顯著差異，與體外結果一致。整體結果顯示，以杏鮑菇菇包青貯取代燕麥後可維持乳量表現，並且不會導致甲烷產量增加。 第二部分副產物試驗採用兩種粗蛋白濃度（15%、17%）下，搭配三種副產物（啤酒粕青貯與豆殼粒）添加量（10%、20%、30%），配製成四種泌乳牛飼糧（CP17×10、CP15×10、CP15×20、CP15×30）。體外發酵結果顯示，於CP15%條件下，隨副產物添加量上升，中洗纖維消化率與甲烷產量皆顯著提升。在相同副產物添加量（10%）下，則以CP17% 組具有較高之中洗纖維消化率與甲烷產量。由發酵液VFA推估之甲烷產量與實測結果具有高度相關性。而在體內試驗中，以面罩測得四組飼糧的甲烷產量無顯著差異，瘤胃液中乙酸產量亦無顯著差異，丙酸產量則呈現CP15×20組高於CP17×10組之趨勢，顯示體內甲烷與VFA結果與體外結果不一致。此差異可能源自副產物取代後飼糧物理特性（如纖維片段大小與結構）改變，導致體外系統無法充分模擬體內實際發酵情況。而關於面罩法未能反映丙酸產量上升可能具有的降甲烷潛力，推測其準確性可能受限於本試驗每日一次餵飼模式下，甲烷排放曲線變異程度較大的影響。 綜合而言，本試驗顯示體外系統在評估副產物飼糧之甲烷排放實際降低效果時可能存在誤差，實際效果仍須透過體內試驗加以驗證。應用面罩法作為短時間採樣技術，其準確性與穩定之餵飼管理模式有關。未來應進一步於穩定飼養管理條件下優化面罩法之測定操作參數，期望建立一套快速、低成本且具實用性的甲烷排放評估技術，而有助於篩選評估農副產物應用於飼糧後之甲烷排放狀況。

Agricultural byproducts have emerged as low-cost, low-carbon alternative ingredients for dairy cattle in response to rising feed import costs and the global trend toward net-zero carbon emissions. When evaluating such ingredients, it is essential to confirm their capacity to maintain production performance while also considering their impact on enteric methane emissions. Although in vitro fermentation is effective for assessing the methane-reducing potential of diets, the actual effects must still be verified through in vivo methods. However, current in vivo methane detection techniques are often costly and inefficient, limiting their applicability in field settings. Therefore, this study aimed to optimize a face mask system in development and evaluate its feasibility for assessing variations in methane emissions from cattle fed agricultural byproduct-based diets. In the first phase, two types of one-way valves were incorporated into the original face mask device to address the abnormally high minute ventilation observed during summer trials. Respiration tests showed that the hand-made valve reduced the cow's respiratory frequency and produced a more stable exhalation pattern than the non-diversion and the space-bag valve. Methane emission data further indicated that the one-way valve design helped mitigate excessive minute ventilation and methane accumulation, thereby improving measurement stability. Flow field modeling revealed a theoretical diversion ratio of 1:4 between the flowmeter and the valve outlet; however, applying this ratio to estimate total minute ventilation produced values substantially higher than those typically observed in dairy cows. This suggests that further calibration is needed through dynamic simulation or practical validation. In the second phase, in vivo and in vitro evaluations of agricultural byproduct-based diets for lactating cows were conducted. The modified face mask method was used to estimate methane emissions at 0, 2–3, and 6 hours post-feeding. In the first trial, Pleurotus eryngii spent mushroom substrate (SMS) silage replaced oat hay in the total mixed ration at three levels (0%, 25%, 40%). In vitro fermentation results showed that increasing SMS inclusion significantly improved neutral detergent fiber digestibility (IVNDFD), while methane production remained statistically unchanged. VFA-predicted methane was highly correlated with measured values, suggesting its potential as an alternative indicator. In the in vivo trial, no significant differences in dry matter intake or milk yield were observed among diets. Methane emissions measured using the face mask method also showed no significant variation, consistent with in vitro findings. Overall, replacing oat hay with Pleurotus eryngii SMS silage maintained production performance without increasing methane production. In the second trial, four diets were formulated with two crude protein (CP) levels (15%, 17%) and three inclusion levels (10%, 20%, 30%) of brewer's spent grain silage and soybean hull pellets. In vitro results showed that under 15% CP, increasing byproduct inclusion significantly increased IVNDFD and methane production. At the 10% inclusion level, the 17% CP diet had higher IVNDFD and methane production than the 15% CP diet. VFA-predicted methane showed strong correlation with measured values, consistent with the first trial. However, in vivo methane emissions did not differ significantly among diets. Rumen acetate concentrations remained similar across diets, while propionate tended to be higher in the CP15×20 group than in CP17×10. These in vivo findings did not align with in vitro results, likely due to variations in diet physical properties (e.g., particle size and structure) after byproduct replacement, which limited the in vitro system's ability to simulate in vivo conditions accurately. Moreover, the once-daily feeding schedule may have caused significant fluctuations in methane emission patterns, limiting the face mask method's ability to detect the potential methane reduction in the CP15×20 group. In conclusion, in vitro systems may introduce bias when assessing the methane-reducing potential of byproduct-based diets, highlighting the necessity of in vivo validation. This study also emphasizes that the accuracy of the face mask method as a short-term sampling technique depends on consistent feeding management. Future efforts should focus on optimizing operational parameters under consistent feeding patterns to develop a rapid, cost-effective, and practical approach for evaluating methane emissions from agricultural byproduct-based diets.