應用三段式體外消化法評估飼糧蛋白質濃度對豬隻糞便排放特性之影響

Abstract

畜牧產業在降低生產成本同時也期望能同時降低環境污染，提供豬隻超過其實際蛋白質需求量的飼糧，不但會增加營養浪費並提高成本，未消化的蛋白質和其他含氮代謝物以排泄物排出體外，也導致隨後的環境污染問題。減少豬隻日糧中的粗蛋白(crude protein, CP)含量並補充結晶胺基酸以滿足相同標準迴腸消化率(standardised ileal digestibility，SID)要求，已成為相應的解決方案。 在豬隻的蛋白質需求研究，以動物飼養試驗需要較長的實驗時間和動物飼養成本，而以體外消化和發酵整合測定，可以在短期內進行多種測試飼糧的評估。但是不同的消化模式和接種源對測試結果具有明顯的差異，因此本試驗透過比較搖瓶與透析兩種消化方法，生長期豬隻飼糧調整以不同CP含量，配合不同階段豬隻來源糞便接種，評估體外消化發酵法在評估蛋白質調整對排放之影響，再配合動物試驗驗證體內外評估指標的關聯。 第一階段體外試驗結果顯示，使用搖瓶和透析法進行消化後，搖瓶法對於氮的消化率方面顯著低於透析法(P < 0.05)，由於搖瓶的消化模式並無法完整的移除消化後產物，因此在模擬體外發酵時可能出現較大差異。使用搖瓶法消化後的產物進行發酵後，不同生長期飼糧調整CP含量的處理組間，其發酵產物以及產氣參數兩個指標皆無顯著差異。原因可能是由於消化方式可能導致的誤差，導致無法正確的推斷出降低飼糧CP含量對於減少排放的影響。而經由透析法消化後再進行體外發酵，所測得的氨態氮、蛋白酶以及尿素酶的活性皆會隨著CP含量降低顯著減少(P < 0.05)；產氣參數中最高產氣發生時間(T max)隨著CP含量下降顯著性上升(P < 0.05)，最大產氣速率(R max)隨著CP含量下降顯著下降(P < 0.05)。揮發性脂肪酸之濃度顯示，丁酸含量在降低CP含量的處理組中顯著較低(P < 0.05)。利用屏東以及宜蘭兩地豬場不同生長期之豬隻糞便接種進行發酵後，其消化與產氣動力結果所呈現的趨勢皆相同。進一步測試降低飼糧CP含量與添加消化酵素之組合處理，配合透析法消化的結果顯示，當飼糧當中添加商用蛋白酶以及非澱粉多醣酶，經體外消化再進行糞便接種發酵後，發酵產物中之氨態氮、蛋白酶活性、尿素酶活性、對甲酚、吲哚以及糞臭素濃度均顯著低於控制組 (P < 0.05)。經由重複實驗結果證明，利用透析法進行消化模式，並搭配新鮮豬糞接種進行體外發酵，能更準確地對飼糧特性進行評估。 動物試驗以起始體重30 kg之生長期豬隻進行，並提供滿足SID之CP＝17％、CP＝15％以及未滿足SID之原牧場的飼糧(控制組，CP＝18％)，經由四週的飼養實驗後，CP17％組生長效率顯著高於CP15％以及控制組(P < 0.05)，豬糞中每日總氮排放量方面，在第二、三週時CP17％組也有顯著較佳的情形，而吲哚、糞臭素以及對甲酚濃度也隨著CP水準下降而顯著降低(P < 0.05)，VFA含量、氨態氮、蛋白酶活性以及尿素酶活性在CP下降時顯著下降(P < 0.05)，整體趨勢也與體外試驗相同。 整合上述體內外試驗結果來看，對豬隻飼糧評估其餵飼對排放的影響時，使用適當的體外的實驗模式進行臭味或含氮廢物排放之篩選，再配合動物實驗進行生長性能的評估，可提升實驗的準確性以及便利性，並減少在動物實驗當中所需的時間及飼養成本，顯示體外試驗的操作結果可以對應到動物實驗當中進行評估使用。

In animal production industry, there is an expectation to reduce production cost and decrease environmental pollution at the same time. Providing diets that over pig actual protein requirements will increase nutritional waste and feeding costs. Undigested proteins and other nitrogen metabolites are excreted from urine and faece then cause subsequent environmental pollution problems. Reducing the crude protein (CP) level in pig diets and supplementing with crystalline amino acids to meet the same standard ileal digestibility (SID) requirements has become a corresponding solution. In the study of pig protein requirements and adjustments, owing to higher time and cost in animal experiment, the in vitro digestion and fermentation integrated trial could provide an effective alternative method to test a variety of diets in a short period of time. However, different digestion model and inoculation source may result in significantly effect on the test results. To evaluate the applicality of the in vitro digestion and fermentation method in assessing the effect of protein adjustments on emissions, the experiment comparing the digestion methods of shake flask and dialysis were tested. Fecal samples from two growth period pig fed different CP levels were collected as inoculation source. Finally, the in vivo feeding experiment was proceeded to proof the effectiveness of the in vitro indicators. The first-stage in vitro test indicated that significantly lower nitrogen digestibility in shake flasks digestion compare with dialysis (P < 0.05), it means that the shake flask digestion cannot complete remove the digest products. It may lead to faulty in vitro fermentation result. It showed no significant differences in the fermentation products and gas production parameters between the treatments of CP levels in the growing pig diets under shaking flask digestion treatment. However, due to the possible misestimate from the digestion mode, it is difficile to correctly conclude the effect of reducing diet CP level on emissions. The fermentation result of samples that digested by dialysis method indicated that the ammonia nitrogen, protease and urease activities were significantly reduced with CP levels decreased (P < 0.05). According to gas production parameters, the highest gas production time (Tmax) significantly increased (P < 0.05) and the maximum gas production rate (Rmax) significantly decresaed with the decreasing of CP levels (P < 0.05). In addition, the concentration butyric acid was significantly lower in the treatments with low CP levels (P < 0.05). The fecal inoculation source from Pingtung and Yilan also resulted in the same trends of digestion and gas production dynamics. In the next study, we combined the decreasing CP levels of diets and digestive enzymes supplementation as multiple-treatment for odor reducing. The results indicated that in the commercial versazyme and NSPase significantly decreased the concentration of ammonia nitrogen, p-cresol, indole and skatole, protease activity and urease activity compared with the control group (P < 0.05). The repeated experiment results implied that the in vitro dialysis digestion and fermentation method could accurately evaluate the utilization characteristics of the diets. The growing pigs which have 30 kg start weight were applied in animal experiment. Three diets including control diet (from original farm without SID modification, CP=18%) and test diets with CP=17% and CP=15% (satisfied the SID requirement) were fed in this trial for four weeks. The results showed that the growth efficiency of the CP17% group was significantly higher than other treatment groups (P < 0.05). Comparing to other treatment groups, the CP17% group also showed significantly lower daily total nitrogen emissions of pig feces in the second and the third week. The concentration of indole, skatole, p-cresol VFA and ammonia nitrogen, protease and urease activities were significantly decreased with the decreasing of diet CP levels. The resulted had the similarity trend with in vitro trial, it indicated that the in vitro results can be corresponding to animal experiments. In conclusion, to evaluate the emission impact of pig diets, the accuracy and convenience of evaluation could be improved by applying the appropriate in vitro experimental models to investigate odor and nitrogen waste emissions before animal experiments. The selected diet with in vitro evaluation could be fed to animal and estimate the growth performance. It could result in the reducing time and feeding costs in animal experiments.