| Abstract: | 百慕達乾草是國內餵飼乳牛常用的牧草，其纖維含量偏高，而且在瘤胃的分解緩慢，可能會限制對動物的養分供應。本論文研究的目的在於探討乳牛瘤胃中添加以木聚醣酵素為主之外源纖維分解酵素，對百慕達乾草瘤胃原位（in situ）纖維分解率、酵素活性變化與發酵產物的影響。 試驗一評估瘤胃中添加不同劑量（0、75或150 g）之外源纖維分解酵素，對乳牛瘤胃酵素活性變化與百慕達乾草原位分解率之影響。結果顯示，添加酵素顯著提高餵飼後1.5小時與4.5 小時之瘤胃木聚醣酵素（xylanase）活性，而且以添加150 g的效果最好；但是對羧甲基纖維素酵素（carboxy-methyl cellulase, CMCase）的活性沒有影響。添加酵素提高飼餵後4.5小時瘤胃原位中洗纖維（neutral detergent fiber, NDF）分解率，其他培養時間（1.5、7.5與24小時）則沒有處理效應。酵素的添加對酸洗纖維（acid detergent fiber, ADF）分解率沒有影響，但顯著提高餵飼後4.5小時之半纖維素分解率，而以150 g組最高。SDS-PAGE電泳圖顯示，外源酵素在瘤胃中可能會快速分解。 試驗二的目的在於探討外源纖維分解酵素與水溶性蛋白質的組合，對瘤胃酵素活性變化、百慕達乾草原位分解率與發酵作用之影響。在體外試驗（in vitro）方面，使用不同劑量（0?0.1 %）外源酵素單獨與瘤胃液共同培養，發現瘤胃液中的CMCase與xylanase活性隨著培養時間的增加而逐漸分解。然而，當額外加入牛血清白蛋白或明膠共同培養時，瘤胃液中的酵素活性分解較為緩慢。藉由酵素動力學模式推算，在明膠使用劑量為0.20 %時，可以減緩酵素活性分解達50 %。在動物試驗方面，使用兩頭配備有瘤胃廔管的荷蘭種母牛，試驗處理分為牛隻餵飼時於瘤胃不添加（對照組）或添加80 g纖維分解酵素、160 g明膠、80 g 酵素搭配160 g明膠，以及160 g酵素與160 g明膠均分為兩次（早上八時和中午十二時）添加。結果發現，試驗的處理降低餵飼後之瘤胃pH 值，與提高氨、還原糖、蛋白質與非氨非蛋白質態氮濃度。瘤胃乙酸與總揮發性脂肪酸濃度也因為試驗處理而提高，其中以酵素與明膠同時添加的效果較單獨添加酵素為佳，而添加兩次酵素與明膠的處理，比添加一次的處理有較高的總揮發性脂肪酸濃度。酵素搭配明膠比單獨添加酵素，提高瘤胃CMCase、xylanase、與外切纖維素分解酵素（avicelase）活性，同時降低蛋白質分解酵素（proteinase）活性，其中以添加兩次酵素搭配明膠的處理效果最高。另外，百慕達乾草的瘤胃原位分解率也有正面的處理效應，試驗處理提高餵飼後百慕達草在瘤胃中乾物質、NDF、ADF與半纖維素分解率，其中效果以混合（酵素與明膠）添加的方式較單獨添加酵素，對改善乾物質、NDF、ADF與半纖維素分解率的效果為優。由SDS-PAGE電泳圖可看出，明膠有減緩外源酵素在瘤胃中的分解，而補充兩次明膠的效果更為明顯。 整體而言，本論文研究的結果顯示，外源纖維分解酵素在瘤胃中，可能是因為瘤胃中蛋白質分解酵素的作用，而快速分解，因而促進瘤胃纖維分解的效果無法持續長久。瘤胃中添加酵素搭配明膠，可以延長酵素在瘤胃中的活性，促進瘤胃纖維分解與發酵作用的效果更為明顯，可能是因為明膠與瘤胃中蛋白質分解酵素發生作用，減少外源纖維分解酵素被破壞。
Bermudagrass hay is often fed to dairy cow. It is high in fiber but slow in ruminal degradation, which may restrict nutrient availability for animal.The research object was to evaluate the effect of adding an exogenous fibrolytic enzyme product, mainly xylanase, on ruminal hay degradation in situ, changes in enzyme activities, and fermentation characteristics. In exp. 1, the effect on ruminal enzyme activities and in situ bermudagrass hay disappearance was estimated by adding different doses (0, 75 or 150 g/d) of a fibrolytic enzyme product to dairy cows. The results show that enzyme treatment increased ruminal xylanase activity at 1.5 h and 4.5 h after feeding. The activity was the highest for 150 g treatment. However, treatment had no effect on ruminal CMCase activity. In situ ruminal degradation of bermudagrass neutral detergent fiber was increased by enzyme addition at 4.5 h after feeding. At other incubation times (1.5, 7.5, and 24 h), there was no treatment effect. In situ acid detergent fiber degradation was unaffected by adding enzymes, but 4.5 h hemicellulose degradation were higher, 150 g treatment being the highest. The SDS-PAGE shows that exogenous enzymes might disappear rapidly in the rumen. In exp. 2, the objective was to investigate the effect of adding exogenous fibrolytic enzymes in combination with soluble protein on changes in ruminal enzyme activity, in situ bermudagrass hay degradation, and fermentation. In vitro trial, different exogenous enzyme doses (0-0.1%) were incubated with rumen fluid. It was found that ruminal CMCase and xylanase activities disappeared as the incubation time increased. However, when BSA or gelatin was incubated together with the enzyme, ruminal enzyme activities disappeared slowly. According to the estimate from enzyme kinetics model, when gelatin dose used was 0.20%, enzyme activities disappearance was reduced by 50%. In animal trial, two ruminally cannulated Holstein cows were used to compare the following treatments: without or with adding 80 g fibrolytic enzyme, 160 g gelatin, 80 g enzyme and 160 g gelatin, and 160 g enzyme and 160 g gelatin divided twice (8:00 a.m. and 12:00). The results show that treatments decreased ruminal pH value after feeding, but ruminal ammonia, reducing sugar, protein, and non-ammonia non-protein nonamino acid contents were increased by treatments. Ruminal acetic acid proportion and total VFA concentrations were also increased by treatments, adding enzyme and gelatin simultaneously being higher than adding enzyme alone. Adding twice produced more ruminal total VFA than once. Ruminal CMCase, xylanase, and avicelase activities were increased and proteinase activity was decreased by adding enzyme and gelatin together than adding enzyme alone, and the effect was greater for adding twice treatment. Treatments increased in situ ruminal DM, NDF, ADF, and hemicellulose degradation of bermudagrass hay after feeding, addition of enzyme and gelatin together being the highest. SDS-PAGE results show that adding gelatin could retard enzyme disappearance in the rumen, and the effect of twice addition was significant. In conclusion, the results of this thesis research show that exogenous fibrolytic enzymes seemed to be degraded by ruminal proteinase and disappeared rapidly in the rumen. Thus, the efficacy on the improvement of ruminal fiber degradation did not prolong. Gelatin appeared to react with ruminal proteinase, and reduced the disappearance of exoganous enzymes in the rumen. Addition of enzyme and gelatin in combination to rumen could prolong enzyme activities in the rumen, with simultaneous enhancement in ruminal fiber degradation and fermentation. |
