添加不同水平黑曲霉对奶牛瘤胃体外发酵参数、甲烷排放及微生物区系的影响

doi:10.16431/j.cnki.1671-7236.2025.02.013

摘要/Abstract

摘要： 【目的】试验旨在研究黑曲霉(Aspergillus niger)对奶牛瘤胃体外发酵、甲烷(CH₄)排放及微生物区系的影响，为黑曲霉作为饲料添加剂在奶牛生产中的合理应用提供理论依据。【方法】采用体外瘤胃发酵装置，共分为4组：A(对照组)、B、C和D组，分别向每组发酵瓶(含人工饲料及培养液)中添加0、15、20、25 mg黑曲霉(孢子浓度≥1.0×10⁹/g)；每种处理设3次重复，另设3个空白组以校正气体和CH₄产量(空白组不含饲料，只有培养液)；在培养开始后的第3、6、9、12、24 h，测量产气量和CH₄产量；培养24 h后，分析发酵液的pH、氨态氮(NH₃-N)、微生物蛋白(MCP)、挥发性脂肪酸(VFA)以及瘤胃微生物群落多样性。【结果】与A组相比，B、C和D组的体外瘤胃发酵液pH显著升高(P＜0.05)，NH₃-N含量显著降低(P＜0.05)，且C和D组的MCP含量显著高于A组(P＜0.05)，而B组则显著低于其他3组(P＜0.05)。各组之间各种VFA含量、乙酸/丙酸比值以及总挥发性脂肪酸(TVFA)含量均无显著差异(P＞0.05)。A组CH₄产量占总产气量比值显著高于B、C和D组(P＜0.05)。16S rRNA测序表明，与A组相比，B组瘤胃微生物Alpha及Beta多样性指数无显著差异(P>0.05)。在瘤胃门水平前10种细菌中，其中变形菌门和拟杆菌门为奶牛瘤胃中的优势菌门，且A、B两组差异不显著(P＞0.05)；螺旋体菌门与丙酸和NH₃-N含量呈显著负相关(P＜0.05)；纤维杆菌门与pH呈显著正相关(P＜0.05)，与戊酸、丁酸、乙酸、CH₄和NH₃-N含量呈显著负相关(P＜0.05)。在瘤胃属水平前10种细菌中，其中普氏菌属和理研菌科RC9为奶牛瘤胃中的优势菌属；与A组相比，B组琥珀酸弧菌属的相对丰度显著降低(P＜0.05)。理研菌科RC9与NH₃-N含量呈显著正相关(P＜0.05)；拟杆菌目RF16细菌属与戊酸、丁酸、乙酸、CH₄和NH₃-N含量呈显著正相关(P＜0.05)。与A组相比，B组细菌和产甲烷菌的绝对含量显著增加(P＜0.05)；B组原虫的绝对含量显著减少(P＜0.05)。【结论】黑曲霉的添加在改善奶牛瘤胃发酵环境的同时，通过调节瘤胃内微生物群落结构，影响VFA、NH₃-N、MCP的产生和CH₄的排放，研究结果揭示了微生物干预措施对提升奶牛健康和生产效率潜在的复杂作用机制。

关键词: 黑曲霉; 瘤胃体外发酵; 甲烷排放; 微生物区系

Abstract: 【Objective】 The purpose of this study was to study the effects of Aspergillus niger on rumen fermentation in vitro,methane emission and microflora of dairy cows,and provide theoretical basis for the rational application of Aspergillus niger as a feed additive in dairy cow production. 【Method】 In vitro rumen fermentation device was used to divide into four groups:Group A (control group),groups B,C and D.0,15,20 and 25 mg of Aspergillus niger (spore concentration ≥1.0×10⁹ /g) were added to the fermentation bottle (including artificial feed and culture medium) of each group,respectively.Each treatment was repeated 3 times,and 3 blank groups were set up to correct the gas and methane content (blank group did not contain feed,only culture medium).Gas and methane production were measured at 3,6,9,12 and 24 h after the beginning of culture.After 24 hours of culture,the pH,ammonium nitrogen (NH₃-N),microbial protein (MCP),volatile fatty acid (VFA) and microbial community diversity of rumen were analyzed. 【Result】 Compared with group A,the pH of rumen fermentation fluid of groups B,C and D were significantly increased (P＜0.05),while the NH₃-N content was significantly decreased (P＜0.05).The MCP content in groups C and D was significantly higher than that in group A (P＜0.05),while that in group B was significantly lower than that in the other three groups (P＜0.05).There were no significant differences in VFA content,acetic acid/propionic acid ratio and TVFA content among all groups (P＞0.05).The ratio of CH₄ production to total gas production in group A was significantly higher than that in groups B,C and D (P＜0.05).16S rRNA sequencing showed that there was no significant difference in rumen microbial Alpha and Beta diversity index between groups B and A (P>0.05).Among the top 10 bacteria in rumen at phylum level,Proteobacteria and Bacteroidetes were the dominant bacteria in rumen of dairy cows,and there was no significant difference between groups A and B (P＞0.05).Spirochaetota had a significant negative correlation with PA and NH₃-N content (P＜0.05).Fibrobacterota was positively correlated with pH (P＜0.05),and negatively correlated with VA,BA,AA,CH₄ and NH₃-N contents (P＜0.05).Among the top 10 bacteria in rumen at genus level,Prevotella and Rikenellaceae_RC9_gut_group were the dominant bacteria in the rumen of dairy cows.Compared with group A,the relative abundance of Succinivibrio in group B was significantly decreased (P＜0.05).Rikenellaceae_RC9_gut_group was positively correlated with NH₃-N content (P＜0.05).There was a significantly positive correlation between the contents of VA,BA,AA,CH₄ and NH₃-N with Bacteroidales_RF16_group (P＜0.05).Compared with group A,the absolute content of bacteria and methanogens in group B was significantly increased (P＜0.05).The absolute content of protozoa in group B was significantly reduced (P＜0.05). 【Conclusion】 Besides improving the rumen fermentation environment of dairy cows,the addition of Aspergillus niger affected the production of VFA,NH₃-N,MCP and methane emission by regulating the microbial community structure in rumen,revealing the potential complex mechanism of microbial intervention to improve the health and production efficiency of dairy cows.

Key words: Aspergillus niger; rumen fermentation in vitro; methane emission; microflora

中图分类号:

S816.7

李若楠, 王昊天, 李和平, 刘智勇, 刘洋, 钟凯. 添加不同水平黑曲霉对奶牛瘤胃体外发酵参数、甲烷排放及微生物区系的影响[J]. 中国畜牧兽医, 2025, 52(2): 624-635.

LI Ruonan, WANG Haotian, LI Heping, LIU Zhiyong, LIU Yang, ZHONG Kai. Effects of Supplemental Levels of Aspergillus niger on Rumen Fermentation Parameters in vitro,Methane Emission and Microflora in Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 624-635.

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