为确保我国牛羊肉和奶源自给率目标的实现，优质饲草年产量需达到1.2亿吨以上，但目前仍有近5000万吨的缺口。苜蓿作为国内重要的优质饲草，被誉为“牧草之王”，尽管其产量逐年提高，但仍无法满足国内市场需求。在此背景下，草木樨作为一种适应性强、饲用成本低且营养价值高的牧草，展现出良好的潜力。然而，草木樨中含有较高水平的抗营养因子——香豆素，这一成分会影响草木樨适口性，家畜过量食用易引起皮下出血等症状，进而影响了草木樨的饲用价值。因此，本研究旨在研究草木樨替代苜蓿喂养湖羊的可行性，研究草木樨对湖羊的生长产性能、肉品质、瘤胃微生物区系及瘤胃上皮的影响。同时，本研究还将挖掘草木樨在湖羊体内的降解过程，并探讨瘤胃微生物对其香豆素的降解作用及机制。将进一步验证预处理草木樨对香豆素降解效率的影响，为香豆素的有效降解提供技术方案。这将为草木樨作为优质饲草的广泛应用提供理论支持和技术保障。

草木樨对湖羊生产性能、肉品质的影响及瘤胃香豆素降解与代谢分析

试验旨在研究草木樨干草对湖羊生产性能、肉品质的影响及瘤胃香豆素降解与代谢分析。选取4月龄体重为30 ± 3.30 kg湖羊20只，随机分为2组（n=10），分别饲喂含苜蓿干草和草木樨干草的全混合日粮，精粗比均为5：5，预饲期为7 d，试验期80 d。结果表明：与苜蓿组相比，饲喂草木樨显著降低了湖羊的终末体重、体增重、平均日采食量和总采食量（P < 0.05）；草木樨组的胴体重和屠宰率显著低于苜蓿组（P < 0.05）；且草木樨组的粗蛋白、中性洗涤纤维和酸性洗涤纤维的表观消化率显著低于苜蓿组（P < 0.05）。在肉品质方面，结果显示草木樨组的湖羊背最长肌、腿肌和臀中肌的剪切力均显著高于苜蓿组（P < 0.05）；通过测定肌肉脂肪酸含量发现，草木樨组的花生四烯酸和饱和脂肪酸显著低于苜蓿组，油酸和单不饱和脂肪酸显著高于苜蓿组（P < 0.05）。在瘤胃发酵特征方面，饲喂草木樨显著增加了氨态氮、总挥发性脂肪酸和丁酸摩尔百分比（P < 0.05）。通过瘤胃乳头相关参数及瘤胃上皮组织形态学的测定，草木樨组的乳头密度、乳头单位面积和乳头总面积显著低于苜蓿组（P < 0.05）；草木樨组的瘤胃乳头角质层显著高于苜蓿组（P < 0.05），HE切片中可观察到草木樨组角质层内存在有核细胞，表明瘤胃上皮角化不全。β多样性结果显示，饲喂草木樨对β多样性有影响（P < 0.05）。LEfSe分析结果表明，在门水平上草木樨组中厚壁菌门（Firmicutes）的相对丰度显著高于苜蓿组，变形菌门（Proteobacteria）的相对丰度显著低于苜蓿组（P < 0.05）。属水平上瘤胃球菌（Ruminococcus）、NK4A214 group、Candidatus Saccharimonas和Christensenellaceae_R-7_group的相对丰度显著高于苜蓿组（P < 0.05）。此外，该研究还发现，香豆素在瘤胃液中被降解。通过比对草木樨组和苜蓿组的差异代谢产物，香豆素的潜在代谢产物有6种：2-甲氧基苯甲酸、对羟基苯甲醛、2-甲氧基苯乙酸、反式肉桂酸、2-甲氧基-4-乙烯基苯酚和间甲基苯酚。综上，草木樨对湖羊生长有负面影响，但有助于改善其肌肉脂肪酸组成，从而影响肉质的口感和营养价值，香豆素在瘤胃中被有效降解。

比较研究瘤胃真菌与细菌对草木樨香豆素的降解效果

本章旨在深入比较瘤胃真菌与细菌对草木樨香豆素的降解效果，探讨不同微生物群落在香豆素降解中的作用及机制。通过比较这两类微生物群落的降解效果，分析其对草木樨香豆素的降解机制。在本研究中，通过对完整瘤胃微生物群和富集瘤胃真菌的降解能力进行比较，发现完整瘤胃微生物群的香豆素降解比例显著高于富集瘤胃真菌组（P < 0.05）。尽管如此，二者的香豆素降解率/干物质降解率比值却相近。同时比较了富集瘤胃真菌与富集瘤胃细菌的体外培养实验结果。结果表明，富集瘤胃细菌的总产气量、底物消失率及香豆素降解率均显著高于富集瘤胃真菌组（P < 0.05）。考虑到完整瘤胃微生物群中细菌数量高出富集真菌3个数量级，表明瘤胃真菌对草木樨香豆素的降解效率较高。当调整到相同数量的微生物数量水平后，瘤胃真菌在香豆素降解中的表现显著优于细菌组（P < 0.05），证实了真菌群落对香豆素降解能力更强。进一步采用Pecoramyces sp. F1真菌进行体外培养，研究其在不同浓度香豆素（0 mmol/L和3 mmol/L）处理下的转录组和代谢途径的变化。结果表明，香豆素处理后，瘤胃真菌的转录组发生了显著变化。共有390个基因显著上调，表明该真菌在香豆素降解过程中发生了基因表达的变化，这些基因与碳水化合物代谢、次生代谢物的生物合成以及核糖体生物发生等过程密切相关。UPLC-MS分析进一步显示，邻香豆酸在发酵的第3天达到高峰，随后逐步转化为草木樨酸。基于这些代谢产物的变化，可以推测香豆素的降解过程是通过真菌中的水解酶将香豆素转化为邻香豆酸，并通过NAD(P)H氧化还原酶进一步转化为草木樨酸。这一发现不仅首次揭示了瘤胃真菌在香豆素降解中的作用，还深入阐明了香豆素降解的关键酶类和代谢途径。

瘤胃真菌发酵液青贮对草木樨饲用价值的影响

本章聚焦于瘤胃真菌发酵液对草木樨青贮效果及体外发酵特性的影响，香豆素降解与营养成分变化。结果表明，随时间延长，各组香豆素降解率渐升，瘤胃真菌组从青贮第1天起显著高于对照组（P < 0.05），60 d时瘤胃真菌组降解率达78.67%，较对照组提高15.99%。草木樨干物质、粗蛋白、可溶性碳水化合物和淀粉含量随处理时间降低，瘤胃真菌组在不同时间点的干物质、粗蛋白、可溶性碳水化合物和淀粉含量与对照组有显著差异（P < 0.05）。而瘤胃真菌组与对照组在青贮过程中的中性洗涤纤维与酸性洗涤纤维含量的变化较小。同时，营养成分保留率随时间递减，瘤胃真菌组在多个时间点的干物质回收率、粗蛋白、可溶性碳水化合物和淀粉保留率显著高于对照组（P < 0.05）。由青贮发酵品质的结果可知，处理组发酵液与对照组在pH、氨态氮和乳酸含量上差异显著（P < 0.05）。感官评价方面，对照组总评12分，等级良好；瘤胃真菌组总评20分，等级优秀。体外瘤胃发酵中，处理组与对照组在pH值、干物质降解率、氨态氮和微生物蛋白浓度方面差异显著（P < 0.05），瘤胃真菌组表现更佳。处理组总挥发性脂肪酸显著高于对照组（P < 0.05），瘤胃真菌组最高。瘤胃真菌组产气量显著高于其他组，处理组产甲烷量高于对照组，但瘤胃真菌组48 h甲烷累计占比量显著低于其他组（P < 0.05）。综上，瘤胃真菌能有效降解草木樨香豆素，改善营养成分保留率、发酵品质和体外发酵特性，降低甲烷占比，具有良好应用前景。

综上所述，香豆素通过影响瘤胃上皮吸收，限制了湖羊的生长，但改善了肉品质（如肌肉脂肪酸组成）。瘤胃液能降解香豆素，其中瘤胃真菌降解效率更高，选用Pecoramyces sp. F1深入研究，发现其通过水解酶和NAD(P)H氧化还原酶降解香豆素。用瘤胃真菌发酵液青贮草木樨，可有效降解香豆素，提升青贮效果与体外发酵特性，为草木樨作为饲草的应用提供理论依据。

In order to ensure the realization of the self-sufficiency rate of beef, mutton and milk in China, the annual output of high-quality forage grass needs to reach more than 120 million tons, but there is still a gap of nearly 50 million tons. As an important forage in China, alfalfa is known as the "king of forage". Although its output increases year by year, it still cannot meet the demand of the domestic market. In this context, Melilotus albus showed good potential as a kind of forage with strong adaptability, low feeding cost and high nutritional value. However, Melilotus albus contains a high level of anti-nutrient factor, coumarin, which will affect the palatability of Melilotus albus, and cause symptoms such as subcutaneous bleeding in livestock, which will affect the feeding value of Melilotus albus. Therefore, the purpose of this study was to explore the feasibility of Melilotus albus instead of alfalfa feeding Hu sheep, and to study the effects of coumarin on the growth performance, meat quality, rumen microflora and rumen epithelium of Hu sheep. At the same time, this study will also explore the degradation process of Melilotus albus in Hu sheep, and explore the effect and mechanism of rumen microorganisms on its coumarin degradation. The effect of pretreatment of Melilotus albus on the degradation efficiency of coumarin will be further verified, and a technical scheme will be provided for the effective degradation of coumarin. This will provide theoretical support and technical guarantee for the wide application of Melilotus albus as a high quality forage.

Effects of Melilotus albus on growth performance, meat quality and rumen coumarin degradation and metabolism of Hu sheep

The experiment was conducted to study the effects of Melilotus albus on growth performance, meat quality and rumen coumarin degradation and metabolism of Hu sheep. Twenty 4-month-old Hu sheep with a body weight of 30 ± 3.30 kg were randomly divided into 2 groups (n = 10) and fed with mixed diets containing alfalfa hay and Melilotus albus hay, respectively, with a ratio of concentrate to crude of 5: 5. The pre-feeding period lasted for 7 days, and the experimental period lasted for 80 days. The results showed as follows: Compared with alfalfa hay group, feeding Melilotus albus significantly decreased the final body weight, body gain, average daily feed intake and total feed intake of Hu sheep (P < 0.05). The carcass weight and slaughter rate of Melilotus albus group were significantly increased (P < 0.05). The apparent digestibility of crude protein, neutral detergent fiber and acid detergent fiber in Melilotus albus group was significantly decreased (P < 0.05). In terms of meat quality, the results showed that the shear force of longissimus dorsi muscle, leg muscle and gluteus media muscle of Hu sheep in Melilotus albus group was significantly increased (P < 0.05). Through the determination of muscle fatty acid content, it was found that arachidonic acid and saturated fatty acid in Melilotus albus group were significantly decreased, oleic acid and monounsaturated fatty acid were significantly increased (P < 0.05). In terms of rumen fermentation characteristics, feeding Melilotus albus significantly increased the percentage of ammonia nitrogen, total volatile fatty acids and butyric acid molar was decreased (P < 0.05). According to rumen papillary-related parameters and rumen epithelial histomorphology, the nipple density, nipple unit area and total nipple area of Melilotus albus group were significantly increased (P < 0.05). Moreover, compared with the control group, the stratum corneum of the rumen papilla in the osmanthus group was significantly decreased (P < 0.05). HE sections observed that nuclear cells were present in the stratum corneum of the osmanthus group, indicating incomplete keratinization of the rumen epithelium. β diversity results showed that feeding Melilotus albus had an effect on β diversity (P < 0.05). The results of LEfSe analysis showed that at the phylum level, the relative abundance of Firmicutes in the alfalfa group was significantly increased, and the relative abundance of Proteobacteria was significantly deceased (P < 0.05). At the genus level, the relative abundances of Ruminococcus, NK4A214 group, Candidatus Saccharimonas and Christensenellace_R7_group were significantly increased(P < 0.05). In addition, the study found that coumarin was degraded in rumen fluid. By comparing the metabolites of Melilotus albus group and alfalfa group, there were 6 potential metabolites of coumarin: p-hydroxybenzaldehyde, 2-methoxyphenylacetic acid, m-methylphenol, trans-cinnamic acid, 2-methoxy4-vinylphenol and 2-methoxybenzoic acid . In conclusion, Melilotus albus has a negative effect on the growth of Hu sheep, but helps to improve the fatty acid composition of muscle, thus affecting the taste and nutritional value of meat, and coumarin is effectively degraded in the rumen.

The degradation effects of rumen fungi and bacteria on Melilotus albus were compared

The purpose of this chapter is to compare the degradation effects of rumen fungi and bacteria on Melilotus albus, and to explore the role and mechanism of different microbial communities in the degradation of Melilotus albus. By comparing the degradation effect of these two microbial communities, the degradation mechanism of melilotus coumarin was analyzed. In this study, by comparing the degradation ability of intact rumen microflora and enriched rumen fungi, it was found that the proportion of coumarin degradation of intact rumen microflora was significantly increased compare to rumen fungi group (P < 0.05). Nevertheless, the coumarin/dry matter degradation ratio of the two was similar. The results of in vitro culture of enriched rumen fungi and enriched rumen bacteria were compared. The results showed that the total gas production, substrate loss rate and coumarin degradation rate of enriched rumen bacteria were significantly increased (P < 0.05). Considering that the number of bacteria in the complete rumen microflora was 3 orders of magnitude decreased , it indicated that rumen fungi had a higher degradation efficiency of coumarin. When adjusted to the same number of microorganisms, the performance of anaerobic fungi in the degradation of coumarin was significantly increased (P < 0.05), which confirmed that the fungal community had a stronger ability to degrade coumarin.Further, rumen fungi was cultured in vitro to study the transcriptomic and metabolic pathway changes of the fungi treated with different concentrations of coumarin (0 mmol/L and 3 mmol/L). The results showed that the transcriptome of rumen fungi was significantly changed after coumarin treatment. A total of 390 genes were significantly upregated, indicating that the gene expression of the fungus was changed during coumarin degradation, and these genes were closely related to carbohydrate metabolism, biosynthesis of secondary metabolites and ribosome biogenesis. UPLC-MS analysis further showed that o-coumaric acid reached its peak on the third day of fermentation, and then gradually converted to melilotic acid. Based on the changes of these metabolites, it can be inferred that the degradation process of coumarin is the conversion of coumarin to o-coumaric acid by hydrolase in fungi, and further conversion to melilotic acid by NAD(P)H oxidoreductase. This discovery not only revealed the role of rumen fungi in coumarin degradation for the

first time, but also clarified the key enzymes and metabolic pathways of coumarin degradation.

Effects of rumen fungi fermentation broth silage on feeding value of Melilotus albus

This chapter focuses on the effects of rumen fungi on the silage effect and in vitro fermentation characteristics, coumarin degradation and nutrient composition changes: The degradation rate of coumarin in rumen fungi group was significantly increased (P < 0.05), and the degradation rate of rumen fungi group was 78.67% at 60 days, which was 15.99% increased. The contents of dry matter, crude protein, soluble carbohydrate and starch in Melilotus albus decreased with the treatment time, and the contents of dry matter, crude protein, soluble carbohydrate and starch in rumen fungi group were significantly different from those in control group at different time points (P < 0.05). The contents of neutral and acid detergent fibers in rumen fungi group and control group changed little during silage. At the same time, the nutrient retention rate decreased with time, and the dry matter recovery, crude protein, soluble carbohydrate and starch retention rates in rumen fungi group were significantly increased compare to control group at multiple time points (P < 0.05). The results of silage fermentation quality showed that there were significant differences in pH, ammonia nitrogen and lactic acid contents between the treatment group and the control group (P < 0.05). In terms of sensory evaluation, the control group scored 12 points, the grade was good; The total score of rumen fungi group was 20 points, and the grade was excellent. In vitro rumen fermentation, there were significant differences in pH value, dry matter degradation rate, ammonia nitrogen and microbial protein concentration between the treatment group and the control group (P < 0.05), and the performance of rumen fungi group was better. Total volatile fatty acids in treatment group was significantly increased (P < 0.05), and the highest value was found in rumen fungi group. The gas production of rumen fungi group was significantly increased, and the methane production of treatment group was increased, but the cumulative methane fraction of rumen fungi group at 48 h was significantly increased (P < 0.05). In conclusion, rumen fungi can effectively degrade Melilotus albus, improve nutrient retention, fermentation quality and in vitro fermentation characteristics, and reduce the proportion of methane, which has a good application prospect.

In summary, coumarin limited the growth of Hu sheep by affecting rumen epithelial absorption, but improved meat quality (such as muscle fatty acid composition). Rumen fluid can degrade coumarin, and rumen fungi have higher degradation efficiency.Rumen fungi was selected for further study, and it was found that coumarin was degraded by hydrolase and NAD(P)H oxidoreductase. Using rumen fungi to silage Melilotus albus can effectively degrade coumarin, improve silage effect and in vitro fermentation characteristics, and provide theoretical basis for the application of Melilotus albus as forage.

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