肉羊产业是畜牧业的重要组成部分，其经济效益受到品种、饲料、环境和管理等因素的影响，且肉羊产业中饲料成本约占养殖成本的60%~70%。蚕豆皮作为蚕豆副产物之一，不仅含有多种常规营养物质，如膳食纤维、粗蛋白、微量元素等，还含有原花青素生物活性物质，可以作为一种比较理想的非常规饲料原料使用。课题组前期在等能等氮的基础上，分别用基础日粮和添加10%、20%、30%蚕豆皮的日粮饲喂湖羊，饲养试验结果表明添加30%蚕豆皮日粮组的湖羊生长性能显著提高。因此，本研究应用HE、RT-qPCR和高通量测序等研究方法，深入探究日粮添加30%蚕豆皮对育肥湖羊肠道形态、免疫屏障基因表达、消化酶活性、肠道微生物，以及肌肉品质、肌纤维特性和肌肉氨基酸组成的影响，进一步阐明日粮添加蚕豆皮改善湖羊生产性能的潜在机制，为开发利用蚕豆皮资源，提高畜牧业经济效益提供科学依据。

1日粮添加蚕豆皮对湖羊小肠形态、免疫屏障功能、消化酶活性和微生物菌群的影响

本试验旨在研究日粮添加30%蚕豆皮对育肥湖羊小肠形态、免疫屏障相关基因表达水平、消化酶活性和微生物菌群多样性、相对丰度及功能的影响。试验选取30只4月龄体重相近（27±2.0kg）湖羊公羊，平均分为2个处理组，每处理组3个重复共15只羊。分别饲喂基础日粮（对照组，CON）和添加30%蚕豆皮日粮（蚕豆皮组，BBS）。试验期60天，含10天预饲期。试验结束后每组各选5只羊屠宰，采集小肠组织及其内容物，应用HE染色观察小肠组织形态，RT-qPCR检测空肠紧密连接蛋白Claudin-1、Occludin、MUC-2、ZO-1和炎症因子IL-6、IL-10、IL-1β、TNF-α mRNA表达量；生化试剂盒检测小肠消化酶活性，高通量测序检测空肠微生物菌群多样性、相对丰度和功能。HE结果表明，与对照组相比，BBS组湖羊十二指肠组织形态无显著变化（P>0.05），回肠绒毛高度和绒毛高度/隐窝深度值显著升高（P<0.05），隐窝深度有下降趋势（P=0.074）。RT-qPCR结果表明：BBS组空肠紧密连接蛋白Claudin-1、Occludin、MUC-2、ZO-1 mRNA表达量无显著变化（P>0.05）；促炎因子IL-1β的mRNA表达量显著降低（P<0.05），IL-6、IL-10和TNF-α mRNA表达量无显著变化（P>0.05）。生化结果表明：小肠α-淀粉酶、胰蛋白酶、脂肪酶活性无显著变化（P>0.05）。微生物测序结果表明：BBS组Shannon指数、Chao1指数和Observed_otus指数显著降低（P<0.05）；门水平上，变形菌门相对丰度显著升高（P<0.05），厚壁菌门、髌骨细菌门和厚壁菌门/拟杆菌门值显著降低（P<0.05）；属水平上，BBS组糖单胞菌属、糖酵解菌属等的相对丰度显著降低（P<0.05）；BBS组丰度差异显著类群降低，且增强了菌群在糖、脂肪和蛋白质代谢途径的富集。因此，日粮添加蚕豆皮可以改善育肥湖羊小肠组织形态，抑制炎症因子表达，降低空肠微生物多样性，抑制有害菌增殖，增强机体糖、脂肪和蛋白质代谢。

2日粮添加蚕豆皮对湖羊肌肉品质、肌纤维特性和氨基酸组成的影响

本试验旨在研究日粮添加蚕豆皮对育肥湖羊肌肉品质、肌纤维组织形态和肌肉氨基酸组成影响。试验分组和试验过程同试验一，试验结束后采集湖羊背最长肌组织。应用pH计、肉色仪、肌肉嫩度仪、压力仪、游标卡尺和硫酸绘图纸分别测定肌肉pH、肉色、剪切力、失水率、GR值和眼肌面积，应用HE染色观察并计算肌纤维横截面积、直径和密度，应用全自动氨基酸分析仪测定肌肉氨基酸组成。结果表明，与对照组相比，BBS组眼肌面积显著增高（P<0.05）；肌纤维横截面积显著降低（P<0.05），肌纤维密度显著增高（P<0.05）；Glu、Arg和NEAA含量显著增高（P<0.05），Val（P=0.086）和EAA（P=0.071）含量较对照组有上升趋势。因此，日粮添加30%蚕豆皮能够提高肌肉嫩度，改善氨基酸组成。

The mutton sheep industry is an important part of animal husbandry, and its economic benefits are affected by species, feed, environment and management, etc and the cost of feed in the mutton sheep industry accounts for about 60% to 70%. Broad bean skin(BBS), one of the by-products of broad bean, contains a variety of conventional nutrients, such as dietary fiber, crude protein, trace elements, etc., and also contains proanthocyanidin bioactive substances, which can be used as an ideal unconventional feed material. Our previous study of diet supplemented with different proportions of BBS, on the basis of isoenergy and nitrogen, showed that the diet supplemented with 30% BBS significantly improved the growth performance of Hu sheep. Therefore, this study applied HE, RT-qPCR and high-throughput sequencing methods to deeply explore the effect of dietary supplementation of 30% BBS on intestinal morphology, digestive enzyme activity, barrier function, microbial diversity, muscle quality, the effect of muscle fiber characteristics and muscle amino acid composition to further elucidate the potential mechanism of dietary BBS the performance of Hu sheep, which will provide a scientific basis for the development and utilization of BBS resources and the improvement of animal husbandry economic benefits.

1 Effects of dietary broad bean skin on small intestine morphology, digestive enzyme activity, immune barrier function and microbial flora in Hu sheep

The purpose of this experiment was to investigate the effects of dietary supplementation of 30% BBS on intestinal morphology, immune barrier-related gene expression levels, digestive enzyme activities, and microbial flora structure and function in fattening Hu sheep. Thirty 4-month-old male Hu sheep with similar body weight (27±2.0kg) were divided into 2 treatment groups(n=15), with 3 replicates in each group, fed with the basal diet (control group, CON) and the diet supplemented with 30% broad bean skin (broad bean skin, BBS), respectively. The trial period is 60 days including a pre-trial period of 10 days. At the end of the experiment, 5 sheep in each group were selected for slaughter, the small intestine tissue and its contents were collected. HE staining was used to observe the intestinal tissue morphology, and RT-qPCR was used to detect the mRNA expression of intestinal tight junction protein including Claudin-1, Occludin, MUC-2 and ZO-1, inflammatory factor including IL-6, IL-10, IL-1β and TNF-α, biochemical kit to detect digestive enzyme activity, high-throughput sequencing to detect jejunum microbiota diversity, relative abundance and function. HE results show that, compared with the control group, the duodenal tissue morphology of Hu sheep in the experimental group had no significant change (P>0.05), the ileal villus height and V/C value increased significantly (P<0.01), and the crypt depth showed a downward trend (P=0.074) in BBS group. RT-qPCR results show that, there was no significant change in the mRNA expression of Claudin-1, Occludin, MUC-2, ZO-1, IL-6, IL-10 and TNF-α(P>0.05), but the mRNA expression of pro-inflammatory factor IL-1β was significantly decreased (P<0.05) in BBS group. Biochemical results showed that, there was no significant change in the activities of α-amylase, trypsin and lipase in the small intestine (P>0.05). Jejunal microbial sequencing results show that, the Shanon index, Chao1 index and Observed_otus index were significantly decreased in BBS group(P<0.05); At the phylum level, the relative abundance of Proteobacteria increased significantly (P<0.05), while Firmicutes, Patescibacteria and F/B values decreased significantly in BBS group(P<0.05); At the genus level, the relative abundances of Candidatus_Saccharimonas, Saccharofermentans, etc, decreased significantly (P<0.05) in BBS group; Significant differences in the abundance of taxa in the BBS group were reduced, and the enrichment of bacteria in sugar, fat and protein metabolism pathways was enhanced. Therefore, adding broad bean skin to the diet can improve the small intestine tissue morphology and immune function, reduce the jejunal microbial diversity, inhibit harmful flora, and enhance the sugar, fat and protein metabolism of fattening Hu sheep.

2 Effects of dietary broad bean skin on muscle quality, muscle fiber tissue morphology and muscle amino acid composition in Hu sheep

The purpose of this experiment was to study the effect of adding broad bean skin to the diet on muscle quality, muscle fiber tissue morphology and muscle amino acid composition in fattening Hu sheep. The experiment grouping and experiment process were the same as experiment 1, after the experiment, the longissimus dorsi muscle of Hu sheep was collected. The pH meter, meat color meter, muscle tenderness meter, pressure meter, vernier caliper and sulfuric acid drawing paper were used to measure muscle pH, meat color, shear force, water loss rate, and GR value respectively, the cross-sectional area, diameter and density of muscle fibers were observed and calculated by HE staining, and the amino acid composition of muscle was determined by automatic amino acid analyzer. The results showed that the eye muscle area of BBS group was higher than that of the control group (P<0.05); In BBS group, the cross-sectional area of muscle fibers was lower (P<0.05), and the density of muscle fibers was higher than that of the control group (P<0.05); The contents of Glu, Arg and NEAA were higher (P<0.05), and the contents of Val (P=0.086) and EAA (P=0.071) tend to increase compared with the control group. Therefore, adding 30% BBS to the diet can increase meat yield and muscle tenderness, and improve the amino acid composition in Hu sheep.

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