饲喂柠条对滩羊血清指标及瘤胃、结肠菌群结构的影响

doi:10.3864/j.issn.0578-1752.2023.14.015

摘要/Abstract

摘要：

【目的】研究日粮中含有10%柠条（Caragana korshinskii Kom.，CK）对滩羊血脂、免疫指标及瘤胃、结肠菌群的影响，为CK替代部分饲料饲喂滩羊提供理论依据。【方法】将12只平均初始体重相似的四月龄滩羊母羔（27.5±3.32 kg），随机分为两组，每组6只。对照组（NC）饲喂基础日粮（30%精料和70%苜蓿干草），试验组（CK）用10% CK替代等量苜蓿干草，自由饮水。两组日粮营养水平相似，CK的纤维含量略高于苜蓿。预试期10 d，正试期60 d。试验结束后采集颈静脉血样品用于检测血液生化指标（血脂因子及免疫因子）；采集瘤胃液、结肠内容物，提取DNA，并利用16S rRNA测序检测瘤胃、结肠内菌群群落多样性，并通过重组质粒，比如月形单胞菌属（Selenomona）、普雷沃氏菌属（Prevotella）、布劳特氏菌属（Blautia）、脱硫弧菌属（Desulfovibrio）、瘤胃球菌属（Ruminococcus）、拟杆菌属（Bacteroides），构建标准曲线，利用实时荧光定量PCR（qPCR）对重要菌属进行绝对定量检测。【结果】正试期结束时，两组滩羊的末重和日均采食量相似，无显著性差异；检测发现，相比于NC组，CK组滩羊体内脂质消化吸收和代谢状况的相关指标呈现不同程度降低，如CK组血清甘油三酯（TRIG）含量显著降低（P<0.05），极低密度脂蛋白（VLDL）含量极显著降低（P<0.01），低密度脂蛋白（LDL）含量有降低的趋势（0.05<P<0.1）。炎性因子和脂解因子白介素6（IL-6）浓度显著增加（P<0.05），炎性细胞因子干扰素γ（IFN-γ）浓度趋于降低（P=0.058）；CK组结肠细菌群落Shannon多样性指数极显著增加（P<0.01），瘤胃细菌群落Chao1指数趋于降低（0.05<P<0.1）；CK组瘤胃内颤螺菌属（Oscillospira）相对丰度趋向降低（P=0.07）；阿克曼菌属（Akkermansia）相对丰度显著降低（P<0.05）；结肠内碳水化合物降解相关菌拟杆菌属、有害菌密螺旋体属（Treponema）的相对丰度趋向降低（P=0.059，P=0.061），与肥胖、便秘、免疫相关的产丁酸菌颤螺菌属的相对丰度显著增加（P<0.05）；两组间瘤胃和结肠内细菌群落均在脂质代谢和其他次级代谢产物的生物合成能力上有显著差异（P<0.05）。皮尔森相关性分析表明，TRIG相对丰度与假丁酸弧菌（Pseudobutyrivibrio）等属（降解纤维也是产丁酸）的相对丰度呈显著正相关（P<0.05）。【结论】柠条部分替代苜蓿对滩羊生长性能无显著影响，但可以调节胃肠道菌群结构，通过改变胃肠道细菌菌群的组成降低了滩羊血液中TRIG、LDL和VLDL的浓度，参与了宿主的脂代谢过程。

关键词: 血脂, 16S rRNA, 瘤胃菌群, 结肠菌群

Abstract:

【Objective】 This study aimed to examine the effects of Caragana korshinskii Kom. (CK) at the rate of 10% of the diet on blood lipids, immune indicators, and rumen and colon microbiota of Tan sheep, so as to provide a theoretical basis for CK being partial Tan sheep forage. 【Method】 Twelve Tan sheep (4-month-old) with an initial average weight of 27.5 ± 3.32 kg were randomly divided into 2 groups (6 in each group): NC group was fed with 30% concentrate and 70% alfalfa; CK group, with 30% concentrate, 60% alfalfa and 10% CK, and drinking water ad libitum. The nutrient composition of the two groups were similar, but the fiber content of CK was slightly higher than that of alfalfa. The preparatory experiment was in 10 days and the formal experiment was lasted for 60 days. At the end of the 60 days, jugular vein blood was collected to measure blood biochemical indexes (blood lipids and immune factors) and rumen fluid, and colon content were collected to detect the changes of flora in rumen and colon, respectively, by 16S rRNA sequencing method. Through construction of the recombinant plasmid, such as Selenomona, Prevotella, Blautia, Desulfovibrio, Ruminococcus and Bacteroides, the absolute quantitative of important genera were detected by real-time quantitative PCR (qPCR) comparing to its standard curve, respectively. 【Result】 At the end of the trial period, the end weight and daily average feed intake of Tan sheep were similar between two groups. Compared with NC group, the indexes related to the lipid absorption and digestion in CK group decreased, such as the content of serum triglyceride (TRIG, P<0.05) and very low density lipoprotein (VLDL, P<0.01) decreased markedly, while the concentration of low density lipoprotein (LDL) tended to decrease significantly (0.05<P<0.1). The inflammatory and lipolytic factor, such as interleukin-6 (IL-6), increased significantly (P<0.05) whereas the inflammatory factor, γ-Interferon (IFN-γ) marked decreased tendentiously (P=0.058). By analyzing microflora of rumen fluid and colon contents, adding CK to diet increased community Shannon diversity index of colonic bacteria communities significantly (P<0.01), while tended to decrease the Chao1 index in rumen (0.05<P<0.1). Compared with NC group, the relative abundance of Oscillospira under CK decreased (P=0.07), while that of Akkermansia notable under CK increased (P<0.05) in rumen. The relative abundance of harmful bacteria, like Treponema, and carbohydrate degradation related Bacteroides significantly decreased (P=0.059, P=0.061), but that of Oscillospira, which a butyric acid producing bacteria related to obesity, constipation and immunity, gained markedly (P<0.05) in colon under CK. It was evidently deduced that the bacterial communities in rumen and colon had significant difference on the capacities for lipid metabolism and biosynthesis of other secondary metabolite between two groups (P<0.05). Based on Pearson correlation analysis, it was showed that the level of serum TRIG was positively correlated with rumen and colonic Pseudobutyrivibrio (P<0.05), which was both fiber degrading bacteria and butyric acid producing bacteria. 【Conclusion】 There was no significant effect of CK partial substitution for alfalfa on the growth performance of Tan sheep, but could reduce the concentrations of serum TRIG, LDL and VLDL through affecting the gastrointestinal tract microbiota, and then participate in the lipid metabolism of the host.

Key words: blood lipids, 16S rRNA, rumen microbiota, colon microbiota

韩晶, 王小琪, 段子渊. 饲喂柠条对滩羊血清指标及瘤胃、结肠菌群结构的影响[J]. 中国农业科学, 2023, 56(14): 2812-2827.

HAN Jing, WANG XiaoQi, DUAN ZiYuan. Effects of Caragana korshinskii Kom. on Serum Indexes, Rumen and Colon Microbiota of Tan Sheep[J]. Scientia Agricultura Sinica, 2023, 56(14): 2812-2827.

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项目 Item	组别 Group
项目 Item	NC组 NC group	CK组 CK group
成分 Ingredient
苜蓿 Alfalfa	70.0	60.0
柠条 Caragana korshinskii Kom		10.0
精料 Commercial concentrate	30.0	30.0
合计 Total	100.0	100.0
营养水平 Chemical composition
干物质Dry matter	90.4	90.4
有机物Organic matter	88.4	89.0
粗蛋白质Crude protein	15.5	14.9
粗脂肪Ether extract	2.0	2.0
酸性洗涤纤维Acid detergent fiber	31.1	32.7
中性洗涤纤维Neutral detergent fiber	49.7	51.8
总能Gross energy (MJ·kg^-1)	15.8	16.0

菌属 Genus	引物 Primers（5'to3'）	扩增长度 Amplicon length (bp)	参考文献 Reference
月形单胞菌属 Selenomonas	F：TGAGTGAAGAAGGGTTTCGGC	511	[21]
月形单胞菌属 Selenomonas	R：TTGAGTTTCAGTCTTGCGACCG	511	[21]
瘤胃球菌属 Ruminococcus	F：GAGTGAAGTAGAGGTAAGCGGAATTC	243	[22]
瘤胃球菌属 Ruminococcus	R：GCCGTACTCCCCAGGTGG	243	[22]
脱硫弧菌属 Desulfovibrio	F：CCGTAGATATCTGGAGGAACATCAG	135	[23]
脱硫弧菌属 Desulfovibrio	R：ACATCTAGCATCCATCGTTTACAGC	135	[23]
布劳特氏菌属 Blautia	F：TCTGATGTGAAAGGCTGGGGCTTA	250	[24]
布劳特氏菌属 Blautia	R：GGCTTAGCCACCCGACACCTA	250	[24]
拟杆菌属 Bacteroides	F：CTGAACCAGCCAAGTAGCG	226	[25]
拟杆菌属 Bacteroides	R：CCGCAAACTTTCACAACTGACTTA	226	[25]
普雷沃氏菌属 Prevotella	F：GGTTCTGAGAGGAAGGTCCCC	121	[26]
普雷沃氏菌属 Prevotella	R：TCCTGCACGCTACTTGGCTG	121	[26]

项目 Item	瘤胃Rumen			结肠Colon
项目 Item	AR组 AR group	BR组 BR group	P值 P value	AG组 AG group	BG组 BG group	P值 P value
好氧型Aerobic	0.033±0.015	0.016±0.011	0.041	0.085±0.073	0.075±0.030	0.589
厌氧型Anaerobic	0.842±0.025	0.880±0.034	0.065	0.754±0.087	0.724±0.025	0.394
兼性厌氧型Facultatively anaerobic	0.034±0.025	0.025±0.011	0.818	0.004±0.001	0.003±0.001	0.394
生物膜形成Forms biofilms	0.051±0.013	0.041±0.014	0.240	0.099±0.084	0.067±0.029	0.937
革兰氏阳性Gram positive	0.442±0.071	0.467±0.097	0.818	0.635±0.063	0.705±0.070	0.132
革兰氏阴性Gram negative	0.558±0.071	0.533±0.097	0.818	0.365±0.063	0.295±0.070	0.132
潜在致病性Potentially pathogenic	0.648±0.072	0.638±0.638	0.818	0.450±0.076	0.385±0.050	0.180

菌属 Genus	瘤胃Rumen			结肠Colon
菌属 Genus	AR组 AR group	BR组 BR group	P值 P value	AG组 AG group	BG组 BG group	P值 P value
脱硫弧菌属 Desulfovibrio	4.75±0.37（×10⁴）	4.19±0.21（×10⁴）	0.01	3.80±0.20（×10³）	3.83±0.13（×10³）	0.83
瘤胃球菌属 Ruminococcus	6.55±0.82（×10⁶）	6.77±0.30（×10⁶）	0.09	7.85±0.66（×10⁷）	7.80±0.38（×10⁷）	0.64
拟杆菌属Bacteroides	4.18±0.72（×10⁴）	4.45±0.57（×10⁴）	0.52	7.00±0.26（×10⁶）	6.88±0.10（×10⁶）	0.36
普雷沃氏菌属 Prevotella	6.88±0.29（×10⁶）	7.17±0.35（×10⁶）	0.19	7.22±0.28（×10⁷）	6.98±0.04（×10⁷）	0.08
月形单胞菌属 Selenomonas	4.86±0.18（×10⁴）	5.43±0.42（×10⁴）	0.02	5.38±0.22（×10⁵）	5.24±0.14（×10⁵)	0.26
布劳特氏菌属 Blautia	4.77±0.22（×10⁴）	4.61±0.26（×10⁴）	0.29	5.76±0.14（×10⁵）	5.49±0.22（×10⁵)	0.09