大蒜油对山羊瘤胃生物氢化、乳脂肪酸组成及乳腺脂肪生成相关基因表达影响的研究
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摘要
随着经济的发展和人们生活水平的不断改善,食品中含有的可能有益于维持健康和抵御疾病的微量成分日益受到重视。高摄入量的饱和脂肪酸能够增加患心血管疾病的风险,然而单不饱和脂肪酸和多不饱和脂肪酸具有降低胆固醇的作用,共轭亚油酸(CLA)也具有潜在的对人体有益的生物学功能。反刍动物产品是人类饮食中CLA的主要来源,因此,改善乳中脂肪酸组成,特别是提高CLA的含量已成为反刍动物营养研究的热点之一。日粮脂肪在瘤胃中经历水解和生物氢化,转运和吸收的脂肪酸在乳腺中还经历脂肪酸重新合成以及去饱和反应,这些反应影响乳中脂肪酸组成。同时,随着促生长抗生素在动物饲料中的禁用,人们逐渐关注植物精油作为其替代物在动物生产上的应用。大蒜具有广谱抗菌性和降血脂的特性,可能对瘤胃生物氢化及乳脂肪酸组成产生影响。因此,本文研究了大蒜油作为瘤胃调控剂对山羊瘤胃发酵、生物氢化、乳脂肪酸组成以及乳腺脂肪生成相关基因表达的影响。
     1大蒜油在不同精粗比底物条件下对瘤胃微生物发酵的影响
     运用随机区组试验设计研究了4种水平的大蒜油(终浓度为0、30、300、3000mg/L发酵液)在4种精粗比底物(干草:精料为10:0、7:3、5:5、3:7)条件下对瘤胃微生物体外厌氧发酵24h的影响。结果显示,24h累计产气量随着大蒜油水平的增加而降低(P≤0.05),每隔3h产气量的动态变化表明大蒜油改变了体外发酵的模式。相对于对照组,大蒜油Gar3000增加了pH值(P≤0.05),而Gar30和Gar300对pH值没有影响(P>0.05)。NH3-N和MCP浓度的变化分别存在着对照组>Gar300>Gar30>Gar3000和Gar30>对照组>Gar3000>Gar300的关系。大蒜油降低了总VFAs浓度(P≤0.05),但是Gar30和Gar300之间没有差异(P>0.05);大蒜油Gar30和Gar300降低乙酸比例和乙酸/丙酸(P≤0.05),增加丙酸和丁酸比例(P≤0.05)。相对于其他发酵底物,大蒜油Gar30和Gar300在高精料下对24h累计产气量和总VFAs浓度(高粗料除外)的抑制作用较小,对NH3-N浓度、乙酸比例和乙酸:丙酸的降低以及丙酸比例的增加比较明显。试验表明,大蒜油抑制体外发酵存在剂量依赖效应,具有延缓体外发酵进程的特点,中等水平的大蒜油(例如30和300mg/L)在高水平精料底物下对瘤胃微生物发酵的调控效果更好。
     2瘤胃灌注大蒜油对山羊瘤胃发酵动力学、脂肪酸组成及生物氢化菌数量的影响
     2.1瘤胃灌注大蒜油对山羊采食量和血液生化指标的影响
     运用交叉设计试验研究了瘤胃灌注大蒜油(0.8g/d)30d对山羊采食量和血液生化指标的影响。结果显示,大蒜油降低了干物质采食量(P≤0.05),处理与采样天之间在采食量方面也存在互作效应(P≤0.05);大蒜油降低了试验期第3d和6d的干物质采食量(P<0.001和P=0.011),但是对其他天的采食量没有显著影响(P>0.05)。大蒜油降低了血清HDL-C、LDL-C和胆固醇浓度(P≤0.01)及血清脂肪酶活力(P=0.048),有增加甘油三酯浓度的趋势(0.050.1)。结果表明,瘤胃灌注大蒜油影响了山羊采食量,这种影响随着作用时间的延长而减弱;大蒜油有降血脂的特性,可能降低了甘油酯的水解。
     2.2瘤胃灌注大蒜油对山羊瘤胃发酵动力学的影响
     通过分析交叉设计试验中试验期第28、29和30d不同时间点的瘤胃发酵参数,研究了瘤胃灌注大蒜油(0.8g/d)对山羊瘤胃发酵动力学的影响。结果显示,大蒜油增加了饲喂前(0h)山羊瘤胃NH3-N和MCP浓度(Ps0.01),对pH值、总VFAs浓度、vFA组成比例及乙酸/丙酸均没有影响(P>0.1)。瘤胃发酵过程中,大蒜油对发酵参数不同时间点的平均效应与0h的作用效果类似(降低山羊pH值(P≤0.05)和总VFAs浓度(0.05     2.3瘤胃灌注大蒜油对山羊瘤胃脂肪酸组成、总细菌区系及生物氢化菌数量的影响
     运用交叉设计试验研究了瘤胃灌注大蒜油(0.8g/d)30d对山羊瘤胃脂肪酸组成、总细菌区系以及生物氢化细菌数量的影响。大蒜油降低了瘤胃C15、C16和SFA比例(P≤0.01),增加了C18、TVA、c9t11CLA、t10c12CLA和PUFA的比例(P≤0.05),有增加MUFA比例的趋势(0.050.1)。结果表明,大蒜油改变了山羊瘤胃脂肪酸组成,通过抑制B. proteoclasticus的生长而不影响Butyrivibrio菌来调控瘤胃生物氢化进程。
     2.4运用16S rRNA基因克隆序列分析瘤胃灌注大蒜油对山羊瘤胃Butyrivibrio菌多样性的影响
     采用特异性引物B395f/B812r筛选16S rRNA基因全序列克隆,建立了山羊瘤胃Butyrivibrio菌的16SrRNA基因克隆库。结果显示,在对照组285个总菌克隆中筛选出37个Butyrivibrio菌克隆(登录号:HQ326588-HQ326624),占瘤胃细菌的12.98%,在大蒜油处理组347个总菌克隆中筛选出38个Butyrivibrio菌克隆(登录号:JN008400-JN008437),占瘤胃细菌的10.95%。基于RDP数据库的序列分析显示,两个克隆库中的克隆归类为Pseudobutyrivibrio属、Butyrivibrio属以及Lachnospiraceae科中其他的属,在大蒜油组的山羊瘤胃Butyrivibrio菌克隆库中还发现部分克隆归类为Ruminococcus属及Ruminococcaceae科中其他的属。序列比对分析显示,在两个克隆库中,克隆数最多的是与Lachnospiraceae bacterium有高相似性的克隆,在对照组和处理组Butyrivibrio菌克隆库中分别占32.43%和21.05%,其次是与Pseudobutyrivibrio ruminis有高相似性的克隆,在对照组和处理组Butyrivibrio菌克隆库中分别占21.62%和15.79%;在大蒜油处理组Butyrivibrio菌克隆库中,与Ruminococcus flavefaciens有高相似性的克隆占了10.53%,而对照组中没有发现与R. flavefaciens有高相似性的克隆。Shannon指数在对照组和大蒜油组山羊瘤胃Butyrivibrio菌克隆库中分别为2.47和2.91。结果表明,Butyrivibrio菌占山羊瘤胃细菌的12.98%,包括Pseudobutyrivibrio属、Butyrivibrio属以及Lachnospiraceae科中其他的属,瘤胃灌注大蒜油降低了Butyrivibrio菌在瘤胃细菌重的比例,但是增加了其遗传多样性。
     3添加大蒜油对奶山羊乳脂肪酸组成及乳腺脂肪生成相关基因表达的影响
     3.1添加大蒜油对奶山羊采食量、血液生化指标及乳成分的影响
     24头早期泌乳中国萨能奶山羊随机分为4组(6头/组,2头/圈),研究其在玉米青贮自由采食条件下饲喂含不同大蒜油水平(0、0.57、1.14、1.71g/kg)的精料(0.79kg/d)30d对采食量、血液生化指标以及乳成分的影响。结果显示,精料的采食量没有受到影响,但是高水平大蒜油(1.35g/d)降低了玉米青贮的采食量(P≤0.05)添加大蒜油对血液生化指标没有显著作用(P>0.05),但是具有降低血清LDL-C和NEFA浓度的趋势(0.050.1)。结果表明,高水平的大蒜油降低了采食量,添加大蒜油对奶山羊血液生化指标和乳成分的调控作用很小。
     3.2添加大蒜油对奶山羊乳脂肪酸组成及乳腺脂肪生成相关基因表达的影响
     24头早期泌乳中国萨能奶山羊随机分为4组(6头/组,2头/圈),研究其在玉米青贮自由采食条件下饲喂含不同大蒜油水平(0、0.57、1.14、1.71g/kg)的精料(0.79kg/d)30d对奶山羊乳脂肪酸组成及乳腺脂肪生成相关基因(ACC、FAS、LPL、SCD、SREBP1和PPARγ)表达的影响。结果显示,添加大蒜油改变了试验期间乳脂肪酸组成,其中对d14-21乳脂肪酸影响比较明显。试验27d后,添加大蒜油降低了乳C14:0、C16:0和SFA脂肪酸的比例(PS0.05),增加了C18:0、C18:1、C18:3、c9t11CLA、MUFA和PUFA的比例以及c9t11CLA/(c9t11CLA+t11C18:1)(P≤0.05)。随着大蒜油添加水平的增加,乳中TVA和t10c12CLA的比例也随之增加,但是差异不显著(P>0.05)。定量分析结果显示,试验处理对奶山羊乳腺脂肪生成相关基因表达的影响不显著(P>0.05)。相关分析显示,SREBP1与FAS基因表达之间有显著正相关(P≤0.01),SCCD与LPL(P≤0.05)和SREBP1(0.05With the improvement of economy and living standard, public interests regarding the microcomponents of food with possiblely beneficial effects on health maintenance and disease prevention are increasing. High consumption of dietary saturated fatty acid could increase the risk of coronary heart disease in humans, while monounsaturated fatty acid and polyunsaturated fatty acid could have cholesterol-lowering effects. Conjugated linoleic acid (CLA) was reported to have potentially positive health effects. Food products from ruminants are major dietary sources of CLA for humans, and thus optimization of the FA composition, especially CLA content in milk, has become an important part of nutritional research in ruminants. Dietary lipids undergo extensive hydrolysis and biohydrogenation in the rumen, and the fatty acids formed were absorbed and transported to mammary gland where de novo fatty acid synthesis and desaturation were carried out in mammary tissues which would affect milk fatty acid profile. At the same time, people's interests regarding the use of plant-derived essential oil have increased due to the abandoned use of growth-promoting antibiotics in animal feeds. Sutides have demonstrated that garlic has a wide spectrum of antibacterial activity and blood lipid-lowering effect which may influence ruminal biohydrogenation and milk fatty acid composition. Therefore, the aim of this study was to investigate the effects of garlic oil as ruminal modifer on ruminal fermentation, biohydrogenation, and milk fatty acid profile and lipogenesis-related gene expression in mammary gland of goats.
     1Effects of garlic oil on in vitro fermentation of substrates with different forage to concentrate ratios by microorganism from rumen of goats
     Different levels of garlic oil (0,30,300, and3000mg/L of culture fluid) were incubated for24h in diluted ruminal fluid with substrates with different ratios of forage to concentrate (10:0,7:3,5:5and3:7) to investigate their effects on rumen microbial fermentation by random block experimental design. Results showed that the24-h cumulative gas production was reduced (P<0.05) with the increasing level of garlic oil. The dynamic changes of gas production per3hours suggested garlic oil altered the mode of in vitro fermentation. Compared with the control, the high level of garlic oil (3000mg/L) increased pH (P≤0.05), while the levels of30and300mg/L had no significant effects on pH (P>0.05). The magnitude of concentrations of NH3-N and MCP followed the order control>300mg/L>30mg/L>3000mg/L and30mg/L> control>3000mg/L>300mg/L, respectively. Compared with the control, garlic oil supplementation reduced total VFAs concentration (P≤0.05), whereas there were no significant differences (P>0.05) between30and300mg/L. The levels of30and300mg/L reduced the proportion of acetate and ratio of acetate to propionate (P≤0.05), and increased the proportions of propionate and butyrate (P≤0.05). Compared with other substrates, the effects of garlic oil (30and300mg/L) on high concentrate substrates had minor depression on24-h cumulative gas production and total VFAs concentration, whereas had more evident effect on reducing NH3-N concentration, acetate proportion and ratio of acetate to propionate, as well as increasing propionate proportion. In conclusion, garlic oil inhibited in vitro ruminal fermentation in a dose-dependent manner accompanied by the effects of alleviating the process of fermentation. The moderate levels (such as30and300mg/L) of garlic oil had better manipulation on rumen microbial fermentation of high concentrate-substrate.
     2Effects of ruminal infusion of garlic oil on ruminal fermentation dynamics, fatty acid profile and biohydrogenation bacterial population of goats
     2.1Effects of ruminal infusion of garlic oil on dry matter intake and blood parameters of goats
     The effects of garlic oil on dry matter intake and blood parameters of goats were investigated by garlic oil infusion (0.8g/d) via ruminal fistulas in a cross-over design with two30-d periods. Results showed that garlic oil reduced DM intake (P≤0.05). And there was interaction between treatment and day for DM intake (P≤0.05). Garlic oil reduced DM intake on d3and6(P<0.001and P=0.011, respectively), but had no significant effect (P>0.05) on that of other days. Garlic oil reduced serum HDL-C, LDL-C, cholesterol concentrations (P≤0.01) and lipase activity (P=0.048), and tended to increase triglycerides concentration (0.050.1). Overall, garlic oil infusion via ruminal fistulas affected DM intake, and the effect diminished over time. Garlic oil had lipid-lowering effect and may reduce the hydrolysis of glycerides.
     2.2Effects of ruminal infusion of garlic oil on ruminal fermentation dynamics of goats
     The effects of garlic oil on ruminal fermentation dynamics were investigated through analyzing features in ruminal content collected at different time on d28,29and30of experimental period from goats treated by garlic oil infusion (0.8g/d) via ruminal fistulas in a cross-over design. Garlic oil increased (P≤0.01) ruminal NH3-N and MCP concentrations, but had no effects (P>0.1) on pH, total VFAs concentration, individual VFA proportion and ratio of acetate to propionate in ruminal content sampled before morning feeding (0h). During ruminal fermentation, the effects of garlic oil on the average of parameters at different time were similar with the effects at0h with the exception of reducing pH (P≤0.05) and total VFAs concentration (0.05     2.3Effects of ruminal infusion of garlic oil on ruminal fatty acid profile, total bacterial community and biohydrogenation bacterial pupolation of goats
     The effects of garlic oil on ruminal fatty acid profile, total bacterial community and biohydrogenation bacterial population of goats were investigated by ruminal infustion of garlic oil (0.8g/d) in a cross-over design in30day period. Garlic oil reduced ruminal C15, C16and SFA proportions (P≤0.01), increased the proportions of C18, TV A, c9t11CLA, t10c12CLA and PUFA (P≤0.05). and tended to increase MUFA proportion (0.05bacterial community. Real-time PCR showed that garlic oil tended to reduce B. proteoclasticus population and the ratio B. proteoclasticus/Butyrivibrio (0.05bacterial population(P>0.1). In conclusion, garlic oil altered ruminal fatty acid profile and the process of ruminal biohydrogenation through depressing the growth of B. proteoclasticus without effect on Butyrivibrio group bacterial population.
     2.4Effects of ruminal infusion of garlic oil on diversity of Butyrivibrio group bacteria using sequence analysis of16S rRNA gene clone from rumen of goats
     Based on Butyrivibrio-specific selection by primers B395f/B812r from full-length16S rRNA gene clones, Butyrivibrio group bacterial clone library from rumen of goats was constructed. The16S rRNA gene clone library showed that the Butyrivibrio group represented about12.98%(37of total285clones) and10.95%(38of total347clones) of ruminal bacteria in the control (accession numbers HQ326588-HQ326624) and garlic oil treatment (accession numbers JN008400-JN008437), respectively. Based on sequence analysis with the RDP library, the clones in both libraries were classified to the genus Pseudobutyrivibrio, Butyrivibrio and others within the family Lachnospiraceae. Some clones classified to the genus Ruminococcus and others within the family Ruminococcaceae were found in the treatment library. Results of sequence blast showed that the dominant clones were affiliated with Lachnospiraceae bacterium (control and treatment,32.43%and21.05%), followed by Pseudobutyrivibrio ruminis (control and treatment,21.62%and15.79%). The clones affiliated with the species Ruminococcus flavefaciens in the treatment library represented10.53%. No clones affiliated with the species Ruminococcus flavefaciens were found in the control. The Shannon indexes of the control and treatment library were2.47and2.91, respectively. Overall, Butyrivibrio group bacteria represented about12.98%in rumen of goats, classified to the genus Pseudobutyrivibrio, Butyrivibrio and others within the family Lachnospiraceae. Garlic oil infusion reduced the proportion, but increased the genetic diversity of Butyrivibrio group bacteria in rumen of goats.
     3Effects of garlic oil supplementation on milk fatty acid profile and lipogenesis-related gene expression in mammary gland of dairy goats
     3.1Effects of garlic oil supplementation on dry matter intake, blood parameters and milk composition of dairy goats
     The effects of garlic oil supplementation on dry matter intake, blood parameters and milk composition were investigated. Twenty four early-lactation Saanen dairy goats of China were paired and randomly allocated to four treatments with six goats each, and were offered corn silage ad libitum and0.79kg/d DM concentrate supplemented with different levels of garlic oil (0,0.57,1.14,1.71g/kg DM concentrate) for30d. Results showed that the concentrate intake was not affected, but the high level of garlic oil supplementation (1.35g/d) reduced corn silage intake (P≤0.05). Garlic oil supplementation had no significant effects on blood parameters (P>0.05), but tended to reduce serum LDL-C and NEFA concentrations (0.050.1) with the exception of tending to increase lactose content (0.05     3.2Effects of garlic oil supplementation on milk fatty acid profile and lipogenesis-related gene expression in mammary gland of dairy goats
     The effects of garlic oil supplementation on milk fatty acid profile and lipogenesis-related genes (ACC, FAS, LPL, SCD, SREBP1and PPARy) expression in mammary gland were investigated. Twenty four early-lactation Saanen dairy goats of China were paired and randomly allocated to four treatments with six goats each, and were offered corn silage ad libitum and0.79kg/d DM concentrate supplemented with different levels of garlic oil (0,0.57,1.14and1.71g/kg DM concentrate) for30d. Results showed that garlic oil supplementation altered milk fatty acid profile with more evident effects during d14-21. Compared with the control, garlic oil supplementation reduced the proportions of C14:0, C16:0and SFA (P≤0.05). and increased C18:0, C18:l, C18:3, c9t11CLA, MUFA and PUFA proportions and the ratio c9tl1CLA/(c9t11CLA+t11C18:1)(P≤0.05) in milk of dairy goats after27d. The proportions of TVA and t10c12CLA in milk increased with the increasing level of garlic oil in concentrate, but the differences were not significant (P>0.05). Real-time PCR analysis showed that there were no significant effects of garlic oil on lipogenesis-related genes mRNA abundance. Correlation analysis showed that there was a positive correlation (P≤0.01) between SREBP1and FAS mRNA abundance. And the mRNA abundance of SCD positively correlated with those of LPL (P≤0.05) and SREBP1(0.05
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