,1, 斯琴毕力格2, 敖长金2The Effects of Artemisia annua Extracts on the Rumen Fermentation in Dairy Cows
WANG LiFang,1, 2, AO ChangJin2第一联系人:
责任编辑: 林鉴非
收稿日期:2018-02-12接受日期:2018-06-12网络出版日期:2018-12-01
| 基金资助: |
Received:2018-02-12Accepted:2018-06-12Online:2018-12-01
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王丽芳, 斯琴毕力格, 敖长金. 黄花蒿提取物对奶牛瘤胃发酵指标的影响[J]. 中国农业科学, 2018, 51(23): 4548-4555 doi:10.3864/j.issn.0578-1752.2018.23.013
WANG LiFang, , AO ChangJin.
0 引言
【研究意义】共轭亚油酸（CLA）是一组含有共轭双键的亚油酸（linoleic acid）的几何异构体和位置异构体的总称[1,2],cis-9、trans-11-CLA被认为是CLA的最主要的活性形式,俗名瘤胃酸,占牛奶中总CLA的80%—90%[3]。CLA具有抗癌、抗糖尿病、减少脂肪沉积和动脉硬化、改变营养分配及调节免疫系统等生物学作用[4,5,6,7,8]。因此,增加乳中cis-9、trans-11-CLA含量成为动物营养学领域的研究热点。【前人研究进展】国内外很多****对增加乳中CLA含量开展了大量研究。研究表明,奶牛日粮中分别添加富含亚油酸及亚麻酸的日粮及浸提的油料籽实和豆油可以增加牛奶中CLA含量[9],添加鱼油可以提高牛奶中CLA含量[10],体外试验表明,稀释率、pH值及鱼油和葵花油对CLA含量有显著影响[11],在体外试验亚油酸C18:2存在的条件下,添加离子载体（尼日利亚菌素,莫能菌素和替曲那新）减少了亚油酸的完全氢化,增加了200%的cis-9,trans-11-CLA[12],奶牛釆食含有菊科植物的牧草或其提取物可以增加牛奶和羊奶中CLA含量[13,14,15,16]。【本研究切入点】多数研究主要集中于如何增加牛奶中CLA的含量,但是对于影响CLA合成的作用机制研究相对较少,特别是对于植物提取物调控牛奶中CLA含量的作用机制研究较少。【拟解决的关键问题】本研究通过在奶牛日粮中添加菊科植物黄花蒿乙醇提取物,研究其对瘤胃中影响CLA合成的溶纤维丁酸弧菌Butyrivibrio fibrisolvens（B.fibrisolvens）和蛋白溶解梭菌Clostridium proteoclasticum （C.proteoclasticum） 的影响,同时探讨对奶牛瘤胃中挥发性脂肪酸和pH值的影响,旨在从瘤胃层次探明黄花蒿乙醇提取物对牛奶中CLA合成的部分作用机制。1 材料与方法
1.1 试验时间和地点
试验于2016年9月5日至10月15日在内蒙古呼和浩特市赛罕区章盖营牧场进行,试验期40 d,前9 d为预饲期,10—40 d为正式期。1.2 试验材料
1.2.1 试验动物及饲养管理 选取15头体重为（600±29）kg、胎次（2—3）、泌乳期（158±3）d及泌乳量（22.8±1.8）kg·d-1相近的荷斯坦奶牛为试验动物。试验牛统一管理,自由采食、自由饮水;每天饲喂2次、挤奶2次（早上4：30、下午4：30）。1.2.2 试验日粮 试验所用精饲料和粗饲料均来自呼和浩特市赛罕区章盖营牧场,粗饲料由苜蓿、羊草、玉米青贮和谷草组成,基础日粮组成及营养水平见表1。
Table 1
表1
表1基础饲粮组成及营养水平（干物质基础）
Table 1
| 饲粮组成（DM） Diet composition | 含量 Content (%) | 营养水平 Nutrient levels | ||
|---|---|---|---|---|
| 原料Ingredients | 干物质DM(kg/（d·头）) | 13.5 | ||
| 精饲料 Concentrated feed | 53.2 | 产奶净能NEL(MJ·kg-1) | 6.4 | |
| 苜蓿 Alfalfa | 10.9 | 粗蛋白 CP（%） | 14.2 | |
| 羊草 Leymus chinensis | 3.6 | 粗脂肪 EE（%） | 2.94 | |
| 青贮 Silage | 25.1 | 中性洗涤纤维NDF（%） | 42.5 | |
| 谷草 Millet straw | 7.2 | 钙 Ca（%） | 0.83 | |
| 磷 P（%） | 0.65 |
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1.2.3 黄花蒿乙醇提取物 黄花蒿乙醇提取物购于南京泽朗公司,提取工艺条件是：乙醇浓度55%,提取温度95℃,提取2 h。GC-MS和LC-MS分析的黄花蒿乙醇提取物活性成分主要包括：倍半萜类35%、芳香族类29%、脂肪酸类6%、甾体类6%、三萜类6%、脂肪族类4%、生物碱类3%、酚类5%、杂环类2%、其他4%（GC-MS）;黄酮类和萜类（LC-MS）。
1.3 试验设计
试验采用完全随机设计,分为3组,分别是对照组和2个试验组,每组5头牛。试验1组黄花蒿乙醇提取物的添加量为96 g/（d·头）,试验2组黄花蒿乙醇提取物的添加量为160 g/（d·头）[16],每天饲喂两次,饲喂时黄花蒿乙醇提取物先与200g精饲料混合均匀让奶牛釆食完再饲喂TMR饲料。前9 d为预饲期,10—40 d为正式期,试验期共40 d。1.4 测定指标
溶纤维丁酸弧菌B.fibrisolvens、蛋白溶解梭菌C.proteoclasticum、瘤胃总菌Total bacteria、pH值、VFA。正式试验期最后一天,每组随机选取3头牛,通过口腔采集瘤胃液,4层纱布过滤后直接测pH值;过滤后的瘤胃液取5 mL加入预先装有1 mL 25%偏磷的离心管中,摇匀,置于-20℃冰箱中冷冻保存待测VFA;分别取过滤后的瘤胃液相、瘤胃固相及瘤胃液全混合物分装于5 mL冻存管,所有样品置于液氮中冷冻保存备测3种细菌。
1.4.1 细菌基因组DNA的提取 DNA提取参照QIAamp DNA Stool Mini Kit试剂盒说明书操作。并利用1.2%琼脂糖凝胶电泳检测所提取的细菌基因组DNA的质量。
1.4.2 标准质粒的制备 分别采用这3种细菌的特异性引物,同时以瘤胃内容物基因组DNA为模板,PCR扩增其特异性片段,利用胶回收试剂盒 （AXYGEN,USA）回收产物,并连接pMD18-T载体（Takara,Japan）,转化入感受态细胞。菌落PCR鉴定阳性克隆子测序鉴定。根据测序结果,利用质粒提取试剂盒（AXYGEN,USA）提取含有目的片段的标准质粒,并利用Qubit 3.0（Invitrogen,USA）测定质粒DNA溶液浓度,计算标准质粒的拷贝数。
1.4.3 标准曲线的制备 把标准质粒DNA进行10倍梯度系列稀释制作标准样品和待测样品一起扩增,得出标准曲线,根据所得标准曲线计算出样品中的基因拷贝数,最后以基因拷贝数每μL为单位进行分析。
1.4.4 荧光定量 本研究通过荧光定量的方法对3种菌的基因进行定量。基因引物序列见表2,引物浓度均为10 mmol·L-1。qPCR反应在专用的PCR八连管（Axygen, USA）中进行,25 μL体系,反应体系见表3。所有样品做3个重复,使用Takara试剂盒。RT-PCR 的反应程序为：95℃变性10 min,在95℃变性10 s,55℃退火30 s,72℃延伸30 s,循环40次,荧光数据采集在延伸时进行。实时定量PCR在FTC-3000荧光定量PCR仪中进行。
Table 2
表2
表2瘤胃氢化菌PCR的引物序列
Table 2
| 细菌 Target bacterium | 序列(5’ to 3’) Primer sequence | 扩增片段Product size（bp） | 退火温度Annealing temperature (℃) |
|---|---|---|---|
| 瘤胃总菌 Total bacteria | F:CGGCAACGAGCGCAACCC R:CCATTGTAGCACGTGTGTAGCC | 147 | 55 |
| 溶纤维丁酸弧菌 B.fibrisolvens | F:GCCTCAGCGTCAGTAATCG R:GGAGCGTAGGCGGTTTTAC | 187 | 55 |
| 蛋白溶解梭菌 C.proteoclasticum | F:TCCGGTGGTATGAGATGGGC R:GTCGCTGCATCAGAGTTTCCT | 185 | 55 |
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Table 3
表3
表3PCR体系
Table 3
| 试剂 Reagents | 体积 Volume (μL) |
|---|---|
| SRBR Premix Ex TaqTMⅡ（2×） | 12.5 |
| 正向引物Forward | 1 |
| 反向引物Reverse | 1 |
| DNA | 5 |
| ddH2O | to 25 |
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1.4.5 B.fibrisolvens的计算 B.fibrisolvens（%）= B.fibrisolvens / Total bacteria×100;
C.proteoclasticum的计算：C.proteoclasticum（%）=C.proteoclasticum / Total bacteria×100。
1.4.6 VFA测定 参照文献[17]方法测定VFA含量。
1.5 数据处理
试验数据采用Excel2007初步整理后,采用SAS 9.2进行方差分析（ANOVA）,采用DUNCAN法进行多重比较,P<0.05 作为差异显著的判断标准。2 结果
2.1 对瘤胃pH值和VFA的影响
从表4可以看出,与对照组相比,奶牛日粮中添加黄花蒿乙醇提取物可以增加瘤胃中pH值,其中两个试验组pH值分别增加了2.63%和8.61%,但差异不显著（P>0.05）;添加黄花蒿乙醇提取物均降低了瘤胃中VFA的含量,除异戊酸和戊酸低剂量黄花蒿乙醇提取物与对照组差异不显著外,其余各组VFA均与对照组差异显著（P<0.05）。其中两个试验组乙酸含量分别降低了36.27%和57.25%,丙酸含量分别降低了28.01%和45.81%,丁酸含量分别降低了17.86%和42.86%,异戊酸含量分别降低了23.35%和52.10%,戊酸含量分别降低了5%和50%,且VFA降低与黄花蒿乙醇提取物添加量呈剂量效应关系。Table 4
表4
表4黄花蒿乙醇提取物对瘤胃内pH值和VFA的影响
Table 4
| 项目 Items | 对照组 Control group | 试验1组 Experimental group 1 | 试验2组 Experimental group 2 | SEM | P 值 P value |
|---|---|---|---|---|---|
| pH | 6.85 | 7.03 | 7.44 | 0.24 | 0.116 |
| 乙酸 Acetate acid | 42.41a | 27.03b | 18.13c | 1.75 | <0.0001 |
| 丙酸 Propionic acid | 12.42a | 8.93b | 6.73b | 1.02 | 0.004 |
| 丁酸 Butyrate | 7.00a | 5.75b | 4.00c | 0.24 | <0.0001 |
| 异戊酸 Isovaleric acid | 1.67a | 1.28ab | 0.80c | 0.21 | 0.0165 |
| 戊酸 Valerate | 0.80a | 0.76a | 0.40b | 0.05 | 0.0003 |
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2.2 对瘤胃中溶纤维丁酸弧菌的影响
从表5可以看出,与对照组相比,奶牛日粮中添加黄花蒿乙醇提取物均增加了瘤胃液相和瘤胃液混合物中B.fibrisolvens的相对百分比,两个试验组分别增加了瘤胃液相中B.fibrisolvens 37.04%和40.74%,增加了瘤胃液混合物中B.fibrisolvens 83.3%和55.56%,差异显著（P<0.05）;降低了瘤胃固相中B. fibrisolvens的相对百分比,与对照组相比,两个试验组B. fibrisolvens的相对百分比分别降低了12.5%和18.75%,但是差异不显著（P>0.05）。Table 5
表5
表5黄花蒿乙醇提取物对瘤胃溶纤维丁酸弧菌的影响
Table 5
| 对照组 Control group | 试验1组 Experimental group 1 | 试验2组 Experimental group 2 | P 值 P value | |
|---|---|---|---|---|
| 瘤胃液相 Rumen liquid | 0.27b | 0.37a | 0.38a | 0.0325 |
| 瘤胃固相 Rumen solid | 0.16 | 0.14 | 0.13 | 0.8451 |
| 瘤胃液全混合物 Rumen mixture | 0.18b | 0.33a | 0.28a | 0.0127 |
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2.3 对瘤胃中蛋白溶解梭菌的影响
从表6可以看出,与对照组相比,奶牛日粮中添加黄花蒿乙醇提取物均降低了瘤胃液相和瘤胃固相中C.proteoclasticum的相对百分比,两个试验组分别降低了瘤胃液相中C.proteoclasticum 5.03%和1.12%,降低了瘤胃固相中C.proteoclasticum 22.69%和4.20%,差异显著（P<0.05）;增加了瘤胃液混合物中C.proteoclasticum的相对百分比,与对照组相比,两个试验组C.proteoclasticum的相对百分比分别 增加了54.49%和16.67%,其中试验1组显著高于对照组（P<0.05）,试验2组与对照组差异不显著（P>0.05）。Table 6
表6
表6黄花蒿乙醇提取物对瘤胃蛋白溶解梭菌的影响
Table 6
| 对照组 Control group | 试验1组 Experimental group 1 | 试验2组 Experimental group 2 | P 值 P value | |
|---|---|---|---|---|
| 瘤胃液相 Rumen liquid | 1.79 | 1.70 | 1.77 | 0.9661 |
| 瘤胃固相 Rumen solid | 2.38 | 1.84 | 2.28 | 0.4201 |
| 瘤胃液全混合物 Rumen mixture | 1.56b | 2.41a | 1.82ab | 0.0601 |
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3 讨论
3.1 瘤胃发酵参数
VFA是瘤胃发酵的主要指标之一,是碳水化合物在瘤胃中发酵的主要产物,可提供反刍动物总能量需要的70%—80%,且VFA含量与反刍动物能量转化效率呈正相关,与pH值呈负相关。本试验结果表明,添加黄花蒿乙醇提取物显著降低了瘤胃中VFA的含量,增加了pH值,提示黄花蒿乙醇提取物可能会影响能量转化效率,影响瘤胃发酵。本研究结果与相关研究结果不完全一致。BAYAT等[18,19]研究报道,奶牛日粮中添加荠蓝油、活酵母、肉豆蔻酸、菜籽油、红花油和亚麻油对瘤胃pH值和VFA没有显著影响;而本试验显著降低了瘤胃中VFA的含量,增加了pH值,导致这种差异的可能是因为添加物不同所致,本试验添加的是植物提取物,而上述研究中添加的是植物油等。本试验也与一些试验结果相同,王丽芳[16]研究结果表明,在奶山羊日粮中添加不同剂量的黄花蒿乙醇提取物均降低了瘤胃中乙酸、丙酸、丁酸等VFA各采样时间点的平均浓度,增加了pH平均值,与本试验结果一致,说明黄花蒿乙醇提取物对不同奶畜瘤胃发酵的影响相似。
3.2 瘤胃中B. fibrisolvens和C. proteoclasticum
反刍动物产品中 CLA 主要来源于两个途径,分别是外源途径和内源途径,外源途径就是亚油酸在瘤胃B.fibrisolvens等分泌的异构化酶作用下,异构化为cis-9,trans-11-CLA,内源途径就是瘤胃不饱和脂肪酸生物氢化形成的中间产物反式油酸（trans-11-18：1,TVA）进入乳腺中,在△9-去饱和酶的作用下生成 CLA[20],C. proteoclasticum可以把TVA转化为C18:0饱和脂肪酸[21],近来,C. proteoclasticum被归类为Butyrivibrio proteoclasticus,在现在的研究中,C. proteoclasticum 进一步被归类为B. proteoclasticus[22]。可以看出,B. fibrisolvens对 cis-9,trans-11-CLA的生成具有正调控作用,而C. proteoclasticum对cis-9,trans-11-CLA的生成具有负调控作用。因此通过营养调控措施,增加瘤胃中B. fibrisolvens的含量,降低C. proteoclasticum的含量有利于cis-9,trans-11-CLA的生成。本试验通过营养调控措施,对黄花蒿乙醇提取物调控cis-9,trans-11-CLA合成的两种细菌B. fibrisolvens和C. proteoclasticum的研究结果表明,添加黄花蒿乙醇提取物可以增加瘤胃液相和瘤胃液全混合物中B.fibrisolvens的相对百分比,降低了瘤胃固相中B. fibrisolvens的相对百分比。茅慧玲[23]研究报道,添加低比例杭白菊茎叶增加了瘤胃液相中B. fibrisolvens的含量,降低了瘤胃固相中B. fibrisolvens的含量,与本试验结果相一致;但是对于C. proteoclasticum研究结果却与本试验结果不同,本试验添加黄花蒿乙醇提取物降低了瘤胃液相和瘤胃固相中C. proteoclasticum的相对百分比,但增加了瘤胃液全混合物中的相对百分比,而茅慧玲[23]研究表明,添加中比例杭白菊茎叶增加了瘤胃液相中C. proteoclasticum的含量,但是对瘤胃固相中C. proteoclasticum的含量没有影响。导致这种差异的原因可能是这两种植物所含的活性成分不完全相同所致,杭白菊茎叶的主要活性成分是黄酮类和挥发油类物质[23],而本试验所用黄花蒿乙醇提取物的主要活性成分除黄酮类还包括萜类和芳香族类物质。RAMOS- MORALES等[24]研究结果表明,大蒜精油中丙基丙硫代亚磺酸酯（PTS）显著增加了瘤胃液中B. fibrisolvens的丰度（P<0.05）,降低了C. proteoclasticum的丰度（P>0.05）,与本试验结果基本一致;RAMOS- MORALES等[25]通过体外试验研究表明,添加0.02 g·L-1蓖麻酸对B. fibrisolvens没有影响,但降低了C. proteoclasticum的含量,有助于CLA的积累,主要原因是蓖麻酸阻止了C. proteoclasticum的生物氢化作用所致,与本试验结果一致。
其他一些相关研究与本研究结果也存在异同点,周薇等[26]研究表明,添加α-亚麻酸在调控CLA生成的过程中,降低了瘤胃液中B. fibrisolvens和C. proteoclasticum的含量,且呈剂量依赖性,而本研究的结果添加黄花蒿乙醇提取物增加了瘤胃液中B. fibrisolvens的含量,与上述研究结果不一致,但是降低C. proteoclasticum的含量与上述研究结果相似,但是本研究这两种菌与黄花蒿乙醇提取物没有剂量效应关系,与上述研究结果也不相符。刘仕军等[27]研究表明,添加鱼油和葵花油抑制了B. fibrisolvens的生长,与本试验结果不一致。史浩亭等[28]研究表明,添加苏子油和橡胶籽油在高剂量（植物油添加量为饲料干物质含量的3%和4%）时均降低了瘤胃液中B. fibrisolvens的相对百分比。RAMOS-MORALES等[29] 体外试验研究表明,添加0.025 g·L-1斑鸠菊酸降低了B. fibrisolvens的含量,添加0.05 g·L-1斑鸠菊酸降低了B. fibrisolvens和C. proteoclasticum的含量,与本试验结果不完全相同。本试验结果与以上结果不同的可能原因是添加物不同,本试验添加的是植物提取物,而以上试验添加的是植物油或脂肪酸。本试验总体趋势是增加瘤胃中B. fibrisolvens的相对百分比,降低瘤胃中C. proteoclasticum的相对百分比,有利于调控CLA的生成,本试验结果与项目组在奶牛饲养试验中增加牛奶中CLA含量的结果相一致[30]。
另外,目前对CLA合成的调控作用在瘤胃层次研究通常只选取瘤胃液相和固相,本研究对瘤胃液相、瘤胃固相和瘤胃液全混合物均进行了研究,结果表明,B.fibrisolvens和C.proteoclasticum在瘤胃不同内容物中表达量不同,这提示在未来的研究中,选择不同瘤胃内容物,结果不相一致。
4 结论
奶牛日粮中添加黄花蒿乙醇提取物影响瘤胃发酵;对瘤胃不同内容物中B. fibrisolvens和C. proteoclasticum影响不同,总体趋势是增加瘤胃中B. fibrisolvens的相对百分比,降低瘤胃中C. proteoclasticum的相对百分比,有利于调控共轭亚油酸的生成。参考文献 原文顺序
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被引期刊影响因子
DOI:10.1007/s11745-998-0293-8URLPMID:9832075 [本文引用: 1]
Abstract Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH3. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c,11t-octadecadienoic acid (18:2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag+ -HPLC), CLA isomers were resolved into seven trans,trans (5-9%), three cis/trans (10-13%), and five cis,cis (<1%) peaks, totaling 15, in addition to that of the 9c,11t-18:2 (78-84%). The FAME of total cheese lipids were fractionated by semipreparative Ag+ -HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m + 2 allylic ion and the m + 3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7t,9c-18:2 and the previously unreported 11t,13c-18:2 and 12c,14t-18:2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10,12-18:2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag+ -HPLC columns are also presented.
DOI:10.1007/s11745-998-0273-zURLPMID:9727611 [本文引用: 1]
The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis -octadecadienoic acid (18∶2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis -18∶2 isomer was resolved chromatographically as the methyl ester by silver ion-high-performance liquid chromatography (Ag + -HPLC); it eluted after the major 9 cis , 11 trans -18∶2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices in-vestigated by Ag + -HPLC, the 7 trans, 9 cis -18∶2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis -18∶2 was shown to elute near the leading edge of the major 9 cis , 11 trans -18∶2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography-electron ionization mass spectrometry (GC-EIMS). The double bond positions were at Δ7 and Δ9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC-direct deposition-Fourier transform infrared as evidenced from the doublet at 988 and 949 cm 611 and absorptions at 3020 and 3002 cm 611 . The 7 trans, 9 cis -18∶2 configuration was established by GC-EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy-and 11-hydroxyoctadec-9 cis enoates with BF 3 , in methanol.
DOI:10.1007/s11745-998-0279-6URLPMID:9727617 [本文引用: 1]
Kramer JK, Parodi PW, Jensen RG, Mossoba MM, Yurawecz MP, Adlof RO.
DOI:10.1093/carcin/8.12.1881URL [本文引用: 1]
DOI:10.1016/0021-9150(94)90034-5URLPMID:7980704 [本文引用: 1]
Abstract Conjugated linoleic acid (CLA) consists of a series of positional and geometric dienoic isomers of linoleic acid that occur naturally in foods. CLA exhibits antioxidant activity in vitro and in vivo. To assess the effect of CLA on atherosclerosis, 12 rabbits were fed a semi-synthetic diet containing 14% fat and 0.1% cholesterol for 22 weeks. For 6 of these rabbits, the diet was augmented with CLA (0.5 g CLA/rabbit per day). Blood samples were taken monthly for lipid analysis. By 12 weeks total and LDL cholesterol and triglycerides were markedly lower in the CLA-fed group. Interestingly, the LDL cholesterol to HDL cholesterol ratio and total cholesterol to HDL cholesterol ratio were significantly reduced in CLA-fed rabbits. Examination of the aortas of CLA-fed rabbits showed less atherosclerosis.
DOI:10.1006/bbrc.1998.8303URLPMID:9535724 [本文引用: 1]
Conjugated linoleic acid (CLA) is a naturally occurring fatty acid which has anti-carcinogenic and anti-atherogenic properties. CLA activates PPARα in liver, and shares functional similarities to ligands of PPARγ, the thiazolidinediones, which are potent insulin sensitizers. We provide the first evidence that CLA is able to normalize impaired glucose tolerance and improve hyperinsulinemia in the pre-diabetic ZDF rat. Additionally, dietary CLA increased steady state levels of aP2 mRNA in adipose tissue of fatty ZDF rats compared to controls, consistent with activation of PPARγ. The insulin sensitizing effects of CLA are due, at least in part, to activation of PPARγ since increasing levels of CLA induced a dose-dependent transactivation of PPARγ in CV-1 cells cotransfected with PPARγ and PPRE X 3-luciferase reporter construct. CLA effects on glucose tolerance and glucose homeostasis indicate that dietary CLA may prove to be an important therapy for the prevention and treatment of NIDDM.
DOI:10.1006/bbrc.1994.1157URLPMID:8117267 [本文引用: 1]
Abstract The ability of conjugated linoleic acid to prevent endotoxin-induced growth suppression was examined. Mice fed a basal diet or diet with 0.5% fish oil lost twice as much body weight after endotoxin injection than mice fed conjugated linoleic acid. By 72 hours post injection, mice fed conjugated linoleic acid had body weights similar to vehicle injected controls; however, body weights of basal and fish oil fed mice injected with endotoxin were reduced. Conjugated linoleic acid prevented anorexia from endotoxin injection. Splenocyte blastogenesis was increased by conjugated linoleic acid.
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DOI:10.1016/j.anifeedsci.2004.08.008URL [本文引用: 1]
The effect of fish oil (FO) on lipolysis and apparent biohydrogenation of linoleic (C18:2n-6, LA), linolenic (C18:3n-3, LNA), eicosapentaenoic (C20:5n-3, EPA) and docosahexaenoic (C22:6n-3, DHA) acid was studied in 6 and 24 h in vitro incubations with diluted sheep rumen contents (50 ml) as inoculum and six substrates (0.50 g) containing compound feeds based on barley, sugar beet pulp and soybean meal. Feeds containing sunflower oil (SO, 80 g/kg DM) or linseed oil (LO, 80 g/kg DM), as C18:2n-6 and C18:3n-3 source, respectively, were further enriched with three levels of fish oil (0, 20 and 40 g/kg DM), whereas addition of lard (40, 20 and 0 g/kg DM) ensured equal amounts of added fat (120 g/kg DM) in all feeds. Lipolysis was determined by the difference in triacylglycerol before and after incubation, whereas apparent biohydrogenation was calculated by the decrease of free polyunsaturated fatty acids (PUFA). FO had no influence on the degree of lipolysis or apparent biohydrogenation of C18:2n-6 and C18:3n-3, or on lipolysis of EPA and DHA in the different incubations. Biohydrogenation of EPA and DHA, however, was reduced with increasing FO dose. Compared to C18:2n-6 and C18:3n-3, lipolysis as well as biohydrogenation of EPA and DHA were significantly lower. Although C18:2n-6 and C18:3n-3 disappeared to the same extent, the amount of stearic acid (C18:0), the end-product of their biohydrogenation, was significantly lower in substrates with FO supplementation. Higher amounts of t11C18:1 and t11c15C18:2 in incubations with these substrates suggest that FO mainly inhibits the final step of biohydrogenation of C18:2n-6 and C18:3n-3. Addition of FO to diets containing C18:2n-6 or C18:3n-3 could increase ruminal accumulation and, hence, duodenal flow of t11C18:1. This could result in a higher uptake by the tissues and more substrate for the endogenous synthesis of c9t11CLA.
DOI:10.3168/jds.S0022-0302(07)71580-1URLPMID:17235173 [本文引用: 1]
The objective of this study was to evaluate the effect of solids dilution rate (SDR) and oil source [soybean oil (SBO) or linseed oil (LSO)] on the ruminal production of -9, -9, -desaturase.
DOI:10.1051/gse:19980208URLPMID:9535354 [本文引用: 1]
We measured effects of continuous vs twice-daily feeding, the addition of unsaturated fat to the diet, and monensin on milk production, milk composition, feed intake, and CO2-methane production in four experiments in a herd of 88 to 109 milking Holsteins. Methane and CO2 production increased with twice-daily feeding, but the CO2:CH4 ratio remained unchanged. Soybean oil did not affect the milkfat percentages, but fatty acid composition was changed. All saturated fatty acids up to and including 16:0 decreased (P < .01), whereas 18:0 and trans 18:1 increased (P < .001). The 18:2 conjugated dienes also increased (P < .01) when the cows were fed soybean oil. Monensin addition to the diet at 24 ppm decreased methane production (P < .01); the CO2:CH4 ratios reached 15, milk production increased (P < .01), and milkfat percentage and total milkfat output decreased (P < .01), as did feed consumption, compared with cows fed diets without monensin (P < .05). Milk fatty acid composition showed evidence of depressed ruminal biohydrogenation: saturated fatty acids (P < .05) decreased and 18:1 increased (P < .001); most of the increase was seen in the trans 18:1 isomer. As with soybean oil feeding, addition of monensin also increased (P < .05) the concentration of conjugated dienes. The monensin feeding trial was repeated 161 d later with 88 cows, of which 67 received monensin in the diet in the first trial and 21 cows were newly freshened and had never received monensin. Methane production again decreased (P < .05), but this time the CO2:CH4 ratio did not change and all other monensin-related effects were absent. The ruminal microflora in the cows that had previously received monensin seemed to have undergone some adaptive changes and no longer responded as before.
DOI:10.1016/S0958-6946(02)00062-6URL [本文引用: 1]
DOI:10.1016/j.livprodsci.2005.09.014URL [本文引用: 1]
An experiment was carried out to evaluate the effect on milk and cheese fatty acid composition of the inclusion of Chrysanthemum coronarium L., (Asteracea) into dairy sheep diet. Plots sown either with a binary mixture consisting of Lolium rigidum Gaudin and Medicago polymorpha (LM) or a ternary mixture including the above species and C. coronarium were (LMC) grazed by groups of Sarda dairy ewes during May. The botanical composition of diet, as measured by n-alkane method, showed that 34% of LMC group diet consisted of Chrysanthemum flowers and leaves. Exposure of sheep to this non-conventional forage did not affect animal performance and milk composition (fat and casein, P > 0.05) but resulted in different milk and cheese fatty acid composition. In particular conjugated linoleic acid, vaccenic acid and linoleic acid were all higher in LMC than LM groups ( P < 0.05). The probable main reason was the higher proportion of linoleic acid in Chrysanthemum forage and hence in LMC than LM diet.
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DOI:10.7685/j.issn.1000-2030.1982.04.010URL [本文引用: 1]
本文介绍了一种应用气相色谱较快速、简便、精确测定瘤胃挥发性脂肪酸(VFA)的方法。瘤胃液仅在存有一种酸性吸附剂的氯仿溶液中(并含巴豆酸作为内标),即可萃取出所含全部VFA。样品由预处理到测出总酸及其组成克分子百分率为时不到10分钟。主要VFA的回收率可达99.51—100.40％。 应用本法在四头乳山羊上的实验结果表明:所得数据与原蒸馏法分析以及国外研究的结果基本一致。
DOI:10.7685/j.issn.1000-2030.1982.04.010URL [本文引用: 1]
本文介绍了一种应用气相色谱较快速、简便、精确测定瘤胃挥发性脂肪酸(VFA)的方法。瘤胃液仅在存有一种酸性吸附剂的氯仿溶液中(并含巴豆酸作为内标),即可萃取出所含全部VFA。样品由预处理到测出总酸及其组成克分子百分率为时不到10分钟。主要VFA的回收率可达99.51—100.40％。 应用本法在四头乳山羊上的实验结果表明:所得数据与原蒸馏法分析以及国外研究的结果基本一致。
DOI:10.3168/jds.2014-7976URLPMID:25726099 [本文引用: 1]
The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53卤7 d in milk) fitted with rumen cannula were used in a 4 4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 1010 cfu/d (treatments A and B), or supplements of 60g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.
DOI:10.3168/jds.2017-13545URLPMID:29224879 [本文引用: 1]
The sense of smell is likely to influence the behaviour of domestic and captive animals in a wide range of management and housing situations. In domestic cattle, there may be unexploited potential for using odours and olfaction in the management; however, published studies on bovine olfactory capacity are scarce. By applying an olfactory Habituation/Dishabituation test developed for rodents,... [Show full abstract]
DOI:10.1038/sj.ijo.0801389URLPMID:10958825 [本文引用: 1]
Conjugated linoleic acid (CLA) is a naturally occurring anticarcinogen found in milk fat and body fat of ruminants. Although CLA is an intermediate in ruminal biohydrogenation of linoleic acid, we hypothesized that its primary source was from endogenous synthesis. This would involve Delta(9)-desaturase and synthesis from trans-11 18:1, another intermediate in ruminal biohydrogenation. Our first experiment supplied lactating cows (n = 3) with trans-11 18:1 by abomasal infusion and examined the potential for endogenous synthesis by measuring changes in milk fat CLA. By d 3, infusion of trans-11 18:1 resulted in a 31% increase in concentration of cis-9, trans-11 CLA in milk fat, demonstrating that an active pathway for endogenous synthesis of CLA exists. Our second experiment examined the quantitative importance of endogenous synthesis of CLA in lactating cows (n = 3) by abomasally infusing a putative stimulator (retinol palmitate) or an inhibitor (sterculic oil) of Delta(9)-desaturase. Infusion of retinol palmitate had no influence on milk fatty acid desaturation, and yield of CLA in milk fat was not altered. However, sterculic oil infusion decreased the concentration of CLA in milk fat by 45%. Consistent with Delta(9)-desaturase inhibition, the sterculic oil treatment also altered the milk fat concentration of other Delta(9)-desaturase products as indicated by the two- to threefold increase in the ratios of 14:0 to 14:1(,) 16:0 to 16:1 and 18:0 to cis-18:1. Using changes in the ratio of 14:0 to 14:1 as an indication of the extent of Delta(9)-desaturase inhibition with the sterculic oil treatment, an estimated 64% of the CLA in milk fat was of endogenous origin. Overall, results demonstrate that endogenous synthesis of CLA from trans-11 18:1 represented the primary source of CLA in milk fat of lactating cows.
DOI:10.1111/j.1365-2672.1964.tb05056.xURLPMID:14219019 [本文引用: 1]
Abstract Polan, C. E. (North Carolina State of the University of North Carolina, Raleigh), J. J. McNeill, and S. B. Tove. Biohydrogenation of unsaturated fatty acids by rumen bacteria. J. Bacteriol. 88:1056-1064. 1964.-A simple, rapid, specific assay for the biohydrogenation of unsaturated fatty acids was developed. With this assay, it was shown that washed suspensions of mixed rumen bacteria hydrogenate linoleic and oleic acids. Butyrivibrio fibrisolvens, a common rumen bacterium, is capable of hydrogenating linoleic acid to octadecenoic acid but not to stearic acid. Complete anaerobiosis is required, and with mixed rumen bacteria more activity is obtained in an atmosphere of hydrogen than of either nitrogen or helium, whereas carbon dioxide is inhibitory. The extent of biohydrogenation varies with the season of the year, and a variable stimulatory effect is obtained upon the addition of boiled rumen fluid. Biohydrogenation activity in B. fibrisolvens is markedly dependent upon the age of the organism and concentration of cells used in the medium. The presence of certain other rumen bacteria, which by themselves are incapable of carrying out the biohydrogenation reaction, can prevent loss of activity of B. fibrisolvens due to age or dilution. Two systems are involved in the complete hydrogenation of linoleic acid: one specific for the conversion of linoleic acid to a monoenoic acid, and the other for the hydrogenation of a monoenoic acid to stearic acid.
DOI:10.1099/ijs.0.65845-0URLPMID:18768601 [本文引用: 1]
It is proposed that Clostridium proteoclasticum be reclassified as Butyrivibrio proteoclasticus comb. nov. on the basis of phylogenetic position, DNA G+C content and physiological traits. Phylogenetic analyses based on 16S rRNA gene sequences from an extensive range of taxa within clostridial rRNA subcluster XIVa grouped C. proteoclasticum together with isolates of the genus Butyrivibrio, though this species was genetically distinct from the extant Butyrivibrio species examined. The DNA G+C content of C. proteoclasticum was originally erroneously reported as 28 mol%. However the genome sequence of the type strain of C. proteoclasticum, strain B316(T), and HPLC analysis estimate the DNA G+C content as 40 mol%, which is within the range reported for strains of Butyrivibrio. C. proteoclasticum was distinguishable from other species of the genus Butyrivibrio as the 16S rRNA gene from strain B316(T) shared less than 97 % sequence similarity with sequences from the type strains of Butyrivibrio species. C. proteoclasticum was also able to convert linoleic acid to stearic acid, in contrast to other species of Butyrivibrio. Physiological characteristics, including carbon source utilization, volatile fatty acid production and proteinase activities, were assessed for a panel of representative strains of the genera Butyrivibrio and Pseudobutyrivibrio and C. proteoclasticum. These data, together with the phylogenetic analyses, support the reclassification of Clostridium proteoclasticum as a separate species within the genus Butyrivibrio, Butyrivibrio proteoclasticus comb. nov. (type strain B316(T)=ATCC 51982(T)=DSM 14932(T)).
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DOI:10.2527/jas.2011-4670URLPMID:22829608 [本文引用: 1]
Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for conjugated linoleic acid (CLA) synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9, cis-12-18:2) or with a combination of LA and RA, or LA and castor oil (LA, RA and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA, rumenic acid (cis-9, trans-11-18:2), accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. B. proteoclasticus, but not B. fibrisolvens, metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition, whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were lower (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo are now required to confirm the potential of these additives.
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DOI:10.1016/j.anifeedsci.2016.10.002URL [本文引用: 1]
Substituted long-chain fatty acids may be useful dietary supplements to suppress ruminal biohydrogenation of unsaturated fatty acids (UFA) and thereby increase the flow of UFA to meat and milk. The aim of this study was to determine ifVernonia galamensis(ironweed, a member of the sunflower family) and vernolic acid (cis-12,13-epoxy-cis-9-octadecenoic acid), the main constituent of the seed oil, affected the biohydrogenation of linoleic acid (LA;cis-9,cis-12-18:2) to rumenic acid (CLA;cis-9,trans-11-18:2), vaccenic acid (VA;trans-11-18:1) and stearic acid (SA; 18:0) by ruminal microorganisms. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet were incubatedin vitrowith LA (0.2g/L) 0.2g/L vernolic acid or 5g/L of dried flowers or leaves ofV. galamensis, either alone or combined. Vernolic acid had a substantial effect on LA metabolism, causing decreases incis-9,trans-11 CLA and VA accumulation as well as SA production (P<0.05). Vernolic acid inhibited growth of the rumen fatty acid-biohydrogenating bacterium,Butyrivibrio fibrisolvensbut notB. proteoclasticusat 0.025g/L; neither species grew at 0.05g/L. An inhibition of the metabolism of LA as well as a decrease in the accumulation ofcis-9,trans-11 CLA and a slowdown in its metabolism were observed in the presence of flowers, leaves and a combination of both (P<0.05). However, only incubations with flowers, either alone or with leaves, resulted in higher accumulation of VA (P 0.05). Vernolic acid, constituted 2% of the total fatty acid content of flowers whereas it was not detected in leaves ofV. galamensis.Thus, the greater accumulation of VA observed with flowers ofV. galamensiswas probably due to other components rather than vernolic acid. It was concluded that vernolic acid andV. galamensisinhibit the biohydrogenation of LAin vitro.V. galamensiscould potentially be used as an additive to alter ruminal biohydrogenation, leading to greater concentrations ofcis-9,trans-11 CLA in meat and milk. Evaluation ofV. galamensis in vivois required to confirm the presentin vitroobservations.
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