大豆黄酮、亚麻籽木脂素和伊普异黄酮对山羊瘤胃代谢影响的研究
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摘要
本文以山羊为研究对象,采用体外培养、体内动物实验两种方法研究大豆黄酮、亚麻籽木脂素和伊普异黄酮对反刍动物瘤胃代谢的影响,重点研究了伊普异黄酮对山羊瘤胃代谢及瘤胃液性激素含量的影响,并对亚麻籽木脂素对山羊瘤胃代谢的影响进行了初步研究,本研究分三大部分:
第一部分大豆黄酮对山羊瘤胃代谢影响的体外研究
实验选用3头健康、体况基本一致装有永久性瘤胃瘘管的本地成年雌性山羊的瘤胃液,采用简易人工瘤胃装置。剂量效应实验:各培养瓶分别添加大豆黄酮使其在培养液中浓度为0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,39℃恒温厌氧培养,10h后收集培养液。动态培养实验:按照培养时间设2h,4h,6h,8h,12h小时5种处理,每个处理分为对照组和实验组(大豆黄酮组),实验组浓度为100 mg/L。
结果表明,大豆黄酮可直接作用于瘤胃微生物,影响瘤胃发酵,添加大豆黄酮可使培养液中pH值下降,改变瘤胃氨氮浓度和微生物蛋白浓度,促进瘤胃氮代谢。TVFA的浓度无明显变化,但可改变各挥发性脂肪酸的浓度和组成,特别使乙酸浓度增加,改变了乙酸和丙酸的比例,说明大豆黄酮改变了瘤胃微生物的发酵类型。在体外条件下,大豆黄酮可被瘤胃微生物降解为雌马酚。
第二部分木脂素对山羊瘤胃代谢影响的体外研究
实验选用3头健康、体况基本一致装有永久性瘤胃瘘管的本地成年雌性山羊的瘤胃液,采用简易人工瘤胃装置。剂量效应实验:各培养瓶分别添加亚麻籽木脂素使其在培养液中浓度为0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,39℃恒温厌氧培养,10h后收集培养液。动态培养实验:按照培养时间设2h,4h,6h,8h,12h小时5种处理,每个处理分为对照组和实验组(亚麻籽木脂素组),实验组浓度为100 mg/L。剂量效应实验结果显示,亚麻籽木脂素50 mg/L、100 mg/L时pH分别下降0.85%(对照组6.27±0.02,VS实验组6.25±0.02,P＜0.05)、2.05%(6.02±0.01,VS 5.89±0.03,P＜0.01)。NH_3-N浓度分别下降12.31%(1.793±0.032 VS 1.572±0.057,P＜0.05)、24.29%(1.745±0.043 VS 1.321±0.097,P＜0.01)。MCP浓度分别上升25.83%(70.595±2.164 VS88.831±2.329,P＜0.05)、8.16%(67.312±4.269 VS 72.806±4.086,P＞0.05)。动态培养结果显示,亚麻籽木脂素组发酵6h、12h时pH分别下降1.35%(6.49±0.04 VS 6.32±0.02,P＜0.01)、2.11%(6.49±0.04 VS 6.02±0.05,P＜0.01)。NH_3-N浓度6h时下降11.72%(1.058±0.090 VS 0.934±0.061,P＜0.01)、12h时上升71.55%(0.952±0.035 VS1.633±0.075,P＜0.01)。总挥发性脂肪酸(TVFA)在6h后显著高于对照组。乙酸(A)比例下降,丙酸(P)比例上升,结果P:A比例明显升高。在体外条件下,亚麻籽木脂素可被瘤胃微生物降解为END和ENL。
第三部分伊普异黄酮对山羊瘤胃代谢及瘤胃液性激素含量的影响。
本部分实验旨在研究伊普异黄酮(Ipriflavone,Ip,7-异丙基异黄酮)对山羊瘤胃代谢以及瘤胃液雌二醇和睾酮含量的影响。包括两系列试验:
系列1体外试验
瘤胃液来自三只装有永久性瘤胃瘘管的本地成年雌性山羊,以0.6 g草粉和0.3 g精料为底物,研究不同浓度IP(0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L),以及IP浓度为100 mg/L时不同培养时间(2h,4h,6h,8h,12h)时对山羊瘤胃代谢的影响。
剂量效应实验结果表明,培养液中IP浓度为100 mg/L和200 mg/L时能显著加强瘤胃代谢,pH水平显著下降,氨氮(NH3-N)浓度和微生物蛋白(MCP)浓度显著升高;动态实验结果表明,4h、8h、12h pH值分别较对照组下降0.68%(P＜0.01)、0.49%(P＜0.05)、2.05%(P＜0.01),对照组NH3-N浓度8h后呈下降趋势,实验组持续上升,在12h较对照组升高12.50%(P＜0.01),对照组和实验组MCP浓度变化规律基本一致,均在培养4h后持续下降,在整个培养过程中,实验组微生物蛋白含量较对照组有升高的趋势。
系列2动物试验
实验选用4只装有永久性瘤胃瘘管的本地成年山羊,采用自身对照设计,对照期饲喂精料,实验期在精料中添加IP(100 mg/100 g精料),于饲喂前及采食后2、4、6、8、10小时采集瘤胃液并测定各相关指标;结果表明,IP有加强瘤胃氮代谢趋势;使乙酸浓度升高(P＜0.01),乙酸比例上升(P＜0.01),丙酸比例下降(P＜0.05),丙酸/乙酸比值下降(P＜0.01);乙酸含量提高了9.66%,乙酸比例上升7.20%,丙酸比例下降5.45%,丙酸/乙酸下降12.31%;瘤胃液中17β-雌二醇和睾酮含量显著上升(P＜0.05)。
提示,伊普异黄酮可以直接作用于瘤胃微生物,加强瘤胃氮代谢,同时能够影响瘤胃的糖代谢,显著提高乙酸的含量和比例,改变瘤胃微生物发酵类型;伊普异黄酮可以提高瘤胃液17β-雌二醇和睾酮的水平。
Goat were used to study the effect of daidzein,linseed lignans and ipriflavone on ruminal metabolism in rumen fluid in goat by the method in vitro and in vivo.Effects of ipriflavone on ruminal metabolism and concentration of sex hormones in rumen in goat were studied importantly.At the same time,we also probed into effects of linseed lignans on ruminal metabolism.This study had three parts:
Part 1:Effects of daidzein on ruminal metabolism in vitro
Ruminal fluid,collected from three adult female goats with permanent rumen fistulae, was incubated in anaerobic media in vitro.In order to confirm the best dose,daidzein was added into the incubation,and the final concentration were 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L.The samples were collected after 10h for anaerobic-incubation(39℃). Furthermore,when the concentration of daidzein was 100 mg·L~(-1),the samples of 2h,4h,6h, 8h,12 h were collected.
Results showed that daidzein could affect ruminal fermentation directly and had an interaction between daidzein and rumen microorganisms.Daidzein could lower the levels of pH,change the concentration of NH_3-N and MCP,increase ruminal nitrogen metamolism, the concentration of TVFA were not affected,but alter the fermentation pattern in the rumen, raise the concentration of acetate(A) specially,change the ratio of P to A,besides,daidzein could be metabolized into equol in vitro.
Part 2:Effects of linseed lignans on ruminal metabolism in vitro
Ruminal fluid,collected from three adult female goats with permanent rumen fistulae, was incubated in anaerobic media in vitro.In order to confirm the best dose,SDG was added into the incubation,and the final concentration were 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L.The samples were collected after 10h for anaerobic-incubation(39℃). Furthermore,when the concentration of SDG was 100 mg·L~(-1),the samples of 2h,4h,6h,8h, 12h were collected.
In the dose experiment,results have shown that at the concentration of 50 mg·L~(-1) and 100 mg·L~(-1),levels of pH,concentration of NH_3-N were reduced by 0.85%(6.27±0.02 VS 6.25±0.02,P＜0.05),2.05%(6.02±0.01 VS 5.89±0.03,P＜0.01),12.31%(1.793±0.032 mg·100mL~(-1) VS 1.572±0.057 mg·100mL~(-1),P＜0.05),24.29%(1.745±0.043 mg·100mL~(-1) VS 1.321±0.097 mg·100mL~(-1),P＜0.01).concentration of MCP were increased by 25.83%(170.595±2.164μg·mL~(-1) VS 88.831±2.329μg·mL~(-1),P＜0.05), 8.16%(67.312±4.269μg·mL~(-1)VS 72.806±4.086μg·mL~(-1),P＞0.05),respectively.
The dynamic experiment have shown that at 6h and 12h,levels of pH were lowered by 1.35%(6.49±0.04 VS 6.32±0.02,P＜0.01),2.11%(6.49±0.04 VS 6.02±0.05, P＜0.01),respectively,while at 6h the concentration of NH_3-N were inhibited by 11.72% (1.058±0.090 mg·100mL~(-1) VS 0.934±0.061 mg·100mL~(-1),P＜0.01),however,at 12h were increased by 71.55%(0.952±0.035 mg·100mL~(-1) VS 1.633±0.075 mg·100mL~(-1), P＜0.01).The concentration of total volatile fatty acids was obviously higher after 6h,the acetate(A) percent decreased,and the propionate(P) percent increased,in results,the ratio of P to A obviously increased.Linseed lignans could be metabolized into END and ENL in vitro.
Part 3:Effeets of Ipriflavone on Ruminal Metabolism and Concentration of Sex Hormones in rumen in Goat
This experiment was conducted to study the effects of ipriflavone on ruminal metabolism and concentration of 17β-estradiol and testosterone in rumen in goat.This study consisted of two series:
Series 1:in vitro
Ruminal fluid was incubated in anaerobic media in vitro with straw powder(0.6 g) and concentrates(0.3 g) as the substrate.Ipriflavone was added into the incubation with the final concentrations at 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,respectively. Furthermore,when the concentration of ipriflavone was 100 mg·L~(-1),the samples of 2h,4h, 6h,8h,12h were collected.
In the dose experiment,results showed that iptiflavone on 100 mg/L and 200 mg/L significantly enhanced ruminal metabolism.Levels of pH were reduced,the concentration of NH_3-N and MCP were increased obviously.The dynamic experiment shown that at 4 h,8 h and 12 h,levels of pH were lowered by 0.68%(P＜0.01),0.49%(P＜0.05),2.05% (P＜0.01) respectively.In control,after 6h the concentration of NH_3-N had downtrend, while,in experiment,the concentration of NH_3-N kept raising.At 12h were increased 12.50%(P＜0.01),the concentration of MCP had the same change of downtrend after 4h both in control and in experiment,In the whole incubation,the concentration of MCP was increased in experiment than that in control.
Series 2:in vivo
Four local adult goats with permanent rumen fistulae were conducted into two periods by self-control design,control period and experiment period.In experiment period, ipriflavone was added into concentrates(100 mg/100g concentrates).Criterias were tested every two hours from 2h to 10h before feeding and after feeding.
Results showed that ipriflavone increased ruminal nitrogen metamolism in tendency, raised the concentration of acetate(A) and the percentage of acetate(P＜0.01),lowed the percentage of propionate(P)(P＜0.05) and the ratio of P:A significantly(P＜0.01);the concentration of acetate was raised 9.66%,the percentage of acetate was raised 7.20%,the percentage of propionate was lowed 5.45%,the ratio of P to A was lowed 12.31%, Ipriflavone could significantly increase the concentration of 17β-estradiol and testosterone in ruminal fluid of goat(P＜0.05).
These resulds may suggest that an interaction between ipriflavone and rumen microorganisms occurred.Ipriflavone could enhance nitrogen metabolism in rumen,affect carbohydrates metabolism,alter the fermentation pattern,and significantly increase the levels of 17β-estradiol and testosterone in ruminal fluid.
第一部分大豆黄酮对山羊瘤胃代谢影响的体外研究
实验选用3头健康、体况基本一致装有永久性瘤胃瘘管的本地成年雌性山羊的瘤胃液,采用简易人工瘤胃装置。剂量效应实验:各培养瓶分别添加大豆黄酮使其在培养液中浓度为0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,39℃恒温厌氧培养,10h后收集培养液。动态培养实验:按照培养时间设2h,4h,6h,8h,12h小时5种处理,每个处理分为对照组和实验组(大豆黄酮组),实验组浓度为100 mg/L。
结果表明,大豆黄酮可直接作用于瘤胃微生物,影响瘤胃发酵,添加大豆黄酮可使培养液中pH值下降,改变瘤胃氨氮浓度和微生物蛋白浓度,促进瘤胃氮代谢。TVFA的浓度无明显变化,但可改变各挥发性脂肪酸的浓度和组成,特别使乙酸浓度增加,改变了乙酸和丙酸的比例,说明大豆黄酮改变了瘤胃微生物的发酵类型。在体外条件下,大豆黄酮可被瘤胃微生物降解为雌马酚。
第二部分木脂素对山羊瘤胃代谢影响的体外研究
实验选用3头健康、体况基本一致装有永久性瘤胃瘘管的本地成年雌性山羊的瘤胃液,采用简易人工瘤胃装置。剂量效应实验:各培养瓶分别添加亚麻籽木脂素使其在培养液中浓度为0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,39℃恒温厌氧培养,10h后收集培养液。动态培养实验:按照培养时间设2h,4h,6h,8h,12h小时5种处理,每个处理分为对照组和实验组(亚麻籽木脂素组),实验组浓度为100 mg/L。剂量效应实验结果显示,亚麻籽木脂素50 mg/L、100 mg/L时pH分别下降0.85%(对照组6.27±0.02,VS实验组6.25±0.02,P＜0.05)、2.05%(6.02±0.01,VS 5.89±0.03,P＜0.01)。NH_3-N浓度分别下降12.31%(1.793±0.032 VS 1.572±0.057,P＜0.05)、24.29%(1.745±0.043 VS 1.321±0.097,P＜0.01)。MCP浓度分别上升25.83%(70.595±2.164 VS88.831±2.329,P＜0.05)、8.16%(67.312±4.269 VS 72.806±4.086,P＞0.05)。动态培养结果显示,亚麻籽木脂素组发酵6h、12h时pH分别下降1.35%(6.49±0.04 VS 6.32±0.02,P＜0.01)、2.11%(6.49±0.04 VS 6.02±0.05,P＜0.01)。NH_3-N浓度6h时下降11.72%(1.058±0.090 VS 0.934±0.061,P＜0.01)、12h时上升71.55%(0.952±0.035 VS1.633±0.075,P＜0.01)。总挥发性脂肪酸(TVFA)在6h后显著高于对照组。乙酸(A)比例下降,丙酸(P)比例上升,结果P:A比例明显升高。在体外条件下,亚麻籽木脂素可被瘤胃微生物降解为END和ENL。
第三部分伊普异黄酮对山羊瘤胃代谢及瘤胃液性激素含量的影响。
本部分实验旨在研究伊普异黄酮(Ipriflavone,Ip,7-异丙基异黄酮)对山羊瘤胃代谢以及瘤胃液雌二醇和睾酮含量的影响。包括两系列试验:
系列1体外试验
瘤胃液来自三只装有永久性瘤胃瘘管的本地成年雌性山羊,以0.6 g草粉和0.3 g精料为底物,研究不同浓度IP(0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L),以及IP浓度为100 mg/L时不同培养时间(2h,4h,6h,8h,12h)时对山羊瘤胃代谢的影响。
剂量效应实验结果表明,培养液中IP浓度为100 mg/L和200 mg/L时能显著加强瘤胃代谢,pH水平显著下降,氨氮(NH3-N)浓度和微生物蛋白(MCP)浓度显著升高;动态实验结果表明,4h、8h、12h pH值分别较对照组下降0.68%(P＜0.01)、0.49%(P＜0.05)、2.05%(P＜0.01),对照组NH3-N浓度8h后呈下降趋势,实验组持续上升,在12h较对照组升高12.50%(P＜0.01),对照组和实验组MCP浓度变化规律基本一致,均在培养4h后持续下降,在整个培养过程中,实验组微生物蛋白含量较对照组有升高的趋势。
系列2动物试验
实验选用4只装有永久性瘤胃瘘管的本地成年山羊,采用自身对照设计,对照期饲喂精料,实验期在精料中添加IP(100 mg/100 g精料),于饲喂前及采食后2、4、6、8、10小时采集瘤胃液并测定各相关指标;结果表明,IP有加强瘤胃氮代谢趋势;使乙酸浓度升高(P＜0.01),乙酸比例上升(P＜0.01),丙酸比例下降(P＜0.05),丙酸/乙酸比值下降(P＜0.01);乙酸含量提高了9.66%,乙酸比例上升7.20%,丙酸比例下降5.45%,丙酸/乙酸下降12.31%;瘤胃液中17β-雌二醇和睾酮含量显著上升(P＜0.05)。
提示,伊普异黄酮可以直接作用于瘤胃微生物,加强瘤胃氮代谢,同时能够影响瘤胃的糖代谢,显著提高乙酸的含量和比例,改变瘤胃微生物发酵类型;伊普异黄酮可以提高瘤胃液17β-雌二醇和睾酮的水平。
Goat were used to study the effect of daidzein,linseed lignans and ipriflavone on ruminal metabolism in rumen fluid in goat by the method in vitro and in vivo.Effects of ipriflavone on ruminal metabolism and concentration of sex hormones in rumen in goat were studied importantly.At the same time,we also probed into effects of linseed lignans on ruminal metabolism.This study had three parts:
Part 1:Effects of daidzein on ruminal metabolism in vitro
Ruminal fluid,collected from three adult female goats with permanent rumen fistulae, was incubated in anaerobic media in vitro.In order to confirm the best dose,daidzein was added into the incubation,and the final concentration were 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L.The samples were collected after 10h for anaerobic-incubation(39℃). Furthermore,when the concentration of daidzein was 100 mg·L~(-1),the samples of 2h,4h,6h, 8h,12 h were collected.
Results showed that daidzein could affect ruminal fermentation directly and had an interaction between daidzein and rumen microorganisms.Daidzein could lower the levels of pH,change the concentration of NH_3-N and MCP,increase ruminal nitrogen metamolism, the concentration of TVFA were not affected,but alter the fermentation pattern in the rumen, raise the concentration of acetate(A) specially,change the ratio of P to A,besides,daidzein could be metabolized into equol in vitro.
Part 2:Effects of linseed lignans on ruminal metabolism in vitro
Ruminal fluid,collected from three adult female goats with permanent rumen fistulae, was incubated in anaerobic media in vitro.In order to confirm the best dose,SDG was added into the incubation,and the final concentration were 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L.The samples were collected after 10h for anaerobic-incubation(39℃). Furthermore,when the concentration of SDG was 100 mg·L~(-1),the samples of 2h,4h,6h,8h, 12h were collected.
In the dose experiment,results have shown that at the concentration of 50 mg·L~(-1) and 100 mg·L~(-1),levels of pH,concentration of NH_3-N were reduced by 0.85%(6.27±0.02 VS 6.25±0.02,P＜0.05),2.05%(6.02±0.01 VS 5.89±0.03,P＜0.01),12.31%(1.793±0.032 mg·100mL~(-1) VS 1.572±0.057 mg·100mL~(-1),P＜0.05),24.29%(1.745±0.043 mg·100mL~(-1) VS 1.321±0.097 mg·100mL~(-1),P＜0.01).concentration of MCP were increased by 25.83%(170.595±2.164μg·mL~(-1) VS 88.831±2.329μg·mL~(-1),P＜0.05), 8.16%(67.312±4.269μg·mL~(-1)VS 72.806±4.086μg·mL~(-1),P＞0.05),respectively.
The dynamic experiment have shown that at 6h and 12h,levels of pH were lowered by 1.35%(6.49±0.04 VS 6.32±0.02,P＜0.01),2.11%(6.49±0.04 VS 6.02±0.05, P＜0.01),respectively,while at 6h the concentration of NH_3-N were inhibited by 11.72% (1.058±0.090 mg·100mL~(-1) VS 0.934±0.061 mg·100mL~(-1),P＜0.01),however,at 12h were increased by 71.55%(0.952±0.035 mg·100mL~(-1) VS 1.633±0.075 mg·100mL~(-1), P＜0.01).The concentration of total volatile fatty acids was obviously higher after 6h,the acetate(A) percent decreased,and the propionate(P) percent increased,in results,the ratio of P to A obviously increased.Linseed lignans could be metabolized into END and ENL in vitro.
Part 3:Effeets of Ipriflavone on Ruminal Metabolism and Concentration of Sex Hormones in rumen in Goat
This experiment was conducted to study the effects of ipriflavone on ruminal metabolism and concentration of 17β-estradiol and testosterone in rumen in goat.This study consisted of two series:
Series 1:in vitro
Ruminal fluid was incubated in anaerobic media in vitro with straw powder(0.6 g) and concentrates(0.3 g) as the substrate.Ipriflavone was added into the incubation with the final concentrations at 0 mg/L,10 mg/L,50 mg/L,100 mg/L,200 mg/L,respectively. Furthermore,when the concentration of ipriflavone was 100 mg·L~(-1),the samples of 2h,4h, 6h,8h,12h were collected.
In the dose experiment,results showed that iptiflavone on 100 mg/L and 200 mg/L significantly enhanced ruminal metabolism.Levels of pH were reduced,the concentration of NH_3-N and MCP were increased obviously.The dynamic experiment shown that at 4 h,8 h and 12 h,levels of pH were lowered by 0.68%(P＜0.01),0.49%(P＜0.05),2.05% (P＜0.01) respectively.In control,after 6h the concentration of NH_3-N had downtrend, while,in experiment,the concentration of NH_3-N kept raising.At 12h were increased 12.50%(P＜0.01),the concentration of MCP had the same change of downtrend after 4h both in control and in experiment,In the whole incubation,the concentration of MCP was increased in experiment than that in control.
Series 2:in vivo
Four local adult goats with permanent rumen fistulae were conducted into two periods by self-control design,control period and experiment period.In experiment period, ipriflavone was added into concentrates(100 mg/100g concentrates).Criterias were tested every two hours from 2h to 10h before feeding and after feeding.
Results showed that ipriflavone increased ruminal nitrogen metamolism in tendency, raised the concentration of acetate(A) and the percentage of acetate(P＜0.01),lowed the percentage of propionate(P)(P＜0.05) and the ratio of P:A significantly(P＜0.01);the concentration of acetate was raised 9.66%,the percentage of acetate was raised 7.20%,the percentage of propionate was lowed 5.45%,the ratio of P to A was lowed 12.31%, Ipriflavone could significantly increase the concentration of 17β-estradiol and testosterone in ruminal fluid of goat(P＜0.05).
These resulds may suggest that an interaction between ipriflavone and rumen microorganisms occurred.Ipriflavone could enhance nitrogen metabolism in rumen,affect carbohydrates metabolism,alter the fermentation pattern,and significantly increase the levels of 17β-estradiol and testosterone in ruminal fluid.
引文
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