热应激对奶牛、奶山羊体内内毒素含量的影响及缓解热应激的营养技术研究

【作者】 王立志；

【导师】 王康宁；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 本研究旨在探索缓解奶牛热应激的方法。首先比较研究了冬季和夏季奶牛血浆内毒素浓度、MDA含量、SOD和GSH-Px活性的差异。在此基础上,以安装有门静脉、肠系膜静脉、颈动脉血管插管的奶山羊为模型,考察门静脉净吸收的内毒素主要来源,热应激对门静脉和肠系膜静脉内毒素的净吸收量、血浆流量、MDA流量、TAP、SOD、GSH-Px、CAT酶活流量的影响,以及补饲酵母培养物、VE对干物质采食量、肛温和呼吸频率、门静脉、颈动脉内毒素浓度和门静脉内毒素净吸收量的影响。根据以上的研究结果,最后以奶牛为实验动物,考察日粮中添加酵母硒以及添加酵母培养物对遭受热应激的奶牛的影响。以期以增强机体抗氧化能力、降低内毒素吸收的方法来达到缓解奶牛热应激的目的。主要的研究内容及结果如下：1、热应激对奶牛血液中内毒素含量和抗氧化指标的影响的研究选用16头健康的中国荷斯坦奶牛,分为夏季组(8月)和冬季组(12月)两个处理,每个处理8个重复,每个重复1头牛。当夏季组奶牛牛舍温湿度指数(THI)>72的时间连续达到15天,冬季组当牛舍THI<72的时间连续达到15天时,颈静脉采血15mL制备血浆,检测血浆中内毒素、超氧化物歧化酶(SOD)、总抗氧化能力(TAP)、谷胱甘肽过氧化物酶(GSH—Px)的活性,以及丙二醛(MDA)的含量。实验结果表明：夏季热应激导致奶牛血清内毒素、MDA含量极显著(p<0.01)升高,SOD活性极显著(p<0.01)降低,TAP和GSH-Px活性显著(p<0.05)降低。2、热应激对奶山羊门静脉和肠系膜静脉内毒素净吸收量和氧化还原指标的影响研究选取3只体况良好,头胎,平均体重30kg的萨能奶山羊,安装门静脉、肠系膜静脉、颈动脉慢性血管插管进行实验。待手术实验羊恢复正常后开始正式实验,将羊单笼饲养于温控室内,控制室内温度为24℃,湿度为55%。正式实验的第4天上午08：00时开始灌注PAH工作液,2小时后第一次采血,以后每隔2小时采一次血,连续采6次,6次平均值作为温度适中区组值,第1、3、6次采血后测定一次肛温和呼吸频率,3次平均值作为温度适中区组值。实验的第6天上午08：00时再次开始灌注对氨基马尿酸(PAH)工作液,同时将室内温度提高到35℃,湿度55%,2小时后第一次采血,以后每隔2小时采一次血,连续采12次,进行24小时热应激实验,第1、6、12次采血后测定一次肛温和呼吸频率,3次平均值作为热应激组值。实验结果表明：热应激导致奶山羊干物质采食量极显著地(P<0.01)下降,肛温和呼吸频率都极显著(P<0.01)升高。随热应激时间的延长,门静脉和肠系膜静脉血浆流量持续降低,颈动脉、门静脉和肠系膜静脉内毒素浓度持续升高。肠系膜静脉和门静脉内毒素的净吸收量在遭受热应激4小时时显著增加,并随遭受热应激时间的延长呈持续增加的趋势。整个热应激期门静脉内毒素净吸收量每小时平均较正常值增加了227.92%,肠系膜静脉增加了279.05%。无论在温度适中区还是热应激期,门静脉吸收的内毒素都主要来源于非肠系膜组织。热应激造成肠系膜静脉血浆SOD、GSH-PX、CAT,门静脉血浆GSH-PX、CAT活力浓度显著(p<0.05)或极显著(P<0.01)升高,但其流量在第4小时左右就显著降低,第16小时左右达到最低值后又逐渐升高,并在较正常值低的一个水平达到稳定。肠系膜静脉血浆TAP浓度在热应激的大部分时间点与正常组差异不显著,但是其流量在热应激初期呈持续下降的趋势,最后在一个较低水平达到稳定；门静脉和肠系膜静脉血浆MDA的浓度和流量在热应激期间呈现先降低再逐渐增加的规律。实验结论：采食5：5精粗比日粮的奶山羊,无论是在温度适中区还是在热应激情况下,门静脉净吸收的内毒素主要来源于非肠系膜组织。热应激导致门静脉和肠系膜静脉血浆流量、TAP、SOD、GSH-Px、CAT酶活流量极显著降低(P<0.01),内毒素的净吸收量极显著增加,且随热应激时间的延长,内毒素的吸收量持续增加。热应激过程中奶山羊胃肠道通透性显著增加发生在氧化损伤之前。3、补饲酵母培养物、VE缓解奶山羊热应激效果的研究选取3只体况良好,头胎,平均体重30kg的萨能奶山羊,安装门静脉、颈动脉慢性血管插管进行实验。采用3*3拉丁方实验设计,设对照组(C组,饲喂基础日粮)、VE组(VE组,在基础日粮基础上每天每头补饲VEl00IU)、酵母组(Y组,在基础日粮基础上每天每头补饲酵母培养物15克,)3个处理组。每期实验为期4天,两期之间间隔4天。酵母和维生素E每天早晨08：00一次性补饲。待手术实验羊恢复正常后开始正式实验,将羊单笼饲养于温控室内,日粮精粗比控制在5：5左右,自由采食,记录采食量。实验期控制室内湿度为55%,温度08：00-20：00为35℃,20：00-08：00为24℃,每期实验的第4天13：00时开始灌注PAH工作液,2小时后第一次采血,以后每隔1小时采一次血,连续采12次,采血期间每两小时测定一次肛温和呼吸频率。间隔期饲喂下一期实验日粮,间隔期控制室内湿度为55%,温度为24℃。实验结果表明：各处理间血浆流速、干物质采食量差异不显著(P>0.05)。热应激期和全期各处理间肛温和呼吸频率平均值,差异都不显著(P>0.05),但在温度适中区各时间点,C组肛温都极显著(P<0.01)高于VE组和Y组,个别时间点的呼吸频率,C组值显著(p<0.05)或极显著(P<0.01)高于VE组和Y组。内毒素净吸收量,热应激期的平均值,C组极显著(P<0.01)高于VE组和Y组；温度适中区平均值,三个处理间差异均不显著(P>0.05)；全期平均值,C组极显著(P<0.01)高于VE组,Y组与其它两组间的差异均不显著。门静脉血浆SOD活力浓度,热应激期的平均值,C组极显著(P<0.01)高于VE组和Y组；温度适中区平均值,C组显著(p<0.05)高于VE组；全期平均值,C组极显著(P<0.01)高于VE组和Y组。门静脉血浆TAP浓度,热应激期的平均值,C组极显著(P<0.01)低于VE组,显著(p<0.05)低于Y组；温度适中区平均值,C组显著(p<0.05)低于VE组,Y组和C组、VE组的差异均不显著(P>0.05)；全期平均值,C组极显著(P<0.01)低于VE组,Y组显著(p<0.05)低于VE组,Y组和C组差异不显著(P>0.05)。门静脉血浆MDA的浓度,热应激期的平均值,C组极显著(P<0.01)高于VE组和Y组,Y组极显著(P<0.01)高于VE组；温度适中区平均值,C组极显著(P<0.01)高于VE组和Y组,VE组和Y组的差异不显著(P>0.05)；全期平均值,C组极显著(P<0.01)高于VE组和Y组,Y组极显著(P<0.01)高于VE组。实验结论：热应激时补饲VE、酵母培养物具有降低奶山羊肛温,门静脉和颈动脉内毒素浓度,减少门静脉内毒素净吸收量和氧化损伤的作用。4、补饲酵母培养物和酵母硒缓解奶牛热应激效果的研究实验采用单因素实验设计,将健康无病、产后120天至200天、产奶量18kg头·d至22kg头·d的48头经产中国荷斯坦奶牛,根据胎次、泌乳天数、实验前-周产奶量相近原则随机分到4个处理组,分别为对照组C组,Se-Y组,YC组,Se-Y+YC组,每组6个重复,每个重复2头牛。对照组C组饲喂基础日粮,Se-Y组在基础日粮基础上每千克干物质加酵母硒0.30g, YC组在基础日粮基础上每千克干物质加酵母培养物4g,Se-Y+YC组在基础日粮基础上每千克干物质加酵母硒0.30g,酵母培养物4g。实验为期47天,其中预饲期7天,正式实验期40天。正式实验期每两天观测一次温度和相对湿度,每10天测定一次牛群干物质采食量,肛温和呼吸频率,每10天测定一次产奶量,每20天采集一次乳样分析乳成分,每10天颈静脉采血15mL用于分析血浆MDA、内毒素含量以及SOD、GSH—Px的活力。实验结果表明：正式实验期的舍内温热环境对奶牛已构成了热应激。各实验组较对照组奶产量都有所提高,其中Se-Y组提高了5.67%(p>0.05),YC组提高了2.06%(p>0.05),Se-Y+YC组提高了8.25%差异显著(p<0.05)；乳脂率和乳糖含量个处理组差异不显著(p>0.05),但各实验组乳蛋白率都有一定程度提高,其中Se-Y组,YC组,Se-Y+YC组较对照组乳蛋白率分别提高4.0%(p>0.05),7.7%(p<0.05)与9.6%(p<0.05)。各实验组肛温都比对照组低,其中Se-Y组较对照组低0.33℃(p>0.05),YC组较对照组低0.27℃(p>0.05),Se-Y+YC组较对照组低0.47℃(p<0.05)。呼吸频率和干物质采食量各处理间差异不显著。GSH-Px酶活浓度,YC组、Se-Y+YC组极显著(P<0.01)高于YC组、C两组。SOD酶活浓度各处理间差异不显著(p>0.05)。血浆MDA含量Se-Y组、Se-Y+YC组较对照组C组极显著降低(p<0.01),YC组较C组显著(p<0.05降低。血浆中内毒素含量,Se-Y组,YC组,Se-Y+YC组较对照组C组极显著(P<0.01)降低,其它三组组间差异不显著(P>0.05)。研究结论：在热应激情况下,补饲酵母培养物能显著增加奶牛乳蛋白含量,显著降低血液中丙二醛含量,极显著降低血液内毒素浓度；补饲酵母硒能极显著提高奶牛血液中硒含量和谷胱甘肽过氧化物酶的活性,极显著降低血液中丙二醛含量和内毒素浓度；联合补饲酵母硒与酵母培养物能显著提高奶牛奶产量和乳蛋白含量,显著降低肛温,极显著提高奶牛血液中硒含量和谷胱甘肽过氧化物酶的活性,极显著降低血液中丙二醛含量和内毒素浓度。它们联合作用效果优于其每一种单独运用。综上所述,夏季热应激导致奶牛血浆内毒素、MDA含量极显著升高,抗氧化能力显著降低。门静脉吸收的内毒素主要来源于非肠系膜组织。热应激导致门静脉和肠系膜静脉血浆抗氧化能力、血浆流量极显著降低,内毒素的净吸收量极显著增加。热应激过程中胃肠道通透性增加发生在氧化损伤之前。补饲酵母硒、酵母培养物都能在一定程度上缓解奶牛热应激,二者联合应用效果更显著。更多还原

【Abstract】 The aim of this research was to search methods to alleviate heat stress in dairy cattle. The difference between endotoxin concentration、MDA concentration、activity of SOD and GSH-Px in plasma of diary cattle in winter and summer were conduct investigated.On this basis,dairy goats with indwelling catheters in aportal vein, mesenteric vein, carotid artery were used as a model to investigate where the endotoxin in portal vein comes from,the effect of heat stress on the net absorbtion of endotoxin across MDV and PDV,plasma flux,,MDA、TAP、SOD、GSH-Px、CAT flux,and the relationship between gastrointestinal permeability changes and oxidative stress in heat situation.Then Three Saanen dairy goats with indwelling catheters in the portal vein, and carotid were used to investigate the effect of yeast culture、VE supplement on dry matter intake, rectum temperature and respiration frequency,net absorbtion of endotoxin across PDV,and the concentration of endotoxin in portal vein and carotid in heat situation,the dynamic changes of concentration of MDA、TAP、SOD in portal were also investigate.At last,dairy cattles were used to investigate the effect of yeast culture and selenium yeast supplement on dairy cattles subjected to heat stress. The main research contents and results are as follows:1、The investigate about the effect of heat stress on blood endotoxin level and anti-oxidation level in dairy cattle16 healthy Holstein cows were divided into the summer group (in August) and winter group (12 months), each treatment with eight repetitions, a cow each repetition.Summer, when the dairy cow-house temperature and humidity index (THI)> 72 up to 15 consecutive days, winter, when the cow-house THI<72 up to 15 consecutive days,15mL jugular vein blood was taken.endotoxin level in plasma, Superoxide Dismutase (SOD), total antioxidant activity (TAP), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content in plasma were detected. The results show that:in summer, cow plasma endotoxin, MDA content was highly significantly (p<0.01)higher, SOD activity was highly significantly (p<0.01) lower, TAP and GSH-Px activity was significantly (p<0.05) lower than in winter. 2、Effect of heat stress on endotoxin and redox flux across mesenteric-drained and portal-drained viscera of dairy goatThree Saanen first lactation dairy goats were surgically fitted with indwelling catheters in the portal vein, the mesenteric vein and carotid. After 3-week recovering the goats were subject to a 6-day experiment stage. On the third day of experiment stage, all the goats were blood sampled 6 times from 8.00 to 20.00 at 2-hr interval. Room temperature was controlled at 24℃during recovering and the first 5 days of experiment stage. On the 6 day of experiment stage, all the goats were blood sampled 12 times from 8.00 at 2-hr interval, and room temperature was adjusted to 35℃at the beginning of the day and kept for 24 hr. With the prolonged heat stress, plasma flux rate in portal and mesenteric vein kept on decreasing, while plasma endotoxin concentration in arterial, portal and mesenteric venous kept on increasing. Net absorption of endotoxin across MDV and PDV began increasing after 4-hr exposure in heat environment. Average net absorption of endotoxin (EU/hr) across MDV and PDV in heat stress group during the whole period of heat stress increased by 279.05% and 227.92% compared to thermo-neutral group. Plasma concentration of GSH-Px and CAT in mesenteric and portal vein, and that of SOD in mesenteric vein, increased significantly during heat stress. Plasma concentration of TAP in mesenteric vein at different time of exposure in heat environment in heat stress group did not differ from the mean value in thermo-neutral group group, while that in portal vein at different time of heat exposure in heat stress group was always lower than the mean value in thermo-neutral group. Main conclusions were:1) net absorption of indotoxin in portal vein is mainly from Non- mesenteric tissues both in heat stress and in thermo-neutral condition; 2) Heat stress may lead to the significant decrease of plasma, TAP, SOD, GSH-Px, and CAT flux across PDV and MDV, and the significant increase of indotoxin flux across PDV and MDV; 3) the increase of gastrointestinal permeability in dairy goats during heat stress may not be induced by the increase of oxidative stress.3、The research about the effect of Supplementary feeding yeast culture, VE on dairy goats in heat stress situation Three Saanen (first lactation、average body weight 30 kg)dairy goats were surgically fitted with indwelling catheters in the portal vein,and carotid.3*3 Latin square experimental design was used.The three treatments were control group(C, fed basal diet),VE group (VE,on the base of basic diet each goat was supplementary fed 100IU VE per day), yeast group (Y, on the base of basic diet each goat was supplementary fed 15 grams of yeast culture per day).Each experiment lasted 4 days,4-day interval between the two. Yeast and vitamin E were fed at 8:00 every morning.After recovered,the goats were housed in single-cage in temperature-controlled room, the ratio of concentrate to roughage is about 5:5, record feed intake. The indoor humidity was controlled and maintained at 55% in all the experiment period,the temperature was 35℃during 08:00-20:00,24℃during20:00-08:OO.The 4th day of each exeperiment PAH was infused at 13:00 pm,2 hours later blood was taken,and then blood was taken at intervals of 1 hour,last for 12 times, rectum temperature and respiration frequency were measured at interval or 2 houes.During the interal of two trial, the indoor humidity was 55%, the temperature was 24℃.The results showed that: among the treatments,the plasma flow rate, dry matter intake was no significant difference (P> 0.05). rectum temperature and respiration frequency during the heat stress period and the whole term of the deal, the difference is not significant (P> 0.05), but in thermoneutral zone, the average rectal temperature of C group was significantly (p<0.05) higher than the VE group, and in thermoneutral zone,at different time points, the average rectal temperature of C group were significantly (P <0.01) higher than the VE group and Y group.the average respiration rate in thermoneutral zone, there was no significant difference (P> 0.05) among the treatments, but at some individual time points, C group was significantly (p<0.05) or very significantly (P<0.01) higher than the VE group and Y group. The net absorption of endotoxin, throughout the trial period, each time point, C group was the highest, VE was the lowest, Y group was middle; During heat stress period, the average net absorption of endotoxin,, C group was significantly (P<0.01) higher than the VE group and Y group; In thermoneutral zone, on average, three treatments were not significantly different (P> 0.05); the average of the whole term, C group was significantly (P<0.01) higher than the VE group, the difference between Y group and the other two groups were not significant.the average SOD activity concentrations of portal vein plasma,in heat stress period, C group was significantly (P<0.01) higher than the VE group and Y groups; In thermoneutral zone, on average, C group was significantly (p<0.05) higher than the VE group; The average of the whole term, C group was significantly (P<0.01) higher than the VE group and Y group.TAP concentration of portal vein plasma, each time point of the entire trial period, C group was the lowest, VE group was the highest, Y group was the middle; In heat stress period, the average, C group was highly significantly (P<0.01) lower than the VE group,was significant (p<0.05) lower than the Y group, Y group was significantly (P <0.01) lower than the VE group; In thermoneutral zone, on average,C group was significantly (p<0.05) lower than the VE group, the differences betweenY group and C group, Ygroup and VE Group were not significant (P> 0.05); the whole term,on average, C group was significantly (P<0.01) lower than the VE group, Y group was significantly (p<0.05) lower than the VE group, the difference betweenY group and C group was not significantly (P> 0.05).each time point of throughout the trial period, MDA concentration of the portal vein plasma, C group was the highest, VE group was the lowest, Y group was the middle; in heat stress period, on average, C group was highly significantly (P<0.01) higher than the VE group and Y group, Y group was highly significantly (P<0.01) higher than the VE group; in thermoneutral zone, C group was highly significantly (P<0.01) higher than the VE group and Y group,there was no significant difference (P> 0.05)betweenVE group and Y group; the whole term,on average, C group was highly significantly (P<0.01) higher than the VE groups and Y group, Y group was highly significantly (P<0.01) higher than the VE group. Experiment Conclusion:during heat stress, supplementary feeding VE, yeast culture could reduce rectal temperature, endotoxin concentrations in portal vein and carotid arteries, the net portal absorption of endotoxin and oxidative damage.4、The study about the effect of supplementary feeding yeast culture and yeast selenium on alleviating heat stress in dairy cows In this study, single-factor experimental design was used.Forty eight during d 120 to d 200 after postpartum, health, milk yield were 18kg to 22kg Holstein dairy cows in mid-lactation, which have similar milk yield, lactation period and parity,were divided randomLy into four groups, each treatment with six repetitions, two cows each repetition.:Se-Y group (on the base of basal diet,0.3g selenium yeast was added per kilogram of dry matter), YC group (on the base of basal diet,4g yeast culture was added per kilogram of dry matter), C group (fed the basal diet) and Se-Y+YC group (on the base of basal diet,4g yeast culture and 0.3g selenium yeast was added per kilogram of dry matter). The total experimental time were 47 days, include 7 days of preliminary experimental period and 40 days of formal experimental period.During the formal experiment period the temperature and relative humidity were recorded every two days, dry matter intake、rectal temperature and respiratory frequency were measured every 10 days. every 10 days milk yield was measured, every 20 days, milk composition were analysed, every 10 days 15mL blood was taken from Jugular vein for the analysis of plasma MDA, endotoxin content and SOD, GSH-Px activity. The results show that:in the formal experiment period, According to temperature and humidity index of, dairy cattle had been in a heat stress condition.The experiment group than the control group had increased in milk production, in which Se-Y group increased 5.67%(p> 0.05), YC group increased 2.06%(p> 0.05), Se-Y+YC group increased 8.25%(p<0.05); milk fat and lactose content among different groups were no significant difference (p> 0.05), but milk protein content of each experiment group were increased to some extent, in which Se-Y group, YC group, Se-Y+YC group than the control group,the rate of milk protein were increased by 4.0%(p> 0.05),7.7%(p<0.05) and 9.6%(p<0.05).The rectal temperature of experiment group was lower than the control group, in which Se-Y group was 0.33℃(p> 0.05)lower than control group, YC group was 0.27℃(p> 0.05)lower than the control group, Se-Y+YC group was 0.47℃(p<0.05)lower than the control group. Respiratory rate and dry matter intake among the treatments were no significant differences. GSH-Px activity concentration, Se-Y group, Se-Y+YC groups was significant (P<0.01) higher than YC group, C groups. SOD activity levels among the treatments were no significant difference (p> 0.05). Plasma MDA concentration, A, Se-Y+YC group was highly significantly (p<0.01) lower than C group, YC group was significantly (p<0.05 lower than C group. Plasma endotoxin concentrate, Se-Y group, YC group, Se-Y+YC group was highly significantly (P<0.01) lower than C group, the other three groups had no significant difference (P> 0.05).Study result:during heat stress period, added yeast culture in the diet can significantly increase the cows milk protein content and significantly reduce the blood MDA content, highly significantly low blood endotoxin concentration; during heat stress period, added yeast selenium in the diet can significantly increase Cows blood selenium content and glutathione peroxidase activity,highly significantly decreas blood MDA content and endotoxin concentration;during heat stress period, yeast seleniumand the combined effects of yeast culture can significantly increase milk yield and milk protein content, significantly low rectal temperature, significantly increase blood selenium content and glutathione peroxidase activity, significantly reduce MDA content in blood and endotoxin concentrations. Their combined effects was more effective than each of their separate application.In summary, heat stress caused decreased anti-oxidation function of dairy cows, increased plasma endotoxin levels. Enhance the body’s antioxidant capacity, supplementary feeding yeast culture, can reduce the absorption of endotoxin in the heat stress situations, thus to some extent mitigate the impact of heat stress on dairy cows.更多还原