【作者】 李鹏；

【导师】 沈向真；

【作者基本信息】 南京农业大学 ， 兽医硕士（专业学位）， 2013， 硕士

【摘要】 奶产业是我国重要的发展产业之一,也是改善居民膳食结构的重要战略性产业,奶业的发展水平既是一个国家农业发展水平高低的重要标志,也是一个国家人民生活水平高低的重要指标。我国由于缺少优质的粗饲料,因此精饲料常常被大量使用以满足动物机体对营养的需求,但是大量饲喂高精料日粮会使瘤胃内发生异常发酵,严重时会发生急性瘤胃酸中毒,慢性时会发生亚急性瘤胃酸中毒,尤其是后者,由于其发病率高、临床症状不明显,常常会对生产造成十分巨大的损失。组胺是一种自体活性物质,正常生理条件下身体含有微量组胺,不会对自体产生危害,当反刍动物大量采食精料后,瘤胃内会产生大量的组胺,肝脏是组胺代谢的主要场所,当外周血中组胺含量过高时,将增加血管的通透性,可以直接或间接引起各种炎症,在奶牛中常见的如乳房炎、蹄叶炎等,这些疾病将会降低奶牛的生产能力,再加上我国的反刍动物日粮水平的日粮精粗比在4：6和6：4之间,因此,探讨在日粮精粗比为4：6和6：4下对组胺在奶山羊瘤胃内生成和肝脏中代谢的影响具有很高的学术价值和实用价值。试验Ⅰ、瘤胃液和血液中组胺测定方法的建立本试验采用RP-HPLC来测定组胺,采用紫外检测器检测,样品经0.5mol·L-1的盐酸提取后,用丹磺酰氯进行柱前衍生,色谱柱为Agilent ZORBAX 80A Extend- C18 (4.6mm×250mm,5μm),流动相A为乙腈,流动相B为0.1%乙酸铵,采用等梯度洗脱,流动相比例为A：B=80：20,流速：1.0mL min-1,进样量：20μ1,柱温：30℃,紫外检测波长：254 nm,最后得到标准曲线为y=0.0008x+0.0568,R2=0.9997,线性范围为：0.1～100μmol·L-1,检测限为：0.015μmol·L-1,回收率为：95.45%,因此,成功建立了使用RP-HPLC来测定瘤胃液和血液中组胺的方法。试验Ⅱ、日粮精粗比对奶山羊瘤胃液内pH值和组胺生成的影响采用装有瘤胃瘘的4只奶山羊随机分为两组,每组2只进行交叉试验,分别饲喂日粮精粗比为4：6和6：4等能等氮的饲料,每周期为3周,共2期,分别在第21天通过瘤胃瘘管采集采食前和采食后第2、4、6和8小时的瘤胃液。结果显示：(1)饲喂日粮精粗比为6：4的奶山羊瘤胃内pH值低于饲喂日粮精粗比为4：6下的pH值,并且在高精料日粮下其体内pH值的下降速率和幅度都大于低精料日粮。(2)在高精料日粮下瘤胃内pH值低于6.0持续了6个小时,提示奶山羊发生了SARA。(3)在高精料日粮下,瘤胃内组胺含量升高,并且差异极其显著(P<0.01)。试验Ⅲ、日粮精粗比对组胺在奶山羊肝脏中代谢的影响采用装有门静脉和肝静脉插管的4只奶山羊随机分为两组,每组2只进行交叉试验,饲喂周期及采样时间同试验Ⅱ,结果显示：(1)在高精料日粮条件下静脉血液内组胺浓度有升高的趋势,说明高精料日粮增加了血液中组胺的含量。(2)不同日粮下门静脉中的组胺浓度高于肝静脉中的组胺浓度,且差异显著(P<0.05),这说明肝脏对组胺有清除作用。(3)在高精料日粮条件下肝脏对组胺的清除率升高,但差异不显著。试验Ⅳ、日粮精粗比对肝脏中组胺受体mRNA表达的影响采用6只奶山羊随机分为两组,每组3只进行交叉试验,饲喂周期及采样时间同试验Ⅱ,结果显示：(1)在奶山羊肝脏中,组胺H1受体可能在肝脏内起主要作用。(2)高精料日粮条件下组胺H1受体的表达量有升高的趋势。综上表明,在我国目前反刍动物日粮水平条件下,饲喂大量高精料日粮瘤胃内组胺含量显著升高,血液中组胺含量也有升高的趋势,组胺H1受体的表达量升高,肝脏对组胺的清除率升高但是不显著。更多还原

【Abstract】 The dairy industry is one of the important development industry in our country, but also is an important strategic industries to improve the diet, the level of development of the dairy industry is not only an important symbol of a national agricultural development level, but also an important indicator of the national people’s living standards. Due to the lack of high-quality roughage, concentrated feed is often extensive used to meet the needs of the animal body’s nutrition, but high concentrate diet causes rumen abnormal fermentation, severe cases may occurrence the Acute ruminal acidosis (ARA), chronic cases may occurrence the Subacute ruminal acidosis (SARA), especially the latter, due to the high incidence and subclinical, may cause enormous economic losses to production.Histamine is a kind of self-body active substances and trace amounts of histamine is not harmful to body under normal physiological conditions. When the rumen occurs abnormal fermentation will produce large amounts of histamine which is degraded in the liver, when high levels of histamine in the peripheral blood, causing a variety of diseases, such as mastitis, laminitis and so on, these diseases will reduce the production capacity of the cows. In addition, most of the dairy farms use the dietary levels which concentrate-roughage ration is between 4:6 and 6:4 in our country, so research pH changes and the formation of histamine in the rumen and histamine metabolism in the liver have great academic values and practical value under the dietary forage to concentrate-roughage ratio of 4:6 and 6:4.Test I Setting up a method for determination of histamine concentration in the rumen fluid and blood. Histamine was determined by RP-HPLC and UV detector in this test, samples were extracted by 0.5mol·L-1 hydrochloric acid, then pre-column derivatization with dansyl chloride, the chromatogram column was Agilent ZORBAX 80A Extend-C18 (4.6 mm×250 mm,5μm), mobile phase A was acetonitrile and mobile phase B was 0.1% ammonium acetate, using gradient elution, mobile phase ratio was A:B=80:20, rate was 1.0ml/min, the injection volume was 20μL, Column temperature was 30℃, detection wavelength was 254nm, resulted standard curve:y=0.0008x+0.0568, R2= 0.9997, linear range:0.1μmol·L-1 to 100μmol·L-1, detection limit:0.015μmol·L-1, recovery rate:95.45%, therefore, the success of setting up a method for determination of histamine concentration in the rumen fluid and blood.Test Ⅱ Effects of dietary forage ratio on the pH values and histamine generated in the rumen.4 dairy goats which were fitted with rumen fistula used for crossover experiment, fed dietary forage to concentrate-roughage ratio of 4:6 and 6:4 respectively by two periods which has equal energy and nitrogen. Three weeks for one period, then on 21 days rumen fluid samples were respectively collected before feeding and after feeding on 2, 4,6 and 8 h. The results showed that:(1) The rumen fluid pH of dairy goats which were fed dietary forage to concentrate ratio of 4:6 was lower than that of the ratio of 4:6, the decline rate and magnitude of the pH value which were fed the high-concentrate greater than the low-concentrate diet. (2) The rumen fluid pH values of dairy goats which were fed forage to concentrate-roughage ratio of 6:4 below 6 for six hours, suggested that the dairy goats may have occurred SARA. (3) The rumen fluid histamine content of dairy goats which were fed forage to concentrate ratio of 6:4 was higher than that of the ratio of 4:6(P<0.01), suggestive of histamine may promote the occurrence and development of SARA.Test Ⅲ Effects of dietary forage ratio on histamine metabolism in liver.4 dairy goats which were fitted with portal vein catheterization and hepatic vein catheterization used for experiment, feeding cycles and the sampling time the same as the second experiment. The results showed that:(1) Histamine concentrations of venous blood tended to increase which was fed by the high concentrate diet, it reveals high concentrate diet increased the content of histamine in the blood. (2) The histamine concentration in the portal vein was higher than the concentration of histamine in the hepatic vein (P<0.05), this showed that the liver had the ability to clear the histamine. (3) The liver clearance rate of dairy goats which was fed the high concentrate diet was higher than that of fed low concentrate diet, but the difference was not significant.Test IV Effects of dietary forage ratio on the expression of histamine receptor mRNA in liver.6 dairy goats used for crossover trial, feeding cycles and the sampling time the same as the second experiment. The results showed that:(1) Histamine receptor H1 may play a dominant role of histamine metabolism in the liver of dairy goats. (2) The expression rate of histamine receptor H1 under fed the high concentrate diet was higher than that of low concentrate diet, but the difference was not significant.In summary, feeding high concentrate diets may lead to SARA occurred on dairy goats, histamine content in the rumen was significantly increased, histamine concentration in the blood and the expression rate of the histamine receptor H1 have the tendency of increase, the liver clearance rate of dairy goats was not significantly increased in our current dietary levels. The elevated histamine concentration may play an important role to promote the occurrence and development of SARA.更多还原