| Modern dairy industries need a large and extensive use of concentrate to meet the energy needs and lactating consumption in high-yielding dairy cows. Subacute ruminal acidosis (SARA) is a very commen disease in dairy cows, sufficient evidence has showed that prevalence of SARA is related to the histamine in animals’ body. When SARA happens, the microorganism in rumen would multiply massively. And their metabolite would induce the increase of mRNA expression of inflammatory factor such as COX-2、TNF-α、IL-1, then active NF-κB signaling pathway. In the current study, the cattle were fed with high or low concentrate ration to induce SARA in accord with normal animal feeding mode. By testing the changes in histamine concentration of the blood and mRNA expression of COX-2、TNF-α、IL-1 in liver, the study is aimed to discover the mechanism of SARA on molecular level and make a reference for the treatment and prevention in the future.1. Histamine levels in portal vein and hepatic vein of high concentrate group and low concentrate group.Six middle-lactation Holsteins cattle were divided into two groups, permanent liver blood fistula was fixed to every cattle’s portal and hepatic vein. One group was fed with a high concentrate, another with the regular ration (control or low concentrate group). Histamine concentration was tested by cattle-histamine-Elisa-kit (Shanghai Kexing,201310) and microplate reader.The color reading under 450 nm of the microplate reader, absorbance value and histamine concentration was conducted as horizontal and vertical axis. Portal and hepatic histamine mean content in SARA group is 19.99 ± 0.21μg/L and 8.55 ± 0.58μg/L, which is significant higher than that in control group (18.12±0.35 μg/L; 9.01± 0.56 μg/L).This demonstrated that high histamine content in the blood may be responsible for its degenerate rate in liver to maintain the normal level in the body.2. Expression of COX-2、NF-KB、IL-6、TNF-α in high concentrate group and low concentrate group.Three dairy cattle were induced SARA by feeding high level concentrate and blood samples were collected by the atificial fistula operated on portal and hepatic vein. Another three cattle feeding normal ration were treated as control group. Collect the liver tissue through liver biopsy, then mRNA expression of COX-2, NF-κB, IL-6 and TNF-a was determined by relative qualification real-time PCR. The results show that mRNA expression of COX-2, NF-κB, IL-6 and TNF-a in the liver was significantly increased in treatment group (p<0.05)3. Absolute quantification of COX-2 in high concentrate group and low concentrate group.Total RNA in liver samples was extracted and reverse-transcript into cDNA. The DNA was amplified by PCR and separated by agarose gel electrophoresis. The stripe was collected and purified by gel recycle kit. Use TAKARA PMD-19T vector kit to transfer DNA fragment into plasmid, and transform it into competent cells. These cells were cultured and screened by LB broth containing 50 μg/ml ampicillin. DNA were extracted by TaKaRa MiniBEST Plasmid Purification KitVet 2.0 kit and acted as standard samples when diluted into 10,102,103,104,105,106,107 and 108 times. The difference value is 3630 and by an average rate of 225%. By detecting COX-2 expression in the liver through real-time PCR, this study revealed that the high concentrate group has a significant high level than the control one. |
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