Global gene expression profiling of the effect of probiotics supplementation in dairy cows

In animal production, the use of probiotics supplements to promote animal health is increasing. The objective of this study was to assess the impact of probiotics administration on global gene expression in dairy cows. Lactating Holstein-Friesian cows (n=10) from the North Carolina Agricultural and Technical State University dairy herd were used for the study. Treatment was a 50 ml oral drench of FASTtrak microbial pack (Probiotics) (Conklin Company, Kansas City, MO) at the recommended dose in sterile endotoxin-free water or sterile endotoxin-free water only (control). This treatment was carried out for 60 days. Whole blood was collected at the beginning (Day 0) and end of the study (Day 60) for microarray analysis. We employed microarray expression profiling as a discovery platform to identify genes with potential association with probiotics supplementation in cows. Gene expression analysis identified 10,859 differentially expressed genes- 1168 upregulated genes and 9691 downregulated gene. Results for pathway analysis showed significant pathways associated with innate immunity such as the Toll-like receptor (TLR) pathway, inflammation response and Wingless (Wnt) signaling pathway. Real-time PCR was used to validate the expression of the Wnt signaling pathway and immune response genes. Probiotic treatment impacted global gene expression, and particularly, the expression of immune response and Wnt signaling pathway genes. Oral administration of probiotics to dairy cows impacts global gene expression and particularly the expression of innate immune genes in dairy cows. Ten animals were enrolled in the study and an initial blood sample was collected (Day 0). Animals (n=5) received either oral supplement of a commercial probiotic under the name FASTtrak microbial pack (Conklin Company, Kansas City, MO) or water only (control animals) for 60 days. Blood samples were collected at the end of the study from probiotics-treated and control animals for RNA extraction and microarray analysis. In vitro effect of lipopolysaccharide (LPS), endotoxin treatment was evaluated using blood samples collected from probiotics-treated animals (Day 60 samples) to serve as a positive control array. A pooled sample was generated by taking an equal concentration of RNA from experimental animals in each group. Pooled samples from each group were hybridized on Agilent one-color bovine v2 bovine (v2) 4x44K array slides.

在畜牧生产中，益生菌补剂用于促进动物健康的应用愈发普遍。本研究旨在评估饲喂益生菌对泌乳期荷斯坦-弗里生奶牛（Holstein-Friesian）全基因表达的影响。本研究选用北卡罗来纳州农业与技术州立大学奶牛群中的10头泌乳期荷斯坦奶牛作为试验对象。试验组奶牛经口灌服50ml以无菌无内毒素水配制的推荐剂量FASTtrak微生物包（益生菌，康克林公司Conklin Company，密苏里州堪萨斯城），对照组仅灌服无菌无内毒素水，试验周期为60天。分别于试验开始当日（第0天）与试验结束当日（第60天）采集全血样本，用于基因芯片（microarray）分析。本研究采用基因芯片表达谱作为筛选平台，以鉴定与奶牛益生菌补饲存在潜在关联的基因。基因表达分析共鉴定出10859个差异表达基因，其中1168个基因上调表达，9691个基因下调表达。通路分析结果显示，与固有免疫相关的显著通路包括Toll样受体（Toll-like receptor, TLR）通路、炎症应答通路以及Wnt信号通路。采用实时荧光定量PCR（real-time PCR）对Wnt信号通路与免疫应答相关基因的表达水平进行验证。试验结果表明，益生菌处理可影响奶牛的全基因表达，尤其对免疫应答与Wnt信号通路相关基因的表达具有调控作用。给泌乳期荷斯坦奶牛经口饲喂益生菌，可改变其全基因表达谱，特别是固有免疫相关基因的表达水平。本研究共纳入10头试验奶牛，于第0天采集初始血液样本。将试验牛随机分为两组，每组5头：一组饲喂市售FASTtrak微生物包益生菌补剂，另一组仅灌服无菌水（对照组），饲喂周期均为60天。试验结束时，分别采集益生菌处理组与对照组奶牛的血液样本，用于RNA提取与基因芯片分析。为设置阳性对照芯片，本研究利用试验结束当日（第60天）采集的益生菌处理组奶牛血液样本，评估了脂多糖（lipopolysaccharide, LPS）即内毒素处理的体外效应。通过将各组试验动物的RNA按等浓度混合制备混合样本，将各组混合样本点样于安捷伦（Agilent）单通道牛v2 4x44K基因芯片玻片进行杂交。