Serial hepatic gene expression profiling in Angus steers during feed restriction and realimentation

Growing ruminants maintained under dietary restriction for extended periods will exhibit compensatory growth when reverted to ad libitum feeding. This period of compensatory growth is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify genetic mechanisms contributing to these physiological changes, 8 month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n=6) for a period of 12 weeks. All steers were then fed ad libitum for the remaining 8 weeks of the experiment (realimentation period). Liver was biopsied from each animal at days -14, +1 and +14 relative to realimentation for RNA extraction and gene expression analysis by microarray hybridization. Steers were assigned randomly to one of two treatment groups, control or feed-restricted, and housed indoors in individual pens. Steers were acclimated to their pens for 5 d prior to starting the experimental treatments. Feed was offered once daily between 0630 and 0930 and orts from the previous day's feeding were collected and weighed to estimate actual intake. Control animals were fed ad libitum throughout the 20-wk experimental period. Feed-restricted steers were offered 60-70% of intake of control animals for 12 wks to target a limited rate of gain of approximately 0.5 kg/d. Restricted steers were then fed ad libitum for the remaining 8 wks of the experiment (realimentation period). During the first 3 d of realimentation, feed offered to both treatment groups was divided into two equal rations to gradually adjust restricted animals to full intake. Water was offered ad libitum throughout the experimental period. Approximately 200 mg of liver tissue was collected from each steer by needle biopsy using a Tru-Cut biopsy needle at -14, +1, +14 d relative to realimentation. Liver samples were immediately frozen in liquid nitrogen and stored at -80C until RNA isolation. Total RNA was isolated from 36 liver samples using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Samples were DNase-treated using the TURBO DNA-free kit (Ambion, Inc., Austin, TX) according to manufacturer’s instructions, followed by column purification using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quality and concentration of RNA were assessed using a 2100 Bioanlayzer (Agilent Technologies, Palo Alto, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Probe labeling, hybridizations of probes to the oligo microarray, and array scanning were performed by the Roche NimbleGen Systems, Inc. Microarray Core Facility in Reykjavik, Iceland according to standard procedures (Madison, WI; http://www.nimblegen.com).

长期处于日粮限制饲养的生长反刍动物，在恢复自由采食（ad libitum feeding）后会表现出补偿性生长（compensatory growth）。该补偿性生长阶段与饲料转化率提升、基础能量需求降低以及循环代谢激素浓度变化密切相关。为阐明介导上述生理变化的遗传机制，研究人员将8月龄阉牛随机分为两组（每组n=6）：自由采食组（对照组），以及采食量为对照组60%~70%的限饲组，限饲周期为12周。随后所有阉牛在实验剩余的8周内均恢复自由采食，即复饲阶段（realimentation period）。在复饲前14天（第-14天）、复饲后第1天和第14天，对每头动物进行肝脏活检，用于RNA提取及基因表达的微阵列杂交（microarray hybridization）分析。所有阉牛先随机分配至对照组或限饲组，单栏舍饲。正式实验开始前，让阉牛适应圈舍5天。每日06:30至09:30投喂一次饲料，收集前一日的饲槽残料并称重以计算实际采食量。对照组在整个20周的实验周期内均自由采食。限饲组阉牛在前12周的采食量为对照组的60%~70%，以实现约0.5kg/d的目标限速增重。随后限饲组阉牛在实验剩余8周内恢复自由采食，即复饲阶段。在复饲的前3天，两组的投喂饲料均分为两等份日粮，以逐步让限饲组动物适应全量采食。整个实验期间均自由提供饮水。使用Tru-Cut活检针通过穿刺法从每头阉牛体内采集约200mg肝脏组织，采样时间点为复饲前后的-14、+1、+14天。肝脏样本立即置于液氮中速冻，并保存于-80℃环境直至RNA提取。使用TRIZOL Reagent（Invitrogen Corp., Carlsbad, CA）从36份肝脏样本中提取总RNA。按照制造商说明书，使用TURBO DNA-free kit（Ambion, Inc., Austin, TX）对样本进行DNase处理，随后使用RNeasy Mini Kit（Qiagen, Valencia, CA）进行柱纯化。使用2100 Bioanalyzer（Agilent Technologies, Palo Alto, CA）和ND-1000分光光度计（NanoDrop Technologies, Wilmington, DE）评估RNA的质量与浓度。探针标记、探针与寡核苷酸微阵列杂交以及芯片扫描均由冰岛雷克雅未克的罗氏NimbleGen Systems, Inc.微阵列核心设施按照标准流程完成（Madison, WI; http://www.nimblegen.com）。