Effects on the hepatic transcriptome due to caloric restriction are not altered by milking frequency. Bos taurus

The ruminant liver has multiple roles in the dairy cow and many of these are crucial in nutrient supply during lactation. Reduced feed intake alters the expression of many genes and pathways in the liver, inducing a period of negative energy balance. Once-daily milking is a management strategy to reduce the effects of periods of negative energy balance so the objective of this study was to determine if once-daily milking altered hepatic gene transcription during a period of negative energy balance induce by caloric restriction. Multiparous Holstein-Friesian and Holstein-Friesian x Jersey cows (n = 120) were grazed on pasture and milked twice daily (2X) from calving until 34 ± 6 days in milk (mean ± standard deviation). Cows were then allocated to one of four treatments in a 2 x 2 factorial arrangement. Treatments consisted of two milking frequencies (2X or once daily; 1X) and two feeding levels for three weeks: adequately fed (AF), consuming 14.3 kg dry matter intake/cow per d, or underfed (UF) consuming 8.3 kg dry matter intake /cow per d. After the treatment period, all cows were fed to target grazing residuals ? 1600 kg DM/cow per d and milked 2X for 20 wk. Liver tissue was collected from 12 cows per treatment by subcutaneous biopsy at 3 wk relative to treatment start, RNA extracted and transcript abundance of genes quantified. Overall design: 48 animals were enrolled in this study, a liver sample from 45 animals was used for microarray analysis. A reference design was used for hybridisation, whereby each of the 46 samples were hybridised to individual microarrays along with a pooled reference sample. The reference sample was generated by taking equal concentrations of each of the experimental samples.

反刍动物肝脏在奶牛体内具有多种生理功能，其中多数功能对泌乳期的营养供给至关重要。采食量降低会改变肝脏内大量基因的表达及通路活性，诱发一段时期的负能量平衡（negative energy balance）。每日一次挤奶是一种可减轻负能量平衡负面影响的饲养管理策略，因此本研究旨在探究：在热量限制（caloric restriction）诱导的负能量平衡期间，每日一次挤奶是否会改变肝脏的基因转录水平。

本研究共纳入120头经产荷斯坦-弗里生奶牛以及荷斯坦-弗里生×泽西牛杂交牛，自产犊至泌乳期34±6天（平均值±标准差）期间均采用牧场放牧饲养，并以每日两次挤奶（2X）的方式进行挤奶。随后将奶牛按照2×2析因设计分为4个处理组。处理组设置包括两种挤奶频率：每日两次（2X）与每日一次（1X），以及两种饲喂水平，试验周期为3周：充分饲喂组（AF），每头奶牛每日干物质采食量为14.3 kg；限饲组（UF），每头奶牛每日干物质采食量为8.3 kg。试验期结束后，所有奶牛均按照目标放牧残留量≥1600 kg DM/头·天进行饲喂，并恢复每日两次挤奶，持续20周。

于试验开始后第3周，通过皮下穿刺活检从每个处理组的12头奶牛体内采集肝脏组织，提取RNA并对基因转录本的丰度进行定量。

整体实验设计：本研究共纳入48头实验动物，其中45头的肝脏样本用于基因芯片（microarray）分析。本研究采用参考杂交设计：将46个样本分别与混合参考样本共同进行芯片杂交。该参考样本通过将所有试验样本按等浓度混合制备得到。