Abdelatty A M, Iwaniuk M E, Garcia M, Moyes K M, Teter B B, Delmonte P, Kadegowda A K G, Tony M A, Mohamad F F, Erdman R A
Animal and Avian Sciences Department, University of Maryland, College Park 20742; Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt 12211.
Animal and Avian Sciences Department, University of Maryland, College Park 20742.
J Dairy Sci. 2017 May;100(5):4000-4013. doi: 10.3168/jds.2016-11130. Epub 2017 Feb 23.
Investigations of the temporal changes in mammary gene expression that occur during sudden diet change have been limited by the use of mammary tissue as the source of RNA because of the invasive nature of mammary biopsy procedures. However, the cytosolic crescent, present in 1% of the largest milk fat globules, contains mammary epithelial cell RNA that has become trapped between the inner and outer milk fat globule membranes during final formation and secretion of milk fat into the lumen of the mammary alveoli. We hypothesized that cytosolic crescent RNA extracted from milk fat could be used as an alternative source of mammary epithelial cell RNA to measure the immediate temporal changes in gene expression as a result of changes in diet. In this experiment, feed restriction was used to mimic the state of negative energy balance observed in early lactation and induce a rapid change in milk fat yield and lipogenic gene expression. Ten multiparous Holstein dairy were fed a basal diet ad libitum during a 14-d preliminary period followed by a 4-d experimental period where 5 cows remained on ad libitum feeding and 5 cows were fed at 60% of their d 8-14 intakes (restricted) on d 15 to 18 and then returned to ad libitum feeding on d 19 to 21. Milk samples were collected from each milking on d 13 to 20 and the milk fat was immediately isolated, mixed with Trizol LS, and stored at -80°C for subsequent extraction of RNA that was used for measurement of gene expression. Feed restriction tended to increase milk fat percentage. However, total milk and milk fat production were reduced by 21 and 18%, respectively. Consistent with increased use of body fat for milk synthesis, serum nonesterified fatty acids increased 6-fold (0.78 mEq/L in the feed restriction vs. 0.13 mEq/L ad libitum group), whereas the milk fatty acids <C16 decreased and ≥C16 increased by 6.5 percentage units. The lipogenic genes ACACA, FASN, and SCD1, and the transcription factor SREBF1were downregulated by 59, 36, 35, and 43%, respectively, during the feed restriction period. In addition, effects of feed restriction by day within period were detected for the genes AGPAT6, GPAM, BTN1A, and SREBF1, suggesting that temporal (day-to-day) changes in gene expression could also be detected. The results of this experiment were consistent with previous feed restriction studies in which researchers used the mammary biopsy technique to study gene expression in the mammary gland. We concluded that RNA isolated from milk fat could be used as an alternative approach to investigate rapid temporal changes in lipogenic gene expression.
由于乳腺活检程序具有侵入性，以往利用乳腺组织作为RNA来源来研究突然改变饮食期间乳腺基因表达的时间变化受到了限制。然而，在最大的乳脂肪球中有1%存在胞质新月体，其中含有乳腺上皮细胞RNA，这些RNA在乳脂肪最终形成并分泌到乳腺腺泡腔的过程中被困在了乳脂肪球的内外膜之间。我们推测，从乳脂肪中提取的胞质新月体RNA可作为乳腺上皮细胞RNA的替代来源，用于测量因饮食变化导致的基因表达的即时时间变化。在本实验中，采用限饲来模拟早期泌乳期出现的负能量平衡状态，并诱导乳脂肪产量和生脂基因表达的快速变化。10头经产荷斯坦奶牛在14天的预试验期内自由采食基础日粮，随后进入4天的试验期，其中5头奶牛继续自由采食，另外5头奶牛在第15至18天按其第8至14天采食量的60%进行限饲，然后在第19至21天恢复自由采食。在第13至20天，每天采集牛奶样本，立即分离乳脂肪，与Trizol LS混合，并储存于-80°C，随后用于提取RNA以测量基因表达。限饲倾向于提高乳脂肪百分比。然而，总牛奶产量和乳脂肪产量分别降低了21%和18%。与增加身体脂肪用于乳合成一致，血清非酯化脂肪酸增加了6倍（限饲组为0.78 mEq/L，自由采食组为0.13 mEq/L），而乳中<C16脂肪酸减少，≥C16脂肪酸增加了6.5个百分点。在限饲期，生脂基因ACACA、FASN和SCD1以及转录因子SREBF1分别下调了59%、36%、35%和43%。此外，还检测到限饲对AGPAT6、GPAM、BTN1A和SREBF1基因在不同日内的影响，表明基因表达的时间（每日）变化也可被检测到。本实验结果与之前的限饲研究一致，在那些研究中，研究人员使用乳腺活检技术来研究乳腺中的基因表达。我们得出结论，从乳脂肪中分离的RNA可作为一种替代方法，用于研究生脂基因表达的快速时间变化。
