This study applied a quantitative proteomics approach along with bioinformatics analyses to investigate changes in the plasma proteome of normal and overconditioned dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets until dry-off to reach targeted differences in BCS and back fat thickness (BFT) until dry-off. At dry-off, normal BCS cows had a BCS <3.5 (minimum, 2.75) and BFT <1.2 cm (minimum, 0.58), and the high BCS cows had a BCS >3.75 (maximum, 4.50) and BFT >1.4 cm (maximum, 2.90). The proteomics study used a subset of 5 animals from each group. These cows were selected based on their circulating concentrations of fatty acids (FA) on d 14 postpartum and β-hydroxybutyrate (BHB) on d 21 postpartum, representing the greater or the lower extreme values within their BCS group, respectively. The high BCS subset (HE-HBCS) had 4.50 < BCS > 3.75, FA = 1.17 ± 0.46 mmol/L, and BHB = 2.15 ± 0.42 mmol/L (means ± SD), and the low BCS subset (LE-NBCS) had 3.50 < BCS > 2.75, FA = 0.51 ± 0.28 mmol/L, and BHB = 0.84 ± 0.17 mmol/L. Plasma samples from d -49, +7, and +21 relative to parturition were used for proteome profiling by applying the quantitative tandem mass tags (TMT) approach. Nondepleted plasma samples were subjected to reduction and digestion and then labeled with TMT 10plex reagents. High-resolution liquid chromatography-tandem mass spectrometry analysis of TMT-labeled peptides was carried out, and the acquired spectra were analyzed for protein identification and quantification. In total, 254 quantifiable proteins (criteria: 2 unique peptides and 5% false discovery rate) were identified in the plasma samples. From these, 24 differentially abundant proteins (14 more abundant, 10 less abundant) were observed in the LE-NBCS cows compared with the HE-HBCS cows during the transition period. Plasma α-2-macroglobulins were more abundant in HE-HBCS versus LE-NBCS cows at d +7 and +21. Gene Ontology enrichment analyses of differentially abundant proteins revealed that the acute inflammatory response, regulation of complement activation, protein activation cascade, and regulation of humoral immune response were the most enriched terms in the LE-NBCS group compared with the HE-HBCS group. In addition, we identified 24 differentially abundant proteins (16 in the LE-NBCS group, and 8 in the HE-HBCS group) during the transition period. The complement components C1q and C5 were less abundant, while C3 and C3d were more abundant in LE-NBCS compared with HE-HBCS cows. Overall, overconditioning around calving was associated with alterations in protein pathways related to acute inflammatory response and regulation of complement and coagulation cascades in transition cows.

中文翻译：

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从怀孕后期到早期泌乳的过渡过程中，正常和过度调节奶牛的血浆蛋白质组学分析和途径分析。

这项研究将定量蛋白质组学方法与生物信息学分析一起应用，以研究过渡期内正常奶牛和过度适应奶牛血浆蛋白质组的变化。在预期产犊日期的前15周，根据当前和先前的身体状况评分（BCS）选择了38头荷斯坦牛，并分配给高BCS组或正常BCS组（每头19头）。他们接受不同的饮食直到干透为止，以达到目标的BCS和背脂厚度（BFT）的差异直到干透为止。晾干时，正常BCS奶牛的BCS <3.5（最小，2.75）和BFT <1.2 cm（最小，0.58），高BCS奶牛的BCS> 3.75（最大，4.50），BFT> 1.4 cm（最高2.90）。蛋白质组学研究使用了每组5只动物的子集。根据产后第14天的脂肪酸（FA）和产后第21天的β-羟基丁酸酯（BHB）的循环浓度选择这些奶牛，分别代表其BCS组中的较高或较低的极值。高BCS子集（HE-HBCS）为4.50 <BCS> 3.75，FA = 1.17±0.46 mmol / L，BHB = 2.15±0.42 mmol / L（均值±SD），低BCS子集（LE-NBCS）具有3.50 <BCS> 2.75，FA = 0.51±0.28 mmol / L和BHB = 0.84±0.17 mmol / L。通过应用定量串联质量标签（TMT）方法，将来自分娩的d -49，+ 7和+21的血浆样品用于蛋白质组分析。对未消耗的血浆样品进行还原和消化，然后用TMT 10plex试剂标记。对TMT标记的肽段进行了高分辨率液相色谱-串联质谱分析，并对获得的光谱进行了分析，以进行蛋白质鉴定和定量。在血浆样品中总共鉴定出254种可定量蛋白质（标准：2种独特的肽和5％的错误发现率）。从这些结果来看，与过渡期HE-HBCS奶牛相比，LE-NBCS奶牛观察到24种差异丰富的蛋白质（丰富度高14种，丰富度低10种）。在d +7和+21时，HE-HBCS奶牛的血浆α-2-巨球蛋白比LE-NBCS奶牛丰富。对差异丰富的蛋白质进行的基因本体论富集分析显示，急性炎症反应，补体激活调节，蛋白激活级联，与HE-HBCS组相比，LE-NBCS组中体液免疫反应的调节和调节是最丰富的术语。此外，我们在过渡期间发现了24种差异丰富的蛋白质（LE-NBCS组为16种，HE-HBCS组为8种）。与HE-HBCS奶牛相比，LE-NBCS的补体成分C1q和C5含量较低，而C3和C3d含量较高。总体而言，产犊前后的过度调节与过渡期母牛急性炎症反应以及补体和凝血级联调节相关的蛋白质途径改变有关。与HE-HBCS奶牛相比，LE-NBCS的补体成分C1q和C5含量较低，而C3和C3d含量较高。总体而言，产犊前后的过度调节与过渡期母牛急性炎症反应以及补体和凝血级联调节相关的蛋白质途径改变有关。与HE-HBCS奶牛相比，LE-NBCS的补体成分C1q和C5含量较低，而C3和C3d含量较高。总体而言，产犊前后的过度调节与过渡期母牛急性炎症反应以及补体和凝血级联调节相关的蛋白质途径改变有关。