Shokry Engy, Pereira Julião, Marques Júnior Jair Gonzalez, da Cunha Paulo Henrique Jorge, Noronha Filho Antônio Dionísio Feitosa, da Silva Jessica Alves, Fioravanti Maria Clorinda Soares, de Oliveira Anselmo Elcana, Antoniosi Filho Nelson Roberto
Laboratório de Métodos de Extração e Separação (LAMES), Instituto de Química (IQ), Universidade Federal de Goiás (UFG), Campus Samambaia, Goiânia, Goiás, Brazil.
Escola de Veterinária e Zootecnia, Universidade Federal de Goiás (UFG), Campus Samambaia, Goiânia, Goiás, Brazil.
PLoS One. 2017 Aug 25;12(8):e0183538. doi: 10.1371/journal.pone.0183538. eCollection 2017.
Important metabolic changes occur during transition period of late pregnancy and early lactation to meet increasing energy demands of the growing fetus and for milk production. The aim of this investigation is to present an innovative and non-invasive tool using ewe earwax sample analysis to assess the metabolic profile in ewes during late pregnancy and early lactation. In this work, earwax samples were collected from 28 healthy Brazilian Santa Inês ewes divided into 3 sub-groups: 9 non-pregnant ewes, 6 pregnant ewes in the last 30 days of gestation, and 13 lactating ewes ≤ 30 days postpartum. Then, a range of metabolites including volatile organic compounds (VOC), amino acids (AA), and minerals were profiled and quantified in the samples by applying headspace gas chromatography/mass spectrometry, high performance liquid chromatography/tandem mass spectrometry, and inductively coupled plasma-optical emission spectrometry, respectively. As evident in our results, significant changes were observed in the metabolite profile of earwax between the studied groups where a remarkable elevation was detected in the levels of non-esterified fatty acids, alcohols, ketones, and hydroxy urea in the VOC profile of samples obtained from pregnant and lactating ewes. Meanwhile, a significant decrease was detected in the levels of 9 minerals and 14 AA including essential AA (leucine, phenyl alanine, lysine, isoleucine, threonine, valine), conditionally essential AA (arginine, glycine, tyrosine, proline, serine), and a non-essential AA (alanine). Multivariate analysis using robust principal component analysis and hierarchical cluster analysis was successfully applied to discriminate the three study groups using the variations of metabolites in the two stress states (pregnancy and lactation) from the healthy non-stress condition. The innovative developed method was successful in evaluating pre- and post-parturient metabolic changes using earwax and can in the future be applied to recognize markers for diagnosis, prevention, and intervention of pregnancy complications in ewes.
在妊娠后期和泌乳早期的过渡期会发生重要的代谢变化，以满足不断生长的胎儿增加的能量需求以及产奶的能量需求。本研究的目的是提出一种创新的非侵入性工具，通过对母羊耳垢样本进行分析，来评估妊娠后期和泌乳早期母羊的代谢概况。在这项工作中，从28只健康的巴西圣伊内斯母羊收集耳垢样本，这些母羊分为3个亚组：9只未怀孕的母羊、6只处于妊娠最后30天的怀孕母羊以及13只产后30天内的泌乳母羊。然后，分别通过顶空气相色谱/质谱法、高效液相色谱/串联质谱法和电感耦合等离子体发射光谱法，对样本中的一系列代谢物进行分析和定量，这些代谢物包括挥发性有机化合物（VOC）、氨基酸（AA）和矿物质。正如我们的结果所示，在所研究的组之间，耳垢的代谢物谱发生了显著变化，从怀孕和泌乳母羊获得的样本的VOC谱中，未酯化脂肪酸、醇、酮和羟基脲的水平显著升高。同时，检测到9种矿物质和14种氨基酸的水平显著下降，其中包括必需氨基酸（亮氨酸、苯丙氨酸、赖氨酸、异亮氨酸、苏氨酸、缬氨酸）、条件必需氨基酸（精氨酸、甘氨酸、酪氨酸、脯氨酸、丝氨酸）和一种非必需氨基酸（丙氨酸）。使用稳健主成分分析和层次聚类分析的多变量分析成功地应用于区分这三个研究组，利用两种应激状态（怀孕和泌乳）与健康非应激状态下代谢物的变化。所开发的创新方法成功地利用耳垢评估了产前和产后的代谢变化，并且未来可应用于识别母羊妊娠并发症的诊断、预防和干预标志物。
