Abstract
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.
Highlights
Several metabolomic approaches have been conducted for the study of different classes of small-sized metabolites reflecting the key metabolic pathways involved in the transition period between late pregnancy and early lactation in ruminants [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
Our results show significant elevation in levels of non-esterified fatty acids (NEFA) with different extents in pregnant and lactating ewes including acetic acid (8); decanoic acid (42); tridecanoic acid (57); tetradecanoic acid (58); hexadecanoic acid (66); octadecanoic acid (80) (P
NEFA are released into the blood circulation and serve as an important source of energy [31]
Summary
Several metabolomic approaches have been conducted for the study of different classes of small-sized metabolites reflecting the key metabolic pathways involved in the transition period between late pregnancy and early lactation in ruminants [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16] The majority of these studies utilized traditional biological fluids such as blood, plasma, serum, etc. It is composed of proteins, lipids, glycopeptides, amino acids (AA), short and long chain fatty acids (saturated and unsaturated), aromatic and long chain hydrocarbons, steroids, volatile organic compounds (VOC), and minerals in addition to some environmental pollutants [18]
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