^1HNMR-Based metabolomic profiling method to develop plasma biomarkers for sensitivity to chronic heat stress in growing pigs

<div><p>The negative impact of heat stress (HS) on the production performances in pig faming is of particular concern. Novel diagnostic methods are needed to predict the robustness of pigs to HS. Our study aimed to assess the reliability of blood metabolome to predict the sensitivity to chronic HS of 10 F1 (Large White × Creole) sire families (SF) reared in temperate (TEMP) and in tropical (TROP) regions (n = 56±5 offsprings/region/SF). Live body weight (BW) and rectal temperature (RT) were recorded at 23 weeks of age. Average daily feed intake (AFDI) and average daily gain were calculated from weeks 11 to 23 of age, together with feed conversion ratio. Plasma blood metabolome profiles were obtained by Nuclear Magnetic Resonance spectroscopy (^1HNMR) from blood samples collected at week 23 in TEMP. The sensitivity to hot climatic conditions of each SF was estimated by computing a composite index of sensitivity (I_<i>sens</i>) derived from a linear combination of <i>t</i> statistics applied to familial BW, ADFI and RT in TEMP and TROP climates. A model of prediction of sensitivity was established with sparse Partial Least Square Discriminant Analysis (<i>s</i>PLS-DA) between the two most robust SF (n = 102) and the two most sensitive ones (n = 121) using individual metabolomic profiles measured in TEMP. The sPLS-DA selected 29 buckets that enabled 78% of prediction accuracy by cross-validation. On the basis of this training, we predicted the proportion of sensitive pigs within the 6 remaining families (n = 337). This proportion was defined as the predicted membership of families to the sensitive category. The positive correlation between this proportion and I_<i>sens</i> (r = 0.97, P < 0.01) suggests that plasma metabolome can be used to predict the sensitivity of pigs to hot climate.</p></div

Additional file 2 of Time course of the response to ACTH in pig: biological and transcriptomic study

Distribution of the rank of the significant adjusted P -values in the tests for DE transcripts between t = 0 and t = + 1, t = 0 and t = + 4 and t = 0 and t = + 24 ‘.pdf’ file. P-values are smaller at t=+1 and t=+4 than at t=+24 implying that the transcripts were overall more differentially expressed between t=0 and t=+1 and between t=0 and t=+4 than between t=0 and t=+24. (PDF 4 kb

Additional file 4 of Time course of the response to ACTH in pig: biological and transcriptomic study

Complete list of enriched GO (Biological process (BP), Molecular function (MF) and Cellular Component (CC) for 34 genes for which L/G ratio had a significant effect ‘.xls’ file. Features the GO items, the corresponding functions, the class of ontology, the number of genes in the input list (enriching a GO and total number) and in the reference list (enriching a GO and total number), the raw and the 679 adjusted Fisher’s exact test P-value and the list of genes. (XLS 7 kb

Correlations analysis.

<p>(A) The plot shows correlations between the MR of SF to sensitive group and the I_<i>sens</i> (r = 0.96, P < 0.0001 for Pearson correlation test). (B) The plot shows correlations of MR and I_<i>sens</i> against the mean feed conversion rate (FCR), average daily weight gain (ADG), weight, rectal temperature (RT), skin temperature (ST), back fat thickness (ABFT) per SF both in TEMP and TEMP climate. Blue and red circles showed positive or negative correlation respectively. (C) The plot shows correlations between the median values selected buckets with a VIP > 1 per SF in TEMP climate and mean values per SF of BW, ADG, ADFI, FCR, ST, RT, ABFT, MR, and I_<i>sens</i> either in TEMP or in TROP climate. The size of circle is proportional to the absolute value of R coefficient of correlation of Pearson. The significance of correlation is P < 0.05 (Pearson correlation test) and the crosses indicate non-significant P values (P > 0.05).</p

sPLS-DA between robust vs sensitive groups.

<p>(A) Projection of animals on the 3 components of the <i>s</i>PLS-DA on metabolomic data between robust (blue) and sensitive groups (orange). Spheres are a 3D representation of confidence ellipses (level 95%). (B) VIP (variable importance projection) plot that shows the variable importance in the <i>s</i>PLS-DA model over the 3 components. Variables with a VIP > 1 (red line) were considered as highly important predictors in <i>s</i>PLS-DA. The blue and orange bars are the buckets that showed a significant higher median value (Wilcoxon test: FDR < 0.05) in the robust or in the sensitive group, respectively. The grey bars indicate buckets that are not significantly different between the robust and the sensitive group (Wilcoxon test: FDR > 0.05). (C) Frequencies stability of selection of variable across the 100 models of CV.</p

Statistical workflow.

<p>Statistical approach that lead to: 1) assess sensitivity of each SF to TROP climate according to a composite index of sensitivity (I_<i>sens</i>) that took into account the between-climate variations of average daily feed intake (ADFI), body weight (BW) at 23 weeks and rectal temperature (RT) at 23 weeks, 2) Build a supervised classification model on metabolomic data between the robust and climate sensitive groups by <i>s</i>PLS-DA, 3) predict the membership rate (MR) of the other SF to the sensitive group using <i>s</i>PLS-DA. The relevance of MR interpretation as predictive index of sensitivity was confirmed through its highly significant correlation with index of sensitivity (I_<i>sens</i>).</p

Phenotypic means of robust and sensitive groups in TEMP and TROP climates.

<p>Phenotypic means of robust and sensitive groups in TEMP and TROP climates.</p

Differential expression results among small intestine segments and Ileal Peyer's patches.

<p>Differential expression results among small intestine segments and Ileal Peyer's patches.</p

Venn diagram of the expressed genes along the 4-different tissues (duodenum, jejunum, ileum and ileal Peyer's patches).

<p>Out of the 19,910 expressed genes, 17, 027 genes were co-expressed between tissues. Venn diagram was plotted by using Venny, an interactive tool for comparing lists.</p

Canonical pathways significantly modulated when comparing different tissues.

<p>Statistical significance of pathway modulation was calculated via a right-tailed Fisher's Exact test in Ingenuity Pathway. Only canonical pathways that presented a −log <i>P</i>-values exceeding 1.30 (FDR <i>q</i>-values <0.05) were preserved. The down-regulated and up-regulated genes for each molecular pathway are presented and the colours indicate the different tissue contrasts.</p