Comparison of microarray and qPCR data for eight genes in pigs divergently selected for low or high residual feed intake (RFI).

<p>Transcriptomic differences between the two lines were validated by qPCR at 132 and 74 days of age (onset of the growing period). For values related to microarray, the highest P-value is reported when several probes are differentially expressed for a unique gene (*P < 0.05; **P < 0.01; ***P<0.001). Fold change value is expressed as the expression ratio of low RFI to high RFI samples; ratio was inversed and preceded by a minus sign for value less than 1 (i.e., a ratio of 0.5 is expressed as -2). <i>CD40</i>, tumor necrosis factor receptor superfamily member 5; <i>GPX3</i>, glutathione peroxidase 3; <i>OAZ3</i>, ornithine decarboxylase antizyme 3; <i>DGAT2</i>, diacylglycerol O-acyltransferase 2; <i>NMI</i>, N-myc interactor; <i>TRAF6</i>, TNF receptor-associated factor 6; <i>SLPI</i>, secretory leukocyte peptidase inhibitor; <i>PSEN1</i>, presenilin 1.</p

Comparison of microarray and qPCR data for three genes in pigs fed the HF or LF diet.

<p>For values related to microarray, the highest P-value is reported when several probes are differentially expressed for a unique gene (*P < 0.05; **P < 0.01; ***P < 0.001). Fold change value is expressed as the expression ratio of HF (high fiber high fat, n = 24) to LF (low fiber low fat, n = 24) diets; ratio was inversed and preceded by a minus sign for value less than 1 (i.e., a ratio of 0.5 is expressed as -2). <i>LCN2</i>, lipocalin 2; <i>CPT1A</i>, Carnitine palmitoyltransferase 1A; <i>PSAP</i>, prosaposin.</p

^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

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

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

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

Clusterization of haplotypes reconstructed at the OARX locus.

<p>(A) Haplotypes determined in the French Grivette sheep population. (B) Haplotypes determined in the Polish Olkuska sheep population. 87 markers located in the interest OARX region (45 Mb–55 Mb) were selected to reconstruct haplotypes. Each column represents one SNP and each line represents one haplotype. For one marker (<i>i</i>) allele 1 is in red (Grivette) or green (Olkuska) in controls, respectively or black in cases, (<i>ii</i>) allele 2 is in white when the phase was unambiguous and (<i>iii</i>) dark grey colour represents unphased SNP. Haplotypes were ordered to distinguish controls versus cases and clusterized to classify similar clades of haplotypes. Markers with evidence of association at significance levels are marked with a star or a hash in French Grivette and Polish Olkuska sheep populations, respectively. In both breeds, the specific haplotype preferentially selected in highly prolific ewes (cases) is symbolized by red (Grivette) and green (Olkuska) boxes. The <i>BMP15</i> gene (48140251 bp–48146740 bp) is located between markers named OARX_6082722 and OARX_56342973.</p

Functional effects of <b><i>FecX^Gr</i></b><b> and </b><b><i>FecX^O</i></b><b> mutations on the BMP15 activity.</b>

<p><i>In vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/− 100 ng of recombinant human BMP15 (Control +/− rhBMP15) or wild-type human BMP15 expressing vector (WT) or the 2 different BMP15 variant vectors (<i>BMP15^T317I (FecX^Gr)</i>; <i>BMP15^N337H (FecX^O)</i>) obtained by directed-mutagenesis. Results are expressed as Means±SD of the relative light unit (RLU) from 3 independent experiments in triplicate for each condition. Pairwise statistical comparisons using a one-way ANOVA test between means were performed and results of statistic test are symbolized by stars: * = p<5E⁻⁰²; ** = p<1E⁻⁰² and *** = p<1E⁻⁰³.</p

Genotypic distributions of <b><i>FecX^Gr</i></b><b> and </b><b><i>FecX^O</i></b><b> mutations.</b>

<p>(A) Genotypic distribution of the <i>BMP15^T317I (FecX^Gr)</i> in the French Grivette sheep population for the litter size phenotype. (B) Genotypic distribution of the <i>BMP15^N337H (FecX^O)</i> in the Polish Olkuska sheep population for the ovulation rate phenotype. (C) Genotypic distribution of the <i>BMP15^T317I (FecX^Gr)</i> in the French Grivette sheep population for the ovulation rate phenotype. (D) Genotypic distribution of the <i>BMP15^N337H (FecX^O)</i> in the Polish Olkuska sheep population for the litter size phenotype. The means LS or OR in breeds are firstly presented then ewes were ordered according to their genotype at the mutation of interest. Means±SD for prolificacy were calculated for the 3 groups of genotype and are noted into each histogram bar. Number of ewes counted per group of genotype is mentioned (n). Pairwise statistical comparisons using a one-way ANOVA test between means of genotype's clades were performed and results of statistic test are symbolized by stars. p: * = p<5E⁻⁰²; ** = p<1E⁻⁰² and *** = p<1E⁻⁰³.</p