Summary of the experimental design of the somatic cell preparation and the various treatments applied, i.e. storage in milk or PBS for 72 h, variation of the centrifugation rates and heat treatment.

<p>Summary of the experimental design of the somatic cell preparation and the various treatments applied, i.e. storage in milk or PBS for 72 h, variation of the centrifugation rates and heat treatment.</p

Flow cytometry scatter pattern for the identification of differential somatic cells in blood-milk mix cell suspension (1/1, v/v) and corresponding isotype control sample, respectively.

<p>(A) FSC/SSC dotplot of cell suspension. All somatic cells (in yellow) in cell suspension (B) were identified by CD45/PerCp+. The subpopulation of cell suspension in APC/FITC dotplot (C), macrophages (red), PMNs (blue) and lymphocytes (green) are identified by CD14/APC+ gate in APC/SSC plot (D), CH138A/FITC+ gate in FITC/SSC plot (E), and FSC/SSC size/granularity gate (F), respectively.</p

Flow cytometry identification of differential somatic cell count and simultaneous their quantification of all cells and each cell type.

<p>(A)(B)(C) fresh blood cell suspension, (D)(E)(F) milk cell suspension after 80°C × 30 min and (G)(H)(I)milk cell suspension conserved at 4°C in PBS pH 7.4. The cell subpopulations of each sample were shown in APC/FITC dotplot (A)(D)(G), the viable and non-viable population of all cells and each subpopulation were shown in histogram-Vioblue scatter (B)(E)(H) and (C)(F)(I), respectively.</p

Cell viability with various centrifugation rates (400, 1500, 3000, 5000×<i>g</i> during 10 min at 4°C) comparing to the reference milk cell suspension without supplementary centrifugation (Ref).

<p>(A) Boxplot and whiskers of flow cytometry results with milk somatic cell suspensions incubated with specific antibodies and vioblue live/dead kit (n = 4); (B) Cell viability of each type cell for 400×<i>g</i> (white bars), 1500×<i>g</i> (hatched bars), 3000×<i>g</i> (grey bars), 5000×<i>g</i> (dark bars) centrifugations by flow cytometry; (C) Mean proportion of macrophages (dark sectors), PMNs (grey sectors) and lymphocytes (white sectors) in cell samples under various centrifugation with different gravitational velocities. Means with different superscripts (a-d) differ significantly (<i>P</i><0.05).</p

Adipocyte differentiation and cellularity.

<p>Abbreviation used: LD, <i>longissimus dorsi</i> muscle; PRAT, perirenal fat; SCAT dorsal subcutaneous adipose tissue.</p><p>^aPigs (42 d of age) were fed either a methionine-deficient (MD) diet or a control (CTRL) diet providing an adequate level of methionine for 10 days. Values are least square means together with residual standard deviation (RSD).</p><p>^bmRNA levels (arbitrary units) of target genes were measured by qPCR.</p><p>^cAdipocyte mean area (μm²) was assessed on histological cross-section of adipose tissue. Total number of adipocyte was estimated per gram of adipose tissue.</p><p>Adipocyte differentiation and cellularity.</p

mRNA levels of anti- and pro-oxidant genes in tissues.

<p>Abbreviation used: LD, <i>longissimus dorsi</i> muscle; PRAT, perirenal fat; SCAT dorsal subcutaneous adipose tissue.</p><p>^a Pigs (42 d of age) were fed either a methionine-deficient (MD) diet or a control (CTRL) diet providing an adequate level of methionine for 10 days. Values are least square means together with residual standard deviation (RSD).</p><p>^b mRNA levels (arbitrary units) of target genes were measured by qPCR.</p><p>mRNA levels of anti- and pro-oxidant genes in tissues.</p

Antioxidant capacities in plasma.

<p>Pigs (42 d of age) were fed either a methionine-deficient (MD) or a CTRL diet providing an adequate level of methionine for 10 days (n = 6 per diet). Values are least square means together with residual standard deviation (RSD). Differences between groups were significant at ***<i>P</i>≤0.001; **<i>P</i>≤0.01 and *<i>P</i>≤0.05. Activity level of total superoxide dismutase (SOD) was expressed in units per mL (U/mL). The antioxidant activities (mmol or μmol equivalent Trolox/L) were determined by 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the Ferric Reducing Ability of Plasma (FRAP) tests.</p

Oxidative enzyme activities.

<p>Abbreviation used: CS, citrate synthase; HAD, β-hydroxy-acyl-CoA dehydrogenase; LD, <i>longissimus dorsi</i> muscle; LDH, lactate dehydrogenase; PRAT, perirenal fat; SCAT dorsal subcutaneous adipose tissue.</p><p>^a Pigs (42 d of age) were fed either a methionine-deficient (MD) diet or a control (CTRL) diet providing an adequate level of methionine for 10 days. Values are least square means together with residual standard deviation (RSD).</p><p>^b Activities were expressed in μmol per min and per mg proteins.</p><p>Oxidative enzyme activities.</p

Ingredients and chemical composition of experimental diets.

<p>Abbreviation used: TSAA, total sulfur amino acid.</p><p>^a MD, a diet deficient in methionine; CTRL, a control diet.</p><p>^b Adjusted for 87.3% of dry matter.</p><p>^c Values were calculated from Sauvant et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130514#pone.0130514.ref019" target="_blank">19</a>].</p><p>^d SID, standardized ileal digestible amino acids. The values were obtained from the calculated ileal digestibility values of the ingredients combined with the measured amino acid contents.</p><p>Ingredients and chemical composition of experimental diets.</p

Lipid content and lipogenic enzyme activities.

<p>Abbreviation used: G6PDH, glucose-6-phosphate dehydrogenase; LD, <i>longissimus dorsi</i> muscle; PRAT, perirenal fat; SCAT dorsal subcutaneous adipose tissue.</p><p>^a Pigs (42 d of age) were fed either a methionine-deficient (MD) diet or a control (CTRL) diet providing an adequate level of methionine for 10 days. Values are least square means together with residual standard deviation (RSD).</p><p>^b Lipid content was expressed in g per 100 g of tissue fresh weight. Activities were expressed in nmoles of NADPH produced (malic enzyme, G6PDH) or consumed (fatty acid synthase) per min and per mg cytosolic proteins.</p><p>Lipid content and lipogenic enzyme activities.</p