Hematocrit and Plasma Metabolites.

<p>Effect of heat stress (HS) and pair-feeding (PF) on (A) hematocrit, (B) plasma glucose, (C) plasma β-hydroxybutyric acid (BHBA), (D) plasma non-esterified fatty acids. In experimental period 1 (P1) all animals were kept at thermoneutral conditions (THI = 59.7) with ad-libitum feeding for six days. During period 2 (P2), HS cows (red lines) were heat-stressed (THI = 76.1), whereas PF cows (blue lines) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap, dashed lines) and again post-partum (pp, solid lines). All data given as mean ± SEM; for P1 the mean of the last days of period 1 is given as used for baseline correction. BHBA: β-hydroxybutyric acid; NEFA: non-esterified fatty acids. Numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6.</p

Catecholamines.

<p>Effect of heat stress (HS) and pair-feeding (PF) on (A) plasma noradrenaline and (B) adrenaline. In experimental period 1 (P1) all animals were kept at thermoneutral conditions (THI = 59.7) with ad-libitum feeding for six days. During period 2 (P2), HS cows (red lines) were heat-stressed (THI = 76.1), whereas PF cows (blue lines) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap, dashed lines) and again post-partum (pp, solid lines). All data given as mean ± SEM; for P1 the mean of the last days of period 1 is given as used for baseline correction. Numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6.</p

Indirect Calorimetry.

<p>Effect of heat stress (HS) and pair-feeding (PF) on (A) heat production per metabolic weight (HP/mBW), (B) net carbohydrate oxidation per metabolic weight (COX/mBW), (C) net fat oxidation per metabolic weight (FOX/mBW), (D) COX/FOX ratio. In experimental period 1 (P1, black) all animals were kept at thermoneutral conditions (TN, THI = 59.7) with ad-libitum feeding (AL) for six days. During period 2 (P2), HS cows (red) were heat-stressed (THI = 76.1), whereas PF cows (blue) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap) and again post-partum (pp). Cows underwent a 24-hours indirect calorimetry analysis on day 5 of each period. Numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6; all data given as sums of continuous measurements over 24 hours on day 6 of each period; ^<b>+</b>P < 0.05 in Wilcoxon signed rank sum test for paired samples of the same group; there were no group differences between independent samples of the same reproductive stage and period according to the exact Wilcoxon-Mann-Whitney test (P > 0.05).</p

Conductance and Water Condensate.

<p>Effect of heat stress (HS) and pair-feeding (PF) on (A) conductance and (B) water condensate. In experimental period 1 (P1, black) all animals were kept at thermoneutral conditions (TN, THI = 59.7) with ad-libitum feeding (AL) for six days. During period 2 (P2), HS cows (red) were heat-stressed (THI = 76.1), whereas PF cows (blue) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap) and again post-partum (pp). Data acquired during the 24-hour indirect calorimetry analysis on day 5 of each period. (A) For conductance analysis, numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6; (B) For water condensate measurement, numbers of animals analyzed per group: HSap n =, 6 PFap n = 6, HSpp n = 4, PFpp n = 4; ^<b>+</b>P < 0.05 in Wilcoxon signed rank sum test for paired samples of the same group; there were no differences in groups of the same reproductive stage in P1 (P > 0.05) according to the exact Wilcoxon-Mann-Whitney test; ^<b>*</b>P < 0.05 in the exact Wilcoxon-Mann-Whitney test for group differences of the same reproductive stage in P2.</p

Nutritional and Vital Data.

<p>(A) Temperature humidity index (THI, solid or dashed lines), and dry matter intake (DMI; dotted lines; given as mean of both heat-stressed (HS, red) and pair-fed groups (PF, blue), respectively) during the six days of P1 and P2 and the transition day (TD). Effects of HS and PF on (B) daily milk yield, (C) rectal temperatures, (D) respiratory rate, (E) heart rate and (F) Water intake/DMI. In experimental period 1 (P1) all animals were kept at thermoneutral conditions (THI = 59.7) with ad-libitum feeding for six days. During period 2 (P2), HS cows (red lines) were heat-stressed (THI = 76.1), whereas PF cows (blue lines) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap, dashed lines) and again post-partum (pp, solid lines). All data given as mean ± SEM; for P1 the mean of the last days of period 1 is given in Fig 1B-F as used for baseline correction; numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6.</p

Plasma Urea and Creatinine.

<p>Effect of heat stress (HS) and pair-feeding (PF) on (A) plasma urea, (B) plasma creatinine, (C) urea/creatinine ratio. In experimental period 1 (P1) all animals were kept at thermoneutral conditions (THI = 59.7) with ad-libitum feeding for six days. During period 2 (P2), HS cows (red lines) were heat-stressed (THI = 76.1), whereas PF cows (blue lines) were pair-fed in thermoneutrality (THI = 60.0) for six days, once ante partum (ap, dashed lines) and again post-partum (pp, solid lines). All data given as mean ± SEM; for P1 the mean of the last days of period 1 is given as used for baseline correction. Numbers of animals analyzed per group: HSap n = 7, PFap n = 6, HSpp n = 6, PFpp n = 6.</p

Components and chemical analysis of TMR diets ante and post-partum.

<p>TMR total mixed ration; DM dry matter</p><p>Components and chemical analysis of TMR diets ante and post-partum.</p

Conjugated linoleic acids effects on systemic insulin sensitivity in primiparous vs. pluriparous cows during lactation.

<p>Glucose and insulin concentration in serum and calculated Revised Quantitative Insulin Sensitivity Check Index (RQUICKI) in cows receiving conjugated linoleic acids (CLA, Lutrell® Pure, BASF SE, Ludwigshafen, Germany) at 100 g/day or a control fat supplement (Silafat®, BASF SE) from day 1 until day 182 postpartum [pluriparous cows (Control, n = 10 and CLA, n = 11) and primiparous cows (Control, n = 4 and CLA, n = 5)] in Trial 1. RQUICKI  = 1/[log(glucose) + log(insulin) + log(NEFA)], <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Holtenius1" target="_blank">[26]</a>. Supplementation with CLA did not interfere with the parameters measured in primiparous cows. Area between vertical lines corresponds to the CLA supplementation period (means ± SEM).</p

Characteristics of the primers and the real-time PCR conditions.

<p>¹ Adiponectin, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Lemor1" target="_blank">[45]</a>.</p><p>² Adiponectin receptor 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Lemor1" target="_blank">[45]</a>.</p><p>³ Adiponectin receptor 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Lemor1" target="_blank">[45]</a>.</p><p>⁴ Leptin, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Yuen1" target="_blank">[93]</a>.</p><p>⁵ Leptin receptor isoform b, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Lemor1" target="_blank">[45]</a>.</p><p>⁶ Leptin receptor.</p><p>⁷ Peroxisome proliferator-activated receptor <i>gamma2.</i></p><p>⁸ Peroxisome proliferator-activated receptor <i>gamma</i>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086211#pone.0086211-Hosseini1" target="_blank">[94]</a>.</p><p>⁹ Interleukin-6.</p><p>¹⁰ Tumor necrosis factor-α.</p><p>¹¹ bp: base pairs.</p><p>¹² Cq: Quantification cycle.</p><p>¹³ Based on slaughter experiment.</p><p>¹⁴ Initial denaturation  = 10 min at 95°C; denaturation  = 30 s at 95°C; extension  = 30 s at 72°C, except for <i>TNF-α</i>, <i>PPAR</i>γ2, <i>PPAR</i>γ, <i>LEPR</i>, and <i>LEPRB</i> (60 s at 72°C).</p><p>¹⁵ Based on liver and s.c. fat biopsies from Trial 1.</p

Longitudinal mRNA expression of genes related to insulin sensitivity in pluriparous cows during lactation I.

<p>Longitudinal mRNA expression of adiponectin receptors (ADIPOR1 and ADIPOR2) in liver and s.c. tail head biopsies of dairy cows together with adiponectin (ADIPOQ) and interleukin-6 (IL-6) mRNA abundance in s.c. adipose tissue and in liver of Trial 1, respectively. Cows were fed with conjugated linoleic acids (CLA, Lutrell® Pure, BASF SE, Ludwigshafen, Germany) at 100 g/day CLA or a control fat supplement (Silafat®, BASF SE) from day 1 until day 182 postpartum in Trial 1. Primiparous cow samples are not included (There are no sample in the CLA group at days 1, 70, 182, and 224) [Control: n = 10, CLA: n = 11]. Cumulative mRNA expression of both control and CLA is shown because CLA effect was insignificant. For normalisation, lipoprotein receptor-related protein 10 (LRP10), RNA Polymerase II (POLR2A) and eukariotic translation initiation factor 3 (EIF3K) in liver and LRP10, glyceraldehyde-phosphate-dehydrogenase (GAPDH), and POLR2A in s.c. adipose tissue were used as reference genes. Different letters indicate significant differences between days relative to parturition (<i>P</i>≤0.05; mean ± SEM). Area between vertical lines corresponds to the CLA supplementation period.</p