J A A Pires
email: jose.pires@inra.fr

K Pawlowski

J Rouel

C Delavaud

G Foucras

P Germon

C Leroux

Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early

Keywords: dairy cow, negative energy balance, inflammation, LPS

Interpretive Summary, Pires et al., page XX. After calving, dairy cows often experience nutritional deficits and depend on body reserves to support milk production. Suboptimal nutrition may explain increased occurrence of inflammatory diseases such as mastitis that impact animal welfare and profitability of dairy farms. We studied the effects of insufficient nutrition on the ability of cows to respond to inflammation. Modifications in blood composition during inflammation differed, and suggest that underfed cows elicit metabolic adaptations in order to support short-term inflammation response. This response may impose a burden to the dairy cow and impact their adaptive capacity to early lactation.

INTRODUCTION

Dairy cows experience profound shifts in hormonal and nutritional status during the periparturient period, rely on extensive mobilization of body fat, proteins, and bone minerals to support the onset of lactation, and often present altered plasma metabolite and mineral profiles [START_REF] Pires | Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows[END_REF][START_REF] Valldecabres | Effect of prophylactic oral calcium supplementation on postpartum mineral status and markers of energy balance of multiparous Jersey cows[END_REF]. Concomitantly, multiple aspects of immune function are modified and the occurrence of metabolic disorders and inflammatory diseases peaks during this period. For instance, leucocyte function is impaired in early lactation compared to midlactation [START_REF] Shuster | Bacterial growth, inflammatory cytokine production, and neutrophil recruitment during coliform mastitis in cows within ten days after calving, compared with cows at midlactation[END_REF], and in cows with increased markers of negative energy balance [START_REF] Kremer | Severity of Experimental Escherichia coli Mastitis in Ketonemic and Nonketonemic Dairy Cows[END_REF][START_REF] Hammon | Neutrophil function and energy status in Holstein cows with uterine health disorders[END_REF]. Leucocyte dysfunction is associated with development of metritis and severity of experimental mastitis [START_REF] Kremer | Severity of Experimental Escherichia coli Mastitis in Ketonemic and Nonketonemic Dairy Cows[END_REF][START_REF] Shuster | Bacterial growth, inflammatory cytokine production, and neutrophil recruitment during coliform mastitis in cows within ten days after calving, compared with cows at midlactation[END_REF][START_REF] Hammon | Neutrophil function and energy status in Holstein cows with uterine health disorders[END_REF]. Furthermore, clinical mastitis during early lactation may impact reproduction and functional longevity [START_REF] Albaaj | High somatic cell counts and changes in milk fat and protein contents around insemination are negatively associated with conception in dairy cows[END_REF][START_REF] Hertl | Does clinical mastitis in the first 100 days of lactation 1 predict increased mastitis occurrence and shorter herd life in dairy cows?[END_REF].

Complex homeorhetic and homeostatic adaptations need to take place during early lactation to orchestrate the partitioning of limiting nutrients towards the mammary gland. These include the establishment of insulin resistance in peripheral tissues and enhanced hepatic glucose output via glycogenolysis and gluconeogenesis [START_REF] Bell | Adaptations of glucose metabolism during pregnancy and lactation[END_REF]Bauman, 1997, Doepel et al., 2009). Experimental acute immune activation imposes a glucose drain that may overwhelm the adaptive capacity of the animal and disrupt homeostasis [START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF]. Accordingly, ovine genetic lines selected for Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 4 divergent milk SCC also present divergent metabolic profiles in response to negative energy balance in early lactation. The high SCC sheep strain experience a greater metabolic burden during experimental energy restriction than the low SCC strain, further suggesting a link between immune function and metabolic robustness [START_REF] Bouvier-Muller | Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis[END_REF].

Feed restriction models have been used extensively to study the ramifications of nutrient deficit, lipomobilization and related metabolic deviations on biological functions at various lactation stages [START_REF] Veenhuizen | Metabolic changes in blood and liver during development and early treatment of experimental fatty liver and ketosis in cows[END_REF][START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF][START_REF] Gross | Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation[END_REF][START_REF] Bjerre-Harpøth | Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation[END_REF]. On the other hand, lipopolysaccharide (LPS) challenges have been employed as a model to study the mechanisms and factors modulating Gram-negative bacteriaassociated mastitis in dairy cows [START_REF] Paape | The bovine neutrophil: Structure and function in blood and milk[END_REF][START_REF] Schukken | Host-response patterns of intramammary infections in dairy cows[END_REF], including the interactions with nutritional status [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF], diet fatty acid composition [START_REF] Greco | Effects of altering the ratio of dietary n-6 to n-3 fatty acids on performance and inflammatory responses to a lipopolysaccharide challenge in lactating Holstein cows[END_REF] and effects of immune activation on metabolic and mineral homeostasis (Waldron et al., 2003a, Waldron et al., 2003b[START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF]. Challenges with LPS have the advantage of inducing repeatable, self-resolved inflammatory responses, with both local and systemic effects, while avoiding the adverse consequences of an infection [START_REF] Hoeben | Role of endotoxin and TNF-α in the pathogenesis of experimentally induced coliform mastitis in periparturient cows[END_REF][START_REF] Vernay | Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows[END_REF].

Relatively few studies have focused on experimentally-induced undernutrition, the resulting negative energy balance, and responses to intramammary immune challenges in vivo [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF][START_REF] Bouvier-Muller | Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis[END_REF]. Nonetheless, research employing feedrestricted midlactation cows as an experimental model have failed to show unequivocal associations between undernutrition and alterations of the immune response at whole animal level [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF].

Immune dysfunction in early lactation cows is driven in part by the prioritization of nutrient partitioning towards milk secretion and altered metabolic milieu resulting from intense lipomobilization and ketosis, and potential deficiencies in key nutrients such as glucose, calcium and specific amino acids [START_REF] Kremer | Severity of Experimental Escherichia coli Mastitis in Ketonemic and Nonketonemic Dairy Cows[END_REF][START_REF] Hammon | Neutrophil function and energy status in Holstein cows with uterine health disorders[END_REF], Martinez et al., 2014, Moyes, Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 5 2015). However, large nutrient imbalances typical of early lactation are difficult to reproduce later in lactation via feed restriction. For instance, the metabolic and hormonal responses to severe nutrient restriction are of lesser amplitude after peak compared to early lactation [START_REF] Gross | Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation[END_REF][START_REF] Bjerre-Harpøth | Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation[END_REF]. Plasma BHB increased when nutrient intake was restricted in early lactation cows by feeding a high-straw diet, but was not affected in mid and late lactation, despite marked negative energy balance and corresponding shifts in plasma glucose and NEFA [START_REF] Bjerre-Harpøth | Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation[END_REF]. We hypothesized that experimentally-induced undernutrition would modify animal responses to mammary inflammation in early lactation cows. A high-straw diet was employed as a restriction model because its low nutrient density and digestibility decrease nutrient intake without the need to limit the quantity of feed offered. The experiment was performed during early lactation because the metabolic deviations in response to nutritional challenges are greatest during this period. The objective was to assess the effects of nutrient restriction on whole-animal responses to an intramammary LPS challenge in early lactation cows.

MATERIALS AND METHODS

Animals, Diets, LPS Challenge and Housing

All procedures were approved by the regional ethics committee on animal experimentation (APAFIS #2018062913565518).

Seventeen multiparous Holstein-Friesian cows were studied from the last 3 weeks of gestation until 7 weeks post-partum. From 24 to 27 ± 3 DIM (mean ± SD), cows were either allowed continuous ad libitum intake of a lactation TMR (CONT, n = 9), or were fed a TMR France) diluted in 10 ml of sterile saline (CDM Lavoisier, Paris, France) containing 0.5 mg/mL of BSA (cell culture grade, endotoxin free, A9576, Sigma-Aldrich, Saint-Quentin-Fallavier, France).

After morning milking, teats were cleaned and the tips swabbed with cotton containing 70%

ethanol. The LPS solution prepared extemporaneously in a sterile environment was injected into the teat cistern via the teat canal, using a sterile disposable syringe fitted with a teat cannula, and the udder was massaged briefly.

Cows were housed in free-stalls equipped with individual feed bunks and automatic gates, except from one week before and until 3 days after the LPS challenge, when they were housed in a tie-stall barn to acclimate, facilitate frequent sampling and animal care. Cows were milked twice daily at approximately 0900 and 1600 h, had fresh feed offered once daily after morning milking and free access to drinking water.

Phenotyping Early Lactation and Responses to Undernutrition

Offered feed and refusals were weighted, and subsamples were collected four days per week to determine DM content after 48 h at 80 °C, and calculate DMI [START_REF] Lerch | Rapeseed or linseed supplements in grass-based diets: Effects on dairy performance of Holstein cows over 2 consecutive lactations[END_REF]. Pre-and postpartum TMR samples were collected weekly, pooled, and analyzed for nutrient composition by wet chemistry (Table 1). Energy balance was estimated as energy intake minus requirements for maintenance and production, calculated according to the INRA system (INRA, 2007) Jugular blood samples were collected at -1, -0.5, 1, 2, 4, 6, 10 and 24 h relative to LPS injection. Plasma concentrations at 72 h relative to initiation of feed restriction were calculated by averaging values at 1 and 0.5 h before LPS administration. Rectal temperature was recorded at the time of each blood sampling on the day of LPS injection using an electronic thermometer (Veterinär-Thermometer SC 12, SCALA Electronic GmbH, Stahnsdorf, Germany).

All blood samples were drawn into evacuated tubes containing EDTA (1.95 mg/mL; Terumo Europe NV, Leuven, Belgium) and centrifuged at 1,400×g for 15 min at 4°C. Plasma was conserved at -20°C until analysis for glucose (glucose oxidase method), BHB (D-Beta-Hydroxybutyrate-Dehydrogenase method), urea (glutamate dehydrogenase method; Thermo Electron SAS, France) and NEFA (Acyl-CoA synthase method; Wako, Sodioda, France) using an automatic analyzer (ARENA 20XT, Thermo Fisher Scientific, Cergy Pontoise, France), insulin using a RIA, as previously described [START_REF] Lerch | Rapeseed or linseed supplements in grass-based diets: Effects on dairy performance of Holstein cows over 2 consecutive lactations[END_REF], and cortisol [START_REF] Boissy | Effects of Androgen Treatment on Behavioral and Physiological Responses of Heifers to Fear-Eliciting Situations[END_REF].

Intra-and inter-assay coefficients of variation were 1.4 and 3.1 % for glucose, 2.1 and 3.0% for NEFA, 4.5 and 5.5% for BHB, 5.9 and 8.5% for urea, and 6.9 and 11.8% for insulin, respectively.

Biopsies of liver and mammary gland (LPS-infused rear quarter) were performed 24 h after LPS injection, after plasma and quarter milk collection, reported elsewhere.

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446

Statistical Analyses and Calculations

Statistical analyses were performed using SAS (version 9.4; SAS Institute Inc., Cary, NC), separately for data collected prepartum, postpartum, and during the time of LPS challenge.

Repeated measures data were analyzed by mixed models that included the fixed effects of Diet, Time, and Diet by Time interaction, the random effect of Cow, and Kenward-Rogers adjustment for calculation of denominator degrees of freedom. The Schwarz's Bayesian criterion was used to compare the fitting of different covariance structures, including spatial power, AR(1), ARH(1) and CS. Heterogeneous variance for each Diet was tested whenever suggested by residual plots.

Significant interactions of Diet and Time were explored using the SLICE and PDIFF options of the LSMEANS statement. Areas under the curve (AUC) during the 10 and 24 h post LPS challenge were calculated by the incremental (positive and negative) method for plasma metabolites and insulin, and by the positive incremental method for cortisol, after discounting for baseline concentrations [START_REF] Cardoso | Technical note: Comparison of 3 methods for analyzing areas under the curve for glucose and nonesterified fatty acids concentrations following epinephrine challenge in dairy cows[END_REF]) using Microsoft Excel (2013), and analyzed as non-repeated variables. Basal plasma metabolite and insulin concentrations were calculated by averaging values from samples collected at 1 h and 0.5 h prior to LPS injection. Logarithmic transformation of response variables was used whenever needed to comply with the assumptions of normality and homoscedasticity of residuals. When transformation was necessary, least squares means (LSMEANS) and standard error of the mean (SEM) were estimated from untransformed values, whereas P-values reflect statistical analysis of transformed data. Values reported are LSMEANS and SEM, unless otherwise stated. The significance level was predefined as P ≤ 0.05, and trends toward significance at 0.05 < P ≤ 0.10.

RESULTS

Prepartum

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019 1A) and plasma BHB (0.56 vs 0.49 ± 0.03) compared to CONT during the 3 weeks prepartum. Plasma NEFA concentration increased during late gestation (Time effect, P = 0.01) and was 0.092, 0.124 and 0.183 ± 0.021 mM on weeks -3, -2 and -1 relative to calving, respectively. Dry matter intake was 15.2, 14.8 and 13.7 ± 0.53 kg (Time effect, P = 0.11) during the same period.

Early Lactation and Responses to Undernutrition

Postpartum DMI, milk yield, fat and protein content and yield, NEL balance, BCS and BW did not differ between the groups before diet change (Figures 1A,1B and Supplementary Figures 1A,1B,2A,2B,2C and 2D). Feeding a TMR containing 48% straw induced a sudden decrease of DMI and negative energy balance, with milk yield decreasing significantly at the second day of restriction and thereafter (Figures 1A,1C and 1B).

Plasma NEFA, glucose, urea and insulin concentrations did not differ until diet change at 24 and2E). Nonetheless, a trend for greater BHB was observed on the first week of lactation for REST compared to CONT cows (Figure 2B). Per design, after nutrient restriction, the REST cows presented significantly greater concentrations of plasma NEFA, BHB and urea, and lower concentrations of glucose and insulin, compared to CONT cows (Figure 2).

± 3 DIM (Figures 2A, 2C, 2D
Therefore, the metabolic profile was profoundly altered at 72 h after dietary treatments (Table 2, Figure 2). Plasma cortisol did not differ between REST and CONT groups during the first 72 h of dietary treatments (data not shown).

Milk composition and component yield are presented in Supplementary Figure 2. Milk fat percentage increased during feed restriction and returned to pre-restriction concentrations on the same day of refeeding the regular lactation diet (Supplementary Figure 2A). Lactose content was depressed during the last 3 days of restriction (Supplementary Figure 2E). Milk, fat, protein and Body condition score decreased postpartum but did not differ significantly between treatments throughout the study (Supplementary Figure 1A). Nonetheless, BW was lower at the end of restriction period for REST than control (P = 0.01; Supplementary Figure 1B), but BW differences were not observed thereafter.

Responses to Intramammary LPS challenge

The inflammatory challenge modified some production variables during the 24 h following the intramammary LPS injection in CONT cows. Control cows decreased DMI (-2.5 kg DM; P < 0.01; Figure 1A), milk protein yield (P < 0.01), lactose content (P = 0.02), and tended to decrease protein content (P = 0.07) on the day of the LPS challenge (Supplementary Figures 2C, 2D and2E).

In CONT cows, milk fat content was greatest during the 48 h following LPS injection (P < 0.05) compared all other DIM, and milk fat yield was increased (P < 0.05) during the same period (Supplementary Figures 2A and2B). In REST cows, milk production tended to decrease (-5.9 kg; P = 0.07) on the day following the LPS challenge (Figure 1B). Milk fat content increased (P = 0.04), whereas lactose concentration and protein yield decreased (P < 0.001) on the day of LPS challenge, as observed for CONT (Supplementary Figures 2A,2E, and 2D, respectively).

Composite milk SCC increased sharply on the day of LPS challenge in both CONT and REST cows (Figure 1D), but SCC was greater for REST (6. 91E6 vs 1.91E6 ± 337E3 cells/mL, P = 0.01; Diet × Time interaction: P = 0.04). Furthermore, REST cows secreted a greater total number somatic cells in milk than CONT on the day of LPS injection (1.35E11 vs 0.67E11 ± 0.203E11 cells; P = 0.03). Composite milk SCC returned to pre-challenge values after 31 and 34 DIM for REST and CONT, respectively.

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019

12

The increment in rectal temperature above pre-LPS challenge values and measured rectal temperature are presented in Figure 3A and Supplementary Figure 3A, respectively. Rectal temperature after 72 h of diet treatments, measured before LPS injection, did not differ between groups (38.3 vs 37.8 ± 0.2 °C for CONT and REST, respectively; Supplementary Figure 3A). For both treatments, temperature increased significantly at 4, 6 and 10 h after LPS injection (Figure 3A, Supplementary Figure 3A). The average temperature during the day of LPS challenge (measured at 0, 1, 2, 4, 6 10 and 24 h) was lower for REST than CONT (38.4 vs 38.9 ± 0.14 °C, P = 0.03; Supplementary Figure 3A). Nonetheless, restriction did not alter temperature increment relative to pre-LPS challenge values (Figure 3A). Maximal temperature increment was +2.1 ± 0.15 °C, at 6 h after LPS injection.

Plasma cortisol increment from baseline in response to LPS and plasma cortisol concentrations are presented in Figure 3B and Supplementary Figure 3B, respectively. Plasma cortisol concentrations did not differ between REST and CONT before the LPS challenge (19.1 vs 24.5 ± 7.3 ng/mL, respectively), peaked in samples collected 4 h after LPS injection (64.5 ± 5.0 ng/mL) and returned to baseline at 10 h post-injection (Supplementary Figure 3B). Nonetheless, REST had a greater cortisol increment above baseline in response to LPS than CONT (P = 0.05;

Figure 3B), and cortisol response AUC was greater when samples collected at 24 h were considered (344 vs 175 ng/mL ×24 h, P = 0.02; 13 concentration was significantly greater than basal at 1 h after LPS challenge and reached maximal concentration after 6 h, with a 2.7-fold increase compared to pre-challenge values. For REST, the maximal insulin concentration was reached after 10 h, with a 1.9-fold increase compared to prechallenge concentrations. Therefore, insulin AUC in response to LPS challenge was smaller in REST compared to CONT cows (P < 0.001, Table 2). Nonetheless, absolute changes in NEFA, BHB and glucose were greater in the REST group, as shown by AUC in Table 2. Plasma glucose was lower for REST than CONT on the day of LPS challenge (Diet effect: P = 0.02; Figure 4B).

When looking at Time effects within each dietary treatment, plasma glucose concentrations increased overtime for REST cows, becoming greater than baseline values at 10 h post LPS injection (P < 0.01; Figure 4A). Meanwhile, in the CONT group, glucose concentration decreased initially below baseline at 4 h (P < 0.01) and rebounded above baseline at 10 h (P < 0.05; Figure 4A). Plasma NEFA concentration decreased sharply after LPS, from 1.672 and 0.371 (P < 0.001) to a nadir of 0.599 ± 0.12 and 0.101 ± 0.10 mM at 4 h after injection, for REST and CONT, respectively, corresponding to 36 and 22% of pre-challenge concentrations (Figure 4C; Diet and Diet × Time interaction, P < 0.001). In the REST group, plasma BHB was 2.98 mM before the LPS challenge, and decreased significantly between 1 to 10 h after LPS injection, reaching a nadir of 1.78 ± 0.43 mM at 10 h. In contrast, BHB was 0.69 mM before LPS injection in CONT, increased significantly above baseline between 1 to 10 h after LPS, reaching 1.23 ± 0.22 mM at 6 h postchallenge (Diet and Diet × Time interaction, P ≤ 0.001; Figure 4D). Plasma urea concentration increased over time after LPS injection (P < 0.01) for both CONT and REST groups, and was significantly greater at 1, 2, and 4 h post-challenge compared to before LPS challenge. A Diet × Time interaction was not observed during this period (P = 0.71; data not shown). At 24 h after LPS challenge (corresponding to 96 h of dietary treatments), plasma insulin, BHB, and glucose had returned to pre-LPS challenge concentrations for both CONT and REST, whereas in REST, plasma NEFA and urea tended to decrease below the pre-LPS challenge concentrations.

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446

14

DISCUSSION

This study assessed whether undernutrition and resulting metabolic imbalance modify the inflammatory response in early lactation cows, as previous research involving midlactation cows showed limited effects of undernutrition on animal-level responses to LPS and S. uberis challenges [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF]. Despite the extreme negative energy balance induced in early lactation cows, few of the inflammation indicators measured in this study differed between normal-fed and restricted cows. Nonetheless, metabolic responses to LPS to differed between the two treatments, and suggest a prioritization of nutrient partition to sustain immune response during an acute intramammary inflammation.

Early Lactation and Responses to Nutrient Restriction

Production and most indicators of metabolic status did not differ between CONT and REST during the first 3 weeks of lactation before restriction. Exceptions were a trend for greater plasma BHB on week 1 observed in REST, and increased milk content in cis-9 18:1, which is potentially derived from adipose tissue, and decreased in FA synthetized de novo (Σ 10:0 to 15:0) during the first two weeks of lactation (data not shown). These differences may be explained by the trend for greater prepartum BCS observed in REST cows.

The experimental undernutrition model employed in the present study (diet containing 48% straw, DM basis) induced repeatable decreases in DMI, energy balance, milk production, and rapid modifications of metabolite and hormonal profiles, and is in agreement with previous research employing a diet containing 60% straw [START_REF] Bjerre-Harpøth | Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation[END_REF]. Per design, the metabolic status was profoundly altered in REST before initiation of LPS challenge (i.e., elevated NEFA, BHB, and low insulin and glucose concentrations in plasma). For these reasons, the nutritional challenge during early lactation may be better suited to assess the ramifications of undernutrition

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 15 and metabolic imbalance on immune activation at whole animal level, compared to later in lactation [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF].

Responses to Intramammary LPS

Immune Activation. Immune activation following the intramammary LPS injection was confirmed by increased rectal temperature, composite SCC, foremilk milk IL-8, IL-1β, TNF-α and CXCL3

concentrations, and anecdotal observation of mammary gland symptoms (e.g., swelling, hardness, redness, and soreness) and modified quarter milk appearance (clots and yellowish color).

Concerning the effect of diet on indicators of inflammatory response, only cortisol response AUC and composite milk SCC differed between CONT and REST. These findings in early lactation cows are in agreement with previous research during midlactation involving partial feed restriction and experimental inflammation by LPS or Streptococcus uberis infection [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF][START_REF] Moyes | Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation[END_REF].

Restricted cows had greater composite milk SCC the day following LPS injection than CONT, but the biological implications of this observation are uncertain. The SCC difference is not explained by a concentration effect due to differences in milk volume because the total number of secreted somatic cells was higher in REST than in CONT cows on the same day, despite their lower milk yield. Our results contrast with those obtained in midlataction cows restricted to 80% of energy requirements for 2 weeks, followed by an intramammary LPS challenge, where no difference in quarter milk SCC was observed [START_REF] Perkins | Clinical responses to intramammary endotoxin infusion in dairy cows subjected to feed restriction[END_REF], and to decreased SCC response to LPS when hyperketonemia was induced in midlactation cows via prolonged i.v. BHB perfusion (Zarrin et al., 2014a). Several factors may explain the enhanced SCC and total SC secretion in REST cows. Mammary epithelial integrity may be altered in early lactation cows undergoing extreme feed-restriction, potentially facilitating the transfer of plasma constituents and PMN into alveolar milk. Moderate feed restriction promotes exfoliation of epithelial cells in milk, and

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 16 mammary epithelium permeability increases during severe restriction (Stumpf et al., 2013, Herve et al., Accepted). Intramammary LPS reduces the alveoli blood-milk barrier integrity [START_REF] Wellnitz | Short communication: Differential immunoglobulin transfer during mastitis challenge by pathogen-specific components[END_REF], via the opening of tight junctions and potential cell damage [START_REF] Wellnitz | Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid[END_REF].

Restricted cows may have been in a proinflammatory state due to extremely low DMI, high plasma NEFA and ketosis. In fact, previous research has shown that selected plasma markers of inflammation are increased before the onset and during ketosis in periparturient cows [START_REF] Abuajamieh | Inflammatory biomarkers are associated with ketosis in periparturient Holstein cows[END_REF]. Some fatty acids at high concentrations are agonists of TLR4, the pathogen recognition receptor on host cells that interacts with LPS to elicit the inflammatory response (Shi et al., 2006, Sordillo and[START_REF] Sordillo | The nexus between nutrient metabolism, oxidative stress and inflammation in transition cows[END_REF].

Nonetheless, the greater milk SCC observed in REST cows after LPS injection does not imply an enhanced immune response, because leucocyte function was probably modified in REST.

For instance, leucocyte phagocytic activity is impaired in early lactation cows experiencing negative energy balance and elevated plasma NEFA [START_REF] Hammon | Neutrophil function and energy status in Holstein cows with uterine health disorders[END_REF] and the severity of experimental E. coli mammary infection seems more pronounced in ketotic cows [START_REF] Kremer | Severity of Experimental Escherichia coli Mastitis in Ketonemic and Nonketonemic Dairy Cows[END_REF], as leucocyte chemotaxis [START_REF] Suriyasathaporn | β-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes[END_REF] and respiratory burst [START_REF] Hoeben | Elevated levels of β-hydroxybutyric acid in periparturient cows and in vitro effect on respiratory burst activity of bovine neutrophils[END_REF] are impacted by BHB at concentrations lower than those observed in REST cows at the time of LPS challenge.

Metabolic Responses to Immune Activation.

The systemic effects of LPS challenge on fat and carbohydrate metabolism are potentially mediated by transient increases in circulating cortisol, TNF-alpha and other proinflammatory cytokines, that modify secretion of insulin, glucagon and other hormones [START_REF] Steiger | Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers[END_REF][START_REF] Hoeben | Role of endotoxin and TNF-α in the pathogenesis of experimentally induced coliform mastitis in periparturient cows[END_REF], Waldron et al., 2003a[START_REF] Kushibiki | Tumor necrosis factor-alpha-induced inflammatory responses in cattle[END_REF], as well as the response of target tissues to hormones such as insulin (Zarrin et al., 2014b). Direct effects of intramammary LPS on whole body metabolism should have been minimal in our study, because previous research using greater LPS doses did not detect LPS in the systemic circulation

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446

17

after its administration into the mammary gland cistern, and report only short-term increases in plasma LPS after i.v. injection [START_REF] Hoeben | Role of endotoxin and TNF-α in the pathogenesis of experimentally induced coliform mastitis in periparturient cows[END_REF].

The increment in cortisol concentrations above pre-challenge baseline and cortisol response AUC were greater for REST than CONT cows (Figure 3B and Table 2). It is unknown whether these differences in cortisol response have contributed to the metabolic effects of LPS observed in our study. Cortisol concentrations per se did not differ between CONT and REST cows (Supplementary Figure 3B), only the cortisol increment above pre-challenge baseline. The increase in plasma insulin concentration was delayed and insulin AUC in response to LPS was attenuated in nutrient-restricted early lactation dairy cows. This effect may be explained by the hypoglycemic state of REST cows.

Plasma glucose increased gradually after LPS in REST, leading to a positive glucose response AUC, despite a concomitant insulin increase. A transient increase in plasma glucose concentration has been observed in response to LPS injection (Zarrin et al., 2014b[START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF]. Because REST cows were in early lactation, nutrient-restricted and ketotic at the time of LPS challenge, glycogenolysis could not have been a source of glucose, as depletion of liver glycogen reserves precedes experimentally induced ketosis [START_REF] Veenhuizen | Metabolic changes in blood and liver during development and early treatment of experimental fatty liver and ketosis in cows[END_REF]. Previous research suggests that hepatic conversion of propionate to glucose may be upregulated after LPS challenge in fed mid-lactation cows (Waldron et al., 2003a). The increase in plasma urea concentration observed until 4 h after LPS injection in our study probably reflects enhanced reliance on amino acids to sustain inflammatory response and gluconeogenesis [START_REF] Gifford | Growth and development symposium: Impacts of inflammation on cattle growth and carcass merit[END_REF][START_REF] Greco | Effects of altering the ratio of dietary n-6 to n-3 fatty acids on performance and inflammatory responses to a lipopolysaccharide challenge in lactating Holstein cows[END_REF].

Plasma NEFA decreased sharply after LPS injection for both CONT and REST cows. It has been suggested that plasma NEFA changes post LPS challenge are due to hyperinsulinemia [START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF], but a NEFA decrease after LPS injection preceded the plasma insulin increment in REST cows. As a consequence, the typical inverse diurnal relationship between

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 18 plasma NEFA and insulin [START_REF] Allen | Drives and limits to feed intake in ruminants[END_REF] is not observed in REST cows challenged with intramammary LPS. Restricted cows were ketotic at the time of LPS injection. The decrease in plasma BHB post LPS challenge is in agreement with the concomitant reduction in plasma NEFA, may result from downregulated liver ketogenesis, increased ketone utilization or a combination of both, and may constitute a glucose-sparing strategy (Zarrin et al., 2014b[START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF]. The reduction of plasma BHB preceded significant time effects in plasma insulin and glucose, as observed for plasma NEFA. Other factors must have mediated early changes in plasma NEFA and BHB concentrations after LPS challenge in REST cows.

In CONT cows, plasma BHB concentrations increased after LPS, despite decreased NEFA and increased insulin, together with a transient decrease in glucose. In CONT cows, these profiles share common patterns with diurnal variations related to morning feed intake [START_REF] Allen | Drives and limits to feed intake in ruminants[END_REF][START_REF] Piantoni | Feed intake is related to changes in plasma nonesterified fatty acid concentration and hepatic acetyl CoA content following feeding in lactating dairy cows[END_REF], but not in REST cows. Therefore, postprandial effects may have played a role in plasma metabolite and insulin profiles observed in CONT cows during the first hours after LPS challenge because fresh TMR was offered after morning milking. Nonetheless, previous research suggests that metabolic effects of high LPS doses are rather independent of feed intake [START_REF] Steiger | Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers[END_REF]. The potential intake effects should be minor in REST cows due to the low DMI, low concentrate content, and low fermentability of the high-straw TMR. Time-changes in plasma metabolite and insulin concentrations in REST cows should have been driven by LPS-induced inflammation.

The increase in plasma glucose and insulin concentrations concomitant with inflammatory response (i.e., fever, increased cortisol, milk IL-8, IL-1β, TNF-α and CXCL3) indicate the establishment of insulin resistance in both CONT and REST cows. Insulin resistance constitutes a homeorhetic adaptation to support the massive glucose requirements of an acute inflammation response, and is probably mediated by sharp increases in systemic cortisol and inflammation cytokines, such as TNF-alpha (Waldron et al., 2003a[START_REF] Kushibiki | Tumor necrosis factor-alpha-induced inflammatory responses in cattle[END_REF][START_REF] Vernay | Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows[END_REF], Moyes et al., 2014, Zarrin Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 et al., 2014b[START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF]. Restricted cows were probably in an insulin resistant state before the LPS challenge, due to hormonal changes, elevated circulating NEFA [START_REF] Pires | Induction of hyperlipidemia by intravenous infusion of tallow emulsion causes insulin resistance in holstein cows[END_REF] and ceramides [START_REF] Davis | Nutrient restriction increases circulating and hepatic ceramide in dairy cows displaying impaired insulin tolerance[END_REF]. Acute adaptations may have allowed REST cows to direct glucose and other nutrients for immune activation, as suggested by plasma metabolite and insulin profiles during the hours following intramammary LPS injection. Nonetheless, the ability to cope with prolonged or repeated challenges and resulting metabolic modifications may be limited (Waldron et al., 2003a[START_REF] Bradford | Invited review: Inflammation during the transition to lactation: New adventures with an old flame[END_REF][START_REF] Kvidera | Glucose requirements of an activated immune system in lactating Holstein cows[END_REF], for instance in the case of chronic inflammation and intake depression during early lactation, which may disrupt homeostatic capacity of the dairy cow.

19

CONCLUSIONS

Experimentally-induced undernutrition to early lactation Holstein cows had limited effects on animal-level indicators of acute inflammation measured in this study, as previously observed in midlactation cows. The short-term response following intramammary LPS injection suggests modifications of nutrient partitioning to support immune activation via an apparent establishment of insulin resistance in both CON and REST cows, despite intense lipomobilization, ketosis, and limited availability of precursors for glucose synthesis in REST cows. Inflammation during early lactation (acute or chronic) may impose a metabolic burden due to aggravated insulin resistance, utilization of glucose and other limiting nutrients to support immune activation, and further challenge the homeostasis mechanisms of early lactation cows.

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. ( 2019 2. Effects of nutrient restriction on plasma cortisol, insulin and metabolite responses to LPS challenge in early lactation cows. Multiparous Holstein cows were either allowed ad libitum intake of a regular diet throughout the study (Control, n = 9), or underwent 96 h of nutrient restriction (Restricted, n = 8) by receiving a ration composed of 48% (DM-basis) straw from 24 to 27 ± 3 DIM (mean ± SD). One healthy rear mammary quarter was injected with 50 µg of LPS (E. coli 0111:B4) 72 h after initiation of dietary treatments. Blood samples were collected at -1, -0. 5, 1, 2, 4, 6, 10 and24

Figure 2 .

 2 Figure 2. Plasma NEFA (A), BHB (B), glucose (C), urea (D) and insulin (E) concentrations.

Figure 3 .

 3 Figure 3. Effects of nutrient restriction on rectal temperature increment (A), plasma cortisol

  Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 34 Pires et al.

Figure

  LPS challenge † P ≤ 0.01 ‡ P ≤ 0.05 § P ≤ 0.10 Diet: P = 0.02 Time: P < 0.01 Diet × Time: P = 0.25

  At 72 h of differential diet between CONT and REST, one healthy rear mammary quarter was injected with 50 µg of LPS (E. coli O111:B4, Ultrapure LPS-EB, InvivoGen, Toulouse,

containing 48% of chopped barley straw (DM-basis) during 96 h (REST, n = 8). Diet ingredient and nutrient composition are presented in Table

1

. Cows were selected based on previous lactation performance, health records and expected calving date, and randomly allocated to treatments before the initiation of the experiment.

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (

2019

). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 6

Phenotyping Responses to LPS challenge

	refeeding the lactation TMR to cows in the REST group. Body weight and body condition score PA) diluted 1:20000 for 1 h and finally TMB ELISA substrate. Commercial ELISA were used for
	(BCS, 6-point, 0 to 5 scale) were recorded weekly, one day before diet change, and on the last day TNF-α (R&D Systems, Minneapolis, MN) and IL1-ß (Thermo Fisher Scientific, Rockford, IL)
	of restriction. according to the manufacturer's instructions. A number of foremilk samples could not be analyzed
	Blood samples were collected from coccygeal vessels before the morning feeding on for SCC for technical reasons, therefore, quarter milk SCC data were excluded to prevent potential
	Wednesday of week -3, -2, -1, 1, 2, 3, 5, 6 and 7 relative to calving, corresponding to -18, -11, -4, 8, bias. The whole-udder composite milk SCC and total number of somatic cells secreted per day were
	15, 21, 35, 42 and 48 DIM, respectively. The first postpartum blood sample was collected after 3 analyzed instead. Total number of somatic cells secreted in milk per day was calculated by
	DIM. Samples were collected from jugular veins at -24, 24, 48 h relative to initiation of restriction converting milk yield from Kg to liters per day (considering a standard density of 1.033), and
	and at equivalent period for CONT. multiplying the volume of milk by SCC per mL.
	Cows were screened for mastitis one week prior and before the milking preceding the LPS
	challenge using the California mastitis test (Neodis, Rambouillet, France), and SCC analyses of
	foremilk samples collected from the rear quarters (Galilait, Theix, 63122 Saint Genès-Champanelle,
	France). Only cows with SCC lower than 100 000 cells/mL in a rear quarter were included in the
	study. Foremilk samples were collected from the LPS challenged quarters immediately before the
	morning milking that preceded the LPS injection (time 0), and at 4, 6, 10 and 24 h after LPS
	injection. These quarter milk samples were analyzed by ELISA for IL-8, TNF-α, CXCL3 and IL1-
	β. For the ELISA bCXCL3 and bIL-8 assays, the sequence of incubation steps, all performed at
	room temperature, was: affinity-purified Ab to C-terminal peptide of bCXCL3 or bIL-8 (2 µg/mL)
	. Milk yield in PBS overnight; blocking of the plate with 0.5% gelatin in PBS for 1 h; incubation at the
	was recorded daily, milk samples were collected at 4 consecutive milkings each week and analyzed appropriate dilution or a series of twofold dilutions of standard bCXCL3 or bIL-8 for 2h;
	for composition and SCC by near-infrared spectrometry and Fossomatic FC method (Foss Electric, biotinylated IgG fraction of rabbit antiserum to recombinant bCXCL3 (1 µg/mL) or mouse
	Hillerød, Denmark), respectively. Weighted milk component means were computed according to monoclonal Ab to ovine IL-8 (0.5 µg/mL; clone 8M6; Bio-Rad Laboratories, Hercules, CA) for 1h;
	PM/AM production. In order to characterize the responses to feed-restriction, DMI and milk avidin-peroxidase conjugate (Molecular Probes, Thermo Fisher Scientific, Rockford, IL) or
	composition were measured daily in both groups from 2 d prior to restriction until 7 d after peroxidase-conjugated goat Ab to mouse IgG (Jackson Immunoresearch Laboratories, West Grove,
	8
	Comment citer ce document : Comment citer ce document :
	Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019).
	Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited
	effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science,
	102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446

Table 2

 2

	). Milk IL-8, IL-1β, TNF-α and CXCL3 concentrations

and Figures

4A, 4B

, 4C and 4D. Plasma insulin concentration increased for both CONT and REST after LPS challenge (Time effect, P < 0.001; Figure

4A

). Nonetheless, for CONT, insulin Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446

Table 1 .

 1 ). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-1544627 Diet ingredient, nutrient and fatty acid composition. Multiparous Holstein cows were either allowed ad libitum intake throughout the study (Control, n = 9), or underwent 4 days of nutrient restriction (Restricted, n = 8) by receiving a ration composed of 48% (DM-basis) straw from 24 to 27 ± 3 DIM (mean ± SD).Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 28 Table

	Dry-period 1	Lactation	Lactation
		Control	Restriction

  Dry matter intake (DMI, A), milk yield (B), energy balance (C), and somatic cell count (SCC, D). Multiparous Holstein cows were either allowed ad libitum intake of a regular diet throughout the study (Control, n = 9), or underwent 4 days of nutrient restriction(Restricted, n = 8)

	by receiving a ration composed of 48% (DM-basis) straw from 24 to 27 ± 3 DIM (mean ± SD). One
	h relative to LPS injection. healthy rear mammary quarter was injected with 50 µg of LPS (E. coli 0111:B4) 72 h after
	initiation of dietary treatments. P-values for SCC reflect statistical analysis with log-transformed
	data. Values are LSM ± SEM.	Treatments Control Restricted	SEM	P
	Cortisol
	72 h (ng/mL) 1	24.5	19.1	5.8	0.51
	AUC10h 2	122	202	32	0.09
	AUC24h 3	175	344	48	0.02
	Insulin
	72 h 1 (µIU/mL)	17	11	1.6	0.009
	AUC10h 2	174	42	21	<0.001
	AUC24h 3	339	120	44	0.002
	Glucose
	72 h (mM) 2	3.83	2.78	0.23	0.005
	AUC10h 3	-0.94	3.56	0.94	0.003
	AUC24h	1.22	11.78	3.00	0.02
	NEFA
	72 h (mM) 1	0.37	1.67	0.05 to 0.17	<0.001
	AUC10h 2	-1.96	-9.05	0.36 to 9.05	0.002
	AUC24h 3	-2.76	-19.31	0.88 to 3.86	<0.001
	BHB
	72 h (mM) 1	0.69	2.98	0.12 to 0.47	<0.001
	AUC10h 2	3.68	-6.05	1.51	<0.001
	AUC24h 3	6.37	-14.68	3.95	0.002
	Urea
	72 h (mM) 1	4.44	5.77	0.19 to 0.54	0.03
	AUC10h 2	2.26	-1.23	1.26	0.06
	AUC24h 3	0.15	-12.63	3.35	0.01
	1 Average concentration at 1 and 0.5 h prior to LPS challenge.
	2 Area under the curve during first 10 h post LPS (concentration units × 10 h).
	3 Area under the curve during first 24 h post LPS (concentration units × 24 h).

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (

2019

). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 29 Figure 1:

  ). One healthy rear mammary quarter was injected with 50 µg of LPS (E.

	the injected mammary quarter before and at 4, 6, 10 and 24 h relative to LPS administration. Values
	are LSM ± SEM.
	Figure 4. Effects of nutrient restriction on plasma insulin and metabolite concentration in response
	to LPS challenge in early lactation cows. Multiparous Holstein cows were either allowed ad libitum
	intake of a regular diet throughout the study (Control, n = 9), or underwent 96 h of nutrient
	restriction (Restricted, n = 8) by receiving a ration composed of 48% (DM-basis) straw from 24 to
	27 ± 3 DIM (mean ± SD). One healthy rear mammary quarter was injected with 50 µg of LPS (E.
	coli 0111:B4) 72 h after initiation of dietary treatments. Blood samples collected before and at 1, 2,
	4, 6, 10 and 24 h relative to LPS injection. Values are LSM ± SEM.
	coli 0111:B4) 72 h after initiation of dietary treatments. Rectal temperature was recorded and blood
	samples collected before and at 1, 2, 4, 6, 10 and 24 h; foremilk milk samples were collected from
	30

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (

2019

). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows. Journal of Dairy Science, 102 (6), 5347-5360. , DOI : 10.3168/jds.2018-15446 31 Pires et al.

ACKNOWLEDGEMENTS

The authors thank the staff at Herbipôle Research Unit (INRA, UE1414, Theix, France) for animal care and sampling; D. Bany, S. Bes, C. Labonne, E. Tixier, M. Tourret (INRA, UMR1213, Saint-Genès-Champanelle, France) for sample collection and laboratory analyses; P. Rainard 20 (INRA, UMR1282 ISP, Nouzilly, France) for valuable discussions on experimental design; M. Faure and C. Ravel (INRA, UMR1213, Saint-Genès-Champanelle, France) for proposing cortisol sampling schedule and cortisol analyses, respectively. The authors are grateful to the Galilait laboratory (Clermont-Ferrand, France) for milk component, SCC and microbiological analyses. This research was funded by GISA meta-program of INRA (Ruminflame and Longhealth projects).

Comment citer ce document : Pires, J., Pawlowski, K., Rouel, J., Delavaud, C., Foucras, G., Germon, P., Leroux, C. (2019). Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows.