domain: sdv info shs

text:
P A Billa

Y Faulconnier

T Larsen

C Leroux

J A A Pires

suggesting their potential use as biomarkers of nutritional status. RUNNING HEAD: MILK METABOLITES DURING UNDERNUTRITION Milk metabolites as noninvasive indicators of nutritional status of midlactation Holstein and Montbéliarde cows

Interpretative Summary, Billa et

INTRODUCTION

Milk composition is modulated by a diversity of factors, including genetics, lactation stage, nutrition and health status, therefore, milk is an obvious source of biomarkers for the monitoring of dairy ruminants. Early lactation is characterized by a rapid increase in milk yield, mobilization of body protein and fat reserves, negative energy balance (NEB), and modifications in milk protein, fat and fatty acid composition. Complex homeorhetic and homeostatic adaptations are required to the direct limiting nutrients towards the mammary gland and support milk synthesis during early lactation [START_REF] Bell | Adaptations of glucose metabolism during pregnancy and lactation[END_REF]. The occurrence of metabolic disorders related to energy metabolism is typical during this period, and plasma concentrations of non-esterified fatty acids (NEFA) and BHB are classic indicators for herd troubleshooting [START_REF] Oetzel | Monitoring and testing dairy herds for metabolic disease[END_REF]. Experimental feed restriction models are used to induce nutrient deficits and NEB at different stages of lactation, assess the production and metabolic responses (Gross et al., 2011a;[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]Friggens et al., 2016), and the effects of NEB on various biological functions of the dairy cow [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] Abdelatty | Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows[END_REF][START_REF] Pires | Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows[END_REF]. Metabolic responses to feed restriction models are of greater amplitude during early lactation compared to later stages [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], but midlactation period is more convenient to run experimental protocols and less prone to confounding due to the dynamic nature of early lactation (Contreras et al., 2016).

The reliance on body reserve mobilization for milk synthesis during early lactation is largely driven by genetics (Friggens et al., 2013). Breed differences between Holstein (HOLS) and Montbéliarde (MONT) cows have been described in various production systems (Dillon et al., 2003;[START_REF] Pomiès | Once-a-day milking of Holstein and Montbéliarde cows for 7 weeks in mid-lactation[END_REF][START_REF] Pires | Physiological adaptations and ovarian cyclicity of Holstein and Montbéliarde cows under two low-input production systems[END_REF]. Holstein cows prioritize milk, fat, Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

protein and lactose secretion compared with MONT (Dillon et al., 2003;[START_REF] Pomiès | Once-a-day milking of Holstein and Montbéliarde cows for 7 weeks in mid-lactation[END_REF][START_REF] Pires | Physiological adaptations and ovarian cyclicity of Holstein and Montbéliarde cows under two low-input production systems[END_REF], but experience greater BCS loss and metabolic deviations during early lactation (Dillon et al., 2003;[START_REF] Pires | Physiological adaptations and ovarian cyclicity of Holstein and Montbéliarde cows under two low-input production systems[END_REF]. Therefore, we hypothesized that breed effects might be used as a model to induce different production and metabolic responses to a nutritional challenge.

Milk is a source of novel indicators of nutritional status of dairy cows. Milk sampling is easy to perform in dairy operations, and can be automated for inline analyses. Certain molecules of intermediary metabolism are present in milk and may be indicators of physiological and nutritional state of dairy cows (Gross and Bruckmaier, 2019). For instance, milk is classically used to monitor ketosis by cow-side tests [START_REF] Oetzel | Monitoring and testing dairy herds for metabolic disease[END_REF] and by automated inline BHB measurements. Milk glucose, glucose-6 phosphate, and acid uric acid concentrations are modulated by diet digestibility and correlated with DMI in midlactation cows [START_REF] Larsen | Minor milk constituents are affected by protein concentration and forage digestibility in the feed ration[END_REF]. Furthermore, milk glucose and glucose-6 phospate concentrations vary according to DIM [START_REF] Larsen | Are free glucose and glucose-6-phosphate in milk indicators of specific physiological states in the cow?[END_REF][START_REF] Zachut | Milk glucose-6phosphate dehydrogenase activity and glucose-6-phosphate are associated with oxidative stress and serve as indicators of energy balance in dairy cows[END_REF]Ferris et al., 2018).

Milk concentrations of these metabolites may reflect modifications of metabolic pathways in mammary epithelial cells, including glucose utilization for glycolysis and lactose synthesis [START_REF] Chaiyabutr | Changes in the concentrations.of the minor Constituents of goat's milk during starvation and on refeeding of the lactating animal and their relationship to mammary gland metabolism[END_REF], glucose-6-phosphate and isocitrate to produce reducing potential (i.e.; NADPH) for de novo fatty acid synthesis (Garnsworthy et al., 2006;[START_REF] Chaiyabutr | Changes in the concentrations.of the minor Constituents of goat's milk during starvation and on refeeding of the lactating animal and their relationship to mammary gland metabolism[END_REF], and to counterbalance oxidative stress associated with FA oxidation [START_REF] Zachut | Milk glucose-6phosphate dehydrogenase activity and glucose-6-phosphate are associated with oxidative stress and serve as indicators of energy balance in dairy cows[END_REF]. Milk uric acid originates in part from ruminal digestion of purine bases and has been suggested as an indicator of microbial protein synthesis [START_REF] Larsen | Fluorometric determination of uric acid in bovine milk[END_REF]. Milk glutamate and free amino acid content may reflect the availability and metabolism of amino acids.

The effects of NEB and breed on concentrations of minor milk metabolites are still insufficiently documented. We hypothesized that concentrations of selected metabolites in

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

milk are modified during periods of NEB, and may constitute novel indicators of energy balance and metabolic status of dairy cows. Thus, the objective was to determine the effects of NEB induced by partial feed restriction on milk concentrations of selected metabolites, assess potential differences between midlactation HOLS and MONT cows, and evaluate their relationships with classic indicators of metabolic status and lipomobilization.

MATERIALS AND METHODS

Experimental Design, Animals, Diets and Housing

All procedures were approved by the ethic committee on animal experimentation During CONT, WEEK1 and WEEK2 periods, all cows were allowed ad libitum intake of a TMR (Table 1). During REST period, feed allowance was reduced to meet 50% of individual NE L requirements calculated from BW, feed intake and milk production and composition recorded before restriction (INRA, 2007). Cows had free access to water and were housed in a free stall barn equipped with automatic feed bunks that control individual access and weight feed intake (CRFI, Biocontrol, Rakkestad, Norway). Gates were programmed to divide individual daily feed allowance in 4 equal portions in 6-h periods.

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Cows ate 3 ± 0.9 kg of hay ( 58 Morning milk samples were collected to determine metabolite concentrations on d -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 18 relative to initiation of feed restriction (Figure 1), before distribution of fresh TMR, and conserved at -20°C until analyses.

Enzymatic-fluorometric methods were used to quantify milk content of BHB [START_REF] Larsen | Fluorometric Determination of β-Hydroxybutyrate in Milk and Blood Plasma[END_REF], uric acid [START_REF] Larsen | Fluorometric determination of uric acid in bovine milk[END_REF], isocitrate [START_REF] Larsen | Fluorometric determination of free and total isocitrate in bovine milk[END_REF], glucose and glucose-6-phosphate [START_REF] Larsen | Fluorometric determination of free glucose and glucose 6-phosphate in cows' milk and other opaque matrices[END_REF], glutamate and free amino groups (NH 2 ) [START_REF] Larsen | Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk[END_REF]. Morning and evening milk samples were collected on d -2, -1, 1, 2, 3, 4, 5, 6 and 13 to determine milk fatty acid (FA) composition by gas chromatography, as previously described [START_REF] Lerch | Rapeseed or linseed supplements in grass-based diets: Effects on milk fatty acid composition of Holstein cows over two consecutive lactations[END_REF]. Briefly, samples were lyophilized and pooled according PM and AM production, to provide daily composite samples for each cow. Samples were methylated and injected into 7890A GC system CN10271102 Series gas chromatograph equipped with a flame ionization detector (Agilent technologies, Santa Clara, California, USA). Peaks were routinely identified through comparison of retention times with FA methyl ester standards. Peak integration was conducted using Chemstation software (Agilent technologies, Santa Clara, California, USA).

Blood Sampling and Analyses. Jugular blood samples were collected on d -3, -2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13 and 18 concentrations were quantified by enzymatic-fluorometric methods [START_REF] Larsen | Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk[END_REF].

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

Statistical Analyses

Statistical analyses were performed using SAS enterprise guide (Version 9.4; SAS Institute INC, Cary, NC). Daily data was analyzed as repeated measures by mixed models that included day, breed and their interaction as fixed effects, cow as random effect, and Kenward-Roger adjustment for calculation of degrees of freedom. The Schwarz's Bayesian criterion was used to compare the fitting of different variance-covariance structures, including spatial power, AR (1), ARH (1) and CS. The means for each variable within animal and period were calculated in order to compare periods (CONT, REST, WEEK1 and WEEK2) and explore breed by period interactions. The models included the fixed effects of period, breed and their interaction, and the random effect of cow. Significant time effects (i.e., day or period) and breed by time interactions were explored by the Fisher's protected least significant difference using the PDIFF and SLICE options of the LSMEANS statement. Residuals were checked for normality and homoscedasticity. Heterogeneous variance was tested whenever suggested by residual plots. Least squares means (LSM) and standard error of the mean (SEM) were estimated from untransformed values, whereas P-values may reflect statistical analysis of logtransformed data when transformation was necessary. Relationships among variables were explored by Spearman rank correlations. Linear regressions between energy balance and milk metabolite concentrations were analyzed using PROC REG procedure of SAS. The significance level was predefined as P ≤ 0.05 and trends toward significance at 0.05 < P ≤ 0.10.

RESULTS AND DISCUSSION

Production Responses

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

A significant period effect was observed for DMI, energy balance, milk and milk component yields (Table 2 and Supplemental Figure 1). Per design, energy balance became negative (-42.5 ± 4.4 MJ/d) and milk, fat and protein yields decreased during REST. Energy balance returned to pre-restriction values during WEEK1, and DMI, fat and protein yields during WEEK2. The effect of restriction on milk, fat and protein yields are in accordance with previous studies in midlactation Holstein cows (Gross et al., 2011a;[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][START_REF] Pires | Effects of abomasal infusion of nicotinic acid on responses to glucose and β-agonist challenges in underfed lactating cows[END_REF].

A significant breed effect was observed for DMI, milk, protein and lactose yields, which were greater in HOLS than in MONT cows (Table 2). Energy balance and fat yield tended to be greater in HOLS than in MONT. Nonetheless, milk yield differences between HOLS and MONT during CONT (+17% for HOLS) were less marked than reported in previous studies during mid lactation (+21%; [START_REF] Pomiès | Once-a-day milking of Holstein and Montbéliarde cows for 7 weeks in mid-lactation[END_REF]Ferlay et al., 2010).

Differences among studies could be explained by feeding systems, milking frequency, and the lactation stage.

Plasma Metabolite and Insulin Concentrations

Plasma metabolite and insulin concentrations are presented in Table 3 andSupplemental Figures 2 and3 The increase in plasma NEFA concentrations during REST reflects lipomobilisation (Chilliard et al., 2000a) and is in accordance with previous feed-restriction studies involving mid-and late lactation cows [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]Gross and Bruckmaier, 2011a;[START_REF] Pires | Effects of abomasal infusion of nicotinic acid on responses to glucose and β-agonist challenges in underfed lactating cows[END_REF]. Nonetheless, the increase in plasma NEFA observed during REST in our experiment was smaller than observed in underfed early lactation cows [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][START_REF] Pires | Undernutrition modified metabolic responses to intramammary lipopolysaccharide but had limited effects on selected inflammation indicators in early-lactation cows[END_REF]. Plasma BHB concentrations increased during restriction (Table 3 and Supplemental Figure 2), but remained below the 1.2 mM threshold of subclinical ketosis [START_REF] Leblanc | Metabolic Predictors of Displaced Abomasum in Dairy Cattle[END_REF], which is in agreement with previous research in underfed midlactation cows [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]Gross et al., 2011a;[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]. As plasma BHB originates in part from rumen butyrate [START_REF] Miettinen | Effects of the Ratio of Ruminal Propionate to Butyrate on Milk Yield and Blood Metabolites in Dairy Cows[END_REF], its concentrations observed during REST probably reflect concomitant modifications of DMI and ruminal butyrate synthesis, and incomplete beta-oxidation of mobilized NEFA. Two cows had increased concentrations of BHB the morning after refeeding the TMR at ad libitum intake (d 7; Supplemental Figure 3), which may result from increased DMI and a shift in ruminal butyrate production. Accordingly, insulin was greatest the day after refeeding at ad libitum intake (d 7, Supplemental Figure 2).

The decrease in plasma glutamine and NH 2 concentrations observed during REST may reflect reduced DMI and amino acid absorption. Glutamine and glutamate are intermediates in many anabolic and catabolic pathways, including regulation of metabolic acidosis, lymphocyte proliferation, and casein synthesis [START_REF] Lobley | Glutamine in Animal Science and Production[END_REF]. Plasma glutamine, glutamate and total amino acid concentrations decrease during periods of NEB, such as early lactation [START_REF] Meijer | Free Amino Acids in Plasma and Muscle of High Yielding Dairy Cows in Early Lactation[END_REF] and during experimental feed restriction in midlactation cows (Girard et al., 2019). Decreased supply of amino acids for intestine absorption may have contributed more to these changes than amino acid catabolism, because plasma urea concentration also decreased during REST, as previously suggested (Girard et al., 2019). The

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. ( 2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

increase in plasma glutamate concentration observed during REST (Table 3 andSupplemental Figure 3) may be due in part to spontaneous glutamine deamination.

Holstein cows had greater plasma glucose, glutamate and NH 2 , concentrations than MONT (Supplemental Figures 2 and3). The greater glucose concentrations observed in HOLS compared to MONT contrast with previous research during the first weeks of lactation in low input systems [START_REF] Pires | Physiological adaptations and ovarian cyclicity of Holstein and Montbéliarde cows under two low-input production systems[END_REF]. These discrepancies may be explained by different lactation stage (early vs. midlactation), diet, and undernutrition model (early lactation spontaneous NEB vs. midlactation feed restriction). The greater BHB concentrations observed in HOLS during CONT (Table 3) probably reflect their greater DMI, ruminal butyrate and BHB synthesis compared with MONT, because all animals were in positive energy balance.

Milk Metabolite Concentrations

Significant time effects were observed for milk concentrations of all metabolites studied ( The decrease of milk uric acid concentrations observed during REST is in agreement with results obtained when cows were offered low energy diets [START_REF] Larsen | Minor milk constituents are affected by protein concentration and forage digestibility in the feed ration[END_REF]. Milk BHB concentrations decreased during REST despite a small but significant increase in plasma BHB, which suggests that BHB was preferentially metabolized in the mammary gland. In contrast, BHB concentrations increase both in plasma and milk during early lactation, and both matrices are used to diagnose subclinical ketosis [START_REF] Nielsen | Diurnal Variation and the Effect of Feed Restriction on Plasma and Milk Metabolites in TMR-fed Dairy Cows[END_REF][START_REF] Oetzel | Monitoring and testing dairy herds for metabolic disease[END_REF].

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

Increased milk glucose-6-phosphate and isocitrate, and decreased milk glucose concentrations during REST are in agreement with previous observations in starved goats [START_REF] Chaiyabutr | Changes in the concentrations.of the minor Constituents of goat's milk during starvation and on refeeding of the lactating animal and their relationship to mammary gland metabolism[END_REF] and in early lactation cows [START_REF] Larsen | Are free glucose and glucose-6-phosphate in milk indicators of specific physiological states in the cow?[END_REF][START_REF] Zachut | Milk glucose-6phosphate dehydrogenase activity and glucose-6-phosphate are associated with oxidative stress and serve as indicators of energy balance in dairy cows[END_REF]. Glucose-6phosphate and isocitrate are precursors for NADPH synthesis via the pentose phosphate and isocitrate dehydrogenase pathways, respectively. Increased milk glucose-6-phosphate and isocitrate content during REST may reflect a shift in these pathways in mammary epithelial cells, due to low plasma insulin, decreased de novo FA synthesis and synthesis of other milk components.

Significant breed effects were observed for milk uric acid and isocitrate concentrations when individual data were analyzed by period (Table 4). Nonetheless, only a trend was detected for milk isocitrate concentration when daily data were analyzed as repeated measures, probably because many time points measured after refeeding (WEEK1 and WEEK2) did not differ between HOLS and MONT (Figures 2 and3). Breed by time interactions were not observed for any of the milk metabolites analyzed in this study.

Milk Fatty Acid Concentrations

Milk FA concentrations are presented in Table 5 and Supplemental increased. These modifications in milk FA profile are in agreement with other feed restriction studies (Chilliard et al., 2000b;Gross et al., 2011b;[START_REF] Abdelatty | Effect of short-term feed restriction on temporal changes in milk components and mammary lipogenic gene expression in mid-lactation Holstein dairy cows[END_REF]. The decrease of Σ

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

10:0 to 15:0 and 16:0 during REST reflect a diminution of de novo FA synthesis in mammary gland, due to a decreased availability of precursors (e.g., acetate and propionate) absorbed from rumen (Chilliard et al., 2000b;Gross et al., 2011b). The increase of 18:0, and cis-9 18:1 and ∑ > C16 FA reflect body fat mobilization (Chilliard et al., 2000b;Gross et al., 2011b ;[START_REF] Pires | Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows[END_REF].

The Σ 10:0 to 15:0 and 16:0 decreased gradually until d 3 of restriction (Supplemental Figure 5), indicating a gradual downregulation of de novo FA synthesis. This downregulation would reduce NADPH requirements for mammary gland lipogenesis [START_REF] Bell | Adaptations of glucose metabolism during pregnancy and lactation[END_REF], and may explain the gradual increase in glucose-6-phosphate and isocitrate concentrations during REST, which became significant at 48 h of REST (Figure 2). This pattern may reflect concomitant effects of limiting plasma glucose availability and reduced glucose uptake by mammary gland during REST, downregulation of lactose synthesis [START_REF] Chaiyabutr | Changes in the concentrations.of the minor Constituents of goat's milk during starvation and on refeeding of the lactating animal and their relationship to mammary gland metabolism[END_REF], and decreased NADPH requirements for de novo fatty acid synthesis [START_REF] Chaiyabutr | Changes in the concentrations.of the minor Constituents of goat's milk during starvation and on refeeding of the lactating animal and their relationship to mammary gland metabolism[END_REF]Garnsworthy et al., 2006). Milk glucose-6-phosphate and isocitrate concentrations decreased on d 5 and 6 of REST, before refeeding at ad libitum intake. Epithelial cell homeostatic mechanisms may have reestablished an equilibrium between cytosolic concentrations of glucose 6-phosphate and isocitrate and the activity of metabolic pathways for which they are precursors (e.g., NADPH and lactose synthesis). [START_REF] Zachut | Milk glucose-6phosphate dehydrogenase activity and glucose-6-phosphate are associated with oxidative stress and serve as indicators of energy balance in dairy cows[END_REF] proposed that FA oxidation in mammary cells during periods of lipomobilization would increase the oxidative stress, requiring the upregulation of the pentose phosphate pathway to generate reducing potential to neutralize reactive oxygen species.

Mammary gland gene expression shows a shift towards increased reliance on β-oxidation for energy and sparing of glucose in underfed early lactation cows [START_REF] Pawłowski | Mammary Gland Transcriptome and Proteome Modifications by Nutrient Restriction in Early Lactation Holstein Cows Challenged with Intra-Mammary Lipopolysaccharide[END_REF].

Oxidative stress may have occurred in our study, inducing a gradual depletion of glucose-6- phosphate and isocitrate on d 5 and 6 of restriction. Indicators of oxidative stress were not measured in the current study.

Comment
The decrease in ∑ OBCFA < C16 observed during restriction (Table 5 and Supplemental Figure 4) may be explained by reduced ruminal synthesis and incorporation of absorbed FA into milk fat. The increase in milk ∑ OBCFA C>16 content (data not shown)

during REST suggests that these FA were mobilized from adipose tissue. The majority of milk OBCFA originates from digested rumen bacteria. Branched chain FA are synthesized by elongation of carbon chains originating from branched chain amino acids (e.g. valine, leucine and isoleucine), and part of the odd chain FA are synthesized by elongation of propionate and valerate. A smaller proportion is synthesized de novo from elongation of proponiate [START_REF] Vlaeminck | Use of Odd and Branched-Chain Fatty Acids in Rumen Contents and Milk as a Potential Microbial Marker[END_REF][START_REF] Vlaeminck | Postruminal synthesis modifies the odd-and branched-chain fatty acid profile from the duodenum to milk[END_REF].

A breed effect was observed for milk 18:0 content, which was greater in HOLS than MONT cows. No breed by time interaction was observed for milk FA concentrations.

Correlation and regression analyses

Correlations are presented in Tables 6 and7 and regressions between energy balance and milk metabolites are presented in Figure 4 and Supplemental Figure 5. All variables presented were significantly correlated with energy balance, except plasma urea concentration and milk total OBCFA content. Among plasma metabolites, NEFA and glucose had the greatest absolute correlation with energy balance (r s = -0.72 and r s = 0.64, respectively; Table 6). Among milk metabolites, milk glucose and glutamate had the greatest absolute correlations with energy balance (r s > 0.60) and plasma NEFA (r s = -0.67, Table 6), which is a classic indicator of lipomobilization. Moreover, milk glucose was correlated with plasma glucose (r s = 0.61) which is also modulated by NEB [START_REF] Meijer | Free Amino Acids in Plasma and Muscle of High Yielding Dairy Cows in Early Lactation[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]Girard et al., 2019). Milk isocitrate and glucose-6-phosphate concentrations were

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

negatively correlated with energy balance (r s = -0.45 and -0.31, respectively; Table 6). Milk glucose and milk glutamate concentrations present the best regressions with energy balance (r² = 0.49 and 0.46, respectively; P < 0.001; Figure 4). Whereas milk glucose-6-phosphate and milk isocitrate present a weak regression with energy balance (r² = 0.10 and 0.26, respectively; P < 0.001). These results suggest that milk glucose and glutamate concentrations may be good indicators of NEB and metabolic status of dairy cows. 1 Intake was limited to meet 50% of NE L requirements during restriction period (REST, d 1 to 6). Cows were allowed ad libitum intake during control period (CONT, d -3 to -1) and after restriction (WEEK1, d 7 to 13; WEEK2, d 14 to 18). a, b, c, d Concern main period effects pooled across both breeds. Period LSMEANS not sharing a common superscript differ (P ≤ 0.05). y, z Breed LSMEANS not sharing a common superscript differ within the period (P ≤ 0.05) , presented when Breed × Period effect was significant. Cows were allowed ad libitum intake during control period (CONT, d -3 to -1) and after restriction (WEEK1, d 7 to 13; WEEK2, d 14 to 18). a, b, c, d Concern main period effects pooled across both breeds. Period LSMEANS not sharing a common superscript differ (P ≤ 0.05).

2 Free amino groups (NH 2 ): Estimation of free amino acid concentration [START_REF] Larsen | Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk[END_REF].

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146. 1.00 (302 )

1 Intake was limited to meet 50% of NE L requirements during restriction period (REST, d 1 to 6). Cows were allowed ad libitum intake during control period (CONT, d -3 to -1) and after restriction (WEEK1, d 7 to 13; WEEK2, d 14 to 18). 2 Free amino groups (NH 2 ): Estimation of free amino acid concentrations [START_REF] Larsen | Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk[END_REF].

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146. BHB) in midlactation Holstein (HOLS; •, solid lines) and Montbéliarde (MONT; ▲, dashed lines) cows. Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18). Values are LSM ± SEM.

Figure 3. Effects of feed restriction on milk concentrations of (A) free amino group (NH 2 ), (B) glutamate and (C) uric acid in midlactation Holstein (HOLS; •, solid lines) and Montbéliarde (MONT; ▲, dashed lines) cows. Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18).Values are LSM ± SEM.

  Figure1). Five cows of each breed were randomly allocated to a group of 10 animals that initiated the experimental protocol one day apart. Mammary and liver biopsies were performed on d 0 and d 6 for complementary studies[START_REF] Billa | Deep RNA-Seq reveals miRNome differences in mammary tissue of lactating Holstein and Montbéliarde cows[END_REF]. The experiment was conducted at the INRA Herbipôle experimental farm of 'Marcenat' (45°18'21'N, 2°50'13'E; 1100 m of altitude; https://doi.org/10.15454/1.5572318050509348E12) during April and early May.

  .9% NDF, 31.9% ADF, 11.5% CP and 5.4 MJ /kg of DM) during the two d after refeeding to provide extra fiber and decrease the risk of ruminal acidosis due to the transition. The ration was analyzed for DM content to calculate individual DMI. Energy balance was estimated according to the INRA system(INRA, 2007), in which NE L is expressed as "unité fourragère lait" (UFL; 1 UFL = 7.12 MJ), as follows: NE intake (UFL) = UFL/kg DM × DMI (kg) -E; with E corresponding to the "digestive interaction", calculated as a function of the percentage concentrate in the diet (% Conc; DM-basis) and UFL intake (i.e., UFL/kg DM × DMI (kg)), using the formula E= (0.00063 × %Conc 2 ) -(0.017 × UFLintake) + (0.002 × UFLintake 2 ). NE production (UFL) = milk yield (kg) × [0.44 + (0.0055 × ( -40 + fat content; g/kg)) + (0.0033*( -31 + protein content; g/kg))]; NE maintenance (UFL) = 0.041 × BW 0.75 × 1.1; Energy balance (UFL) = NE intake -NE maintenance -NE production. Sampling, Measurements and Chemical Analyses Milk Sampling and Analysis. Cows were milked twice daily at approximately 6:30 and 16:00. Milk yield was recorded and milk composition was determined by mid-infrared spectroscopy (LIAL, Aurillac, France) in morning and evening milk samples. Weighted milk component means were computed according to PM/AM production and composition. Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

  Figure 4. Significant time effects were observed for all FA and FA classes, except for total odd and branched chain fatty acids (∑ OBCFA). Milk FA concentrations returned to prechalenge values on d 13, except for OBCFA.Concentrations of FA with 10 to 15 carbons (∑ 10:0 to 15:0), 16:0 and ∑ OBCFA with carbon chain shorter than 16 (∑ OBCFA< C16) decreased during REST, whereas concentration of 18:0 and cis-9 18:1 and of FA with carbon chain greater than 16 (∑ > C16)

  citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

2

  Free amino groups (NH 2 ): Estimation of free amino acid concentration Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

Figure 4 .

 4 Figure 4. Relationship between energy balance and (A) milk glucose (y = 171.0x -62.1, r² = 0.49), (B) natural log of milk glutamate (y = 42.7x + 62.7, r² = 0.46), (C) natural log of milk glucose-6-phosphate (y = -9.5x -37.8, r² = 0.10) and (D) milk isocitrate (y = -458.4 + 70.7, r² = 0.26) in midlactation Holstein (HOLS; •) and Montbéliarde (MONT; ▲) cows ( P < 0.001).Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18).

Billa et al. Figure 1

 1 Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.Journal of Dairy Science, 103 (4), 3133-3146.

  Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

  .-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Supplemental Figure 1. Effects of feed restriction on milk yield (A), DMI (B), energy balance (C), fat (D), protein (E) and lactose (F) yield in midlactation Holstein (HOLS; •, solid lines) and Montbéliarde (MONT; ▲, dashed lines) cows. Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18).Values are LSM .-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. C D Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Supplemental Figure 3. Effects of feed restriction on plasma concentrations of urea (A), free amino groups (NH 2 ) (B), glutamate (C) and glutamine (D) in midlactation Holstein (HOLS; •, solid lines) and Montbéliarde (MONT; ▲, dashed lines) cows. Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18).Values are LSM .-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Supplemental Figure 4. Effects of feed restriction on fatty acid concentrations of sum of FA with 10 to 15 carbons (∑ 10:0 to 15:0) (A), 16:0 (B), 18:0 (C), cis-9 18:1 (D), sum greater than C16 carbon (Σ>C16) (E), sum odd and brain chain fatty acid (∑ OBCFA) (D), sum OBCFA with less than 16 carbon (∑ OBCFA<C16) (G) in midlactation Holstein (HOLS; •, solid lines) and Montbéliarde (MONT; ▲, dashed lines) cows. Fatty acid composition was analyzed in pooled PM/AM milk samples. Intake was limited to meet 50% of NE L requirements during restriction period (d 1 to 6). Cows were allowed ad libitum intake during control period (d -3 to -1) and after restriction (d 7 to 18). Values are LSM ± SEM. .-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

Table 4

 4

, Figures

2 and 3

). Milk BHB, glucose, glutamate, uric acid and NH 2 decreased, whereas glucose-6-phosphate and isocitrate concentrations increased during REST.

Milk glutamate, isocitrate and NH 2 concentrations returned to pre-restriction values on WEEK1. Milk BHB, glucose and uric acid returned to pre-restriction concentrations on WEEK2. Milk glutamate was greater whereas glucose-6-phosphate and isocitrate concentrations were lower on WEEK2 compared to CONT.

Table 7

 7 presents the milk FA correlations with selected variables. Milk FA composition is used as a biomarker of lipomobilization in this study, and was measured in pooled samples of PM/AM milkings, therefore it integrates modifications occurring during 24 h periods, asThe feed restriction model induced NEB and modified plasma metabolites and milk FA concentrations in midlactation cows as expected. The absence of marked breed differences for most variables may be explained by the short duration of the nutritional challenge (6 d)

	isocitrate concentrations were significantly correlated with all reported milk FA and FA
	classes (r s > 0.45 in absolute value), except for total OBCFA, further supporting potential of
	milk glucose, glutamate and isocitrate as biomarkers of NEB and metabolic status of dairy
	cows.

for the calculations of energy balance. Milk ∑ > C16, 18:0 and cis-9 18:1 were negatively correlated with energy balance (r s = -0.54 to -0.56), whereas milk ∑ 10:0 to 15:0, 16:0 and ∑ OBCFA < C16 were positively correlated with energy balance (r s = 0.39 to 0.52). These results were expected due to the known associations among NEB, lipomobilization and milk FA composition

(Gross et al., 2011b;[START_REF] Pires | Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows[END_REF]

. Accordingly, FA potentially synthesized de novo in mammary gland, such as ∑ 10:0 to 15:0 and OBCFA < C16, were positively correlated with plasma glucose, and negatively correlated with plasma NEFA and BHB. Opposite correlations were observed among these plasma metabolites and ∑ > C16, which are FA potentially mobilized from adipose. Moreover, milk glucose, glutamate and

CONCLUSIONS

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

  Chilliard, Y., A. Ferlay, R.M. Mansbridge, and M. Doreau. 2000b. Ruminant milk fat plasticity: nutritional control of saturated, polyunsaturated, trans and conjugated fatty acids. Ann. Zootech. 49:181-205. doi:10.1051/animres:2000117. Garnsworthy, P.C., L.L. Masson, A.L. Lock, and T.T. Mottram. 2006. Variation of Milk Citrate with Stage of Lactation and De Novo Fatty Acid Synthesis in Dairy Cows. J.

	genotype and its expression through time. Anim. Int. J. Anim. Biosci. 7:89-101.
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	Contreras, G.A., K. Thelen, S.E. Schmidt, C. Strieder-Barboza, C.L. Preseault, W. Raphael, Characterizing individual differences in animal responses to a nutritional challenge:
	M. Kiupel, J. Caron, and A.L. Lock. 2016. Adipose tissue remodeling in late-lactation Toward improved robustness measures. J. Dairy Sci. 99:2704-2718.
	dairy cows during feed-restriction-induced negative energy balance. J. Dairy Sci. doi:http://dx.doi.org/10.3168/jds.2015-10162.
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	doi:10.3168/jds.2010-3707.
	Holstein and Swedish-Red × Jersey/Holstein crossbred dairy cows within low-and Gross, J., H.A. van Dorland, R.M. Bruckmaier, and F.J. Schwarz. 2011b. Milk fatty acid
	medium-concentrate grassland-based systems. J. Dairy Sci. 101:7258-7273. profile related to energy balance in dairy cows. J. Dairy Res. 78:479-488.
	doi:10.3168/jds.2017-14107. doi:10.1017/S0022029911000550.
	Friggens, N.C., L. Brun-Lafleur, P. Faverdin, D. Sauvant, and O. Martin. 2013. Advances in Gross, J.J., and R.M. Bruckmaier. 2019. Review: Metabolic challenges in lactating dairy cows

Dillon, P., F. Buckley, P. O'Connor, D. Hegarty, and M. Rath. 2003. A comparison of different dairy cow breeds on a seasonal grass-based system of milk production: 1. Milk production, live weight, body condition score and DM intake. Livest. Prod. Sci. 83:21-33. doi:10.1016/S0301-6226(03)00041-1. Ferlay, A., B. Martin, S. Lerch, M. Gobert, P. Pradel, and Y. Chilliard. 2010. Effects of supplementation of maize silage diets with extruded linseed, vitamin E and plant extracts rich in polyphenols, and morning v. evening milking on milk fatty acid profiles in Holstein and Montbéliarde cows. Animal. 4:627-640. doi:10.1017/S1751731109991224. Ferris, C.P., P.J. Purcell, A.W. Gordon, T. Larsen, and M. Vestergaard. 2018. Performance of predicting nutrient partitioning in the dairy cow: recognizing the central role of Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146. Girard, C.L., N. Vanacker, V. Beaudet, M. Duplessis, and P. Lacasse. 2019. Glucose and insulin responses to an intravenous glucose tolerance test administered to feedrestricted dairy cows receiving folic acid and vitamin B12 supplements. J. Dairy Sci. 102:6226-6234. doi:10.3168/jds.2019-16298. Gross, J., H.A. van Dorland, R.M. Bruckmaier, and F.J. Schwarz. 2011a. Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation. J. Dairy Sci. 94:1820-1830. and their assessment via established and novel indicators in milk. animal 13:s75-s81. doi:10.1017/S175173111800349X. Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows. Journal of Dairy Science, 103 (4), 3133-3146.

Table 1 :

 1 Diet ingredient and nutrient composition Mineral and vitamin contained 4.5% P, 23% Ca, 4.5% Mg, 1% S, 400,000 IU/kg of vitamin A, 100,000 IU/kg of vitamin D3, 1,600 IU/kg of vitamin E, 400 IU/kg of vitamin B1, 1 g/kg of Cu, 5 g/kg of Zn, 4 g/kg of Mn, 0.1 g/kg of I, 40 mg/kg of Co and 24 mg/kg of Se; Galaphos Midi Repro granule, CCPA, Aurillac, France.

	Ingredient (% DM)	Corn silage	66.3
		Barley straw	8.0
		Corn grain	7.6
		Soybean meal	17.4
		Mineral and vitamin mix 1	0.7
	Nutrient	Net Energy (MJ/kg DM)	6.9
	composition	PDI (g/kg DM)	106
	(DM basis)
		CP (% DM)	13.8
		Fat (% DM)	2.4
		Starch (% DM)	23.3
		NDF (% DM)	34.6
		ADF (% DM)	18.7
	1

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

Table 2 .

 2 Effects of feed restriction on energy balance, DMI, milk yield, and milk component yield in midlactation Holstein (HOLS) and Montbéliarde (MONT) cows 1 .

		Period	SEM			P-value
			CONT REST	WEEK 1	WEEK 2		Breed Period	Breed × Period
	Energy	HOLS	37 a	-42 b	34 a	34 a	5	0.09	0.001 0.09
	balance (MJ/d)	MON T	23	-43	23	32	4
	DMI (kg/d)	HOLS MON T	25 a 21	9 c 8	22 b 19	24 a 23	0.8 0.8	0.01	0.001 0.08
	Milk yield	HOLS	29 a	19 d	24 c	27 b	1.2	0.01	0.001 0.25
	(kg/d)	MON T	24	16	21	24	1.1
	Fat yield	HOLS 1030 a	770 b	836 b	964 a	43	0.07	0.001 0.48
	(g/d)	MON T	896	695	739	919	38
	Protein yield	HOLS	883 a	565 c	722 b	844 a	38	0.03	0.001 0.75
	(g/d)	MON T	785	494	628	782	36
	Lactose yield	HOLS 1466 a	966 d	1197 c	1316 b	68	0.01	0.001 0.28
	(g/d)	MON T	1217	797	1007	1180	63
	Body weight (kg)	HOLS MON T	680 a 661	628 c 605	--	661 b 626	21 19	0.35	0.001 0.64
	1 Intake was limited to meet 50% of NE L requirements during restriction period (REST, d 1 to
	6). Cows were allowed ad libitum intake during control period (CONT, d -3 to -1) and after
	restriction (WEEK1, d 7 to 13; WEEK2, d 14 to 18).
	a, b, c, d Concern main period effects pooled across both breeds. Period LSMEANS not sharing a
	common superscript differ (P ≤ 0.05).
	y, z Breed LSMEANS not sharing a common superscript differ within the period (P ≤ 0.05),
	presented when Breed × Period effect was significant.

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

Table 3 .

 3 Effects of feed restriction on plasma metabolite and insulin concentrations in midlactation Holstein (HOLS) and Montbéliarde (MONT) cows 1 .

			Period SEM			P -value
									Breed
			CONT	RES T	WEEK 1	WEEK 2		Breed Period	× Perio
									d
	NEFA	HOLS 0.11 b	0.69 a 0.09 b	0.08 b	0.03	0.93	0.001 0.98
	(mM)	MON T	0.11	0.68 0.09	0.10	0.03
	BHB	HOLS 0.61 y,bc 0.64 a 0.56 ab	0.51 c	0.04	0.53	0.003 0.05
	(mM)	MON T	0.48 z	0.62 0.59	0.52	0.03
	Glucose	HOLS 3.84 b	3.50 c 4.04 ab	3.95 a	0.07	0.08	0.001 0.77
	(mM)	MON T	3.77	3.32 3.93	3.87	0.06
	Glutamat	HOLS 0.10 b	0.11 a 0.09 c	0.09 c	0.003 0.07	0.001 0.17
	e (mM)	MON T	0.09	0.10 0.09	0.09	0.003
	Glutamin	HOLS 0.16 a	0.11 c 0.16 b	0.15 ab	0.01	0.61	0.001 0.13
	e (mM)	MON T	0.16	0.10 0.13	0.15	0.01
	Urea	HOLS 4.61 a	3.50 b 3.71 b	4.63 a	0.41	0.07	0.001 0.3
	(mM)	MON T	5.36	4.61 4.10	4.99	0.38
	NH 2	HOLS 1.43 a	1.21 b 1.47 a	1.48 a	0.04	0.01	0.001 0.19
	(mM) 2	MON T	1.4	1.09 1.28	1.38	0.04
	Insulin	HOLS 20.3 b	8.5 d	25.2 a	16.1 c	1.8	0.13	0.001 0.6
	(µU/ml)	MON T	15.7	7.2	24.5	13.7	1.6

Table 4 .

 4 Effects of feed restriction on milk metabolite concentrations in midlactation Holstein (HOLS) and Montbéliarde (MONT) cows 1 .

		Period	SEM			P-value
							Breed
			CON T	RES T	WEEK 1	WEEK 2	Breed Period	× Perio
							d
	BHB (µM)	HOLS 54.7 b 40.3 c 64.5 a	46.7 b	5.2	0.93	0.001 0.55
		MONT 49.1	41.6 62.0	51.8	4.6
	Glucose (mM) HOLS 0.54 a 0.22 c 0.47 b	0.51 ab	0.03	0.15	0.001 0.17
		MONT 0.47	0.21 0.45	0.45	0.02
	Glucose-6-	HOLS 20.5 b 32.2 a 14.0 c	27.2 a	8.9	0.24	0.001 0.12
	phosphate
	(µM)	MONT 35.5	45.9 18.8	48.7	7.9
	Glutamate	HOLS 0.39 a 0.14 c 0.39 a	0.33 b	0.06	0.77	0.001 0.99
	(mM)	MONT 0.38	0.13 0.38	0.31	0.06
	Isocitrate	HOLS 0.12 c 0.17 a 0.13 c	0.13 b	0.01	0.05	0.001 0.29
	(mM)	MONT 0.14	0.19 0.13	0.16	0.01
	Uric acid	HOLS 0.15 a 0.09 c 0.13 b	0.16 a	0.01	0.003	0.001 0.54
	(mM)	MONT 0.12	0.07 0.09	0.12	0.01
	NH 2 (mM) 1	HOLS 0.81 a 0.71 b 0.84 a	0.83 a	0.04	0.31	0.001 0.72
		MONT 0.79	0.66 0.79	0.79	0.03
	1 Intake was limited to meet 50% of NE L requirements during restriction period (REST, d 1 to 6).

Table 5 .

 5 Effects of feed restriction on milk fatty acid concentrations (g/100g of fatty acids) in midlactation Holstein (HOLS) and Montbéliarde (MONT) cows 1 . Milk fatty acid composition was analyzed in pooled PM/AM samples.Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.Journal of Dairy Science, 103 (4), 3133-3146.

		Period	SEM			P-value
		CON T	RES T	WEEK 1		Breed	Period	Breed × Period
	Σ 10:0 to 15:0 2	HOLS 23.3 a 14.9 b 22.8 a	1.3	0.38	0.001	0.49
		MONT 23.9	14.5 25.3	1.1
	16:0	HOLS 35.0 b 26.6 c 35.2 a	1.1	0.89	0.001	0.18
		MONT 33.6	25.8 37.8	0.9
	18:0	HOLS 7.2 b		11.1 a 8.1 b	0.6	0.02	0.001	0.25
		MONT 6.9		10.4 5.7	0.5
	cis-9 18:1	HOLS 18.0 b 29.6 a 17.5 b	1.6	0.97	0.001	0.4
		MONT 18.7	31.2 15.1	1.4
	Σ >C16 3	HOLS 33.0 b 52.2 a 33.8 b	2.2	0.43	0.001	0.3
		MONT 33.2	53.2 28.3	1.9
	Σ OBCFA	HOLS 3.9		3.7	4.0	0.2	0.37	0.68	0.11
		MONT 3.7		3.8	3.6	0.2
	Σ OBCFA	HOLS 2.2 b		1.8 c	2.5 a	0.2	0.64	0.001	0.81
	<C16 4	MONT 2.1		1.8	2.4	0.1
	1 Intake was limited to meet 50% of NE L requirements during restriction period (REST, d 1 to 6).
	Cows were allowed ad libitum intake during control period (CONT, d -3 to -1) and after
	restriction (WEEK1, d 7 to 13; WEEK2, d 14 to 18).
	a, b, c, d Concern main period effects pooled across both breeds. Period LSMEANS not sharing a
	common superscript differ (P ≤ 0.05).
	2 Sum of FA with between 10 and 15 carbons.
	3 Sum of FA with more than 16 carbons.
	4 Sum of OBCFA with less than 16 carbons.

Table 6 .

 6 Spearman rank correlations among milk metabolites and classic indicators of metabolic status in midlactation Holstein and Montbéliarde cows 1 . Metabolite concentrations were measured in morning milk and plasma samples. Number in brackets correspond to the number of observations (*** P < 0.001; ** P < 0.01; * P < 0.05; † P < 0.1).Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C., Pires, J. (2020). Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.Journal of Dairy Science, 103 (4), 3133-3146.

	Ene	Plas	Plas	Plas	Plas	Plas	Mil	Mil		Mil	Mil	Mil	Mil	Milk
	rgy	ma	ma	ma	ma		ma		k	k		k	k	k	k	NH2 1
	bala	NE	BH	Glu	Glu	NH2	BH	Glu		Glu	Glu	Isoc	Uric
	nce	FA	B	cose	tam	1		B	cose	cose	tam	itrat	acid
					ate							-6-	ate	e
												pho
												sph
												ate
		1.00		-0.72 **	-0.17 *	0.64 **	-	*** 0.	*** 0.48 **	0.63 **	-0.31 **	0.61 **	-0.45 **	0.30 **	0.41 ***
					*			*			*	0.		51	*	*	*	*	*	*
	Energy											34
	balance	(306)		(231			(231		(231		(2		(2	(286	(285	(286)	(285)	(286)	(286	(284
				)			)			)		30		31	)	)	)	)
												)		)
				1.00			0.17 *	-0.53 **	0.	*** -	*** -0.44 **	-0.67 **	0.36 **	-0.67 **	0.48 **	-0.44 **	-0.46 ***
								*			*	45		0.	*	*	*	*	*	*
	Plasma													56
	NEFA			(249			(249		(249		(2		(2	(248	(247	(248)	(247)	(248)	(248	(246
				)			)			)		48		48	)	)	)	)
												)		)
							1.00		-0.33 **	0.	*** -	*** 0.17 ** -0.27 **	-0.09 ns -0.11 †	0.31 **	-0.31 **	-0.06 ns
											*	28		0.	*	*	*
	Plasma													21
	BHB						(249		(249		(2		(2	(248	(247	(248)	(247)	(248)	(248	(246
							)			)		48		48	)	)	)	)
												)		)
									1.00		-	*** 0.	*** 0.35 **	0.61 **	-0.25 **	0.56 **	-0.30 **	0.31 **	0.46 ***
												0.		46	*	*	*	*	*	*
	Plasma											31
	glucose								(249		(2		(2	(248	(247	(248)	(247)	(248)	(248	(246
										)		48		48	)	)	)	)
												)		)
												1.		-	** -0.14	* -0.36 **	0.19 ** -0.39 **	0.33 **	-0.22 **	-0.25 ***
												00		0.	*	*	*	*
	Plasma													17
	glutamate											(2		(2	(247	(246	(247)	(246)	(247)	(247	(245
												48		48	)	)	)	)
												)		)
	Plasma													1.	0.26 **	0.50 **	-0.20 **	0.59 **	-0.34 **	0.50 **	0.44 ***
	NH2 1													00	*	*	*	*	*

Table 7 .

 7 Spearman rank correlations among milk fatty acids and milk metabolites and classic indicators of metabolic status in midlactation Holstein and Montbéliarde cows 1 . Milk fatty acid composition was analyzed in pooled PM/AM milk samples, and metabolite concentrations were measured in morning milk and plasma samples. Number in brackets corresponds to the number of observations. (*** P < 0.001; ** P < 0.01; * P < 0.05; † P < 0.1).

	Σ 10:0 to 15:0 2	16:0	18:0	Cis-9 18:1	Σ >C16 3	Σ <C16 4 OBCFA					Σ OBCFA
	Energy balance			***		***	-0.56 ***	-0.54 ***	-0.55 ***	0.39 ***	-0.09 ns
							(162)		(162)		(162)		(162)	(162)
	Plasma NEFA			***		***	0.61 ***	0.68 ***	0.69 ***	-0.58 ***	-0.09 ns
							(143)		(143)		(143)		(143)	(143)
	Plasma BHB			***		***	0.37 ***	0.43 ***	0.44 ***	-0.48 ***	-0.27 **
							(143)		(143)		(143)		(143)	(143)
	Plasma glucose			***		***	-0.36 ***	-0.45 ***	-0.44 ***	0.28 ***	-0.07 ns
							(143)		(143)		(143)		(143)	(143)
	Plasma glutamate			***		***	0.52 ***	0.52 ***	0.55 ***	-0.55 ***	-0.13 ns
							(143)		(143)		(143)		(143)	(143)
	Plasma NH2 5			***		***	-0.41 ***	-0.55 ***	-0.52 ***	0.51 ***	0.18	*
							(143)		(143)		(143)		(143)	(143)
	Milk BHB			***		***	-0.23	**	-0.38 ***	-0.35 ***	0.15	ns	-0.23 **
							(161)		(161)		(161)		(161)	(161)
	Milk glucose			***		***	-0.57 ***	-0.66 ***	-0.66 ***	0.45 ***	-0.06 ns
							(160)		(160)		(160)		(160)	(160)
	Milk glucose-6-phosphate		ns		ns	0.25	**	0.09	ns	0.15	†	-0.05	ns	0.13 ns
							(161)		(161)		(161)		(161)	(161)
	Milk glutamate			***		***	-0.68 ***	-0.70 ***	-0.71 ***	0.57 ***	0.03 ns
							(160)		(160)		(160)		(160)	(160)
	Milk isocitrate			***		***	0.61 ***	0.56 ***	0.61 ***	-0.47 ***	0.04 ns
							(161)		(161)		(161)		(161)	(161)
	Milk uric acid			***		***	-0.28 ***	-0.48 ***	-0.43 ***	0.53 ***	0.40 ***
							(161)		(161)		(161)		(161)	(161)
	Milk NH2 5			***		***	-0.43 ***	-0.53 ***	-0.52 ***	0.31 ***	-0.16	*
							(160)		(160)		(160)		(160)	(160)
	Σ 10:0 to 15:0 2					***	-0.85 ***	-0.95 ***	-0.96 ***	0.89 ***	0.28 ***
							(162)		(162)		(162)		(162)	(162)
	16:0						-0.78 ***	-0.91 ***	-0.92 ***	0.70 ***	0.04 ns
							(162)		(162)		(162)		(162)	(162)
	18:0						1.00		0.78 ***	0.86 ***	-0.74 ***	-0.10 ns

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C.,

Pires, J. (2020)

. Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146. 2 Sum of FA with between 10 and 15 carbons. 3 Sum of FA with more than 16 carbons. 4 Sum of OBCFA with less than 16 carbons.

5

Free amino groups (NH 2 ): Estimation of amino acid concentration

[START_REF] Larsen | Enzymatic-fluorometric analyses for glutamine, glutamate and free amino groups in protein-free plasma and milk[END_REF]

.

Comment citer ce document : Billa, P.-A., Faulconnier, Y., Larsen, T., Leroux, C.,

Pires, J. (2020)

. Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

Journal of Dairy Science, 103 (4), 3133-3146.

ACKNOWLEDGEMENTS

The authors thank the staff at Herbipôle INRA (UE1414, Marcenat, France) for animal care and sampling; A. Delavaud, S. Bes, D. Chadeyron, E. Tixier and M. Tourret (INRA, UMR1213, Saint-Genès-Champanelle, France) for sample collection and laboratory analyses; J. Clausen and C. Berthelsen (Aarhus University, Tjele, Danemark) for milk metabolite analyses. This research was partially funded by FEDER and Région Auvergne S3 project nr. 23000794, and Compte d'affection Spécial au Développement Agricole et Rural (CASDAR; Paris, France) project nr. 00001908 (Biomarq'lait).