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International audience; In dairy cattle, negative energy balance (NEB) can occur in early lactation or during feed restriction. It can negatively impact animal health and milk production. The aim of this study was to identify NEB biomarkers in milk. We hypothesized that mammary gland metabolism, which is impacted by feeding, can be evaluated through the measure of selected milk metabolite concentrations. Thirty Holstein dairy cows were studied both in early lactation (21 DIM), and in mid-lactation (173 DIM), during a 5-week feed restriction experiment. The feed restriction protocol aimed to reduce milk production by 20%, and to induce body reserves mobilization, i.e., a NEB. Thus, the diet was diluted with wheat hay to reduce the energy density and the diet digestibility by about 10%. Milk samples were collected at 21 DIM, before and during the first week of feed restriction. Data were analyzed by mixed models that included time, parity, and their interaction as fixed effects, and cow as random effect. NEB was observed in early lactation (−5.3 Mcal/d) and several milk metabolite concentrations varied: galactose (−52%, P < 0.001), glucose (−25%, P < 0.001) and glucose-6-phosphate (+291% P < 0.001) compared with during mid-lactation. The accumulation of glucose-6-phosphate may be due to a blockage of the pentose phosphate and isocitrate dehydrogenase pathways. Feed restriction at mid-lactation did not lead to a NEB. Milk production decreased by 10% (P < 0.001). Several milk metabolite concentrations also decreased as early as the first week of feed restriction: galactose (−24%, P < 0.001), glucose (−17%, P < 0.001), glucose-6-phosphate (−9%, P < 0.005), glutamate (−19%, P < 0.001) and isocitrate (−13%, P < 0.001). These variations suggest a global slowdown of mammary epithelial cell metabolism. The feed restriction model used in this study was not as severe as expected because feed intake remained high despite the reduced digestibility. Nonetheless, milk metabolite concentrations were still affected, showing their sensibility to energy intake variations. This study shows that milk metabolite concentration vary both with NEB at early lactation and feed level. Glucose-6-phosphate concentration varies differently between conditions suggesting that its increase may reflect a NEB state. These results need to be validated, and suggest new ways of using milk metabolites as markers of dairy cow energy state

La construction des phénotypes des animaux de rente pendant les premières phases de la vie post-natale est une étape cruciale pour une expression optimale de leur phénotype au stade adulte (multiperformance agronomique). La récente mise en évidence de cellules souches et progénitrices, et de vésicules extracellulaires (VEs), dans le lait de plusieurs espèces (humaine et murine) pose la question du contenu et du devenir de ces éléments cellulaires chez la progéniture, de leurs rôles dans le développement des tissus (participation directe, régulation) et impacts sur la santé tout au long de la vie (robustesse, bien-être). Ce projet, cofinancé par les départements PHASE et GA, participe à la compréhension des mécanismes d’élaboration des phénotypes des animaux de rente en identifiant les éléments cellulaires (cellules totales, cellules souches et VEs) dans les sécrétions lactées (colostrum et lait) de truies, vaches et chèvres laitières. L'apport de ces éléments à la progéniture pouvant avoir un rôle physiologique au début de la vie post-natale, notre protocole expérimental intègre une analyse cinétique grâce à un échantillonnage de «lait» dans les premières heures (0, 3, 6, jusqu’à 24h) après la mise-bas (colostrum), puis au cours des premiers jours de la lactation (jusqu’à 7 jours pour chèvres et vaches, 18 jours pour les truies), sur 6 femelles multipares (IE production laitière et UE3P). Les cellules des sécrétions lactées ont été analysées en cytométrie en flux en utilisant des marqueurs moléculaires spécifiques de typage (combinaison d’anticorps dirigés contre des protéines de surface) ciblant les cellules souches mammaires, les cellules souches mésenchymateuses et les cellules souches hématopoïétiques. Les premiers résultats montrent que, chez la vache et la chèvre, des cellules viables se retrouvent en nombre dans le colostrum dès la mise-bas. Elles diminuent progressivement pendant les 12 heures qui suivent, puis régulièrement tout au long de la semaine de suivi cinétique. Chez la truie, en revanche, le nombre de cellules viables est maximal un jour après la mise-bas. Pour les trois espèces, l’analyse en cytométrie montre que les cellules souches mammaires (0,5% à 1,5% des cellules viables selon l’espèce) sont retrouvées principalement dans le colostrum des premières heures après la mise-bas, devenant quasi inexistantes les jours d'après (< 0,2%). Fait intéressant, une population cellulaire exprimant CD24, une protéine exprimée par certaines cellules souches hématopoïétiques et les lymphocytes B, est fortement présente chez les trois espèces pendant les six premières heures, puis évolue à la baisse au cours de la lactation. Cette population cellulaire mérite une caractérisation plus poussée. A ce jour, le retraitement a posteriori des données de cytométrie des cellules souches mésenchymateuses et hématopoïétiques se poursuit. Cela nous permettra de compléter notre connaissance des différents types de cellules évoluant dans les sécrétions lactées au cours des premiers jours de lactation. Concernant les VEs, nous avons développé la purification par ultracentrifugation différentielle et par chromatographie à exclusion de taille des petites et grandes VEs ainsi que leur analyse physico-chimique par « nanoparticle tracking analysis » et western blot. Chez le bovin, les VEs sont retrouvées dans le lait par centaines de milliard par millilitre de lait écrémé. Cette abondance sera évaluée dans les différentes espèces au cours des phases de lactation et la proportion des sous-classes de VEs sera déterminée suite à leur purification par immunocapture (criblage d’anticorps en cours). En conclusion, le dépouillement partiel des analyses cytométriques et des VEs met notamment en évidence l'existence de cellules souches mammaires dans les sécrétions lactées et suggère la présence de populations cellulaires et vésiculaires spécifiques, potentiellement avantageuses sur le plan physiologique, au(x) premier(s) jour(s) de lactation. Chez les ruminants, nos résultats pourraient venir appuyer des pratiques d’élevage permettant d’améliorer le bien-être et la santé des petits, et la robustesse des animaux adultes. Pour l’espèce porcine, nos résultats pourraient confirmer la nécessité pour les porcelets de consommer le colostrum de leur mère biologique. Enfin, la poursuite de la comparaison des 3 espèces sera déterminante pour établir si ce phénomène de transfert cellulaire et vésiculaire est générique ou spécifique à l’espèce.

En élevage laitier, des stress alimentaires liés à la physiologie de l’animal au début de la lactation ou à des pénuries alimentaires peuvent conduire à un déficit énergétique. Le déficit énergétique impacte directement la composition et la production du lait et parfois même la santé de l’animal. Des outils permettant le suivi du statut énergétique des vaches laitières sont donc essentiels. L’objectif de cette étude était d’observer l’effet de protocoles de restriction alimentaire sur les protéomes et miRNomes du lait afin d’identifier des biomarqueurs potentiels du déficit énergétique chez les vaches. Deux essais de restriction alimentaire ont été menés au milieu de la lactation pour induire un déficit énergétique: une restriction de courte durée mais intense (CI) et une restriction de longue durée mais d’intensité modérée (LM). L’essai CI a impliqué 8 vaches Holstein suivies avant pendant et après restriction. L’essai LM a impliqué 19 vaches Holstein qui ont été alimentées soit ad libitum, dans des conditions témoin (n=9) soit en restriction (n=10). La restriction alimentaire a duré 6 et 29 jours avec un apport en matière sèche réduit de 64 et 20 % de l’apport ad libitum, et a induit un déficit énergétique de -42 et -21 MJ/j, respectivement pour les essais CI et LM. Pour l’étude des protéomes et miRNomes, les échantillons de lait utilisés ont été prélevés avant et 5 jours après le début des périodes de restriction. Les microARN ont été extraits du lait entier, des globules gras, des cellules épithéliales mammaires exfoliées dans le lait et des vésicules extracellulaires avant d’être séquencés. Les protéomes ont été obtenus à partir de lait écrémé par LC-MS-MS. Parmi les 2896 microARN détectés dans le lait, l’abondance de 159 microARN a été affectée par la restriction, principalement dans les vésicules extracellulaires (n=83) et le lait entier (n=85), un peu dans les globules adipeux (n=6), mais pas dans les cellules épithéliales mammaires. Parmi ces 159 microARN, 15 variaient dans deux compartiments différents. 345 protéines ont été identifiées dans le lait, dont 166 pour lesquelles l’abondance a été affectée par la restriction, principalement dans l’essai CI (n=164) et très peu dans l’essai LM (n=8). L’intégration, par multi-level sparse PLS-DA et DIABOLO, de ces données omics avec les données de métabolites du lait, de taux et de productions précédemment obtenues a permis l’identification de 14 marqueurs prédictif du régime alimentaire, communs aux deux essais, qui pourront servir à l’élaboration d’un panel de biomarqueurs du déficit énergétique, utilisable en élevage. Ces expériences ont montré que la restriction alimentaire affecte l’abondance des protéines et des microARN dans le lait. Ces résultats seront vérifiés sur un autre essai en début de lactation et lors d’une restriction alimentaire sans déficit énergétique en milieu de lactation. Ces résultats préliminaires sont prometteurs pour la recherche de biomarqueurs non invasifs du bilan énergétique négatif dans le lait.

International audience

International audience; Methionine (Met) supplementation increases milk, protein and fat yields in cow. We investigated whether this could be partly explained by an increasingflow of milk components in the secretory pathways of mammary epithelial cells. Multiparous Alpine goats at mid lactation (n = 48), grouped by levels of expression of the CSN1S1, were assigned to 4 treatments in a randomized complete block design. Goats were fed a fixed amount of hay and a low (LE, 1.47 Mcal/kg DM) or adequate (AE, 1.54 Mcal/kg DM) energy concentrate combined with 2 levels of metabolizable Met: unbalanced vs. balanced using isopropyl ester of 2-hydroxy-4-methylthio butanoic acid (HMBi 0.24% concentrate DM) to cover 100% of Met requirement, based on cow requirement (INRA, 2007). Treatments were: LE, LEMet (LE, balanced Met), AE and AEMet (AE, balanced Met) for 5 weeks. Goats (23) were slaughtered and mammary tissue was processed for Western blotting using secretory compartment specific markers of membrane traffic. Milk protein yield (P = 0.01) and casein content (P = 0.01) increased in goats fed the Met balanced diets. The amount of the endoplasmic reticulum (ER) markers, Cnx and ERLIN2, decreased (20%, P ≤ 0.05) in goats fed the LE diet. Met balanced diets had the opposite effect on both markers (20%, P ≤ 0.05), as well as on protein disulfide isomerase (45%, P ≤ 0.05). These observations are in agreement with a positive effect of Met on the activity of the ER, the site where protein and lipid are synthesized. On the other hand, a specific marker of the exit site of the Golgi apparatus and secretory vesicles formation (AP1) decreased with the LE diet (25%, P ≤ 0.05) and its highest level was found in goats fed Met balanced diet at AE supply. The higher ßCOP (P = 0.01), a marker of intra Golgi transport, clearly reflected a decrease in membrane transport of LE diets. These data strongly suggest that energy level has a direct impact on membrane traffic in the secretory pathway of mammary epithelial cell while Met improve ER activity and has the tendency to further promote intracellular transport of milk components and, ultimately, their secretion.

International audience; Methionine supplementation is known to modulate milk and protein yields in dairy ruminants. However, the mechanism involved in this phenomenon is still unknown. The cellular and molecular responses in the mammary tissue to methionine (Met) supplementation through HMBi (MetaSmart, Adisseo) at low (LE) and adequate levels (AE) of NEL were investigated according to a 2 × 2 factorial arrangement on 48 multiparous Alpine goats at mid lactation, assigned to a randomized block design in 4 groups during 5 weeks. Each goat was genotyped for CSN1S1 gene to balance each group according to the high or low genotypes for CSN1S1 gene. A fixed amount of hay was distributed per group (1.17 Mcal/kg DM) and a fixed amount of concentrates was distributed individually (1.72 vs. 1.83 Mcal/kg DM in LE and AE, respectively). Metabolizable methionine were 1.95 vs. 2.45% of metabolizable protein in unbalanced (LE and AE) vs. balanced (LEMET and AEMET) diets, respectively. Goats (23) were slaughtered after 5 weeks of treatment. Mammary tissues were collected for the analyses of milk protein mRNA levels by real time RT-PCR and apoptosis by immunohistochemistry using TUNEL assay. A variance analysis was performed with genotype, E, Met and E x Met, E x genotype, Met x genotype interactions as fixed effects and for milk, pre-treatment data yields was used as a covariate. Met supplementation increased milk protein yield (+6.5 g/d; P = 0.03). The treatments did no modify LALBA whereas an interaction E x Met (P = 0.001) were observed for both CSN3 and CSN1S1 mRNA levels showing 2-fold increases with Met supplementation in goats fed AE diet. The supplementation of Met reduced by 51% apoptosis rates in the mammary tissue (P < 0.01). Apoptosis rate was correlated with milk yield (−0.48; P < 0.05). These results suggest that Met increased casein gene expression at AE level and reduced cell death which could partly explain the positive effect of Met supplementation on milk synthesis in the goat mammary tissue.

International audience

International audience; In cattle breeding and farming, epigenetic mechanisms are regarded as a potential source of phenotypic variance unaccounted by genetic selection and could be used to better control the environmental impact on the genetic potential of animals. In this review, we briefly describe epigenetic processes, and address, from research studies mainly conducted at INRAE, the major role of epigenetic factors in the elaboration of phenotype in cattle. Focus is given to environmental factors shaping the epigenome, leading to variable effects on fertility, development, health and dairy production. The potential for using epigenetic markers in precision farming and as a complementary approach to genomic selection is also discussed.; En élevage bovin, une part importante de la variance phénotypique échappe à la sélection génétique, car l’expression des gènes quicontrôlent les caractères d’intérêt est déterminée par des facteurs à la fois génétiques et environnementaux. Une meilleure maîtrise desmécanismes épigénétiques, qui traduisent l’impact environnemental au niveau du génome, permettrait ainsi de mieux contrôler le phénotypedes animaux. Dans cette revue, nous décrivons brièvement les processus épigénétiques, puis nous évoquons, à travers plusieursexemples principalement issus de travaux menés à INRAE, le rôle majeur de l’épigénome dans la construction du phénotype chez le bovin.Nous mentionnons en particulier la façon dont certains facteurs environnementaux modifient les marques épigénétiques, ce qui entraînedes effets variables sur les caractères de fertilité, de développement, de santé et de production laitière. Le potentiel d’utilisation de marqueursépigénétiques dans le cadre d’un élevage de précision et en complément des analyses d’association entre génotype et phénotypemenées en sélection génomique est également discuté.

International audience; Methionine (Met) or energy supplementation increase milk protein and fat yields in cow. Here we investigated whether this could be explained by increasing flow of milk components in the secretory pathways of mammary epithelial cells. Multiparous Alpine goats (mid lactation, n=47), grouped by levels of expression of CSN1S1, were assigned to 4 treatments in a randomised complete block design. Treatments were: LE (Low Energy), LEMet (LE, balanced Met), AE (Adequate Energy) and AEMet (AE, balanced Met) for 5 weeks. Diets consisted in a fixed amount of hay per group with individual amount of energy concentrates (LE vs AE: 1.47 vs 1.54 Mcal/kg DM). Isopropyl ester of 2-hydroxy-4-methylthio butanoic acid (HMBi 0.24% concentrate DM) was incorporated in the LEMet and AEMet concentrates based on 100% cow Met requirement. Goats (n=23) were slaughtered and mammary tissue was processed for Western blotting using secretory compartment specific markers. In goats fed Met balanced diets, milk protein yield and casein content significantly increased (P=0.01), as was previously observed in cow. The amount of the endoplasmic reticulum (ER) markers Calnexin and ERLIN2 decreased (20%, P≤0.05) in goats fed the LE diet. Met balanced diets had the opposite effect on both markers (20%, P≤0.05) and on protein disulphide isomerase (45%, P≤0.05). These observations are in agreement with a positive effect of Met on the activity of the ER, the site where milk specific proteins and lipids are synthesised. On the other hand, a specific marker of the exit site of the Golgi apparatus and secretory vesicles formation (AP1) decreased with the LE diet (25%, P≤0.05) and its highest level was found in goats fed Met balanced diet at AE supply. Concerning ßCOP, a marker of intra Golgi transport, its variation clearly evoked a decrease in membrane transport at LE diets. These data show that energy level has a direct impact on membrane traffic in the secretory pathway of mammary epithelial cell while suggesting that Met improves ER activity and has the tendency to further promote intracellular transport of milk components and, ultimately, their secretion.

International audience

Les stress alimentaires peuvent conduire à un état, nommé déficit énergétique, lors duquel l’énergie apportée par la ration est inférieure aux besoins énergétiques. En élevage laitier ce déficit, qu’il soit physiologique, comme en début de lactation, ou environnemental, en cas de pénurie alimentaire, impacte directement la composition et la production du lait et parfois même la santé de l’animal. Au vu de ces connaissances et sachant que l’alimentation des animaux représente le principal coût de production du lait, l’étude des répercussions d’un stress alimentaire sur la lactation est essentielle. Deux stratégies expérimentales peuvent être choisies pour reproduire ce stress : réduire la quantité de nourriture disponible ou diminuer la qualité énergétique de la ration.Une restriction alimentaire engendrant un déficit énergétique a des répercussions sur l’ensemble du métabolisme de l’animal, entraînant notamment une mobilisation des réserves corporelles, un amaigrissement et une altération de la lactation. La diminution des apports alimentaires a des effets sur la régulation hormonale : une baisse de l’insulinémie peut être observée et la concentration sanguine en hormone de croissance augmente, favorisant ainsi la lipolyse dans le tissu adipeux et la néoglucogenèse dans le foie. Par ailleurs la synthèse hépatique d’insulin-like growth factor-1 diminue, réduisant entre autres le flux sanguin et le métabolisme dans la glande mammaire. L’augmentation de la lipolyse mène à une production plus importante d’acides gras non estérifiés qui, lors d’une baisse de glycémie, peuvent être oxydés dans le foie en corps cétoniques, entraînant un risque de cétose. La diminution du flux sanguin mammaire conduit à une diminution des apports en oxygène et nutriments, et est accompagnée d’une modification de l’activité des cellules épithéliales mammaires et d’un remodelage du tissu mammaire. La production laitière est systématiquement diminuée et la composition du lait est impactée :diminution du taux protéique, principalement liée à une baisse de concentration des caséines, augmentation fréquente du taux butyreux, notamment liée à une plus forte concentration en acides gras longs issus de la lipomobilisation, et diminution de la concentration en lactose. La concentration en cellules somatiques est généralement augmentée lors d’un stress alimentaire, accompagnée d’une augmentation de l’exfoliation des cellules épithéliales mammaires, ce qui pourrait indiquer une perte d’intégrité de l’épithélium mammaire.Les effets du stress alimentaire dépendent fortement de la durée et de l’intensité de la restriction ainsi que du stade de lactation des animaux, de plus, la plupart de ces facteurs reviennent rapidement à la normale suite au retour à un régime non restreint. Cependant, afin d’éviter la perte de production et les troubles métaboliques liés au stress alimentaire, il convient d’identifier des biomarqueurs de restriction alimentaire fiables et précoces et de tendre vers une alimentation de précision et une gestion individualisée des animaux.

National audience; La restriction alimentaire induit une période de perturbation de l’homéostasie de l’animal pouvant provoquer un déficit énergétique et des troubles de santé chez la vache laitière. Dans le projet casdar BioMarq’Lait, nous recherchons des biomarqueurs non invasifs (dans le lait) dans le but de fournir aux éleveurs un moyen d’identifier précocement les individus nécessitant une attention particulière, afin d’adapter leur alimentation au cas par cas, pour éviter l’apparition de troubles métaboliques et de bien-être. Le lait contient des cellules épithéliales mammaires (CEM) progressivement exfoliées de l'épithélium au cours de la lactation. Le taux d’exfoliation des CEM, correspondant au nombre de CEM exfoliées dans le lait par jour, pourrait représenter un biomarqueur indicateur de l’adaptation des vaches laitières à la restriction alimentaire. Pour tester cette hypothèse nous avons mis en place 2 essais sur vaches laitières. Dans un premier essai, dix-neuf vaches laitières Holstein à 77 ± 5 j de lactation et produisant 40 ± 0.7 kg de lait/j ont été nourries pendant 4 semaines à hauteur de 100 % (groupe témoin, n=9) ou de 80 % (groupe restreint, n=10), de leur matière sèche ingérée ad libitum pendant les deux semaines précédant le début de l’expérimentation. Du lait a été collecté après 5, 9 et 27 jours de restriction afin de purifier les CEM par une méthode d’immuno-séparation magnétique et de déterminer leur concentration. Le taux d’exfoliation des CEM a ensuite été obtenu en multipliant la concentration en CEM dans le lait par le volume de lait produit par jour. La perte de production laitière de 3,5 kg de lait par j (- 9 %) chez les vaches restreintes par rapport aux vaches témoins s’est accompagnée d’une augmentation de 65 % du taux d’exfoliation des CEM dans le lait (P

In dairy cows, a long-day photoperiod (LDP) is known to increase milk yield. This increased milk yield is associated with greater concentrations of prolactin (PRL). In contrast, short-day photoperiod (SDP) was shown to increase the rate of mammary epithelial cell (MEC) exfoliation as also observed after an experimentally-induced inhibition of PRL. The aim of this study was to investigate if PRL mediates, at least in part, the effect of photoperiod on the MEC exfoliation process. Eight Holstein dairy cows producing 42 ± 3.7 kg of milk per day were submitted to 4 treatments according to a 4 × 4 latin square design: 2 day lengths (8 h of light/d for SDP or 16 h of light/d for LDP) for 20 d and to a pharmacological treatment (a single i.m. administration of 5.6 mg of cabergoline, an inhibitor of PRL secretion, or vehicle) on d10. Blood samples were collected before, during, and after milking to measure plasma concentration of PRL. Milk samples were collected for milk MEC purification using an immunomagnetic method. Milk protein mRNA levels were analysed in milk purified MEC by qRT-PCR to evaluate MEC metabolic activity. Data were analysed by analysis of variance using SAS and the effect of photoperiod, cabergoline treatment, and the interaction between photoperiod and cabergoline treatment were tested. Photoperiod had no effect (P>0.05) on PRL secretion, milk yield, MEC exfoliation rate and metabolic activity. It was therefore impossible to draw conclusion regarding the potential role of PRL as a mediator of the effect of photoperiod. Cabergoline induced a decrease in PRL secretion, by decreasing both basal concentration and the surge at milking, and induced a decrease in milk yield (P

Once-daily milking (ODM) is known to induce udder inflammation, and could lead to a higher mastitis incidence due to increased milk leakage and extended milk stasis that could promote penetration and multiplication of pathogens. As a fact, ODM increases milk somatic cell counts (SCC) with or without an increased number of clinical mastitis. However, bacteriological analyses are often lacking to describe udder inflammation. Our study aimed to describe the effect of initial health status of the udder on the inflammatory response observed when implementing ODM. The trial used 63 cross-bred Holstein × Normande dairy cows at 78±5 days in milk and comprised a 1-wk control period (cows milked twice daily) and a 3-wk period of ODM (d1, 1st d of ODM). The udder and animal inflammatory status were assessed by measuring SCC 4 d/wk in whole-udder milk, interleukins (IL) 6 and 8 in quarter milk, and haptoglobin in plasma on d0, 1, 2, 3 and 14. The infectious status of quarters was determined at d0, 3 and 14 by milk bacteriological analysis and identification of minor and major pathogens by 16S DNA sequencing. Four initial infectious and inflammatory udder’s classes (57 of the 63 cows) were determined: no infected and inflamed quarters (n=13), no infected quarters but at least one inflamed quarter (n=15), at least one quarter inflamed and infected with a minor pathogen (n=14), and at least one quarter inflamed and infected with a major pathogen (n=15). Milk yield losses due to ODM were higher for cows with inflamed udders compared to non-inflamed udders (25-27% vs 17%, respectively). Implementing ODM increased SCC and IL8. The increase in IL8 was higher for cows infected with a major pathogen. Cows with no infected but inflamed quarters contrasted by showing only a slight increase in SCC with no change in IL8 and haptoglobin. Inflammatory and milk yield responses to ODM thus varied according to the initial udder health status.

In dairy cattle, underfeeding is known to influence cow’s homeostasis by inducing body reserve mobilization and by reducing milk production. However, its impact on cow’s immune responses is less known. This study aimed to examine blood cell responsiveness to ex-vivo bacterial challenges in cows fed a standard or a low energy diet. Fifty-four Prim’Holstein at mid lactation received a standard diet (based on corn silage) from calving to 201 days in milk, and then were fed a lower energy diet (14.5% concentrates replaced by 11.5% straw and 3% dehydrated alfalfa) for 5 wk. Milk yield was recorded daily. Blood samples collected at wk -2, -1 and 1, 2 and 5 after diet change were incubated ex-vivo with heat-killed Escherichia coli or Staphylococcus aureus bacteria, or saline as a control for 23h at 38.5°C. Plasma Interleukin 8 (CXCL8), an indicator of the inflammatory response, was then measured by ELISA. Plasma haptoglobin, glucose, urea and NEFA concentrations were determined. Data were analysed using a mixed model of variance testing the effect of week with a cow week random effect. The decrease in energy diet content was associated with a loss of 3 kg/d milk yield (P < 0.001) and reduced glucose and urea plasma concentrations (P< 0.01) while plasma NEFA and haptoglobin concentrations did not vary. A higher production of CXCL8 was observed after E. coli and S. aureus stimulations during the underfeeding period (P

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In ruminants, the early phase of drying-off is a period of mammary gland involution that is marked by the cessation of prolactin (PRL) release. The speed at which the bovine mammary gland involutes following the abrupt cessation of lactation is directly related to the risk of new intramammary infections.[br/] Objectives[br/] Our aim was to assess the effect of PRL inhibition by cabergoline on the speed of the mammary gland involution, through analysis of the changes of mammary secretion composition.[br/] Materials and methods[br/] Fourteen Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n=7) (Velactis ®, Ceva Sante Animale) or placebo (n=7) at the first day of dryingoff (D0). Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for milk fat, lactose, true protein, alpha-lactalbumin and SCC analysis. Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for RNA extraction and RT-PCR analyses.[br/] Results[br/] As expected, SCC progressively increased whereas lactose content decreased in mammary secretions after drying-off (P < 0.001). The increase in SCC was 2.4 fold higher in cabergoline treated cows than in control cows (P < 0.01). The decrease of lactose content in mammary secretions progressively decreased during involution and was associated with paralleled change in GLUT-1 mRNA level coding the main glucose transporter in the udder. These decreases were faster in cabergoline treated cows compared to controls with lower lactose content in cabergoline treated cows already by D1 than in controls (P < 0.05) and significant decrease in GLUT-1 mRNA levels at D1 and D8 respectively for cabergoline and control treatments compared to D-6 (P ≤ 0.05). Cabergoline treatment tended to increase fat content at D3 after drying-off (P < 0.10). No significant effects of cabergoline treatment were observed both in true protein and in alpha-lactalbumin contents in mammary secretions or in alphalactalbumin and kappa-casein mRNA levels in mammary tissues.[br/] Conclusions[br/] The changes in lactose, SCC and fat in mammary secretions and GLUT-1 mRNA level in the udder, indicate that cabergoline treatment was efficient to hasten the mammary gland involution without affecting milk protein synthesis in the mammary tissue. Cabergoline could facilitate dairy management at the time of dry-off.

ObjectivesIn ruminants, the early phase of drying-off is a period of intense mammary gland involution that is due, in part, to dramatic decline prolactin (PRL) release. The speed at which the bovine mammary gland involutes following the abrupt cessation of lactation is also directly related to the risk of new intramammary infections. Thus, strategies to hasten involution following dry-off could have implications in preventing mastitis and optimizing mammary tissue regenerative processes.Materials and methodsTo assess the effect of prolactin inhibition by cabergoline on mammary gland involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n=7) or placebo (n=7) within 4 hours after the last milking before the drying off at the day of drying-off (D0). Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for lactoferrin and zymography analyses to detect the activity of enzymes such as MMP, matrix metalloproteinases involved in the remodelling of mammary tissue during involution. Mammary epithelial cells (MEC) were also purified from mammary secretions after centrifugation andimmunocytochemical binding in order to evaluate the MEC exfoliation. Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for lactoferrin immunochemistry and zymography analyses.ResultsThe activity of MMP9 increased after drying-off in mammary secretions (P < 0.001). Cabergoline increased the activity of MMP9 (1.7 fold, P < 0.05) in mammary secretions and MMP-2 in mammary tissue after drying-off (1.4 fold, P ≤ 0.01). MEC concentration progressively increased in mammary secretions after drying-off (P < 0.01). Cabergoline induced an increase in MEC concentration (P =0.04). Lactoferrin content progressively increased in mammary secretions during involution. The rise of lactoferrin content in mammary secretions was significant starting at D4 in the cabergoline treated cows (P ≤0.05) whereas it only happened at D8 in controls (P < 0.05). Overall, cabergoline treatment increased lactoferrin content of mammary secretions (P = 0.10). The total lactoferrin immunostaining in the mammary tissue increased after drying-off (P < 0.05). Compared with during lactation, this increase was observed at D1 and D8, respectively for cabergoline treated cows and control cows (P

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Objectives[br/] In ruminants, the early phase of drying-off is a period of intense mammary gland involution that is due, in part, to dramatic decline prolactin (PRL) release. The speed at which the bovine mammary gland involutes following the abrupt cessation of lactation is also directly related to the risk of new intramammary infections. Thus, strategies to hasten involution following dry-off could have implications in preventing mastitis and optimizing mammary tissue regenerative processes.[br/] Materials and methods[br/] To assess the effect of prolactin inhibition by cabergoline on mammary gland involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n=7) or placebo (n=7) within 4 hours after the last milking before the drying off at the day of drying-off (D0). Mammary secretion samples were collected using a teat-cannula once during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off. The mammary secretion samples were used for lactoferrin and zymography analyses to detect the activity of enzymes such as MMP, matrix metalloproteinases involved in the remodelling of mammary tissue during involution. Mammary epithelial cells (MEC) were also purified from mammary secretions after centrifugation and immunocytochemical binding in order to evaluate the MEC exfoliation. Mammary biopsy samples were collected one week before drying-off (D-6), at D1 and at D8 and used for lactoferrin immunochemistry and zymography analyses.[br/] Results[br/] The activity of MMP9 increased after drying-off in mammary secretions (P < 0.001). Cabergoline increased the activity of MMP9 (1.7 fold, P < 0.05) in mammary secretions and MMP-2 in mammary tissue after drying-off (1.4 fold, P ≤ 0.01). MEC concentration progressively increased in mammary secretions after drying-off (P < 0.01). Cabergoline induced an increase in MEC concentration (P =0.04). Lactoferrin content progressively increased in mammary secretions during involution. The rise of lactoferrin content in mammary secretions was significant starting at D4 in the cabergoline treated cows (P ≤0.05) whereas it only happened at D8 in controls (P < 0.05). Overall, cabergoline treatment increased lactoferrin content of mammary secretions (P = 0.10). The total lactoferrin immunostaining in the mammary tissue increased after drying-off (P < 0.05). Compared with during lactation, this increase was observed at D1 and D8, respectively for cabergoline treated cows and control cows (P

International audience; In INRA (2018) feeding system, the first parameter estimated in response to net energy (E) and metabolizable protein (P) supply is milk protein yield (MPY). Milk yield response is then calculated from the MPY response using a linear function. This suggests an important link between milk protein secretion and milk volume. However, MPY and lactose yield did not respond totally in parallel to E and P supplies (Lemosquet et al. 2010. EAAP pub #127: 177; Daniel et al., 2016. Animal: 1). Indeed, milk volume greatly depends on mammary lactose synthesis, the most important osmotic nutrient synthesized within the mammary epithelial cells. The main precursor of lactose is glucose taken up by the mammary gland. Mammary glucose uptake can also contribute to furnish energy through oxidation. This energy is necessary for milk synthesis. Both mammary protein turnover and milk protein synthesis required ATP as energy. In this presentation, partition of mammary glucose uptake between lactose synthesis and other utilizations (mainly oxidation) will be discussed using results from experiments reporting mammary glucose uptake in response to variations in E and P supplies. Increasing P supply or modifying E supply (via propionate or glucose digestive infusions) both increased whole-body glucose rate of appearance (WBRA, i.e. glucose entry rate from gluconeogenesis, intestinal absorption and glycogenolysis). However, in mid-lactation cows, an increased WBRA did not always increase mammary glucose uptake, lactose and milk yields (Lemosquet et al., 2009. JDS: 3244; JDS: 6068). Mammary glucose uptake and lactose yield both significantly increased in 2 experiments among 3 increasing E or intestinal glucose supplies. It was also the case in 3 experiments among 5 increasing P or AA supply. Interestingly, lactose yield increased once without any increase in mammary glucose uptake in response to P supply (Lemosquet et al., 2009. JDS: 6068). In Haque et al. (2015. JDS: 3951), mammary glucose uptake tended to increase in response to an « ideal » EAA profile while lactose yield did not change. Overall, in the 6 experiments considered an increased glucose uptake led to decrease the lactose: glucose uptake ratio, suggesting an increase in glucose utilization in other pathways than lactose synthesis. Using a biochemical model (Abdou Arbi et al. 2015. BMC Systems Biology: 8), we confirmed the large mammary gland flexibility to oxidize glucose to produce energy or to use glucose toward lactose synthesis. Analyses on mRNA in milk mammary epithelial cells suggested that glucose utilization pathways seemed not strongly regulated at transcription level when E and P supplies varied except if an important decrease of both supplies is induced at the beginning of lactation (Boutinaud et al., 2915. Frontiers in Genetics: 00323). Overall, these data suggest that in mid-lactation cows, increasing milk protein synthesis and mammary glucose oxidation in response to increased E and P supplies could be a priority over increasing in lactose yield.

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Once-daily milking is known to modify cell number and activity in the bovine mammary gland. However, in goats, the effect of once-daily milking on mammary cell death is controversial. To assess the effect of once-daily milking on mammary transcriptome and cell turnover and the effect of the duration ofonce-daily milking, 10 goats producing 2.5 kg of milk per day at 100 DIM were divided into 2 groups. All goats were twice daily milked during a pre-experimental period of 2 wk. Then, the goats were once daily milked for the 3 following weeks. Mammary biopsy samples were collected at d −1 and 7 after the start of once-daily milking for one group of 5 goats and at d −1 and 21 for a second group of 5 goats. Cell apoptosis and proliferation rates were analyzed in mammary tissue by immunohistological analyses after TUNEL and PCNA staining, respectively. Ribonucleic acid was extracted from mammary tissues. A transcriptomic analysis using the Agilent Bovine 4x44k microarrays has been performed to compare the effect of once-daily milking on mammary transcript profiles. Data were normalized and statistical significant raw P-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure. A similar reduction in milk yield (−19%) wasobserved in both groups of goats during once-daily milking compared with twice-daily milking. Cell apoptosis was higher during once-daily milking than during twice-daily milking for both groups of goats (P < 0.05) whereas cell proliferation did not vary (P = 0.27). The transcriptomic analysis showed a differential gene expression of 4,039 transcripts, 2,238 and 1,801 transcripts up- and downregulated, respectively, by once-daily milking compared with twice-daily milking. More than 1,000 transcripts were commonly regulated between the 2 groups of goats. IPA analysis showed that these transcripts were part of networks associated with DNA replication, cellular growth and proliferation, and cell-to-cell signaling and interaction in both groups of goats. RT-qPCR analysis of 11 genes confirmed the differential gene expression with a downregulationof genes involved in milk synthesis and an upregulation of genes involved in cellular cycle and apoptosis. These results showed that once-daily milking induce cell turnover in goat mammary tissue, with a small impact of time.

International audience; Diet, in term of quantity and quality, is one of the main factors that affect milk yield and composition in dairy cows. The mammary epithelial cell (MEC) exfoliation process, together with the cell proliferation/apoptosis balance, participates in regulating the MEC number in the mammary gland and thus milk yield. Little is known about the effect of diet in the regulation of MEC number in the mammary tissue. This trial aimed to investigate the effect of feeding level and nature of forage on the MEC exfoliation rate. Sixteen multiparous dairy cows producing 37.0 ± 0.68 kg at 99 ± 4.6 DIM were divided into 4 groups of 4 cows each according to their diet. After a pre-experimental period of 3 wk, cows were fed either 100 or 75% of their ad libitum DMI with diets based on corn silage or conserved grass for 3 wk (Period 1). The feeding level was then reversed for the 3 following weeks (Period 2). Milk samples were collected at the end of the pre-experimental period and 15 d after the beginning of each period for milk MEC purification using an immunomagnetic method to assessthe daily MEC exfoliation rate. The percentage of apoptotic MEC in milk was determined by flow cytometry after annexin V/propidium iodide labeling. Mammary epithelium integrity was assessed by milk Na:K ratio determination. Data were analyzed using the mixed procedure of SAS. After 15 d of treatment, feed restriction decreased milk yield by 10% (P < 0.05) without affecting the MEC exfoliation rate. The Na:K ratio was greater with feed restriction, suggesting a loss of mammary epithelium integrity (P < 0.05). The nature of forage affected neither milk yield nor mammary epithelium integrity, but the daily MEC exfoliation rate was greater incows fed with the corn silage–based diet (P < 0.01). When cows were fed 100%, the percentage of apoptotic cells among exfoliated MEC was lower with the corn silage–based diet than with conserved grass–based diet (P < 0.05). These results show that the MEC exfoliation rate was not affected by a 15-d feed restriction whereas it was affected by the nature of forage. Mammary epithelial cell exfoliation rates did not varyalong with variations of milk yield. The effect of the duration of feed restriction on the MEC exfoliation rate and the effect of the nature of forage on the cell proliferation/apoptosis balance need to be investigated further.

In dairy cows, feed restriction is known to decrease milk yield by reducing the number of mammary epithelial cells (MEC) in the udder, due to an increased rate of MEC apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process that could participate in regulating MEC number. We hypothesized that the decreased number of MEC in the udder of feed-restricted cows might also result froman increase in MEC exfoliation rate. Nineteen Holstein dairy cows producing 40 ± 0.7 kg at 80 ± 4 DIM were divided into 2 groups, control (n = 9) and feed restricted (n = 10). Ad libitum DMI was recorded during a pre-experimental period of 2 wk. For 29 d (period 1), cows were fed either 100 or 80% of their DMI measured during the pre-experimental period. Then, all cows were fed ad libitum for 35 d (period 2). Feed intake andmilk production were recorded daily. Blood and milk samples were collected once during the pre-experimental period and 5, 9, and 27 d after the beginning of periods 1 and 2. Mammary epithelial cells were purified from milk samples using an immunomagnetic method. Lipid mobilization was assessed by plasma NEFA concentration. Mammary epithelium integrity was assessed by plasma lactose concentration, milk Na, andmilk Na:K ratio. Mammary tissue samples were collected by biopsy at the end of each period in order to analyze the rate of cell proliferation and apoptosis. Data were analyzed using the mixed procedure of SAS. Compared with control feeding, feed restriction induced a decrease in DMI by 20% (P < 0.001), an increase in plasma concentration of NEFA (P < 0.001), and a 3-kg/d milk yield loss (P < 0.001) but had no effect on the rate of proliferation and apoptosis in the mammary tissue. Daily MEC exfoliation rate was 65% greater in feed-restricted cows compared with control cows (P < 0.05). Feed restriction had no effect on plasma lactose concentration and milk Na:K ratio but increased milk Na concentration, which does not allow conclusions about the integrity of the mammary epithelium. In period 2, when all cows returned to ad libitum feeding, no significant difference was observed on DMI, milk yield, MEC exfoliation rate, and mammary epithelium integrity between feed-restricted and control cows. These results show that the exfoliation process could play a role in regulating the number of MEC in the udder of dairy cows during a feed restriction without a carryover effect on their milk production.

International audience; Epigenetic modifications such as DNA methylation play a role in regulating gene expression and consequently in biological processes, such as those involved in health and disease. In dairy cows, profound changes occur during the post calving period, leading to immunosuppression and increased susceptibility to diseases. In order to monitor the postpartum health status of cows, one challenge is to describe the methylome of a purified subpopulation of immune cells, such as monocytes, and to determine its alterations/modifications in response to environmental or physiological changes. In this study, genome-wide DNA methylation profiles were obtained from purified monocytes (n=11), Peripheral Blood Mononuclear Cells (PBMC; n=4) sampled at D15 after calving and fibroblasts (n=2) using Reduced Representation Bisulfite Sequencing (RRBS). After sequencing and mapping to the reference genome (UMD3.1 assembly), 34.6% to 40.2% of uniquely mapped reads were obtained. Only CpGs covered between 10X and 500X (CpG10-500), were analyzed, which represented 55 ± 9.8 % of total CpGs with no significant differences between libraries. The chromosomic distribution of CpG10-500 was independent of the chromosome length (pvalue=0.48) but significantly associated with the coding gene content (pvalue=0.0001076). The global CpG10-500 methylation scores varied between cell types (fibroblasts: 47% ± 0.1, monocytes: 54.5% ± 2.9 and PBMC: 54.1% ± 1.8), and 19417 monocyte-specific Differentially Methylated Cytosines (DMCs) corresponding to 1572 DMRs were identified. This study therefore highlights CpGs and regions displaying a specific methylation pattern in monocytes, which could be targeted by epigenetic changes induced by environmental conditions (husbandry, nutrition, infection challenges…).

Caractérisation du processus d’exfoliation des cellules épithéliales mammaires au cours de la traite chez la vache laitière. 23. Rencontres autour des Recherches sur les Ruminants

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The presence of mammary epithelial cells (MEC) in milk indicates that some MEC are shed from the mammary epithelium. The MEC exfoliation process in dairy cows likely participates in the regulation of MEC number in the udder and thus in milk yield variations. The mechanisms that regulate the exfoliation process are not known. However, we can hypothesize that milking, through its mechanical pressure on the mammary epithelium, is a special time for exfoliation of the MEC. In order to characterize MEC exfoliation during milking, 9 multiparous cows were milked and milk samples were collected each minute for composition analysis. The samples collected at the beginning (after 1 and 2 min), in the middle and at the end of milking were used for MEC purification using an immuno-magnetic method. Tight junction opening was assessed by plasma lactose concentration and Na:K ratio in milk. Data were analyzed using the mixed procedure of SAS. Milk flow increased until the 3rd minute and then decreased as the milking progressed (P

International audience; Our objective was to study the evolution of milk composition during the time course of milking. We used 9 dairy cows averaging 56±6 days in milk. Yield, composition (fat, protein, calcium) and free fatty acids (determined by copper soap method) were measured on milk samples collected every min during morning milking (on one day per dairy cow). Statistical analysis was carried out using the Proc Mixed procedurein SAS software with repeated statement. Milk fat content increased during milking (from 6.9 to 62.6 g/ kg). Lactose and protein contents slightly but significantly decreased during milking (from 51.8 to 47.7 g/ kg and from 28.1 to 26.4 g/kg respectively). Milk free fatty acids (Meq/100 g fat) measured after milking decreased during the first 3 minutes and after reached a plateau. Milk free fatty acids measured after 24 hours of storage at 4 °C followed the same trend. Milk lipolysis remained unchanged during milking in our trial. Milk fat globule diameter measured as d4,3 increased at the beginning of milking, but reached a plateau very quickly. Measured as d3,2, it also increased at the beginning of milking and decreased at the end of milking that could suggest a reduction in milk fat globule membrane availability towards the end of milking. Milkcalcium decreased during milking (from 1,205 to 1,147 mg/kg). Intra animal, there was a strong curvilinear relationship between milk fat content and milk fat globule diameter, d4,3 (R2 from 0.67 to 0.98), a positive linear relationship between milk protein content and milk calcium and lactose contents (R2 from 0.32 to 0.95 and from 0.50 to 0.99, respectively) and a negative linear relationship between milk fat globule diameterand milk free fatty acids measured after 24 hours of storage at 4 °C (R2 from 0.13 to 0.97). In conclusion, there was a great change in milk composition during milking and a complete milking is necessary to obtain a representative picture of the secreted milk.

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International audience; Epigenetic modifications such as DNA methylation play a role in regulating gene expression and consequently in biological processes, such as those involved in health and disease. In human, studies provide evidences that the methylome in peripheral blood mononuclear cells (PBMC) can reflect some disease susceptibility. In dairy cows, the post calving period is characterized by profound changes associated with an immunosuppression increasing the susceptibility to diseases. This study aims to quantify the global DNA methylation levels (dMe) by LUminometric Methylation Assay in bovine PBMC and in milk leukocytes and epithelial cells. Parity, milk production and quality, health and diet were reported from 52 Holstein housed at INRA’s experimental dairy farms. Genomic DNAs were extracted from 94 PBMC samples collected at D15 and D60 of lactation. PBMC-dMe varied from 72 to 86%. The proportion of different blood cell types were determined by cytometry and were found stable during the lactation period analyzed. dMe variations do not correlate with blood cell populations. No effect of lactation period, parity or breeding was observed. Moreover, dMe of PBMC was not significantly altered in pathological cows (mastitis or uterine post-partum disease, n=6) in comparison with healthy cows (n=18). For the 24 cows at the two lactation stages, milk leukocytes (ML) and epithelial cells (MEC) were also purified after milk centrifugation and immunomagnetic binding and gDNA extracted. Using the paired samples (PBMC-ML-MEC), dMe was found to be cell type specific: 77.08±1.8% in PBMC vs 65.6±4.1% in ML (significantly different, P

In ruminants, the early phase of drying-off is a period of mammary gland involution where lactose secretion is inhibited and lactoferrin is stimulated. GLUT-1 is a glucose transporter that has a key role in supplying substrate for lactose synthesis. The analysis of the changes in lactose and lactoferrin contents in mammary secretions and in GLUT-1 and lactoferrin contents in the udder can provide valuable information about the speed of the mammary involution. To assess the effect of prolactin inhibition by cabergoline on udder involution, 14 Holstein dairy cows were injected with a single i.m. administration of 5.6 mg cabergoline (n = 7) or placebo (n = 7) within 4 h after the last milking the day of drying off (D0). After D0, hay and water ad libitum was supplied to the cows for 10 d. Mammary secretion samples, collected during lactation (D-6) and at D1, D2, D3, D4, D8 and D14 after the drying-off, were used for lactose and lactoferrin analysis. Mammary biopsy samples, collected at D-6, D1 and D8, were used for GLUT-1 mRNA and lactoferrin analyses. Lactose content of mammary secretions progressively decreased during involution, whereas their lactoferrin content increased. The change in lactose content was associated with paralleled change in GLUT-1 mRNA level in the udder. These decreases were faster in cabergoline treated cows compared with controls with lower lactose content in cabergoline treated cows already by D1 than in controls (P < 0.05) and significant decrease in GLUT-1 mRNA levels at D1 and D8 respectively for cabergoline and control treatments compared with D-6 (P ≤ 0.05). The rise of lactoferrin content in mammary secretions was significant starting at D4 in the cabergoline treated cows (P ≤ 0.05) whereas it only happened at D8 in controls (P < 0.05). Overall, cabergoline treatment decreased GLUT-1 mRNA level (P < 0.05) and increased lactoferrin content (P = 0.10). Similarly, lactoferrin immunostaining intensity in the mammary tissue was higher at D1 than at D-6 in cabergoline treated cows (P ≤ 0.05), whereas it tended to be higher only at D8 in controls (P < 0.10). Our results indicate that cabergoline treatment was efficient to hasten the udder involution and therefore facilitates the dry-off.