Porcine milk exosomes modulate the immune functions of CD14+ monocytes in vitro.

Exosomes mediate near and long-distance intercellular communication by transferring their molecular cargo to recipient cells, altering their biological response. Milk exosomes (MEx) are internalized by immune cells and exert immunomodulatory functions in vitro. Porcine MEx can accumulate in the small intestine, rich in macrophages. No information is available on the immunomodulatory ability of porcine MEx on porcine monocytes, which are known precursors of gut macrophages. Therefore, this study aims at (1) assessing the in vitro uptake of porcine MEx by porcine monocytes (CD14+), and (2) evaluating the in vitro impact of porcine MEx on porcine monocytes immune functions. MEx were purified by ultracentrifugation and size exclusion chromatography. The monocytes' internalization of PKH26-labeled MEx was examined using fluorescence microscopy. Monocytes were incubated with increasing exosome concentrations and their apoptosis and viability were measured. Lastly, the ability of MEx to modulate the cells' immune activities was evaluated by measuring monocytes' phagocytosis, the capacity of killing bacteria, chemotaxis, and reactive oxygen species (ROS) production. MEx were internalized by porcine monocytes in vitro. They also decreased their chemotaxis and phagocytosis, and increased ROS production. Altogether, this study provides insights into the role that MEx might play in pigs' immunity by demonstrating that MEx are internalized by porcine monocytes in vitro and exert immunomodulatory effects on inflammatory functions.

The effect of N-arachidonoylethanolamide administration on energy and fat metabolism of early lactating dairy cows.

The aim of the study was to investigate the effect of N-arachidonoylethanolamide (AEA), an endocannabinoid with orexigenic characteristics, on plasma endocannabinoid concentrations, feed intake, energy balance, lipomobilisation, and hepatic lipid metabolism of early-lactating dairy cows. The experiment involved 10 pairs of Holstein half-sibling cows (end of 2nd-3rd pregnancy). Half-sibs of each pair were randomly assigned to either AEA (n = 10) or control (CON) group (n = 10). From day 1 to 30 postpartum, the AEA group received 5 intraperitoneal injections per week of 3 µg/kg body weight AEA and the CON group 0.9% NaCl. In week 1-3 postpartum, AEA administration had no effect on dry matter intake, body weight, or lipomobilisation, but increased plasma triglyceride concentration on d 21 p.p. and mRNA abundances of genes related to hepatic triglyceride synthesis. In week 4 postpartum, the AEA group showed reduced feed intake and whole-body carbohydrate oxidation, but increased whole-body fat oxidation and hepatic lipid accumulation, likely as a result of a counter-regulatory leptin increase. In conclusion, the present study shows a tissue-specific AEA insensitivity and may point to a leptin-controlled regulation of the ECS in early-lactation.

Mild heat stress-induced adaptive immune response in blood mononuclear cells and leukocytes from mesenteric lymph nodes of primiparous lactating Holstein cows.

Heat stress negatively affects the metabolism and physiology of the bovine gut. However, it is not known whether heat stress induces an inflammatory response in mesenteric lymph nodes (MLN), the primary origin of gut immune cells, and thus contributes to inflammatory processes in the circulation. Therefore, our objective was to elucidate the effects of chronic heat stress on the systemic activation of acute-phase response in blood, proinflammatory cytokine production in peripheral blood mononuclear cells (PBMC), and the activation of the toll-like receptor signaling (TLR) 2/4 pathway in MLN leucocytes and their chemokines and chemokine receptor profiles in Holstein cows. Primiparous Holstein cows (n = 30; 169 ± 9 d in milk) were exposed to a temperature-humidity index (THI) of 60 [16°C, 63% relative humidity (RH)] for 6 d. Thereafter, cows were evenly assigned to 3 groups: heat-stressed (HS; 28°C, 50% RH, THI = 76), control (CON; 16°C, 69% RH, THI = 60), or pair-feeding (PF; 16°C, 69% RH, THI = 60) for 7 d. On d 6, PBMC were isolated and on d 7 MLN. Plasma haptoglobin, TNFα, and IFNγ concentrations increased more in HS than CON cows. Concomitantly, TNFA mRNA abundance was higher in PBMC and MLN leucocytes of HS than PF cows, whereas IFNG mRNA abundance tended to be higher in MLN leucocytes of HS than PF cows, but not for chemokines (CCL20, CCL25) or chemokine receptors (ITGB7, CCR6, CCR7, CCR9). Furthermore, the TLR2 protein expression tended to be more abundant in MLN leucocytes of HS than PF cows. These results suggest that heat stress induced an adaptive immune response in blood, PBMC, and MLN leukocytes involving the acute-phase protein haptoglobin, proinflammatory cytokine production, and TLR2 signaling in MLN leucocytes. However, chemokines regulating the leucocyte trafficking between MLN and gut seem not to be involved in the adaptive immune response to heat stress.

Ultrasound characterization of mammary gland development in heifer calves fed at two different levels until weaning.

Ultrasound technologies allow for a non-invasive assessment of mammary gland (MG) development, the differentiation between the tissue types of the MG, and the evaluation of changes in its composition. This study aimed to work out a detailed description of the different stages of MG development that are visually discernible by ultrasonography for providing a template to classify the different structures. With this basis, the qualitative categorization of the developmental stage, as well as further quantitative assessments via pixel densities in the structures of interest, should be facilitated. Ultrasonic images were acquired from all four quarters of 37 German Holstein heifer calves fed either at a high feeding level of milk replacer (MR; 14% solids) at 10 L/day (1.4 kg MR/day; HI, n = 18) or at a restrictive low level of 5.7 L/day (0.8 kg MR/day; RES, n = 19) until linear weaning from week 13 to 14 of life. Ultrasound MG scans were performed first in week 3 of life, fortnightly from week 8-16, and in week 20 of life, in standing position, of each quarter, using a B-mode ultrasound device equipped with a linear probe (18 MHz). The developmental stages of the mammary gland parenchyma (PAR), visible in ultrasound images, obtained over 20 weeks of life, were categorized, described, and drawn by hand. On this basis, a template for classifying the visible categories of mammary PAR development and its surrounding tissue (SURR), and for measuring their pixel brightness was created thus providing an ultrasonographic atlas of the developing bovine MG, describing 11 categories. The ultrasound images were further classified by PAR structure, and pixel brightness was measured in PAR and SURR by using ImageJ Fiji. The difference in pixel brightness between PAR and SURR, the delta (Δ) pixel value was calculated. With increasing age, higher atlas categories of PAR developmental stages were shown. Pixel values, i.e. the brightness of PAR, its SURR, and Δ pixel value changed with age but were neither affected by the feeding group nor by a group × time interaction. With progressing PAR development, its pixel brightness increased from week 10 to 20 of life, i.e., the PAR became more hyperechoic since it spread and grew into its SURR. The atlas can serve as a template for the categorization and qualitative assessment of MG structures and for the quantitative assessment of PAR's development by measuring pixel brightness. With our study, we could show the structural development in PAR as well as in SURR in MG simultaneously in early life and confirm the spreading of PAR into its SURR by ultrasound scanning.

Effects of dietary supplementation with histidine and ß-alanine on blood plasma metabolome of broiler chickens at different ages.

Histidine is an essential amino acid for broiler chickens and a precursor for the dipeptides carnosine and anserine, but little information is available about its metabolism in modern, fast-growing broilers. We used untargeted metabolomics to investigate the metabolic changes caused by the use of different standardized ileal digestible His:Lys ratios in broiler diets with and without ß-alanine supplementation. A total of 2204 broilers were randomly divided into 96 pens of 23 birds each. The pens were divided into 16 blocks, each containing one pen for all six feeding groups (total of 16 pens per group). These feeding groups were fed three different His:Lys ratios (0.44, 0.54, and 0.64, respectively) without and with a combination of 0.5% ß-alanine supplementation. Five randomly selected chickens of one single randomly selected pen per feeding group were slaughtered on day 35 or 54, blood was collected from the neck vessel, and plasma was used for untargeted metabolomic analysis. Here we show that up to 56.0% of all metabolites analyzed were altered by age, whereas only 1.8% of metabolites were affected by the His:Lys ratio in the diet, and 1.5% by ß-alanine supplementation. Two-factor analysis and metabolic pathway analysis showed no interaction between the His:Lys ratio and ß-alanine supplementation. The effect of the His:Lys ratio in the diet was limited to histidine metabolism with a greater change in formiminoglutamate concentration. Supplementation of ß-alanine showed changes in metabolites of several metabolic pathways; increased concentrations of 3-aminoisobutyrate showed the only direct relationship to ß-alanine metabolism. The supplementation of ß-alanine indicated few effects on histidine metabolism. These results suggest that the supplements used had limited effects or interactions on both His and ß-alanine metabolism. In contrast, the birds' age has the strongest influence on the metabolome.

Investigating circulating miRNA in transition dairy cows: What miRNAomics tells about metabolic adaptation.

In the current study, we investigated dairy cows' circulating microRNA (miRNA) expression signature during several key time points around calving, to get insights into different aspects of metabolic adaptation. In a trial with 32 dairy cows, plasma samples were collected on days -21, 1, 28, and 63 relative to calving. Individually extracted total RNA was subjected to RNA sequencing using NovaSeq 6,000 (Illumina, CA) on the respective platform of IGA Technology Services, Udine, Italy. MiRDeep2 was used to identify known and novel miRNA according to the miRbase collection. Differentially expressed miRNA (DEM) were assessed at a threshold of fold-change > 1.5 and false discovery rate < 0.05 using the edgeR package. The MiRWalk database was used to predict DEM targets and their associated KEGG pathways. Among a total of 1,692 identified miRNA, 445 known miRNA were included for statistical analysis, of which 84, 59, and 61 DEM were found between days -21 to 1, 1 to 28, and 28 to 63, respectively. These miRNA were annotated to KEGG pathways targeting the insulin, MAPK, Ras, Wnt, Hippo, sphingolipid, T cell receptor, and mTOR signaling pathways. MiRNA-mRNA network analysis identified miRNA as master regulators of the biological process including miR-138, miR-149-5p, miR-2466-3p, miR-214, miR-504, and miR-6523a. This study provided new insights into the miRNA signatures of transition to the lactation period. Calving emerged as a critical time point when miRNA were most affected, while the following period appeared to be recovering from massive parturition changes. The primarily affected pathways were key signaling pathways related to establishing metabolic and immune adaptations.

MicroRNA profiling of subcutaneous adipose tissue in periparturient dairy cows at high or moderate body condition.

A growing body of evidence shows that microRNA (miRNA), play important roles in regulating adipose tissue (AT) physiology and function. The objective was to characterize the AT miRNA profile in over-conditioned (HBCS, n = 19) versus moderate-conditioned (MBCS, n = 19) periparturient dairy cows. Tail-head subcutaneous AT biopsied on d -49 and 21 relative to parturition were used for miRNA sequencing. The miR-486 was the most significant miRNA among the upregulated miRNA on d -49, which might be related to more pronounced changes in lipogenesis and altered insulin sensitivity in AT of HBCS cows at dry-off. Comparing HBCS to MBCS on d 21, 23 miRNA were downregulated and 20 were upregulated. The predicted targets of upregulated differentially expressed (DE)-miRNA on d 21 were enriched in different pathways, including pathways related to lysosomes and peroxisomes. The predicted targets of downregulated DE-miRNA on d 21 were enriched in various pathways, including epidermal growth factor receptor, insulin resistance, hypoxia-inducible factor 1 signaling pathway, and autophagy. The results showed that over-conditioning was associated with changes in SCAT miRNA profile mainly on d 21, of which most were downregulated. The enriched pathways may participate in over-conditioning-associated metabolic challenges during early lactation.

Effects of different ratios of omega-6:omega-3 fatty acids in the diet of sows on the proteome of milk-derived extracellular vesicles.

Milk is a nutrient-rich biofluid that contains several biocomponents with distinctive functions, including extracellular vesicles (EV). Milk EV have been associated with the regulation of the newborn's immune system and to influence essential cellular development. The EV proteome comprises the protein constituents and cargo; changes in these compartments could impact their role mediating communication. The ratio of dietary ω-6 to ω-3 polyunsaturated fatty acids (PUFA) is known to affect health and inflammation, and to induce changes in milk fatty acid composition, but no reports have included the milk EV fraction so far. We isolated EV from milk samples obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet or a test diet enriched in ω-3 (ω6:ω3 = 4:1). Small milk-derived EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Using a TMT-based high-resolution quantitative approach, the proteomics analysis revealed variations in the milk EV proteome within the diet groups with differences in the abundance of spondin-2 and 78 kDa glucose-regulated protein. Future studies are encouraged to explore further dietary effects on milk EV composition and their relation to the offspring's development. SIGNIFICANCE: Milk EV are known as key players mediating the regulation of the infant's immune system and growth. The EV proteome comprises the protein constituents and protein cargo, and any changes in this system could impact their role in intercellular communication. This study aimed at evaluating how different ω-6:ω-3 ratios in the maternal diet could translate to the milk EV proteome. This is relevant for basic research, but also has applied aspects in animal nutrition and health and may provide new perspectives for feeding additives.

Plasma proteomics reveals crosstalk between lipid metabolism and immunity in dairy cows receiving essential fatty acids and conjugated linoleic acid.

Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from - 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days - 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status.

Symposium review: Adipose tissue endocrinology in the periparturient period of dairy cows.

The involvement of adipose tissue (AT) in metabolism is not limited to energy storage but turned out to be much more complex. We now know that in addition to lipid metabolism, AT is important in glucose homeostasis and AA metabolism and also has a role in inflammatory processes. With the discovery of leptin in 1994, the concept of AT being able to secrete messenger molecules collectively termed as adipokines, and acting in an endo-, para-, and autocrine manner emerged. Moreover, based on its asset of receptors, many stimuli from other tissues reaching AT via the bloodstream can also elicit distinct responses and thus integrate AT as a control element in the regulatory circuits of the whole body's functions. The protein secretome of human differentiated adipocytes was described to comprise more than 400 different proteins. However, in dairy cows, the characterization of the physiological time course of adipokines in AT during the transition from pregnancy to lactation is largely limited to the mRNA level; for the protein level, the analytical methods are limited and available assays often lack sound validation. In addition to proteinaceous adipokines, small compounds such as steroids can also be secreted from AT. Due to the lipophilic nature of steroids, they are stored in AT, but during the past years, AT became also known as being able to metabolize and even to generate steroid hormones de novo. In high-yielding dairy cows, AT is substantially mobilized due to increased energy requirements related to lactation. As to whether the steroidogenic system in AT is affected and may change during the common loss of body fat is largely unknown. Moreover, most research about AT in transition dairy cows is based on subcutaneous AT, whereas other depots have scarcely been investigated. This contribution aims to review the changes in adipokine mRNA and-where available-protein expression with time relative to calving in high-yielding dairy cows at different conditions, including parity, body condition, diet, specific feed supplements, and health disorders. In addition, the review provides insights into steroidogenic pathways in dairy cows AT, and addresses differences between fat depots where possible.

Longitudinal liver proteome profiling in dairy cows during the transition from gestation to lactation: Investigating metabolic adaptations and their interactions with fatty acids supplementation via repeated measurements ANOVA-simultaneous component analysis.

Repeated measurements analysis of variance - simultaneous component analysis (ASCA) has been developed to handle complex longitudinal omics datasets and combine novel information with existing data. Herein, we aimed at applying ASCA to 64 liver proteomes collected at 4-time points (day -21, +1, +28, and + 63 relative to parturition) from 16 Holstein cows treated from 9 wk. antepartum to 9 wk. postpartum (PP) with coconut oil (CTRL) or a mixture of essential fatty acids (EFA) and conjugated linoleic acid (CLA) (EFA + CLA). The ASCA modeled 116, 43, and 97 differentially abundant proteins (DAP) during the transition to lactation, between CTRL and EFA + CLA, and their interaction, respectively. Time-dependent DAP were annotated to pathways related to the metabolism of carbohydrates, FA, and amino acid in the PP period. The DAP between FA and the interaction effect were annotated to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, retinol metabolism, and steroid hormone biosynthesis. Collectively, ASCA provided novel information on molecular markers of metabolic adaptations and their interactions with EFA + CLA supplementation. Bioinformatics analysis suggested that supplemental EFA + CLA amplified hepatic FA oxidation; cytochrome P450 was enriched to maintain metabolic homeostasis by oxidation/detoxification of endogenous compounds and xenobiotics. SIGNIFICANCE: This report is among the first ones applying repeated measurement analysis of variance-simultaneous component analysis (ASCA) to deal with longitudinal proteomics results. ASCA separately identified differentially abundant proteins (DAP) in 'transition time', 'between fatty acid treatments', and 'their interaction'. We first identified the molecular signature of hepatic metabolic adaptations during postpartum negative energy balance; the enriched pathways were well-known pathways related to mobilizing fatty acids (FA) and amino acids to support continuous energy production through fatty acid oxidation, TCA cycle, and gluconeogenesis. Some of the DAP were not previously reported in transition dairy cows. Secondly, we provide novel information on the mechanisms by which supplemented essential FA and conjugated linoleic acids interact with hepatic metabolism. In this regard, FA amplified hepatic detoxifying and oxidation capacity through ligand activation of nuclear receptors. Finally, we briefly compared the strengths and weaknesses of the ASCA model with PLS-DA and outlined why these methods are complementary.

Liver proteome profiling in dairy cows during the transition from gestation to lactation: Effects of supplementation with essential fatty acids and conjugated linoleic acids as explored by PLS-DA.

This study aimed at investigating the synergistic effects of essential fatty acids (EFA) and conjugated linoleic acids (CLA) on the liver proteome profile of dairy cows during the transition to lactation. 16 Holstein cows were infused from 9 wk. antepartum to 9 wk. postpartum into the abomasum with either coconut oil (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (EFA + CLA). Label-free quantitative proteomics was performed in liver tissue biopsied at days -21, +1, +28, and + 63 relative to calving. Differentially abundant proteins (DAP) between treatment groups were identified at the intersection between a multivariate and a univariate analysis. In total, 1680 proteins were identified at each time point, of which between groups DAP were assigned to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, steroid hormone biosynthesis, glycolysis/gluconeogenesis, and glutathione metabolism. Cytochrome P450, as a central hub, enriched with specific CYP enzymes comprising: CYP51A1 (d - 21), CYP1A1 & CYP4F2 (d + 28), and CYP4V2 (d + 63). Collectively, supplementation of EFA + CLA in transition cows impacted hepatic lipid metabolism and enriched several common biological pathways at all time points that were mainly related to ω-oxidation of fatty acids through the Cytochrome p450 pathway. SIGNIFICANCE: In three aspects this manuscript is notable. First, this is among the first longitudinal proteomics studies in nutrition of dairy cows. The selected time points are critical periods around parturition with profound endocrine and metabolic adaptations. Second, our findings provided novel information on key drivers of biologically relevant pathways suggested according to previously reported performance, zootechnical, and metabolism data (already published elsewhere). Third, our results revealed the role of cytochrome P450 that is hardly investigated, and of ω-oxidation pathways in the metabolism of fatty acids with the involvement of specific enzymes.

Effects of a Maternal Essential Fatty Acid and Conjugated Linoleic Acid Supplementation during Late Pregnancy and Early Lactation on Hematologic and Immunological Traits and the Oxidative and Anti-Oxidative Status in Blood Plasma of Neonatal Calves.

Fatty acids are known for their regulatory role in inflammation and oxidative stress. The present study investigated 38 calves born from dams, abomasally supplemented with coconut oil, essential fatty acids (EFA), conjugated linoleic acid (CLA) or EFA + CLA, according to immunological traits and the oxidative and anti-oxidative status for the first 5 days of life. On day 2 of life, plasma total bilirubin, cholesterol, interleukin 1-ß and ferric ion reducing anti-oxygen power (FRAP) were lower in calves with than without maternal EFA supplementation, and FRAP additionally on day 4. On day 3, the concentrations of reactive oxygen metabolites were higher in calves with than without maternal EFA supplementation and additionally on day 5 together of retinol. Total leucocyte counts were decreased in the EFA group compared to the CLA group on day 5. Lymphocyte proportions decreased from day 1 to 5 only in the EFA + CLA group. On day 2, plasma total protein was higher in CLA and EFA + CLA than in EFA calves. Similarly, CLA calves had higher interleukin 1-ß concentrations compared to EFA + CLA calves. FRAP was decreased by CLA on day 4. Overall, the maternal fatty acid supply affected the inflammatory response and the oxidative and anti-oxidative status of the neonatal offspring.

Comparative proteome profiling in exosomes derived from porcine colostrum versus mature milk reveals distinct functional proteomes.

Exosomes are membranous vesicles of endocytic origin, recently been considered as major players in cell-cell communication. Milk is highly complex, and diverse biocomponents provide adequate nutrition, transfer immunity, and promote adequate neonate development. Milk exosomes are suggested to have a key role in these processes, yet to be further explored, and the alteration of the exosomes' cargo in different stages of lactation stages is important for understanding the factors relevant in nursing and also for improving milk replacer products both for humans and animals. We isolated exosomes from porcine milk in different lactation stages and analyzed their content using a TMT-based high-resolution quantitative proteomic approach. Exosomes were isolated using ultracentrifugation coupled with size exclusion chromatography to enrich milk-derived exosomes in samples obtained at day 0, 7, and 14 after parturition, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Quantitative proteomics analysis revealed different proteome profiles for colostrum exosomes and milk exosomes. The functional analysis highlighted pathways related to the regulation of homeostasis to be upregulated in colostrum exosomes, and pathways such as endothelial cell development and lipid metabolism to be upregulated in mature milk exosomes. This study endorses the importance of exosomes as active biocomponents of milk and provides knowledge for future studies exploring their role in the regulation of immunity and growth of the newborn. SIGNIFICANCE: The identified functional proteome and protein-protein interaction networks identified in our study help to elucidate the role of milk exosomes in different lactation periods. The results generated herein are of relevance for the basic understanding of their impact on the infant's development but also for bringing forward the manufacturing of milk replacers.

Effects of colostrum feeding on the mRNA abundance of genes related to toll-like receptors, key antimicrobial defense molecules, and tight junctions in the small intestine of neonatal dairy calves.

The objective of the present study was to elucidate the effect of feeding either colostrum or milk-based formula on the mRNA abundance of genes related to pathogen recognition [toll-like receptors (TLR1-10)], antimicrobial defense [ß-defensin 1 (DEFB1) and peptidoglycan recognition protein 1 (PGLYRP1)], and tight junctions (claudin 1 = CLDN1, claudin 4 = CLDN4, and occludin = OCLN) in different sections of the small intestine of neonatal calves at d 4 of life. Holstein dairy calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) with comparable nutrient composition but lower contents of several bioactives in the formula than in the respective colostrum group until d 4 of life. Following euthanasia on d 4 (2 h after feeding), tissue samples from the duodenum, jejunum (proximal, middle, and distal), and ileum were collected. The mRNA abundance of the target genes was quantified by quantitative PCR. The mRNA abundance of TLR1, TLR6, TLR9, and TLR10 were greater in COL than in FOR calves. However, the mRNA abundance of TLR2, TLR3, TLR4, TLR5, and TLR7 did not differ between groups. A group × gut region interaction was observed for the mRNA abundance of TLR8 with greater values in duodenum and proximal jejunum of COL than in FOR calves but in the more distal regions, in mid and distal jejunum, and ileum, this diet effect disappeared or was reversed. We observed greater mRNA abundance of TLR1 in the jejunum (middle and distal) and ileum, TLR2, TLR4, TLR6, and TLR9-10 in the distal jejunum and ileum, and of TLR3 in the distal jejunum, and TLR5, TLR7, and TLR8 in the ileum compared with the other gut regions. The mRNA abundance of PGLYRP1, DEFB1, and OCLN did not differ between groups. The mRNA abundance of CLDN1 was greater, but the CLDN4 mRNA tended to be lower in COL than in FOR calves. The mRNA abundance of PGLYRP1 was lower in the distal jejunum and DEFB1 mRNA in the middle jejunum compared with the other gut regions. The mRNA abundances of OCLN and CLDN4 were greater in the duodenum, and of CLDN1 in the middle and proximal jejunum compared with the other gut regions. Overall, the greater mRNA abundance of 5 different TLR, and CLDN1 in most intestinal sections of the COL calves may suggest that feeding colostrum improves immune responsiveness and epithelial barrier function in neonatal calves.

Longitudinal changes in fatty acid metabolism and in the mitochondrial protein import system in overconditioned and normal conditioned cows: A transcriptional study using microfluidic quantitative PCR.

This study investigated the effect of body condition around calving on the hepatic mRNA expression of genes involved in fatty acid (FA) metabolism and mitochondrial protein import system of dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets to reach targeted differences in BCS and backfat thickness (BFT) until dry-off. At dry-off, normal BCS (NBCS) cows had a BCS <3.5 and BFT <1.2 cm, and the high BCS (HBCS) cows had a BCS >3.75 and BFT >1.4 cm. The expression of targeted genes in the liver was assayed by reverse-transcription quantitative real-time PCR using microfluidics integrated fluidic circuit chips on a subset of 5 cows from each group. Liver biopsies were collected at d -49, +3, +21, and +84 relative to parturition. The mRNA abundance of 47 genes related to lipid metabolism including carnitine metabolism, FA uptake and transport, lipoprotein export, carnitine metabolism, mitochondrial and proximal FA oxidation, ketogenesis, AMP-activated protein kinase/mammalian target of rapamycin pathway, and mitochondrial protein import system was assessed in liver tissue. The mRNA abundances of FA binding protein (FABP)6 (in both groups), and FABP1 and solute carrier family 22 member 5 (SLC22A5) in HBCS were upregulated (>1.5-fold change, FC) in early lactation (at d +3 and +21 postpartum) compared with antepartum (d -49), indicating promoted FA uptake and intracellular transport in the liver due to the metabolic adaptations of elevated lipo-mobilization after parturition. The upregulation of SLC22A5 and SLC25A20 after parturition was more pronounced in HBCS than in NBCS cows, suggesting a need for increasing the capacity of FA uptake, and FA transport into the hepatocyte. The increased mRNA abundance of carnitine palmitoyltransferase 1A, after parturition and to a greater extent in HBCS (FC = 4.1) versus NBCS (FC = 2.1) indicates a physiological increase in the capacity of long-chain fatty acyl-CoA entry into the liver mitochondria compared with antepartum (ap; d -49 relative to calving). The greater hepatic mRNA abundance of genes encoding enzymes involved in mitochondrial FA oxidation in HBCS than in NBCS points to an increased rate of mitochondrial ß-oxidation. The hepatic mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 2 and 3-hydroxy-3-methylglutaryl-CoA were upregulated after parturition (d +21/d +3 pp) to a greater extent in HBCS than in NBCS cows, indicating that excess acetyl-CoA generated via ß-oxidation was increasingly used for ketogenesis. We observed for the first time that the mRNA abundance of genes involved in the translocase of the inner membrane (TIM) complex (TIM22 and TIM23) in the hepatic mitochondrial protein import system were undergoing distinct changes during the transition from late pregnancy to early lactation in dairy cows. Even though sample size in this study was relatively small, the results support that overconditioning around calving may contribute to mitochondrial FA overload and greater ketogenesis at the level of transcription in the liver of early lactation cows.

Muscle metabolome and adipose tissue mRNA expression of lipid metabolism-related genes in over-conditioned dairy cows differing in serum-metabotype.

Over-conditioned dairy cows, classified by body condition score (BCS) and backfat thickness (BFT) are less able to metabolically adapt to the rapidly increasing milk yield after parturition. Based on serum metabolome and cluster analyses, high BCS cows (HBCS) could be classified into metabotypes that are more similar to normal (NBCS) cows, i.e., HBCS predicted normal (HBCS-PN) than the HBCS predicted high (HBCS-PH) cows-similar to the concept of obese but metabolically healthy humans. Our objective was to compare muscle metabolome and mRNA abundance of genes related to lipogenesis and lipolysis in adipose tissue between HBCS-PH (n = 13), HBCS-PN (n = 6), and NBCS-PN (n = 15). Tail-head subcutaneous fat was biopsied on d -49, 3, 21, and 84 relative to parturition. Potential differences in the oxidative capacity of skeletal muscle were assessed by targeted metabolomics in M. semitendinosus from d 21. Besides characteristic changes with time, differences in the mRNA abundance were limited to lipogenesis-related genes on d -49 (HBCS-PH > HBCS-PN). The HBCS-PH had more than two-fold higher muscle concentrations of short (C2, C4-OH, C6-OH) and long-chain acylcarnitines (C16, C18, and C18:1) than HBCS-PN, indicating a greater oxidative capacity for fatty acids (and utilization of ketones) in muscle of HBCS-PN than HBCS-PH cows.

Phosphoproteomic Analysis of Subcutaneous and Omental Adipose Tissue Reveals Increased Lipid Turnover in Dairy Cows Supplemented with Conjugated Linoleic Acid.

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.

Characteristics of the Oxidative Status in Dairy Calves Fed at Different Milk Replacer Levels and Weaned at 14 Weeks of Age.

A paradigm shift in the way of rearing heifer calves from restricted feeding and early weaning towards greater feed allowances and later weaning ages is ongoing. We aimed at characterizing the oxidative status in Holstein heifer calves fed with milk replacer (MR) at either a restrictive (RES) or a high (HIGH) level for 14 weeks. We compared two groups: HIGH (10 L MR/d, n = 18) and RES (5.7 L/d, n = 19) from day five until week 14 of life. In blood samples collected at birth, and then fortnightly from week 8-16, and in week 20, the antioxidative capacity measured as ferric reducing ability of plasma (FRAP), oxidative damage of lipids measured as thiobarbituric acid reactive substances (TBARS) and oxidative damage of proteins measured as advanced oxidation products of proteins (AOPP), free radicals measured as reactive oxidative metabolites (dROM), and the glutathione peroxidase (GSH-Px) activity, as well as leptin, adiponectin and haptoglobin were assessed. The time course of these variables during the first 20 weeks of life showed characteristic patterns; group differences were limited to adiponectin, AOPP, and FRAP. RES calves had lower growth rates, showed signs of hunger, but did not differ from HIGH in their intake of solid starter feed and in health status. This work characterizes the changes in oxidative status of dairy calves with increasing age and confirms the benefits of a high feeding plane with regard to welfare and development.

Dietary l-carnitine Supplementation Modifies the Lipopolysaccharide-Induced Acute Phase Reaction in Dairy Cows.

l-carnitine plays an important role in energy metabolism through supporting the transport of activated fatty acids to the subcellular site of ß-oxidation. An acute phase reaction (APR) is known as an energy consuming process. Lipopolysaccharides (LPS) are often used in animal models to study intervention measures during innate immune responses such as APR. Thus, the aim of the study was to investigate the effects of dietary l-carnitine supplementation during an LPS-induced APR in mid-lactating German Holstein cows. Animals were assigned to a control (CON, n = 26) or l-carnitine group (CAR, n = 27, 25 g rumen-protected l-carnitine/cow/d) and received an intravenous injection of LPS (0.5 µg/kg body weight) at day 111 post-partum. Blood samples were collected from day 1 pre-injection until day 14 post-injection (pi). From 0.5 h pi until 72 h pi blood samplings and clinical examinations were performed in short intervals. Clinical signs of the APR were not altered in group CAR except rumen motility which increased at a lower level compared to the CON group after a period of atonia. Group CAR maintained a higher insulin level compared to group CON even up to 72 h pi which might support glucose utilization following an APR.