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.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

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.

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.

Effects of milk replacer meal size on feed intake, growth performance, and blood metabolites and hormones of calves fed milk replacer with or without butyrate ad libitum: A cluster-analytic approach.

This study intended to classify ad libitum-fed calves according to their milk replacer (MR) meal size using the K-means clustering approach. This study aimed to investigate the effects of MR meal size on feed intake, growth performance, and blood metabolic and hormones of ad libitum MR-fed calves. German Holstein calves (16 male and 16 female) were studied from birth until d 77 of age. All calves received first colostrum (2.5 kg) milked from their dams within 2 h after birth. Subsequent colostrum meals (subsequent 4 meals until 2.5 d of age; 2 meals/d) and MR (125 g of powder/L; 21.7% crude protein, 18.6% crude fat) were fed ad libitum by teat bucket until d 10 ± 2 of age. Afterward, calves were housed in group pens with automatic feeders for MR (maximum of 25 L/d) and concentrate from 10 ± 3 d of age. Half of the calves received MR supplemented with butyrate to improve growth performance. Milk intake was stepped down to 2 L/d from wk 9 to 10, and 2 L/d of MR were offered until the end of the study. On d 1, 2, 4, and 7, and then weekly until wk 11 of age, blood samples were collected for measurement of metabolites and hormones related to energy metabolism and growth. The K-means cluster analysis on the MR meal size data collected from the automatic feeder resulted in 3 clusters (n = 14, n = 12, and n = 6). Two clusters with a sufficient cluster size (n = 14 and n = 12) were included for further statistical analysis using repeated measures mixed-model ANOVA. In both clusters, butyrate supplementation was equally distributed and failed to affect a difference in MR meal size. Cluster 1 showed calves with higher MR meal size (HI; 2.2 ± 0.11 L/visit of MR) and cluster 2 with lower meal size (LO; 1.8 ± 0.07 L/visit of MR) supplemented MR without (HIB-; n = 6; LOB-, n = 7) or with 0.33% calcium-sodium butyrate (HIB+; n = 6; LOB+, n = 7). Dry matter intake of MR did not differ between HI and LO, but intakes of concentrate and total dry matter tended to be greater in HI than in LO and increased more distinctly in HI than in LO at the end of the study. The average daily gain (g/d) was greater in HI than in LO. Plasma concentrations of total protein (g/L), albumin (g/L), glucose (mmol/L), urea (mmol/L), insulin (µg/L), and glucagon (ng/L) were higher, and the concentrations of insulin-like growth factor I tended to be higher, in HI than in LO calves. Plasma ß-hydroxybutyrate was higher in LO than in HI at d 63 and lower in calves fed MR with butyrate at d 77. In conclusion, clustering analysis discriminates 2 main groups of calves with different MR meal size and indicates an effect of MR meal size on solid feed intake, growth performance, and metabolic changes.

Consequences of Maternal Essential Fatty Acid and Conjugated Linoleic Acid Supplementation on the Development of Calf Muscle and Adipose Tissue.

Common silage and concentrate-based diets in dairy and beef production may deliver insufficient amounts of essential fatty acids (EFA), thereby also reducing conjugated linoleic acids (CLA) in body tissues and milk. An impaired maternal EFA and CLA supply can have an important impact on calf postnatal development. The current study investigates how maternal supplementation with EFA and CLA affects muscle and adipose tissue development in neonatal calves. Holstein cows (n = 40) were abomasaly supplemented with coconut oil (control), CLA or EFA, or both combined during the transition period. Calves were fed their dam's colostrum until slaughter at day 5 of life. Fatty acid composition and tissue morphology were analyzed. In muscle and adipose tissues, EFA, CLA, and metabolites were elevated, indicating the effective transfer of maternally-supplemented FA to the offspring. Muscle fiber types, fiber nuclei, myosin heavy chain isoform distribution, capillarization, and fat cell size of intramuscular and other adipose tissues did not differ among groups. The results confirm that maternal nutrition during the transition period can alter the FA composition of the calf tissues. This could influence the offspring's development and health in the long-term, even though only minor effects were observed in the neonatal calves' tissue morphology.

Short communication: Colostrum versus formula: Effects on mRNA expression of genes related to branched-chain amino acid metabolism in neonatal dairy calves.

The objective of the current study was to elucidate the effect of feeding colostrum or milk-based formula on the tissue mRNA abundance of the most relevant branched-chain amino acids (BCAA) transporters and catabolizing enzymes in newborn calves. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) with comparable nutrient composition but lower contents of free BCAA, insulin, and insulin-like growth factor-I in the formula than in the respective colostrum for up to 4 d of life. Tissue samples from liver, kidney fat, 3 different muscles [M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM)], as well as duodenum, jejunum, and ileum were collected following euthanasia on d 4 at 2 h after feeding. The plasma-free BCAA were analyzed, and the tissue abundance of solute carrier family 1 member 5 (SLC1A5), SLC7A5, and SLC38A2 as well as mitochondrial isoform of branched-chain aminotransferase (BCATm), branched-chain α-keto acid dehydrogenase E1α (BCKDHA), and branched-chain α-keto acid dehydrogenase E1ß (BCKDHB) were assessed. The preprandial plasma concentrations of free BCAA were affected by time but did not differ between groups. The plasma concentrations of free BCAA decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total BCAA concentrations in FOR than in COL. The mRNA abundances of BCATm, BCKDHA, BCKDHB, as well as BCAA transporters in the liver, were not affected by the diet. In kidney fat, the mRNA abundance of BCAA catabolizing enzymes did not differ between groups, but that of SLC1A5 was lower in FOR than in COL. The mRNA abundance of BCAA catabolizing enzymes in different sections of the small intestine was not affected by the diet, whereas that of SLC7A5 was or tended to be lower in the duodenum, proximal jejunum, and mid jejunum of the COL calves compared with the FOR calves. The mRNA abundance of BCKDHA was lower in MLD and MM but greater in MS for the FOR calves compared with the COL calves. The mRNA abundance of SLC7A5 in MST was lower in FOR than in COL, whereas it was unaffected by the diet in MLD and MM. The differential effect of feeding colostrum on the mRNA abundance of BCKDHA in 3 different muscle tissues might point to a muscle type-specific response. The results also indicate that the colostral BCAA might be favorably used for anabolic metabolism in the small intestine of neonatal calves. Such effects are speculated to be due to the stimulatory effects of growth factors and hormones present in colostrum.

Effects of Dietary Fatty Acids on Bovine Oocyte Competence and Granulosa Cells.

Here we assessed the effects of dietary essential fatty acids on the developmental competence of oocytes in cows and on the functionality of follicular granulosa cells (GC). Lactating German Holstein cows were supplemented from week 9 ante partum (ap) until week 8 post-partum (pp) in four dietary groups designed as (i) control (CTRL: coconut oil), (ii) essential fatty acid (EFA: linseed and safflower oil), (iii) conjugated linoleic acid (CLA: Lutalin®), and (iv) EFA+CLA (mixture of linseed oil, safflower oil and Lutalin®). EFA, CLA or EFA+CLA supplementation did not improve in vitro embryo production. However, higher proportions of α-linolenic acid (ALA) and cis-9, trans-11 CLA were observed in the follicular fluid suggesting the exposure of GC to relatively high levels of ALA and cis-9, trans-11 CLA. Consequently, we tested different concentrations of ALA and cis-9, trans-11 CLA in a bovine GC culture model for their effects on steroid production, marker gene expression and viability. Both fatty acids upregulated CD36 and downregulated the expression of FOXL2, while ALA significantly increased SOX 9 transcript levels. Both ALA and cis-9, trans-11 CLA reduced the CCND2 expression and cis-9, trans-11 CLA induced apoptosis. ALA and cis-9, trans-11 CLA significantly down-regulated the expression of STAR, CYP19A1, FSHR, LHCGR and decreased the 17ß-Estradiol (E2) and progesterone (P4) production. In conclusion, dietary lipids did not improve in vitro embryo production, while ALA and cis-9, trans-11 CLA affected the morphology and functionality of GC. This could suggestively lead to compromised follicle development and ovarian cyclicity in dairy cows.

Dietary Fatty Acids Affect Red Blood Cell Membrane Composition and Red Blood Cell ATP Release in Dairy Cows.

Diets of dairy cows are often based on maize silage (MS), delivering lower amounts of n-3 fatty acids (FA) compared to grass silage-based diets. The fatty acid composition of the cell membrane can affect the cell function. We evaluated the effects of an MS-based diet on bovine red blood cell (RBC) membrane FA composition and dietary effects on controlled ATP release of RBC. In trial 1, German Holstein cows were fed an MS-based total mixed ration for 24 weeks. The FA composition of RBC membranes from repeatedly taken blood samples was analysed in addition to the abundance of the RBC membrane protein flotillin-1, which is involved in, for example, cell signalling. In trial 2, four rumen fistulated MS-fed cows were abomasally infused in a 4 × 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed-safflower oil mix (EFA; rich in n-3 FA), Lutalin®, providing conjugated linoleic acids (CLA) or the combination of the supplements, EFA + CLA) for six weeks, followed by a three-week washout period. In trial 2, we analysed RBC ATP release, flotillin-1, and the membrane protein abundance of pannexin-1, which is involved in ATP release as the last part of a signalling cascade. In trial 1, the total amount of n-3 FA in RBC membranes decreased and the flotillin-1 abundance increased over time. In trial 2, the RBC n-3 FA amount was higher after the six-week infusion period of EFA or EFA + CLA. Furthermore, depending on the dosage of FA, the ATP release from RBC increased. The abundance of flotillin-1 and pannexin-1 was not affected in trial 2. It is concluded that changes of the membrane FA composition influence the RBC function, leading to altered ATP release from intact bovine RBC.

Mammalian target of rapamycin signaling and ubiquitin proteasome-related gene expression in 3 different skeletal muscles of colostrum- versus formula-fed calves.

The rates of protein turnover are higher during the neonatal period than at any other time in postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system are key pathways regulating cellular protein turnover. The objectives of this study were (1) to elucidate the effect of feeding colostrum versus milk-based formula on the mRNA abundance of key components of the mTOR pathway and of the ubiquitin-proteasome system in skeletal muscle of neonatal calves and (2) to compare different muscles. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. The nutrient content in formula and colostrum was similar, but formula had lower concentrations of free branched-chain AA (BCAA) and free total AA, insulin, and insulin-like growth factor (IGF)-I than colostrum. Blood samples were taken from d 1 to 4 before morning feeding and before and 2 h after the last feeding on d 4. Muscle samples from M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM) were collected after slaughter on d 4 at 2 h after feeding. The preprandial concentrations of free total AA and BCAA, insulin, and IGF-I in plasma changed over time but did not differ between groups. Plasma free total AA and BCAA concentrations decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total AA and BCAA concentrations in FOR than in COL. Plasma insulin concentrations increased after feeding in both groups but were higher in COL than in FOR. Plasma IGF-I concentrations decreased in COL, whereas they remained unchanged in FOR after feeding. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in 3 different skeletal muscles was greater in COL than in FOR, whereas that of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) was unaffected by diet. The mRNA abundance of ubiquitin activating enzyme (UBA1) and ubiquitin conjugating enzyme 1 (UBE2G1) enzymes was not affected by diet, whereas that of ubiquitin conjugating enzyme 2 (UBE2G2) was greater (MLD) or tended to be greater (MM) in COL than in FOR. The mRNA abundance of atrogin-1 in MLD and MST was lower in COL than in FOR, whereas that of muscle ring finger protein-1 (MuRF1) was greater (MST) or tended to be greater (MLD). The abundance of MuRF1 mRNA was highest in MST, followed by MLD, and was lowest in MM. The results indicate that colostrum feeding may stimulate protein turnover that may result in a high rate of protein deposition in a muscle type-specific manner. Such effects seem to be mediated by the postprandial increase in plasma insulin.

Quercetin Feeding in Newborn Dairy Calves Cannot Compensate Colostrum Deprivation: Study on Metabolic, Antioxidative and Inflammatory Traits.

Immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. Pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. The flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. The aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. Twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day 1 and 2 and milk replacer thereafter. From day 2 onwards, 7 calves per diet group were additionally fed quercetin aglycone (50 mg/(kg body weight × day)). Blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid A. Trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and F2-isoprostanes were analyzed to evaluate plasma antioxidative status. Expression of tumor necrosis factor, interleukin-1α, interleukin-1ß, serum amyloid A, haptoglobin, fibrinogen, C-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mRNA were measured in liver tissue on day 8. Plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. Plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. Body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formula- than in colostrum-fed groups. Hepatic mRNA expression of tumor necrosis factor was higher after quercetin feeding and expression of C-reactive protein was higher after formula feeding. Data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

BACKGROUND: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates. OBJECTIVE: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves. METHODS: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 µmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes. RESULTS: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves. CONCLUSIONS: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves.

Ontogenic Changes of Villus Growth, Lactase Activity, and Intestinal Glucose Transporters in Preterm and Term Born Calves with or without Prolonged Colostrum Feeding.

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.

Diet effects on glucose absorption in the small intestine of neonatal calves: importance of intestinal mucosal growth, lactase activity, and glucose transporters.

Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters.

Reduced AgRP activation in the hypothalamus of cows with high extent of fat mobilization after parturition.

Agouti-related protein (AgRP), produced by neurons located in the arcuate nucleus of the hypothalamus stimulates feed intake. During early lactation dairy cows increase their feed intake and additionally mobilize their fat reserves leading to increased plasma non-esterified fatty acid (NEFA) concentrations. Since cows with a higher extent of fat mobilization exhibit the lower feed intake, it seems that high NEFA concentrations confine hyperphagia. To test the involvement of AgRP neurons, we investigated 18 cows from parturition until day 40 postpartum (pp) and assigned the cows according to their NEFA concentration on day 40pp to either group H (high NEFA) or L (low NEFA). Both groups had comparable feed intake, body weight, milk yield, energy balance, plasma amino acids and leptin concentrations. Studies in respiratory chambers revealed the higher oxygen consumption and the lower respiratory quotient (RQ) in H compared to L cows. mRNA abundance of neuropeptide Y, peroxisome proliferator-activated receptor-gamma, AMP-activated protein kinase, and leptin receptor in the arcuate nucleus were comparable between groups. Immunohistochemical studies revealed the same number of AgRP neurons in H and L cows. AgRP neurons were co-localized with phosphorylated adenosine monophosphate-activated kinase without any differences between groups. The percentage of cFOS-activated AgRP neurons per total AgRP cells was lower in H cows and correlated negatively with oxygen consumption and NEFA, positively with RQ, but not with feed intake. We conclude that AgRP activation plays a pivotal role in the regulation of substrate utilization and metabolic rate in high NEFA dairy cows during early lactation.

Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.

Trans-10,cis-12 conjugated linoleic acid (CLA) supplementation causes milk fat depression in dairy cows, but CLA effects on glucose metabolism are not clear. The objective of the study was to investigate glucose metabolism, especially endogenous glucose production (eGP) and glucose oxidation (GOx), as well as hepatic genes involved in endogenous glucose production in Holstein cows supplemented either with 50 g of rumen-protected CLA (9% trans-10,cis-12 and 10% cis-9,trans-11; CLA; n=10) or 50 g of control fat (24% C18:2; Ctrl; n=10) from wk 2 before parturition to wk 9 of lactation. Animal performance data were recorded and blood metabolites and hormones were taken weekly from 2 wk before to 12 wk after parturition. During wk 3 and 9 after parturition, glucose tolerance tests were performed and eGP and GOx were measured by [U-(13)C] glucose infusion. Liver biopsies were taken at the same time to measure total fat and glycogen concentrations and gene expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and carnitine palmitoyl-transferase 1. Conjugated linoleic acid feeding reduced milk fat, but increased milk lactose output; milk yield was higher starting 5 wk after parturition in CLA-fed cows than in Ctrl-fed cows. Energy balance was more negative during CLA supplementation, and plasma concentrations of glucose were higher immediately after calving in CLA-fed cows. Conjugated linoleic acid supplementation did not affect insulin release during glucose tolerance tests, but reduced eGP in wk 3, and eGP and GOx increased with time after parturition. Hepatic gene expression of cytosolic phosphoenolpyruvate carboxykinase tended to be lower in CLA-fed cows than in Ctrl-fed cows. In spite of lower eGP in CLA-fed cows, lactose output and plasma glucose concentrations were greater in CLA-fed cows than in Ctrl-fed cows. This suggests a CLA-related glucose sparing effect most likely due to lower glucose utilization for milk fat synthesis and probably because of a more efficient whole-body energy utilization in CLA-fed cows.

Intestinal glucose absorption but not endogenous glucose production differs between colostrum- and formula-fed neonatal calves.

Glucose supply markedly changes during the transition to extrauterine life. In this study, we investigated diet effects on glucose metabolism in neonatal calves. Calves were fed colostrum (C; n = 7) or milk-based formula (F; n = 7) with similar nutrient content up to d 4 of life. Blood plasma samples were taken daily before feeding and 2 h after feeding on d 4 to measure glucose, lactate, nonesterified fatty acids, protein, urea, insulin, glucagon, and cortisol concentrations. On d 2, additional blood samples were taken to measure glucose first-pass uptake (FPU) and turnover by oral [U-(13)C]-glucose and i.v. [6,6-(2)H(2)]-glucose infusion. On d 3, endogenous glucose production and gluconeogenesis were determined by i.v. [U-(13)C]-glucose and oral deuterated water administration after overnight feed deprivation. Liver tissue was obtained 2 h after feeding on d 4 and glycogen concentration and activities and mRNA abundance of gluconeogenic enzymes were measured. Plasma glucose and protein concentrations and hepatic glycogen concentration were higher (P < 0.05), whereas plasma urea, glucagon, and cortisol (d 2) concentrations as well as hepatic pyruvate carboxylase mRNA level and activity were lower (P < 0.05) in group C than in group F. Orally administered [U-(13)C]-glucose in blood was higher (P < 0.05) but FPU tended to be lower (P < 0.1) in group C than in group F. The improved glucose status in group C resulted from enhanced oral glucose absorption. Metabolic and endocrine changes pointed to elevated amino acid degradation in group F, presumably to provide substrates to meet energy requirements and to compensate for impaired oral glucose uptake.

Nutritional programming of gastrointestinal tract development. Is the pig a good model for man?

The consequences of early-life nutritional programming in man and other mammalian species have been studied chiefly at the metabolic level. Very few studies, if any, have been performed in the gastrointestinal tract (GIT) as the target organ, but extensive GIT studies are needed since the GIT plays a key role in nutrient supply and has an impact on functions of the entire organism. The possible deleterious effects of nutritional programming at the metabolic level were discovered following epidemiological studies in human subjects, and confirmed in animal models. Investigating the impact of programming on GIT structure and function would need appropriate animal models due to ethical restrictions in the use of human subjects. The aim of the present review is to discuss the use of pigs as an animal model as a compromise between ethically acceptable animal studies and the requirement of data which can be interpolated to the human situation. In nutritional programming studies, rodents are the most frequently used model for man, but GIT development and digestive function in rodents are considerably different from those in man. In that aspect, the pig GIT is much closer to the human than that of rodents. The swine species is closely comparable with man in many nutritional and digestive aspects, and thus provides ample opportunity to be used in investigations on the consequences of nutritional programming for the GIT. In particular, the 'sow-piglets' dyad could be a useful tool to simulate the 'human mother-infant' dyad in studies which examine short-, middle- and long-term effects and is suggested as the reference model.

Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves.

Glucocorticoids and colostrum feeding influence postnatal maturation of the somatotropic axis. We have tested the hypothesis that dexamethasone (Dexa) affects the somatotropic axis in neonatal calves dependent on colostrum intake. Calves were fed either with colostrum or with a milk-based formula (n = 14/group), and, in each feeding group, one-half of the calves were treated with Dexa (30 micro g. kg body wt-1. day-1). Pre- and postprandial blood samples were taken on days 1, 2, 4, and 5, and liver samples were taken on day 5 of life. Dexa increased insulin-like growth factor (IGF)-I, but decreased growth hormone (GH) and IGF-binding protein (IGFBP)-1 and -2 plasma concentrations and increased GH receptor (GHR) mRNA levels in liver. Dexa increased IGF-I mRNA levels only in formula-fed calves and increased hepatic GHR binding capacity, but only in colostrum-fed calves. Colostrum feeding decreased IGFBP-1 and -2 plasma concentrations and hepatic IGFBP-2 and -3 mRNA levels. In conclusion, Dexa and colostrum feeding promoted maturation of the somatotropic axis. Dexa effects partly depended on whether colostrum was fed or not.