Search

Cryptosporidiosis is one of the main causes of diarrhea in children and young livestock. The interaction of the parasite with the intestinal host cells has not been characterized thoroughly yet but may be affected by the nutritional demand of the parasite. Hence, we aimed to investigate the impact of C. parvum infection on glucose metabolism in neonatal calves. Therefore, N = 5 neonatal calves were infected with C. parvum on the first day of life, whereas a control group was not (N = 5). The calves were monitored clinically for one week, and glucose absorption, turnover and oxidation were assessed using stable isotope labelled glucose. The transepithelial transport of glucose was measured using the Ussing chamber technique. Glucose transporters were quantified on gene and protein expression level using RT-qPCR and Western blot in the jejunum epithelium and brush border membrane preparations. Plasma glucose concentration and oral glucose absorption were decreased despite an increased electrogenic phlorizin sensitive transepithelial transport of glucose in infected calves. No difference in the gene or protein abundance of glucose transporters, but an enrichment of glucose transporter 2 in the brush border was observed in the infected calves. Furthermore, the mRNA for enzymes of the glycolysis pathway was increased indicating enhanced glucose oxidation in the infected gut. In summary, C. parvum infection modulates intestinal epithelial glucose absorption and metabolism. We assume that the metabolic competition of the parasite for glucose causes the host cells to upregulate their uptake mechanisms and metabolic machinery to compensate for the energy losses.

Postnatal metabolism depends on maturation of key metabolic pathways around birth. In this regard, endogenous glucose production is impaired in calves born preterm. Concerning protein metabolism, the rates of protein turnover are greater during the neonatal period than at any other period of postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system (UPS) are considered as the major regulators of cellular protein turnover. The objectives of this study were to investigate (1) the changes in plasma AA profiles, (2) the mRNA abundance of mTOR signaling and UPS-related genes in skeletal muscle, and (3) the mRNA abundance of branched-chain AA (BCAA) catabolic enzymes in skeletal muscle and adipose tissue in neonatal calves with different degree of maturation during the transition to extrauterine life. Calves (n = 7/treatment) were born either preterm (PT; delivered by cesarean section 9 d before term) or at term (T; spontaneous vaginal delivery) and were left unfed for 1 d. Calves in treatment TC were also spontaneously born but were fed colostrum and transition milk for 4 d. Blood samples were collected from all calves at birth and at 24 h of life. Additional blood samples were taken 2 h after feeding (26 h of life) for PT and T calves, and on d 4 of life for TC, to determine plasma glucose, urea, and AA. Tissue samples from 3 muscles [M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM)], and kidney fat were collected following euthanasia at 26 h after birth (PT, T) or on d 4 of life (TC) at 2 h after feeding. The concentrations of the majority of plasma AA (Ala, Gln, Asn, Cit, Lys, Orn, Thr, and Tyr), nonessential AA, and total AA were greater during the first 24 h and also before and 2 h after feeding in PT than in T. The ratio of plasma BCAA to the aromatic AA (Tyr and Phe) was greatest in TC, followed by T, and least in PT. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in MLD and MM was greater in PT and T than in TC. The mRNA abundance of muscle-specific ligases FBXO32 (F-box only protein 32) in the 3 different skeletal muscles and TRIM63 (tripartite motif containing 63) in MLD was greater in PT and T than in TC; in MM, TRIM63 mRNA was greatest in PT. The mRNA for BCKDHA and BCKDHB (the α and β polypeptide of branched-chain α-keto acid dehydrogenase) in kidney fat was elevated in PT and T compared with TC, suggesting a possible enhancement of BCAA oxidation as energy source to cover the energetic and nutritional postnatal demands in PT and T in a starved state. The increased abundances of mTOR-associated signaling factors and muscle-specific ligase mRNA indicate a greater rate of protein turnover in muscles of PT and T in a starved state. Elevated plasma concentrations of several AA may result from enhanced muscle proteolysis and impaired conversion to glucose in the liver of PT 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 [β-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.

We tested the hypothesis that the maternal supply of essential fatty acids (EFA), especially α-linolenic acid, and conjugated linoleic acid (CLA), affects glucose metabolism, the endocrine regulation of energy metabolism and growth, and the intestinal development of neonatal calves. We studied calves from dams that received an abomasal infusion of 76 g/d coconut oil (CTRL; n = 9), 78 g/d linseed oil and 4 g/d safflower oil (EFA; n = 9), 38 g/d Lutalin (BASF SE) containing 27% cis-9,trans-11 and trans-10,cis-12 CLA (CLA; n = 9), or a combination of EFA and CLA (EFA+CLA; n = 11) during the last 63 d of gestation and early lactation. Calves received colostrum and transition milk from their own dam for the first 5 d of life. Insulin-like growth factor (IGF)-I, leptin, and adiponectin concentrations were measured in milk. Blood samples were taken before first colostrum intake, 24 h after birth, and from d 3 to 5 of life before morning feeding to measure metabolic and endocrine traits in plasma. On d 3 of life, energy expenditure was evaluated by a bolus injection of NaH13CO3 and determination of CO2 appearance rate. On d 4, additional blood samples were taken to evaluate glucose first-pass uptake and 13CO2 enrichment after [13C6]-glucose feeding and intravenous [6,6-2H2]-glucose bolus injection, as well as postprandial changes in glucose, nonesterified fatty acids (NEFA), insulin, and glucagon. On d 5, calves were killed 2 h after feeding and samples of small intestinal mucosa were taken for histomorphometric measurements. The concentrations of IGF-I, adiponectin, and leptin in milk decreased during early lactation in all groups, and the concentrations of leptin in first colostrum was higher in EFA than in CTRL cows. Plasma glucose concentration before first colostrum intake was higher in EFA calves than in non-EFA calves and was lower in CLA calves than in non-CLA calves. Plasma IGF-I concentration was higher on d 1 before colostrum intake in EFA calves than in EFA+CLA calves and indicated an overall CLA effect, with lower plasma IGF-I in CLA than in non-CLA calves. Postprandial NEFA concentration was lowest in EFA and CLA calves. The postprandial rise in plasma insulin was higher in EFA than in non-EFA calves. Plasma adiponectin concentration increased from d 1 to d 2 in all groups and was higher on d 3 in CLA than in non-CLA calves. Plasma leptin concentration was higher on d 4 and 5 in EFA than in non-EFA calves. Maternal fatty acid treatment did not affect energy expenditure and first-pass glucose uptake, but glucose uptake on d 4 was faster in EFA than in non-EFA calves. Crypt depth was lower, and the ratio of villus height to crypt depth was higher in the ilea of CLA than non-CLA calves. Elevated plasma glucose and IGF-I in EFA calves immediately after birth may indicate an improved energetic status in calves when dams are supplemented with EFA. Maternal EFA and CLA supplementation influenced postprandial metabolic changes and affected factors related to the neonatal insulin response.

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 β-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.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid (ALA), and conjugated linoleic acid (CLA) supplementation on metabolic and endocrine traits related to energy metabolism, including the somatotropic axis, in mid-lactation dairy cows. Four cows (126 ± 4 d in milk) were used in a dose-escalation study design and were abomasally infused with coconut oil (CTRL; 38.3 g/d; providing saturated fatty acids), linseed and safflower oils (EFA; 39.1 and 1.6 g/d; n-6:n-3 FA ratio = 1:3), Lutalin (CLA; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d of each), or EFA and CLA (EFA+CLA) for 6 wk. The initial dosage was doubled twice after 2 wk, resulting in 3 dosages (dosages 1, 2, and 3). Each cow received each fat treatment at different times. Cows were fed with a corn silage-based total mixed ration providing a low-fat content and a high n-6:n-3 fatty acid ratio. Plasma concentrations of metabolites and hormones (insulin-like growth factor-binding proteins only on wk 0 and 6) were analyzed at wk 0, 2, 4, and 6 of each treatment period. Liver biopsies were taken before starting the trial and at wk 6 of each treatment period to measure hepatic mRNA abundance of genes linked to glucose, cholesterol and lipid metabolism, and the somatotropic axis. The changes in the milk and blood fatty acid patterns and lactation performance of these cows have already been published in a companion paper. The plasma concentration of total cholesterol increased with dosage in all groups, except CLA, reaching the highest levels in EFA+CLA and CTRL compared with CLA. The high-density lipoprotein cholesterol plasma concentration increased in CTRL and was higher than that in EFA and CLA, whereas the concentration of low-density lipoprotein cholesterol increased in a dose-dependent manner in EFA and EFA+CLA, and was higher than that in CLA. Hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA synthase 1 was upregulated in all groups but was highest in EFA+CLA. Expression of sterol regulatory element-binding factor 1 tended to be lowest due to EFA treatment, whereas expression of long chain acyl-CoA-synthetase was lower in EFA than in CTRL. Hepatic mRNA expression of GHR1A tended to be higher in EFA+CLA than in CTRL. The plasma concentration of insulin-like growth factor I increased in CLA, and the plasma IGFBP-2 concentration was lower in EFA+CLA than in CTRL at wk 6. The plasma concentration of adiponectin decreased in EFA+CLA up to dosage 2. Plasma concentrations of albumin and urea were lower in CLA than in CTRL throughout the experimental period. Supplementation with EFA and CLA affected cholesterol and lipid metabolism and their regulation differently, indicating distinct stimulation after the combined EFA and CLA treatment. The decreased IGFBP-2 plasma concentration and upregulated hepatic mRNA abundance of GHR1A in EFA+CLA-supplemented cows indicated the beneficial effect of the combined EFA and CLA treatment on the somatotropic axis in mid-lactation dairy cows. Moreover, supplementation with CLA might affect protein metabolism in dairy cows.

Feeding management of the postnatal and preweaning calf has an important impact on calf growth and development during this critical period and affects the health and well-being of the calves. After birth, an immediate and sufficient colostrum supply is a prerequisite for successful calf rearing. Colostrum provides high amounts of nutrient as well as non-nutrient factors that promote the immune system and intestinal maturation of the calf. The maturation and function of the neonatal intestine enable the calf to digest and absorb the nutrients provided by colostrum and milk. Therefore, colostrum intake supports the start of anabolic processes in several tissues, stimulating postnatal body growth and organ development. After the colostrum feeding period, an intensive milk feeding protocol, that is, at least 20% of BW milk intake/day, is required to realise the calf potential for growth and organ development during the preweaning period. Insufficient milk intake delays postnatal growth and may have detrimental effects on organ development, for example, the intestine and the mammary gland. The somatotropic axis as the main postnatal endocrine regulatory system for body growth is stimulated by the intake of high amounts of colostrum and milk and indicates the promotion of anabolic metabolism in calves. The development of the forestomach is an important issue during the preweaning period in calves, and forestomach maturation is best achieved by solid feed intake. Unfortunately, intensive milk-feeding programmes compromise solid feed intake during the first weeks of life. In the more natural situation for beef calves, when milk and solid feed intake occurs at the same time, calves benefit from the high milk intake as evidenced by enhanced body growth and organ maturation without impaired forestomach development during weaning. To realise an intensive milk-feeding programme, it is recommended that the weaning process should not start too early and that solid feed intake should be at a high extent despite intensive milk feeding. A feeding concept based on intensive milk feeding prevents hunger and abnormal behaviour of the calves and fits the principles of animal welfare during preweaning calf rearing. Studies on milk performance in dairy cows indicate that feeding management during early calf rearing influences lifetime performance. Therefore, an intensive milk-feeding programme affects immediate as well as long-term performance, probably by programming metabolic pathways during the preweaning period.

International audience; 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.

International audience; 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.

Long non-coding RNAs (lncRNAs) holdgene regulatorypotential, but require substantial further functional annotation in livestock. Applying two metabogenomic approaches by combiningtranscriptomicandmetabolomicanalyses, we aimed to identify lncRNAs with potential regulatory function for divergent nutrient partitioning of lactating crossbred cows and to establish metabogenomic interaction networks comprising metabolites, genes and lncRNAs. Through correlation analysis of lncRNA expression with transcriptomic and metabolomic data, we unraveled lncRNAs that have a putative regulatory role in energy and lipid metabolism, the urea andtricarboxylic acid cycles, andgluconeogenesis. EspeciallyFGF21,which correlated with a plentitude of differentially expressed genes, differentially abundant metabolites, as well as lncRNAs, suggested itself as a key metabolic regulator. Notably, lncRNAs in close physical proximity to coding-genes as well as lncRNAs with natural antisense transcripts appear to perform a fine-tuning function in gene expression involved in metabolic pathways associated with different nutrient partitioning phenotypes.

Simple Summary Fatty acids play an important role in the regulation of inflammation and oxidative stress. The birth and the neonatal period are characterized by a high risk of inflammation and an increased production of reactive oxygen species in the calf. The present study deals with the effects of a different maternal fatty acid supply including the supplementation of saturated fatty acids by coconut oil, essential fatty acids, conjugated linoleic acid or a combination of essential fatty acids and conjugated linoleic acid on the immunological and oxidative as well as anti-oxidative status of neonatal calves. Maternal essential fatty acid as well as conjugated linoleic acid supply affected the inflammatory response and the oxidative and anti-oxidative status of the neonatal offspring. Essential fatty acids might have beneficial effects on the prevention of dysregulated inflammation after birth and reduced the plasma bilirubin concentrations in this period. Conjugated linoleic acid and saturated fatty acids might increase the inflammatory response. Similarly, plasma bilirubin increased, which in part might serve as a protector against oxidative stress in the early phase after birth. Abstract 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-β 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-β 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.

Intensive milk feeding and butyrate supplementation in calves stimulate body growth and affect gastrointestinal development. The aim of the present study was to investigate the synergistic effects of ad libitum milk replacer (MR) feeding and butyrate supplementation of MR on rumen and small intestinal growth and on gene expression in the small intestine related to growth and energy metabolism at weaning. Male Holstein calves (n = 32) received colostrum from birth to d 3 of age and MR either ad libitum (Adl) or restrictively (Res; 6 L of MR/d; 12.5% solids) with (AdlB+, ResB+) or without (AdlB−, ResB−) 0.24% butyrate from d 4 until wk 8 of age. From wk 9 to 10, all calves were weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. At d 80, calves were slaughtered, volatile fatty acids were measured in rumen fluid, and rumen and small intestine samples were taken for histomorphometric measurements. The expression of mRNA associated with the local insulin-like growth factor (IGF) system and glucose metabolism as well as lactase and maltase activities were measured in the intestinal mucosa. The small intestine was 3 m longer in Adl than in Res. In the atrium ruminis, papilla width was greater in Res than in Adl. Villus circumference, cut surface, and height in the duodenum, proximal jejunum, and ileum were greater in Adl than in Res and in the proximal, mid, and distal jejunum and ileum were greater in calves treated with butyrate. Crypt depth in the duodenum and proximal jejunum was greater in Adl than in Res and in the ileum was smaller in calves treated with butyrate. The villus height:crypt depth ratio was greatest in AdlB+ calves. In the proximal and mid jejunum, IGF1 mRNA abundance was lower in calves treated with butyrate. In the proximal jejunum, INSR mRNA abundance was greater in Res than in Adl. The abundance of PCK2 mRNA was greater in Res than in Adl in the duodenum and was greatest in ResB− in the mid jejunum. Lactase activity tended to be greater in Res than in Adl and after butyrate treatment in the proximal jejunum. The results indicated an elevated growth of the small intestinal mucosa at weaning due to intensive milk feeding and butyrate supplementation, and the local IGF system was involved in intestinal growth regulation. Rumen development was not affected by butyrate supplementation of MR and was slightly delayed due to ad libitum MR feeding.

Our aim was to compare the energy balance estimated (EBest) according to equations published by various energy feeding systems (German Society for Nutrition Physiology, French National Institute for Agricultural Research, and US National Research Council) and the EB calculated by use of calorimetrically measured heat production (EBhp) of 20 high-yielding (≥10,000 kg/305 d) German Holstein cows at -4 (pregnant, nonlactating) and 2 wk (early lactation) relative to parturition. In addition to heat production, feed and water intake, physical activity (including standing-lying behavior), body weight, body condition score, body temperature, plasma concentrations of fatty acids and β-hydroxybutyrate, milk yield, and milk composition were measured to characterize the metabolic status. The EBhp was balanced [2.74 ± 4.09 MJ of metabolizable energy (ME)/d; ±standard error] before calving, but strongly negative (-84.7 ± 7.48 MJ of ME/d) at wk 2 of lactation. At both time points, EBhp and EBest differed significantly. On average, the equations overestimated the antepartum EB by 33 MJ of ME/d and underestimated the postpartum negative EB by 67 MJ of ME/d, respectively. Because the same ME intake and energy-corrected milk values were used for calculation of EBest and EBhp in our study, we considered that the factors (0.488 to 0.534 MJ of ME/kg0.75) currently used to calculate the ME requirements for maintenance probably underestimate the needs of high-yielding dairy cows, particularly during early lactation. In accord, heat production values determined under standard conditions of thermoneutrality and locomotion restriction amounted to 0.76 ± 0.02 MJ of ME/kg0.75 (4 wk antepartum) and 1.02 ± 0.02 MJ of ME/kg0.75 (2 wk postpartum), respectively. The expected positive correlation between EBhp and DMI was observed in pregnant cows only; however, a bias of 26 MJ of ME/d between mean actual energy intake and ME intake predicted according to German Society for Nutrition Physiology was found in cows at wk 4 antepartum. At both investigated time points, mobilization of tissue energy reserves (reflected by plasma fatty acid concentration) was related to EBhp. In early lactating cows, metabolic body weight (kg0.75) and the percentage of milk fat showed the strongest correlation (correlation coefficient = -0.70 and -0.73) to EBhp. Our findings must be taken into account when experimental data are interpreted because the true energy status might be significantly overestimated when EBest is used.

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.

Prebiotic supplements and high-protein (HP) diets reduce body weight and modulate intestinal microbiota. Our aim was to elucidate the combined effect of an inulin/oligofructose (FOS) and HP diet on body weight gain, energy metabolism and faecal microbiota. Forty male C57BL/6NCrl mice were fed a control (C) diet for 2 weeks and allocated to a C or HP (40 % protein) diet including no or 10 % inulin/FOS (C + I and HP + I) for 4 weeks. Inulin/FOS was added in place of starch and cellulose. Body weight, food intake, faecal energy and nitrogen were determined. Indirect calorimetry and faecal microbiota analysis were performed after 3 weeks on diets. Body weight gain of HP-fed mice was 36 % lower than HP + I- and C-fed mice (P< 0⋅05). Diet digestibility and food conversion efficiency were higher in HP + I- than HP-fed mice (P< 0⋅01), while food intake was comparable between groups. Total energy expenditure (heat production) was 25 % lower in HP + I- than in C-, HP- and C + I-fed mice (P< 0⋅001). Carbohydrate oxidation tended to be 24 % higher in HP- than in HP + I-fed mice (P< 0⋅05). Faecal nitrogen excretion was 31–45 % lower in C-, C + I- and HP + I- than in HP-fed mice (P< 0⋅05). FaecalBacteroides–PrevotellaDNA was 2⋅3-fold higher in C + I- and HP + I- relative to C-fed mice (P< 0⋅05), butClostridium leptumDNA abundances was 79 % lower in HP + I- than in HP-fed mice (P< 0⋅05). We suggest that the higher conversion efficiency of dietary energy of HP + I but not C + I-fed mice is caused by higher digestibility and lower heat production, resulting in increased body mass.

Sufficient glucose availability is crucial for exploiting the genetic potential of milk production during early lactation, and endocrine changes are mainly related to repartitioning of nutrient supplies toward the mammary gland. Long-chain fatty acids, such as essential fatty acids (EFA) and conjugated linoleic acid (CLA), have the potential to improve negative energy balance and modify endocrine changes. In the present study, the hypothesis that combined CLA and EFA treatment supports glucose metabolism around the time of calving and stimulates insulin action and the somatotropic axis in cows in an additive manner was tested. Rumen-cannulated German Holstein cows (n = 40) were investigated from wk 9 antepartum (AP) until wk 9 postpartum (PP). The cows were abomasally supplemented with coconut oil (CTRL, 76 g/d); 78 g/d of linseed and 4 g/d of safflower oil (EFA); Lutalin (CLA, isomers cis-9,trans-11 and trans-10,cis-12 CLA, each 10 g/d); or the combination of EFA+CLA. Blood samples were collected several times AP and PP to determine the concentrations of plasma metabolites and hormones related to glucose metabolism and the somatotropic axis. Liver tissue samples were collected several days AP and PP to measure glycogen concentration and the mRNA abundance of genes related to gluconeogenesis and the somatotropic axis. On d 28 AP and 21 PP, endogenous glucose production (eGP) and glucose oxidation (GOx) were measured via tracer technique. The concentration of plasma glucose was higher in CLA than in non-CLA-treated cows, and the plasma β-hydroxybutyrate concentration was higher in EFA than in non-EFA cows on d 21 PP. The eGP increased from AP to PP with elevated eGP in EFA and decreased eGP in CLA-treated cows; GOx was lower in CLA than in CTRL on d 21 PP. The plasma insulin concentration decreased after calving in all groups and was higher in CLA than in non-CLA cows at several time points. Plasma glucagon and cortisol concentrations on d 21 PP were lower in CLA than non-CLA groups. The glucagon/insulin and glucose/insulin ratios were higher in CTRL than in CLA group during the transition period. Plasma IGF-I concentration was lower in EFA than non-EFA cows on d 42 AP and was higher during the dry period and early lactation in CLA than in non-CLA cows. The IGF binding protein (IGFBP)-3/-2 ratio in blood plasma was higher in CLA than in non-CLA cows. Hepatic glycogen concentration on d 28 PP was higher, but the mRNA abundance of PC and IGFBP2 was lower in CLA than non-CLA cows on d 1 PP. The EFA treatment decreased the mRNA abundance of IGFBP3 AP and PCK1, PCK2, G6PC, PCCA, HMGCS2, IGFBP2, and INSR at several time points PP. Results indicated elevated concentrations of plasma glucose and insulin along with the stimulation of the somatotropic axis in cows treated with CLA, whereas EFA treatment stimulated eGP but not mRNA abundance related to eGP PP. The systemic effects of the combined EFA+CLA treatment were very similar to those of CLA treatment, but the effects on hepatic gene expression partially corresponded to those of EFA treatment.

Sufficient maternal supply of essential fatty acids (EFA) to neonatal calves is critical for calf development. In the modern dairy cow, EFA supply has shifted from α-linolenic acid (ALA) to linoleic acid (LA) due to the replacement of pasture feeding by corn silage-based diets. As a consequence of reduced pasture feeding, conjugated linoleic acid (CLA) provision by rumen biohydrogenation was also reduced. The present study investigated the fatty acid (FA) status and performance of neonatal calves descended from dams receiving corn silage-based diets and random supplementation of either 76 g/d coconut oil (CTRL; n = 9), 78 g/d linseed oil and 4 g/d safflower oil (EFA; n-6/n-3 FA ratio = 1:3; n = 9), 38 g/d Lutalin (BASF SE, Ludwigshafen, Germany) providing 27% cis-9,trans-11 and trans-10,cis-12 CLA, respectively (CLA; n = 9), or a combination of EFA and CLA (EFA+CLA; n = 11) in the last 9 wk before parturition and following lactation. The experimental period comprised the first 5 d of life, during which calves received colostrum and transition milk from their own dam. The nutrient compositions of colostrum and transition milk were analyzed. Plasma samples were taken after birth and before first colostrum intake and on d 5 of life for FA analyses of the total plasma fat and lipid fractions. Maternal EFA and CLA supplementation partly affected colostrum and transition milk composition but did not change the body weights of calves. Most EFA in calves were found in the phospholipid (PL) and cholesterol ester (CE) fractions of the plasma fat. Maternal EFA supplementation increased the percentage of ALA in all lipid fractions of EFA and EFA+CLA compared with CTRL and CLA calves on d 1 and 5, and the increase was much greater on d 5 than on d 1. The LA concentration increased from d 1 to 5 in the plasma fat and lipid fractions of all groups. The concentrations of docosapentaenoic acid, docosahexaenoic acid, and arachidonic acid in plasma fat were higher on d 1 than on d 5, and the percentage of n-3 metabolites was mainly increased in PL if dams received EFA. The percentage of cis-9,trans-11 CLA was higher in the plasma fat of EFA+CLA than CTRL calves after birth. By d 5, the percentages of both CLA isomers increased, leading to higher proportions in plasma fat of CLA and EFA+CLA than in CTRL and EFA calves. Elevated cis-9,trans-11 CLA enrichment was observed on d 5 in PL, CE, and triglycerides of CLA-treated calves, whereas trans-10,cis-12 CLA could not be detected in individual plasma fractions. These results suggest that an altered maternal EFA and CLA supply can reach the calf via the placenta and particularly via the intake of colostrum and transition milk, whereas the n-3 and n-6 FA metabolites partly indicated a greater transfer via the placenta. Furthermore, the nutrient supply via colostrum and transition milk might be partly modulated by an altered maternal EFA and CLA supply but without consequences on calf performance during the first 5 d of life.

Long non-coding RNAs (lncRNAs) can influence transcriptional and translational processes in mammalian cells and are associated with various developmental, physiological and phenotypic conditions. However, they remain poorly understood and annotated in livestock species. We combined phenotypic, metabolomics and liver transcriptomic data of bulls divergent for residual feed intake (RFI) and fat accretion. Based on a project-specific transcriptome annotation for the bovine reference genome ARS-UCD.1.2 and multiple-tissue total RNA sequencing data, we predicted 3590 loci to be lncRNAs. To identify lncRNAs with potential regulatory influence on phenotype and gene expression, we applied the regulatory impact factor algorithm on a functionally prioritized set of loci (n = 4666). Applying the algorithm of partial correlation and information theory, significant and independent pairwise correlations were calculated and co-expression networks were established, including plasma metabolites correlated with lncRNAs. The network hub lncRNAs were assessed for potential cis-actions and subjected to biological pathway enrichment analyses. Our results reveal a prevalence of antisense lncRNAs positively correlated with adjacent protein-coding genes and suggest their participation in mitochondrial function, acute phase response signalling, TCA-cycle, fatty acid &beta, oxidation and presumably gluconeogenesis. These antisense lncRNAs indicate a stabilizing function for their cis-correlated genes and a putative regulatory role in gene expression.

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 E1β (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.

Haptoglobin (Hp), one of the major positive acute phase proteins in cattle, is released in response to proinflammatory cytokines. Colostrum intake might influence the response of the innate immune system, including Hp gene expression. Thus, we hypothesized that plasma concentrations and tissue mRNA expression of Hp in neonatal calves might be influenced by early nutrition in the neonatal calf and would thus be greater if receiving colostrum compared with milk-based formula. Two trials were performed. In trial 1, German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. Blood was sampled from d 1 to 4 before morning feeding and before and 2 h after feeding on d 4. Tissue samples from liver, kidney fat, duodenum, and ileum were collected after slaughter on d 4 at 2 h after feeding. In trial 2, calves born preterm (n = 7) or at term (n = 7) received colostrum only at 24 h post natum. Blood was sampled at birth, and before and 2 h after feeding. Tissue samples from liver and kidney fat were collected after slaughter at 26 h after birth. Blood plasma, colostrum, and formula Hp concentrations were determined using a competitive ELISA. Tissue expression of Hp mRNA was quantified by real-time quantitative PCR. The formula contained much less Hp (≤0.5 µg/mL) than colostrum (69.3, 93.9, and 20.4 µg/mL from d 1 to d 3, respectively). In trial 1, before colostrum or formula feeding, plasma concentrations of Hp were comparable in both groups. Plasma Hp increased in FOR after feeding, resulting in greater or a trend for greater plasma Hp concentrations in FOR than in COL calves. The mRNA abundance of Hp in liver and kidney fat was 3- and 2.2-fold greater in FOR than in COL calves, respectively, whereas duodenal and ileal abundance of Hp mRNA did not differ between groups. In trial 2, plasma Hp concentrations decreased slightly over time in term calves, but they did not differ in both groups before and 2 h after feeding on d 2. The abundance of Hp mRNA in liver was 5.3-fold greater in term than in preterm calves, whereas its abundance in kidney fat did not differ between groups. Contrasting our hypothesis, formula, but not colostrum feeding was associated with greater Hp mRNA abundance in liver and adipose tissue, indicating that the response of innate immune system seems to be modulated by formula feeding because of the lack of immunoglobulin intake. The lower hepatic abundance of Hp mRNA in preterm calves than in term calves may indicate lower synthetic capacity of the liver for Hp in preterm calves shortly after birth.

Dairy cows are exposed to increased inflammatory processes in the transition period from late pregnancy to early lactation. Essential fatty acids (EFA) and conjugated linoleic acid (CLA) are thought to modulate the inflammatory response in dairy cows. The present study investigated the effects of a combined EFA and CLA infusion on the fatty acid (FA) status in plasma lipids, and whether changes in the FA pattern were associated with the acute phase and inflammatory response during late pregnancy and early lactation. Rumen-cannulated Holstein cows (n = 40) were assigned from wk 9 antepartum to wk 9 postpartum to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 g/d of linseed oil and 4 g/d of safflower oil; ratio of oils = 19.5:1; n-6:n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; isomers cis-9,trans-11 and trans-10,cis-12; each 10 g/d), or both (EFA+CLA). Blood samples were taken to measure changes in FA in blood plasma on d -63, -42, 1, 28, and 56, and in plasma lipid fractions (cholesterol esters, free fatty acids, phospholipids, and triglycerides) on d -42, 1, and 56 relative to calving, and in erythrocyte membrane (EM) on d 56 after calving. Traits related to the acute phase response and inflammation were measured in blood throughout the study. Liver samples were obtained for biopsy on d -63, -21, 1, 28, and 63 relative to calving to measure the mRNA abundance of genes related to the inflammatory response. The concentrations of α-linolenic acid and n-3 FA metabolites increased in lipid fractions (especially phospholipids) and EM due to EFA supplementation with higher α-linolenic acid but lower n-3 metabolite concentrations in EFA+CLA than in EFA treatment only. Concentration of linoleic acid decreased in plasma fat toward calving and increased during early lactation in all groups. Concentration of plasma arachidonic acid was lower in EFA- than in non-EFA-treated groups in lipid fractions and EM. The cis-9,trans-11 CLA increased in all lipid fractions and EM after both CLA treatments. Plasma haptoglobin was lowered by EFA treatment before calving. Plasma bilirubin was lower in EFA and CLA than in CTRL at calving. Plasma concentration of IL-1β was higher in EFA than in CTRL and EFA+CLA at certain time points before and after calving. Plasma fibrinogen dropped faster in CLA than in EFA and EFA+CLA on d 14 postpartum. Plasma paraoxonase tended to be elevated by EFA treatment, and was higher in EFA+CLA than in CTRL on d 49. Hepatic mRNA abundance revealed time changes but no treatment effects with respect to the inflammatory response. Our data confirmed the enrichment of n-3 FA in EM by EFA treatment and the inhibition of n-3 FA desaturation by CLA treatment. The elevated n-3 FA status and reduced n-6:n-3 ratio by EFA treatment indicated a more distinct effect on the inflammatory response during the transition period than the single CLA treatment, and the combined EFA+CLA treatment caused minor additional changes on the anti-inflammatory response.

Long noncoding RNAs (lncRNAs) emerged as important regulatory component of mechanisms involved in gene expression, chromatin modification and epigenetic processes, but they are rarely annotated in the bovine genome. Our study monitored the jejunum transcriptome of German Holstein calves fed two different milk diets using transcriptome sequencing (RNA-seq). To identify potential lncRNAs within the pool of unknown transcripts, four bioinformatic lncRNA prediction tools were applied. The intersection of the alignment-free lncRNA prediction tools (CNCI, PLEK and FEELnc) predicted 1,812 lncRNA transcripts concordantly comprising a catalogue of 1,042 putative lncRNA loci expressed in the calves’ intestinal mucosa. Nine lncRNA loci were differentially expressed (DE lncRNAs) between both calf groups. To elucidate their biological function, we applied a systems biology approach that combines weighted gene co-expression network analysis with functional enrichment and biological pathway analysis. Four DE lncRNAs were found to be strongly correlated with a gene network module (GNM) enriched for genes from canonical pathways of remodeling of epithelial adherens junction, tight junction and integrin signaling. Another DE lncRNA was strongly correlated with a GNM enriched for genes associated with energy metabolism and maintaining of cellular homeostasis with a focus on mitochondrial processes. Our data suggest that these DE lncRNAs may play potential regulatory roles in modulating biological processes associated with energy metabolism pathways and cellular signaling processes affecting the barrier function of intestinal epithelial cells of calves in response to different feeding regimens in the pre-weaning period.

The development of the gastrointestinal tract in newborn calves is essential for sufficient nutrient uptake. An intensive milk feeding during the neonatal period may impair the rumen development in calves. The aim of this study was to investigate effects of milk replacer (MR) feeding in unlimited amounts for the first 5 wk of age on the gastrointestinal growth and development in preruminant calves at wk 9 of age. Twenty-eight newborn Holstein and Holstein × Charolais crossbred calves (19 male and 9 female) were fed MR ad libitum (ADLIB) or in restricted amounts (6 L per day; RES) until wk 5 of age. Thereafter, the MR intake of ADLIB was gradually reduced at wk 6 and 7, and all calves received 6 L of MR per day until wk 9 of age. In wk 9, calves were slaughtered and carcass and organ weight as well as rumen papilla size in the atrium, ventral sac, and ventral blind sac, and villus size of the mucosa in the small intestine (duodenum; proximal, mid, and distal jejunum; and ileum) were determined. The expression of mRNA associated with the local insulin-like growth factor (IGF) system was measured in the rumen epithelium. Ad libitum MR feeding increased MR intake and growth in ADLIB without influencing concentrate intake compared with RES. Carcass weight in wk 9 was greater in ADLIB than in RES. The density of the rumen papillae in the atrium and ventral blind sac was greater in RES than in ADLIB calves, but surface area of the epithelium was not different between groups in the investigated regions of the rumen. The mRNA abundance of IGF1 in the atrium tended to be greater and the IGFR1 mRNA abundance in the ventral sac tended to be lower in the ADLIB than in the RES feeding group. The rumen pH and volatile fatty acid concentrations were not affected by MR feeding intensity. In mid-jejunum, villus circumference was greater in ADLIB than in RES calves. In the distal jejunum, villus surface area and the villus height/crypt depth ratio were greater and the villus circumference and height tended to be greater, whereas crypt depth was smaller in ADLIB than in RES calves. The findings from this study indicate that ad libitum MR feeding for 5 wk of age followed by its gradual reduction promotes growth performance without any negative influence on gastrointestinal growth and development in dairy calves at 9 wk of age.

High performing dairy cows experience distinct metabolic stress during periods of negative energy balance. Subclinical disorders of the cow’s energy metabolism facilitate failure of adaptational responses resulting in health problems and reduced performance. The autonomic nervous system (ANS) with its sympathetic and parasympathetic branches plays a predominant role in adaption to inadequate energy and/or fuel availability and mediation of the stress response. Therefore, we hypothesize that indices of heart rate variability (HRV) that reflect ANS activity and sympatho-vagal balance could be early markers of metabolic stress, and possibly useful to predict cows with compromised regulatory capacity. In this study we analysed the autonomic regulation and stress level of 10 pregnant dried-off German Holstein cows before, during and after a 10-h fasting period by using a wide range of HRV parameters. In addition heat production (HP), energy balance, feed intake, rumen fermentative activity, physical activity, non-esterified fatty acids, β-hydroxybutyric acid, cortisol and total ghrelin plasma concentrations, and body temperature (BT) were measured. In all cows fasting induced immediate regulatory adjustments including increased lipolysis (84%) and total ghrelin levels (179%), reduction of HP (−16%), standing time (−38%) and heart rate (−15%). However, by analysing frequency domain parameters of HRV (high-frequency (HF) and low-frequency (LF) components, ratio LF/HF) cows could be retrospectively assigned to groups reacting to food removal with increased or decreased activity of the parasympathetic branch of the ANS. Regression analysis reveals that under control conditions (feeding ad libitum) group differences were best predicted by the nonlinear domain HRV component Maxline (L MAX, R 2=0.76, threshold; TS=258). Compared with cows having L MAX values above TS (>L MAX: 348±17), those with L MAX values below TS (L MAX cows (18.5±0.4 and 47.3 kg/day). From the present study, it seems conceivable that L MAX can be used as a predictive marker to discover alterations in central autonomic regulation that might precede metabolic disturbances.

The objective of the study was to evaluate the effect of prepartum and postpartum (PP) supplementation with 2 isomers of conjugated linoleic acid (CLA) on reproductive parameters and some related metabolic factors in dairy cows. High-producing, multiparous Holstein Friesian cows (n = 60) were allotted to 3 treatment groups: the CLA1 group (n = 20) was supplemented with 70 g of lipid-encapsulated CLA providing 7 g each of cis-9,trans-11 and trans-10,cis-12 CLA from d 21 (d 21) before expected calving until d 7 after artificial insemination (AI), that is, until 77 to 91 d PP; the CLA2 group (n = 20) was supplemented with the same amount of CLA beginning at calving until d 7 after AI; and the control group (n = 20) received an isocaloric, isonitrogenous, and isolipidic diet. Blood samples were taken weekly to measure glucose, insulin, insulin-like growth factor-I (IGF-I), and leptin. Liver biopsy was performed in 10 cows per group for growth hormone receptor 1A and IGF-I mRNA analyses. At d 49 to 63 PP, ovulation was synchronized with the Pre-Synch protocol followed by fixed-time AI. Milk progesterone was monitored from calving until d 35 post-AI. Cows returning to estrus following AI were inseminated. Supplementation with CLA before calving improved the recovery of plasma leptin levels in the early PP period (from the day of calving until wk 3 PP; treatment effect). Later PP (wk 5), plasma IGF-I, and leptin remained significantly higher in both CLA1 and CLA2 groups compared with control, although hepatocellular IGF-I mRNA was not different among groups. Plasma IGF-I levels remained higher in both CLA-treated groups on the day of AI. Growth hormone receptor 1A mRNA levels in hepatic tissue decreased in all groups, reaching a nadir in the first week PP. Days to first PP ovulation did not differ between groups; however, both supplemented groups conceived earlier compared with control (d 97 ± 19, d 97 ± 23, and d 113 ± 30 for CLA1, CLA2, and control, respectively). Plasma progesterone concentration was higher in both supplemented groups on d 2 to 5 following the synchronized ovulation than in controls. We concluded that CLA supplementation around calving alters PP metabolic signals as reflected by higher plasma leptin and IGF-I levels. Conjugated linoleic acid stimulated early luteal function and reduced the PP interval to conception.

Free fatty acid receptors (FFAR) play significant roles in various physiological processes, including energy metabolism, through interaction with their ligands, fatty acids. To determine whether the receptors FFAR1 and FFAR2 are involved in the regulation of liver metabolism during the peripartal period, we selected 13 German Holstein multiparous dairy cows and grouped them as high β-hydroxybutyrate (H-BHB; n = 8) or low β-hydroxybutyrate (L-BHB; n = 5) according to their individual maximum plasma BHB concentration observed within wk 2 or 3 postpartum (H-BHB: >1 mmol/L and L-BHB

Rations including high amounts of corn silage are currently very common in dairy production. Diets with corn silage as forage source result in a low supply of essential fatty acids, such as α-linolenic acid, and may lead to low conjugated linoleic acid (CLA) production. The present study investigated the effects of abomasal infusion of essential fatty acids, especially α-linolenic acid, and CLA in dairy cows fed a corn silage-based diet on performance, milk composition, including fatty acid (FA) pattern, and lipid metabolism from late to early lactation. Rumen-cannulated Holstein cows (n = 40) were studied from wk 9 antepartum to wk 9 postpartum and dried off 6 wk before calving. The cows were assigned to 1 of 4 treatment groups. Cows were abomasally supplemented with coconut oil (CTRL, 76 g/d), linseed and safflower oil (EFA, 78 and 4 g/d; linseed/safflower oil ratio = 19.5:1; n-6/n-3 FA ratio = 1:3), Lutalin (CLA, 38 g/d; BASF SE, Ludwigshafen, Germany; isomers cis-9,trans-11 and trans-10,cis-12 each 10 g/d) or EFA+CLA. Milk composition was analyzed weekly, and blood samples were taken several times before and after parturition to determine plasma concentrations of metabolites related to lipid metabolism. Liver samples were obtained by biopsy on d 63 and 21 antepartum and on d 1, 28, and 63 postpartum to measure triglyceride concentration. Body composition was determined after slaughter. Supplementation of CLA reduced milk fat concentration, increased body fat mass, and improved energy balance (EB) in late and early lactation, but EB was lowest during late lactation in the EFA group. Cows with CLA treatment alone showed an elevated milk citrate concentration in early lactation, whereas EFA+CLA did not reveal higher milk citrate but did have increased acetone. Milk protein was increased in late lactation but was decreased in wk 1 postpartum in CLA and EFA+CLA. Milk urea was reduced by CLA treatment during the whole period. After calving, the increase of nonesterified fatty acids in plasma was less in CLA groups; liver triglycerides were raised lowest at d 28 in CLA groups. Our data confirm an improved metabolic status with CLA but not with exclusive EFA supplementation during early lactation. Increased milk citrate concentration in CLA cows points to reduced de novo FA synthesis in the mammary gland, but milk citrate was less affected in EFA+CLA cows, indicating that EFA supplementation may influence changes in mammary gland FA metabolism achieved by CLA.

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 17β-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.

Colostrum provides high amounts of nutritive and non-nutritive substrates, which are essential for calf nutrition and passive immunization. Colostral growth factors and hormones have beneficial effects on postnatal maturation and may affect substrate utilization and energy expenditure in neonatal calves. We tested the hypothesis that energy metabolism and its endocrine regulation differ during the first 10 d of life in calves fed either colostrum or a milk-based formula with a similar nutrient composition to colostrum, but largely depleted of bioactive substances, for the first 2 d postnatum. Male Holstein calves (n = 18) were fed either pooled colostrum (COL; n = 9) or a milk-based formula (FOR; n = 9) for the first 2 d of life. From d 3 on, all calves received same milk replacer. On d 2 and 7 of life, calves were placed in a respiration chamber for indirect calorimetric measurements to calculate heat production, fat (FOX) and carbohydrate oxidation (COX), as well as respiratory quotient. Blood was sampled on d 1 before first colostrum intake and on d 2, 3, 7, 8, 9, and 10 before morning feeding, to measure plasma concentrations of immunoglobulins, metabolites, and hormones. Additional postprandial blood samples were taken on d 1 and 9 at 30, 60, 120, 240, and 420 min after milk feeding. Liver samples were collected on d 10 of life to determine gene expression related to energy metabolism. Formula-fed calves showed lower plasma concentrations of total protein, immunoglobulins, haptoglobin, leptin, adiponectin, and insulin-like growth factor (IGF) binding protein (IGFBP)-4 during the whole study but temporarily higher plasma concentrations of urea, insulin, glucagon, triglyceride, and cholesterol on the first day after feeding, compared with concentrations in COL. The temporary increase in glucagon, triglyceride, and cholesterol on d 1 reversed on d 2 or 3, showing higher concentrations in COL than in FOR calves. In FOR, IGF-I, IGFBP-2, and IGFBP-3 were lower on d 3 than in COL. Interestingly, FOR calves had higher heat production during respiratory measurements on d 2 and higher body temperature on d 2, 3, and 5 than those of COL. The hepatic mRNA abundance of cytosolic phosphoenolpyruvate carboxykinase was higher in FOR than in COL. Our results indicate that first milk feeding after birth influenced whole-body energy expenditure but not FOX and COX in neonatal calves, and the absorption of colostral leptin and adiponectin might affect insulin sensitivity on d 1 of life.

Background A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. Results In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (

Background: Genomic regions associated with divergent livestock feed efficiency have been found predominantly outside protein coding sequences. Long non-coding RNAs (lncRNA) can modulate chromatin accessibility, gene expression and act as important metabolic regulators in mammals. By integrating phenotypic, transcriptomic, and metabolomic data with quantitative trait locus data in prioritizing co-expression network analyses, we aimed to identify and functionally characterize lncRNAs with a potential key regulatory role in metabolic efficiency in cattle.Materials and Methods: Crossbred animals (n = 48) of a Charolais x Holstein F2-population were allocated to groups of high or low metabolic efficiency based on residual feed intake in bulls, energy corrected milk in cows and intramuscular fat content in both genders. Tissue samples from jejunum, liver, skeletal muscle and rumen were subjected to global transcriptomic analysis via stranded total RNA sequencing (RNAseq) and blood plasma samples were used for profiling of 640 metabolites. To identify lncRNAs within the indicated tissues, a project-specific transcriptome annotation was established. Subsequently, novel transcripts were categorized for potential lncRNA status, yielding a total of 7,646 predicted lncRNA transcripts belonging to 3,287 loci. A regulatory impact factor approach highlighted 92, 55, 35, and 73 lncRNAs in jejunum, liver, muscle, and rumen, respectively. Their ensuing high regulatory impact factor scores indicated a potential regulatory key function in a gene set comprising loci displaying differential expression, tissue specificity and loci overlapping with quantitative trait locus regions for residual feed intake or milk production. These were subjected to a partial correlation and information theory analysis with the prioritized gene set.Results and Conclusions: Independent, significant and group-specific correlations (|r| > 0.8) were used to build a network for the high and the low metabolic efficiency group resulting in 1,522 and 1,732 nodes, respectively. Eight lncRNAs displayed a particularly high connectivity (>100 nodes). Metabolites and genes from the partial correlation and information theory networks, which each correlated significantly with the respective lncRNA, were included in an enrichment analysis indicating distinct affected pathways for the eight lncRNAs. LncRNAs associated with metabolic efficiency were classified to be functionally involved in hepatic amino acid metabolism and protein synthesis and in calcium signaling and neuronal nitric oxide synthase signaling in skeletal muscle cells.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid, and conjugated linoleic acid (CLA) supplementation on fatty acid (FA) composition, performance, and systemic and hepatic antioxidative and inflammatory responses in dairy cows. Four cows (126 ± 4 d in milk) were investigated in a 4 × 4 Latin square and were abomasally infused with 1 of the following for 6 wk: (1) coconut oil (control treatment, CTRL; 38.3 g/d; providing saturated FA), (2) linseed and safflower oil (EFA treatment; 39.1 and 1.6 g/d, respectively; providing mainly α-linolenic acid), (3) Lutalin (BASF, Ludwigshafen, Germany; CLA treatment; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d each), (4) or EFA+CLA. The initial dosage was doubled every 2 wk, resulting in 3 dosages (dosage 1, 2, and 3). Cows were fed a corn silage-based total mixed ration with a high n-6/n-3 FA ratio. Dry matter intake and milk yield were recorded daily, and milk composition was measured weekly. The FA compositions of milk fat and blood plasma were analyzed at wk 0, 2, 4, and 6. The plasma concentration and hepatic mRNA abundance of parameters linked to the antioxidative and inflammatory response were analyzed at wk 0 and 6 of each treatment period. Infused FA increased in blood plasma and milk of the respective treatment groups in a dose-dependent manner. The n-6/n-3 FA ratio in milk fat was higher in CTRL and CLA than in EFA and EFA+CLA. The sum of FA

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 &times, 4 Latin square model with three successively increasing lipid dosages (coconut oil, linseed&ndash, safflower oil mix (EFA, rich in n-3 FA), Lutalin&reg, 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.

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.

Intrauterine growth retardation is associated with metabolic consequences in adulthood. Since our previous data indicate birth weight-dependent effects of feed restriction (R) on protein degradation processes in the liver, it should be investigated whether effects on connective tissue turnover are obvious and could be explained by global changes of histone H3K9me3 and H3K9ac states in regulated genes. For this purpose, female littermate pigs with low (U) or normal (N) birth weight were subjected to 3-week R (60% of ad libitum fed controls) with subsequent refeeding (REF) for further 5 weeks. The 3-week R-period induced a significant reduction of connective tissue area by 43% in the liver of U animals at 98 d of age, which was not found in age-matched N animals. Of note, after REF at 131 d of age, in previously feed-restricted U animals (UR), the percentage of mean connective tissue was only 53% of ad libitum fed controls (UK), indicating a persistent effect. In U animals, R induced H3K9 acetylation of regulated genes (e.g. XBP1, ERLEC1, GALNT2, PTRH2), which were inter alia associated with protein metabolism. In contrast, REF was mostly accompanied by deacetylation in U and N animals. Thus, our epigenetic data may give a first explanation for the observed birth weight-dependent differences in this connective tissue phenotype.

Animal welfare in dairy calf husbandry depends on calf rearing and is probably improved by intensive milk feeding programs. In addition, butyrate supplementation in milk replacer (MR) stimulates postnatal growth and may affect the immune system in calves. We have investigated the combined effects of ad libitum MR feeding and butyrate supplementation on feeding behavior, health, and the immune responses in calves. Holstein calves (n = 64) were examined from birth until wk 11 of age. Calves received MR either ad libitum (Adl) or restrictively (Res) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate supplementation starting on d 4. From wk 9 to 10, all calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Calves were housed in straw-bedded group pens with automatic MR feeders, where feed intake and feeding behavior were documented. Blood was drawn on d 1 before the first colostrum intake; on d 2, 4, and 7; and weekly thereafter until the end of the study to measure plasma concentrations of total protein, albumin, the immunoglobulins IgG1, IgG2, and IgM, and the acute phase proteins fibrinogen, serum amyloid A, and haptoglobin. Liver samples were taken on d 50 and 80 to determine gene expression related to acute phase proteins. Body temperature was measured daily for the first 3 wk, and clinical traits were scored daily. Ad libitum MR feeding resulted in greater MR intake, greater MR intake per meal, slower sucking rate, and greater body weight, but in a lower number of unrewarded visits and lower concentrate intake when compared with Res. Butyrate reduced the sucking rate but increased MR intake per meal. Immunoglobulins in the blood plasma increased after colostrum intake in all calves, with only minor differences among groups throughout the study. Plasma fibrinogen and serum amyloid A increased in the first week of life in all calves, and fibrinogen was greater in Res than in Adl on d 21, 49, and 63. Hepatic gene expression of fibrinogen on d 80 was greater in Adl than in Res. Gene expression of SAA2 was greater on d 50 in Adl than in Res and on d 80 was greater in ResB+ than in ResB-. Body temperature was greater in Adl than in Res during the first 2 wk, but neither MR feeding nor butyrate affected the health status. An improved animal welfare in Adl calves is supported by fewer signs of hunger, but intensive milk feeding and butyrate did not affect the health and immune status of the calves in a consistent manner.