Effects of freestall vs. bedded pack housing on growth performance, health status, and behavioral responses of weaned heifers.

This research evaluates the effect of two housing types, freestalls vs. bedded-pack, on growth performance, health scoring, and behavior of weaned heifers. Twenty-four heifers with no previous experience with freestalls (12 heifers/ treatment) were randomly assigned to the two treatments (freestalls vs. bedded-pack) at d 80 ± 3 of age. Heifers had free access to total mixed ration (TMR) and water throughout the experiment. The TMR intake and health score were recorded daily from d 80 to 110 of age. Body weight and skeletal growth of heifers were measured at the beginning (d 80) and the end (d110) of the experiment. Feeding behaviors were recorded for a period of 22 h and at the same time. Growth performance (ADG, structural growth, and final body weight), overall DMI, and health assessment (fecal score, general appearance, and rectal temperature) were similar between treatments. Behavioral patterns of eating, drinking, standing, lying, and rumination were also similar between treatments. However, in freestall-housed heifers, the proportion of time spent lying inside the stall increased by 8.1%, and time spent lying outside the stall decreased by 9.44% from the beginning to the end of the study (from 80 to 110 d). In summary, our results indicate that weaned heifers showed similar growth performance, behavior, and health when housed in freestall and bedded-pack housing. However, introducing weaned heifers to freestall housing at an early age will prepare them for using the facility.

Changes in the lipidome of water buffalo milk during intramammary infection by non-aureus Staphylococci.

This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

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

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

Effects of replacing steam-flaked corn with shredded sugar beet pulp on feed sorting, behavior, blood metabolites, and growth performance of dairy calves.

This experiment was conducted to investigate the effects of partial replacement of steam-flaked corn (SFC) with shredded sugar beet pulp (SBP) in the starter diet on selective intake (sorting), feeding and chewing behavior, blood biochemical parameters, and growth in newborn female Holstein dairy calves. A total of 48 calves (3 d old; 40.1 ± 0.84 kg body weight; mean ± SE) were randomly assigned to 1 of 2 feeding treatments containing 0 or 25% SBP (percentage of dry matter [DM]) in the starter diet. Calves were weaned on d 61 and remained in the study until d 81. Intake of starter feed and total intake of DM (milk DM + starter feed DM), crude protein, and neutral detergent fiber were increased (P < 0.05) by feeding SBP; however, intake of starch (P < 0.01) and total intake of ether extract (P = 0.03) were decreased with no apparent effect on total intake of ME. Average daily gain, feed efficiency, final weight, and skeletal growth also showed no significant changes. Circulating concentrations of glucose, total protein, and albumin were not affected by partial replacement of SBP with SFC; however, higher concentrations of blood urea-N (P = 0.01) and a lower albumin-to-globulin ratio (P = 0.03) were observed in SBP- vs. SFC-fed calves. Calves fed SBP sorted more for particles retained on the 4.75-mm sieve (P = 0.02) and against particles retained on the 0.6-mm sieve and bottom pan (P < 0.01). Intake of neutral detergent fibers and starch from particles retained on all sieve fractions was increased and decreased (P < 0.01), respectively, by replacing SFC with SBP. Replacement of SBP with SFC was associated with increased meal length and meal size and increased rumination frequency and length, but decreased intervals between rumination (P ≤ 0.01). Calves fed SBP spent more time eating, rumination, and standing and less time lying and non-nutritive oral behaviors (P < 0.01). In general, 25% replacement of SFC with SBP did not affect calf performance but increased time spent rumination and eating and decreased non-nutritive oral behaviors.

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

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

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

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

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.

Profiling of circulating microRNA and pathway analysis in normal- versus over-conditioned dairy cows during the dry period and early lactation.

The objective of this study was to determine the circulating microRNA (miRNA) profile in over-conditioned (HBCS) versus normal-conditioned (NBCS) dairy cows in combination with pathway enrichment analyses during the transition period. Thirty-eight multiparous Holstein cows were selected 15 wk before anticipated calving date based on their current and previous body condition scores (BCS) for forming either a HBCS group (n = 19) or a NBCS group (n = 19). They were fed different diets during late lactation to reach the targeted differences in BCS and backfat thickness until dry-off. A subset of 15 animals per group was selected based on their circulating concentrations of nonesterified fatty acids (on d 14 postpartum) and ß-hydroxybutyrate (on d 21 postpartum), representing the greater or the lower extreme values within their BCS group. Blood serum obtained at d -49 and 21 relative to parturition (3 pools with 5 cows per each group and time point) were used to identify miRNA that were differentially expressed (DE) between groups or time points using miRNA sequencing. No DE-miRNA were discovered between NBCS versus HBCS. Comparing pooled samples from d -49 and d 21 resulted in 7 DE-miRNA in the NBCS group, of which 5 miRNA were downregulated and 2 miRNA were overexpressed on d 21 versus -49. The abundance of 5 of these DE-miRNA was validated in all individual samples via quantitative PCR and extended to additional time points (d -7, 3, 84). Group differences were observed for miR-148a, miR-122 as well as miR-455-5p, and most DE-miRNA (miR-148a, miR-122, miR-30a, miR-450b, miR-455-5p) were downregulated directly after calving. Subsequently, the DE-miRNA was used for bioinformatics analysis to identify putative target genes and the most enriched biological pathways. The most significantly enriched pathways of DE-miRNA were associated with cell cycle and insulin signaling as well as glucose and lipid metabolism. Overall, we found little differences in circulating miRNA in HBCS versus NBCS cows around calving.

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.

Discovery of different metabotypes in overconditioned dairy cows by means of machine learning.

Using data from targeted metabolomics in serum in combination with machine learning (ML) approaches, we aimed at (1) identifying divergent metabotypes in overconditioned cows and at (2) exploring how metabotypes are associated with lactation performance, blood metabolites, and hormones. In a previously established animal model, 38 pregnant multiparous Holstein cows were assigned to 2 groups that were fed differently to reach either high (HBCS) or normal (NBCS) body condition score (BCS) and backfat thickness (BFT) until dryoff at -49 d before calving [NBCS: BCS < 3.5 (3.02 ± 0.24) and BFT < 1.2 cm (0.92 ± 0.21), mean ± SD; HBCS: BCS > 3.75 (3.82 ± 0.33) and BFT > 1.4 cm (2.36 ± 0.35)]. Cows were then fed the same diets during the dry period and the subsequent lactation, and maintained the differences in BFT and BCS throughout the study. Blood samples were collected weekly from 7 wk antepartum (ap) to 12 wk postpartum (pp) to assess serum concentrations of metabolites (by targeted metabolomics and by classical analyses) and metabolic hormones. Metabolic clustering by applying 4 supervised ML-based classifiers [sequential minimal optimization (SMO), random forest (RF), alternating decision tree (ADTree), and naïve Bayes-updatable (NB)] on the changes (d 21 pp minus d 49 ap) in concentrations of 170 serum metabolites resulted in 4 distinct metabolic clusters: HBCS predicted HBCS (HBCS-PH, n = 13), HBCS predicted NBCS (HBCS-PN, n = 6), NBCS predicted NBCS (NBCS-PN, n = 15), and NBCS predicted HBCS (NBCS-PH, n = 4). The accuracies of SMO, RF, ADTree, and NB classifiers were >70%. Because the number of NBCS-PH cows was low, we did not consider this group for further comparisons. Dry matter intake (kg/d and percentage of body weight) and energy intake were greater in HBCS-PN than in HBCS-PH in early lactation, and HBCS-PN also reached a positive energy balance earlier than did HBCS-PH. Milk yield was not different between groups, but milk protein percentage was greater in HBCS-PN than in HBCS-PH cows. The circulating concentrations of fatty acids (FA) increased during early lactation in both groups, but HBCS-PN cows had lower concentrations of ß-hydroxybutyrate, indicating lower ketogenesis compared with HBCS-PH cows. The concentrations of insulin, insulin-like growth factor 1, leptin, adiponectin, haptoglobin, glucose, and revised quantitative insulin sensitivity check index did not differ between the groups, whereas serum concentrations of glycerophospholipids were lower before calving in HBCS-PH than in HBCS-PN cows. Glycine was the only amino acid that had higher concentration after calving in HBCS-PH than in HBCS-PN cows. The circulating concentrations of some short- (C2, C3, and C4) and long-chain (C12, C16:0, C18:0, and C18:1) acylcarnitines on d 21 pp were greater in HBCS-PH than in HBCS-PN cows, indicating incomplete FA oxidation. In conclusion, the use of ML approaches involving data from targeted metabolomics in serum is a promising method for differentiating divergent metabotypes from apparently similar BCS phenotypes. Further investigations, using larger numbers of cows and farms, are warranted for confirmation of this finding.

Alterations of the acylcarnitine profiles in blood serum and in muscle from periparturient cows with normal or elevated body condition.

The objective of the current study was to characterize muscle and blood serum acylcarnitine (AcylCN) profiles and to determine the mRNA abundance of muscle carnitine acyltransferases in periparturient dairy cows with high (HBCS) and normal body condition (NBCS). Fifteen weeks antepartum, 38 pregnant multiparous Holstein cows were assigned to 2 groups that were fed differently to reach the targeted BCS and backfat thickness (BFT) until dry-off at -49 d before calving (HBCS: BCS >3.75 and BFT >1.4 cm; NBCS: <3.5 and <1.2 cm). Thereafter, both groups were fed identical diets. Blood samples and biopsies from the semitendinosus muscle were collected on d -49, 3, 21, and 84 relative to calving. Actual BCS at d -49 were 3.02 ± 0.24 and 3.82 ± 0.33 (mean ± SD) for NBCS and HBCS, respectively. In both groups, serum profiles showed marked changes during the periparturient period, with decreasing concentrations of free carnitine and increasing concentrations of long-chain AcylCN. Compared with NBCS, HBCS had greater serum long-chain AcylCN in early lactation, which may point to an insufficient adaptation of their metabolism in response to the metabolic load of fatty acids around parturition. The muscle concentrations of C5-, C9-, C18:1-, and C18:2-AcylCN were lower and those of C14:2-AcylCN were greater in HBCS than in NBCS cows. The mRNA abundance of carnitine palmitoyltransferase (CPT)1, muscle isoform (CPT1b) and CPT2 increased from d -49 to early lactation (d 3, d 21), followed by a decline to nearly antepartum values by d 84; this change was not affected by group. In conclusion, over-conditioning around calving seems to be associated with mitochondrial overload, which can result in incomplete fatty acid oxidation in dairy cows.

Plasma proteomic profiling and pathway analysis of normal and overconditioned dairy cows during the transition from late pregnancy to early lactation.

This study applied a quantitative proteomics approach along with bioinformatics analyses to investigate changes in the plasma proteome of normal and overconditioned dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets until dry-off to reach targeted differences in BCS and back fat thickness (BFT) until dry-off. At dry-off, normal BCS cows had a BCS <3.5 (minimum, 2.75) and BFT <1.2 cm (minimum, 0.58), and the high BCS cows had a BCS >3.75 (maximum, 4.50) and BFT >1.4 cm (maximum, 2.90). The proteomics study used a subset of 5 animals from each group. These cows were selected based on their circulating concentrations of fatty acids (FA) on d 14 postpartum and ß-hydroxybutyrate (BHB) on d 21 postpartum, representing the greater or the lower extreme values within their BCS group, respectively. The high BCS subset (HE-HBCS) had 4.50 < BCS > 3.75, FA = 1.17 ± 0.46 mmol/L, and BHB = 2.15 ± 0.42 mmol/L (means ± SD), and the low BCS subset (LE-NBCS) had 3.50 < BCS > 2.75, FA = 0.51 ± 0.28 mmol/L, and BHB = 0.84 ± 0.17 mmol/L. Plasma samples from d -49, +7, and +21 relative to parturition were used for proteome profiling by applying the quantitative tandem mass tags (TMT) approach. Nondepleted plasma samples were subjected to reduction and digestion and then labeled with TMT 10plex reagents. High-resolution liquid chromatography-tandem mass spectrometry analysis of TMT-labeled peptides was carried out, and the acquired spectra were analyzed for protein identification and quantification. In total, 254 quantifiable proteins (criteria: 2 unique peptides and 5% false discovery rate) were identified in the plasma samples. From these, 24 differentially abundant proteins (14 more abundant, 10 less abundant) were observed in the LE-NBCS cows compared with the HE-HBCS cows during the transition period. Plasma α-2-macroglobulins were more abundant in HE-HBCS versus LE-NBCS cows at d +7 and +21. Gene Ontology enrichment analyses of differentially abundant proteins revealed that the acute inflammatory response, regulation of complement activation, protein activation cascade, and regulation of humoral immune response were the most enriched terms in the LE-NBCS group compared with the HE-HBCS group. In addition, we identified 24 differentially abundant proteins (16 in the LE-NBCS group, and 8 in the HE-HBCS group) during the transition period. The complement components C1q and C5 were less abundant, while C3 and C3d were more abundant in LE-NBCS compared with HE-HBCS cows. Overall, overconditioning around calving was associated with alterations in protein pathways related to acute inflammatory response and regulation of complement and coagulation cascades in transition cows.

Technical note: Rapid field test for the quantification of vitamin E, ß-carotene, and vitamin A in whole blood and plasma of dairy cattle.

Fast and easy tests for quantifying fat-soluble vitamins such as vitamin E and vitamin A, as well as ß-carotene, in whole blood without a need to preprocess blood samples could facilitate assessment of the vitamin status of dairy cattle. The objective of this study was to validate a field-portable fluorometer/spectrophotometer assay for the rapid quantification of these vitamins in whole blood and plasma of dairy cows and calves. We measured the concentrations of vitamin E and ß-carotene in whole blood and plasma from 28 dairy cows and 11 calves using the iCheck test (BioAnalyt GmbH, Teltow, Germany) and compared the results with the current analytical standard (HPLC) in 2 independent laboratories, one at the University of Potsdam (Germany) and at one at DSM Nutritional Products Ltd. (Kaiseraugst, Switzerland). For vitamin A, the HPLC measurements were done only in the laboratory in Germany. The whole-blood concentrations of vitamin E as determined by iCheck (blood-hematocrit-corrected) ranged from 1.82 to 4.99 mg/L in dairy cows and 0.34 to 3.40 mg/L in calves. These findings were moderately correlated (R2 = 0.66) with the values assessed by HPLC in dairy cattle (cows + calves). When calves were excluded, the correlation was higher (R2 = 0.961). The ß-carotene and vitamin A values obtained by the reference method HPLC were highly correlated with the iCheck methods in whole blood (R2 = 0.99 and 0.88, respectively). In plasma, we observed strong correlations between the concentrations assessed by iCheck and those of HPLC for vitamin E (R2 = 0.97), ß-carotene (R2 = 0.98), and vitamin A (R2 = 0.92) in dairy cattle (cows + calves). For vitamin E, ß-carotene, and vitamin A, we compared the relationship between the differences obtained by the iCheck assay and the HPLC measurements, as well as the magnitude of measurements, using Bland-Altman plots to test for systematic bias. For all 3 vitamins, the differences values were not outside the 95% acceptability limits; we found no systematic error between the 2 methods for all 3 analytes.

Metabolomics meets machine learning: Longitudinal metabolite profiling in serum of normal versus overconditioned cows and pathway analysis.

This study aimed to investigate the differences in the metabolic profiles in serum of dairy cows that were normal or overconditioned when dried off for elucidating the pathophysiological reasons for the increased health disturbances commonly associated with overconditioning. Fifteen weeks antepartum, 38 multiparous Holstein cows were allocated to either a high body condition (HBCS; n = 19) group or a normal body condition (NBCS; n = 19) group and were fed different diets until dry-off to amplify the difference. The groups were also stratified for comparable milk yields (NBCS: 10,361 ± 302 kg; HBCS: 10,315 ± 437 kg; mean ± standard deviation). At dry-off, the cows in the NBCS group (parity: 2.42 ± 1.84; body weight: 665 ± 64 kg) had a body condition score (BCS) <3.5 and backfat thickness (BFT) <1.2 cm, whereas the HBCS cows (parity: 3.37 ± 1.67; body weight: 720 ± 57 kg) had BCS >3.75 and BFT >1.4 cm. During the dry period and the subsequent lactation, both groups were fed identical diets but maintained the BCS and BFT differences. A targeted metabolomics (AbsoluteIDQ p180 kit, Biocrates Life Sciences AG, Innsbruck, Austria) approach was performed in serum samples collected on d -49, +3, +21, and +84 relative to calving for identifying and quantifying up to 188 metabolites from 6 different compound classes (acylcarnitines, AA, biogenic amines, glycerophospholipids, sphingolipids, and hexoses). The concentrations of 170 metabolites were above the limit of detection and could thus be used in this study. We used various machine learning (ML) algorithms (e.g., sequential minimal optimization, random forest, alternating decision tree, and naïve Bayes-updatable) to analyze the metabolome data sets. The performance of each algorithm was evaluated by a leave-one-out cross-validation method. The accuracy of classification by the ML algorithms was lowest on d 3 compared with the other time points. Various ML methods (partial least squares discriminant analysis, random forest, information gain ranking) were then performed to identify those metabolites that were contributing most significantly to discriminating the groups. On d 21 after parturition, 12 metabolites (acetylcarnitine, hexadecanoyl-carnitine, hydroxyhexadecenoyl-carnitine, octadecanoyl-carnitine, octadecenoyl-carnitine, hydroxybutyryl-carnitine, glycine, leucine, phosphatidylcholine-diacyl-C40:3, trans-4-hydroxyproline, carnosine, and creatinine) were identified in this way. Pathway enrichment analysis showed that branched-chain AA degradation (before calving) and mitochondrial ß-oxidation of long-chain fatty acids along with fatty acid metabolism, purine metabolism, and alanine metabolism (after calving) were significantly enriched in HBCS compared with NBCS cows. Our results deepen the insights into the phenotype related to overconditioning from the preceding lactation and the pathophysiological sequelae such as increased lipolysis and ketogenesis and decreased feed intake.

Diurnal variation of NMR based blood metabolites in calves fed a high plane of milk replacer: a pilot study.

BACKGROUND: Blood profiles have been used to monitor herd health status, diagnose disorders, and predict the risk of diseases in cattle and calves. Characterizing plasma metabolites in dairy calves could provide further insight into daily metabolic variations and the mechanisms that lead to metabolic diseases. In addition, by understanding physiological ranges of plasma metabolites relative to meal and the time of feeding in healthy animals, veterinarians can accurately diagnose abnormalities with a blood test. For diagnostic purposes, nuclear magnetic resonance (NMR) spectroscopy shows promise as a new and reliable method to determine a large number of blood metabolites simultaneously. RESULTS: Results demonstrated that the concentration of specific metabolites in plasma (i.e., lysine, isoleucine, leucine, tyrosine, glutamine, creatine, and 1-methylhistidine) fluctuated around meal times, while others (i.e., glutamic acid, methanol, formic acid, and acetic acid) maintained a stable temporal concentration. In addition to temporal changes in concentration, results also characterized differences for overall plasma metabolite concentrations; for example, methionine had the lowest (38 µM) while glutamine had the highest concentration (239 µM) amongst plasma AA. This is the first report describing how the plasma metabolome changes during 24-h period in young calves fed an elevated plane of milk replacer twice daily. CONCLUSIONS: Data from this pilot study will help to establish reference standards for future metabolic diagnostics in dairy calves. In addition, this pilot study illustrated that feeding milk replacer may influence plasma metabolite concentrations. With the rapid implementation of blood metabolomics in monitoring animal health, it is then important to consider the time of feeding during the day when interpreting metabolomics analysis results.