Blood and liver telomere length, mitochondrial DNA copy number, and hepatic gene expression of mitochondrial dynamics in mid-lactation cows supplemented with l-carnitine under systemic inflammation.

The current study was conducted to examine the effect of l-carnitine (LC) supplementation on telomere length and mitochondrial DNA copy number (mtDNAcn) per cell in mid-lactation cows challenged by lipopolysaccharide (LPS) in blood and liver. The mRNA abundance of 31 genes related to inflammation, oxidative stress, and the corresponding stress response mechanisms, the mitochondrial quality control and the protein import system, as well as the phosphatidylinositol 3-kinase/protein kinase B pathway, were assessed using microfluidics integrated fluidic circuit chips (96.96 dynamic arrays). In addition to comparing the responses in cows with or without LC, our objectives were to characterize the oxidative and inflammatory status by assessing the circulating concentration of lactoferrin (Lf), haptoglobin (Hp), fibrinogen, derivates of reactive oxygen metabolites (dROM), and arylesterase activity (AEA), and to extend the measurement of Lf and Hp to milk. Pluriparous Holstein cows were assigned to either a control group (CON, n = 26) or an LC-supplemented group (CAR; 25 g LC/cow per day; d 42 ante partum to d 126 postpartum (PP), n = 27). On d 111 PP, each cow was injected intravenously with LPS (Escherichia coli O111:B4, 0.5 µg/kg). The mRNA abundance was examined in liver biopsies of d -11 and +1 relative to LPS administration. Plasma and milk samples were frequently collected before and after the challenge. After LPS administration, circulating plasma fibrinogen and serum dROM concentrations increased, whereas AEA decreased. Moreover, serum P4 initially increased by 3 h after LPS administration and declined thereafter irrespective of grouping. The Lf concentrations increased in both groups after LPS administration, with the CAR group showing greater concentrations in serum and milk than the CON group. After LPS administration, telomere length in blood increased, whereas mtDNAcn per cell decreased; however, both remained unaffected in liver. For mitochondrial protein import genes, the hepatic mRNA abundance of the translocase of the mitochondrial inner membrane (TIM)-17B was increased in CAR cows. Moreover, TIM23 increased in both groups after LPS administration. Regarding the mRNA abundance of genes related to stress response mechanisms, 7 out of 14 genes showed group × time interactions, indicating a (local) protective effect due to the dietary LC supplementation against oxidative stress in mid-lactating dairy cows. For mtDNAcn and telomere length, the effects of the LPS-induced inflammation were more pronounced than the dietary supplementation of LC. Dietary LC supplementation affected the response to LPS primarily by altering mitochondrial dynamics. Regarding mRNA abundance of genes related to the mitochondrial protein import system, the inner mitochondrial membrane translocase (TIM complex) seemed to be more sensitive to dietary LC than the outer mitochondrial membrane translocase (TOM complex).

Development of a subacute ruminal acidosis risk score and its prediction using milk mid-infrared spectra in early-lactation cows.

A routine monitoring for subacute ruminal acidosis (SARA) on the individual level could support the minimization of economic losses and the ensuring of animal welfare in dairy cows. The objectives of this study were (1) to develop a SARA risk score (SRS) by combining information from different data acquisition systems to generate an integrative indicator trait, (2) the investigation of associations of the SRS with feed analysis data, blood characteristics, performance data, and milk composition, including the fatty acid (FA) profile, (3) the development of a milk mid-infrared (MIR) spectra-based prediction equation for this novel reference trait SRS, and (4) its application to an external data set consisting of MIR data of test day records to investigate the association between the MIR-based predictions of the SRS and the milk FA profile. The primary data set, which was used for the objectives (1) to (3), consisted of data collected from 10 commercial farms with a total of 100 Holstein cows in early lactation. The data comprised barn climate parameters, pH and temperature logging from intrareticular measurement boluses, as well as jaw movement and locomotion behavior recordings of noseband-sensor halters and pedometers. Further sampling and data collection included feed samples, blood samples, milk performance, and milk samples, whereof the latter were used to get the milk MIR spectra and to estimate the main milk components, the milk FA profile, and the lactoferrin content. Because all measurements were characterized by different temporal resolutions, the data preparation consisted of an aggregation into values on a daily basis and merging it into one data set. For the development of the SRS, a total of 7 traits were selected, which were derived from measurements of pH and temperature in the reticulum, chewing behavior, and milk yield. After adjustment for fixed effects and standardization, these 7 traits were combined into the SRS using a linear combination and directional weights based on current knowledge derived from literature studies. The secondary data set was used for objective (4) and consisted of test day records of the entire herds, including performance data, milk MIR spectra and MIR-predicted FA. At farm level, it could be shown that diets with higher proportions of concentrated feed resulted in both lower daily mean pH and higher SRS values. On the individual level, an increased SRS could be associated with a modified FA profile (e.g., lower levels of short- and medium-chain FA, higher levels of C17:0, odd- and branched-chain FA). Furthermore, a milk MIR-based partial least squares regression model with a moderate predictability was established for the SRS. This work provides the basis for the development of routine SARA monitoring and demonstrates the high potential of milk composition-based assessment of the health status of lactating cows.

Targeted assessment of the metabolome in skeletal muscle and in serum of dairy cows supplemented with conjugated linoleic acid during early lactation.

In the dairy cow, late gestation and early lactation are characterized by a complexity of metabolic processes required for the homeorhetic adaptation to the needs of fetal growth and milk production. Skeletal muscle plays an important role in this adaptation. The objective of this study was to characterize the metabolome in skeletal muscle (semitendinosus muscle) and in serum of dairy cows in the context of the physiological changes occurring in early lactation and to test the effects of dietary supplementation (from d 1 in milk onwards) with conjugated linoleic acids (sCLA; 100 g/d; supplying 7.6 g of cis-9,trans-11 CLA and 7.6 g of trans-10,cis-12 CLA per cow/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). The metabolome was characterized in skeletal muscle samples collected on d 21 and 70 after calving in conjunction with their serum counterpart using a targeted metabolomics approach (AbsoluteIDQ p180 kit; Biocrates Life Sciences AG, Innsbruck, Austria). Thereby 188 metabolites from 6 different compound classes (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and hexoses) were quantified in both sample types. In both groups, dry matter intake increased after calving. It was lower in sCLA than in CTR on d 21, which resulted in reduced calculated net energy and metabolizable protein balances. On d 21, the concentrations of dopamine, Ala, and hexoses in the skeletal muscle were higher in sCLA than in CTR. On d 21, the changed metabolites in serum were mainly long-chain (>C24) diacyl phosphatidylcholine PC (PC-aa) and acyl-alkyl phosphatidylcholine (PC-ae), along with lysophosphatidylcholine acyl (lysoPC-a) C26:1 that were all lower in sCLA than in CTR. Supplementation with CLA affected the muscle concentrations of 22 metabolites on d 70 including 10 long-chain (>C22) sphingomyelin (SM), hydroxysphingomyelin [SM(OH)], PC-aa, and PC-ae along with 9 long-chain (>C16) lysoPC-a and 3 metabolites related to amino acids (spermine, citrulline, and Asp). On d 70, the concentrations of lysoPC-a C18:2 and C26:0 in serum were higher in the sCLA cows than in the CTR cows. Regardless of treatment, the concentrations of Ile, Leu, Phe, Lys, His, Met, Trp, and hydroxybutyrylcarnitine (C4-OH) decreased, whereas those of ornithine, Gln, and trans-4-hydroxyproline (t4-OH-Pro) increased from d 21 to 70 in muscle. The significantly changed metabolites in serum with time of lactation were 28 long-chain (>C30) PC-ae and PC-aa, 7 long-chain (>C16) SM and SM(OH), along with lysoPC-a C20:3 that were all increased. In conclusion, in addition to other significantly changed metabolites, CLA supplementation mainly led to changes in muscle and serum concentrations of glycerophospholipids and sphingolipids that might reflect the phospholipid compositional changes in muscle. The metabolome changes observed in sCLA on d 21 seem to be, at least in part, due to the lower DMI in these cows. The changes in the muscle concentrations of AA from d 21 to 70, which coincided with the steady energy and MP balances, might reflect a shift of protein synthesis/degradation balance toward synthesis.

Effects of dietary l-carnitine supplementation on the response to an inflammatory challenge in mid-lactating dairy cows: Hepatic mRNA abundance of genes involved in fatty acid metabolism.

This study aimed at characterizing the effects of dietary l-carnitine supplementation on hepatic fatty acid (FA) metabolism during inflammation in mid-lactating cows. Fifty-three pluriparous Holstein dairy cows were randomly assigned to either a control (CON, n = 26) or an l-carnitine supplemented (CAR; n = 27) group. The CAR cows received 125 g of a rumen-protected l-carnitine product per cow per day (corresponding to 25 g of l-carnitine/cow per day) from d 42 antepartum (AP) until the end of the trial on d 126 postpartum (PP). Aside from the supplementation, the same basal diets were fed in the dry period and during lactation to all cows. In mid lactation, each cow was immune-challenged by a single intravenous injection of 0.5 µg of LPS/kg of BW at d 111 PP. Blood samples were collected before and after LPS administration. The mRNA abundance of in total 39 genes related to FA metabolism was assessed in liver biopsies taken at d -11, 1, and 14 relative to LPS (d 111 PP) and also on d 42 AP as an individual covariate using microfluidics integrated fluidic circuit chips (96.96 dynamic arrays). In addition to the concentrations of 3 selected proteins related to FA metabolism, acetyl-CoA carboxylase α (ACACA), 5' AMP-activated protein kinase (AMPK), and solute carrier family 25 member 20 (SLC25A20) were assessed by a capillary Western blot method in liver biopsies from d -11 and 1 relative to LPS from 11 cows each of CAR and CON. On d -11 relative to LPS, differences between the mRNA abundance in CON and CAR were limited to acyl-CoA dehydrogenase (ACAD) very-long-chain (ACADVL) with greater mRNA abundance in the CAR than in the CON group. The liver fat content decreased from d -11 to d 1 relative to the LPS injection and remained at the lower level until d 14 in both groups. One day after the LPS challenge, lower mRNA abundance of carnitine palmitoyltransferase 1 (CPT1), CPT2, ACADVL, ACAD short-chain (ACADS), and solute carrier family 22 member 5 (SLC22A5) were observed in the CAR group as compared with the CON group. However, the mRNA abundance of protein kinase AMP-activated noncatalytic subunit gamma 1 (PRKAG1), ACAD medium-chain (ACADM), ACACA, and FA binding protein 1 (FABP1) were greater in the CAR group than in the CON group on d 1 relative to LPS. Two weeks after the LPS challenge, differences between the groups were no longer detectable. The altered mRNA abundance before and 1 d after LPS pointed to increased transport of FA into hepatic mitochondria during systemic inflammation in both groups. The protein abundance of AMPK was lower in CAR than in CON before the LPS administration. The protein abundance of SLC25A20 was neither changing with time nor treatment and the ACACA protein abundance was only affected by time. In conclusion, l-carnitine supplementation temporally altered the hepatic mRNA abundance of some genes related to mitochondrial biogenesis and very-low-density lipoprotein export in response to an inflammatory challenge, but with largely lacking effects before and 2 wk after LPS.

Gain and loss of subcutaneous and abdominal adipose tissue depot mass of German Holstein dairy cows with different body conditions during the transition period.

Subcutaneous adipose tissue (SCAT) and abdominal adipose tissue (AAT) depots are mobilized during the fresh cow period (FCP) and early lactation period (ELP) to counteract the negative energy balance (NEB). Earlier studies suggested that fat depots contribute differently to lipomobilization and may vary in functionality. Differences between the adipose depots might influence the development of metabolic disorders. Thus, the gain and loss of subcutaneous and abdominal adipose depot masses in Holstein cows with lower and higher body condition (mean body condition scores: 3.48 and 3.87, respectively) were compared in the period from d -42 to d 70 relative to parturition in this study. Animals of the 2 experimental groups represented adequately conditioned and overconditioned cows. Estimated depot mass (eDM) of SCAT, AAT, retroperitoneal, omental, and mesenteric adipose depots of 31 pluriparous German Holstein cows were determined via ultrasonography at d -42, 7, 28, and 70 relative to parturition. The cows were grouped according to the eDM of SCAT on d -42 [low body condition (LBC) group: n = 16, mean eDM 8.6 kg; high body condition (HBC) group: n = 15, mean eDM 15.6 kg]. Average daily change (prepartum gain and postpartum loss) in depot masses during dry period (DP; from d -42 to d 7), FCP (d 7 to d 28), and ELP (d 28 to d 70) were calculated and daily dry matter intake and lactation performance recorded. Cows of this study stored about 2 to 3 times more fat in AAT than in SCAT depots. After parturition, on average more adipose tissue mass was lost from the AAT than the SCAT depot (0.23 kg/d vs. 0.14 kg/d). Cows with high compared with low body condition had similar gains in AAT (0.33 kg/d) and SCAT (0.14 kg/d) masses during the DP but mobilized significantly more adipose tissue mass from both depots after calving (AAT, HBC vs. LBC: 0.30 vs. 0.17 kg/d; SCAT, HBC vs. LBC: 0.19 vs. 0.10 kg/d). Correlation analysis indicated a functional disparity between AAT and SCAT. In the case of AAT (R2 = 0.36), the higher the gain in adipose mass during DP, the higher the loss in FCP, but this was not the case for SCAT. During FCP, a greater NEB resulted in greater loss of mass from SCAT (R2 = 0.18). In turn, greater mobilization of SCAT mass led to a higher calculated feed efficiency (R2 = 0.18). However, AAT showed no such correlations. On the other hand, during ELP, loss of both SCAT and AAT mass correlated positively with feed efficiency (R2 = 0.35 and 0.33, respectively). The results indicate that feed efficiency may not be an adequate criterion for performance evaluation in cows during NEB. Greater knowledge of functional disparities between AAT and SCAT depots may improve our understanding of excessive lipomobilization and its consequences for metabolic health and performance of dairy cows during the transition period.

Modeling reticular and ventral ruminal pH of lactating dairy cows using ingestion and rumination behavior.

The prevention and control of metabolic and digestive diseases is an enormous challenge in dairy farming. Subacute ruminal acidosis (SARA) is assumed to be the most severe feed-related disorder and it impairs both animal health and economic efficiency. Currently, ruminal pH as well as variables derived from the daily pH curve are the main indicators for SARA. The objective of this study was to explain the daily pH course in the ventral rumen and reticulum of dairy cows using ingestion pattern and rumination behavior data gathered by automated data recording systems. The data of 13 ruminally fistulated lactating cows were collected at the experimental station of the Friedrich-Loeffler-Institut (Brunswick, Germany). The data included continuous pH measurements, which were recorded simultaneously in the reticulum by pH-measuring boluses and in the ventral rumen by a separate data logger. In addition, rumination behavior was measured using jaw movement sensors, and feed and water intakes were recorded by transponder-assisted systems. Milk yield and body weight were determined during and after each milking, respectively. For statistical evaluation, the data were analyzed using time-series modeling with multiple linear mixed regressions. Before applying the developed mathematical statistical modeling, we performed a plausibility assessment to ensure data quality. The major part of the mathematical statistical modeling consisted of data preparation, where all variables were transformed into a uniform 1-min resolution. Signal transformations were used to model individual feed and water intakes as well as rumination behavior events over time. Our results indicated that diurnal pH curves of both the reticulum and ventral rumen could be predicted by the transformed feed and water intake rates. Rumination events were associated with a marginal temporal increase in pH. We observed that the pH of the ventral rumen was delayed by approximately 37 min compared with that of the reticulum, which was therefore considered in the modeling. With the models developed in this study, 67.0% of the variance of the reticular pH curves and 37.8% of the variance of the ruminal pH curves could be explained by fixed effects. We deduced that the diurnal pH course is, to a large extent, associated with the animal's individual feed intake and rumination behavior.

Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation.

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and ß (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

Insulin signaling and insulin response in subcutaneous and retroperitoneal adipose tissue in Holstein cows during the periparturient period.

Adipose tissue response to endocrine stimuli, such as insulin, is crucial for metabolic adaptation at the onset of lactation in dairy cows. However, the exact molecular mechanisms behind this response are not well understood. Thus, the aim of this study was to determine the dynamics in protein expression and phosphorylation of key components in insulin signaling in subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissues of Holstein dairy cows. Furthermore, by ex vivo examinations, response to insulin was assessed in SCAT and RPAT at different time points during the periparturient period. Biopsy samples were taken 42 d prepartum, and 1, 21, and 100 d postpartum. Insulin and glucose concentrations were measured in blood serum in consecutive serum samples from d -42 until d +100. After parturition, the majority of the key components were downregulated in both adipose tissues but recovered by d +100. The extent of hormone-sensitive lipase phosphorylation increased postpartum and remained high throughout the experimental period. Strong differences in molecular response were observed between the 2 depots. The RPAT expressed a remarkably greater extent of AMP-activated kinase phosphorylation compared with SCAT, indicating that AMP-activated kinase as an energy sensor is highly active particularly in RPAT in times of energy scarcity. Consequently, this depot expressed a greater extent of hormone-sensitive lipase phosphorylation over the whole experimental period. Insulin response after parturition appeared to be greater in RPAT too, due to the significantly greater expression of the insulin receptor at d +21 and +100. Although insulin concentrations in plasma were low postpartum, the depot-specific changes in molecular modulation of insulin signaling and insulin response suggested that both adipose tissue depots studied were contributing to the periparturient homeorhetic adaptation, although most likely to a different extent.

Acylcarnitine profiles in serum and muscle of dairy cows receiving conjugated linoleic acids or a control fat supplement during early lactation.

Acylcarnitines (ACC) are formed when fatty acid (FA)-coenzyme A enters the mitochondria for ß-oxidation and the tricarboxylic acid cycle through the carnitine shuttle. Concentrations of ACC may vary depending on the metabolic conditions, but can accumulate when rates of ß-oxidation exceed those of tricarboxylic acid. This study aimed to characterize muscle and blood serum acylcarnitine profiles, to determine the mRNA abundance of muscle carnitine acyltransferases, and to test whether dietary supplementation (from d 1 in milk) with conjugated linoleic acids (CLA; 100 g/d; each 12% of trans-10,cis-12 and cis-9,trans-11 CLA; n = 11) altered these compared with control fat-supplemented cows (CTR; n = 10). Blood samples and biopsies from the semitendinosus musclewere collected on d -21, 1, 21, and 70 relative to parturition. Serum and muscle ACC profiles were quantified using a targeted metabolomics approach. The CLA supplement did not affect the variables examined. The serum concentration of free carnitine decreased with the onset of lactation. The concentrations of acetylcarnitine, hydroxybutyrylcarnitine, and the sum of short-chain ACC in serum were greater from d -21 to 21 than thereafter. The serum concentrations of long-chain ACC tetradecenoylcarnitine (C14:1) and octadecenoylcarnitine (C18:1) concentrations were greater on d 1 and 21 compared with d -21. Muscle carnitine remained unchanged, whereas short- and medium-chain ACC, including propenoylcarnitine (C3:1), hydroxybutyrylcarnitine, hydroxyhexanoylcarnitine, hexenoylcarnitine (C6:1), and pimelylcarnitine were increased on d 21 compared with d -21 and decreased thereafter. In muscle, the concentrations of long-chain ACC (from C14 to C18) were elevated on d 1. The mRNA abundance of carnitine palmitoyltransferase 1, muscle isoform (CPT1B) increased 2.8-fold from d -21 to 1, followed by a decline to nearly prepartum values by d 70, whereas that of CPT2 did not change over time. The majority of serum and muscle short- and long-chain ACC were positively correlated with the FA concentrations in serum, whereas serum carnitine and C5 were negatively correlated with FA. Time-related changes in the serum and muscle ACC profiles were demonstrated that were not affected by the CLA supplement at the dosage used in the present study. The elevated concentrations of long-chain ACC species in muscle and of serum acetylcarnitine around parturition point to incomplete FA oxidation were likely due to insufficient metabolic adaptation in response to the load of FA around parturition.

Changes in tissue abundance and activity of enzymes related to branched-chain amino acid catabolism in dairy cows during early lactation.

Branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes the irreversible oxidative decarboxylation of branched-chain α-keto acids. This reaction is considered as the rate-limiting step in the overall branched-chain amino acid (BCAA) catabolic pathway in mammals. For characterizing the potential enzymatic involvement of liver, skeletal muscle, adipose tissue (AT), and mammary gland (MG) in BCAA metabolism during early lactation, tissue and blood samples were examined on d 1, 42, and 105 after parturition from 25 primiparous Holstein cows. Serum BCAA profiles were analyzed and the mRNA and protein abundance as well as the activity in the different tissues were assessed for the BCAA catabolic enzymes, partly for the branched-chain aminotransferase and completely for BCKDH. Total BCAA concentration in serum was lowest on d 1 after parturition and increased thereafter to a steady level for the duration of the experiment. Pronounced differences between the tissues were observed at all molecular levels. The mRNA abundance of the mitochondrial isoform of branched-chain aminotransferase (BCATm) was greatest in AT as compared with the other tissues studied, indicating that AT might be an important contributor in the initiation of BCAA catabolism in dairy cows. From the different subunits of the BCKDH E1 component, only the mRNA for the ß polypeptide (BCKDHB), not for the α polypeptide (BCKDHA), was elevated in liver. The BCKDHA mRNA abundance was similar across all tissues except muscle, which tended to lower values. Highest BCKDHA protein abundance was observed in both liver and MG, whereas BCKDHB protein was detectable in these tissues but could not be quantified. Adipose tissue and muscle only displayed abundance of the α subunit, with muscle having the lowest BCKDHA protein of all tissues. We found similarities in protein abundance for both BCKDH E1 subunits in liver and MG; however, the corresponding overall BCKDH enzyme activity was 7-fold greater in liver compared with MG, allowing for hepatic oxidation of BCAA transamination products. Reduced BCKDH activity in MG associated with no measurable activity in AT and muscle may favor sparing of BCAA for the synthesis of the different milk components, including nonessential AA. Deviating from previously published data on BCAA net fluxes and isotopic tracer studies in ruminants, our observed results might in part be due to complex counter-regulatory mechanisms during early lactation.

Interrelations between the rumen microbiota and production, behavioral, rumen fermentation, metabolic, and immunological attributes of dairy cows.

Different studies have shown a strong correlation between the rumen microbiome and a range of production traits (e.g., feed efficiency, milk yield and components) in dairy cows. Underlying dynamics concerning cause and effect are, however, still widely unknown and warrant further investigation. The aim of the current study was to describe possible functional interrelations and pathways using a large set of variables describing the production, the metabolic and immunological state, as well as the rumen microbiome and fermentation characteristics of dairy cows in early lactation (n = 36, 56 ± 3 d in milk). It was further hypothesized that the feed intake-associated behavior may influence the ruminal fermentation pattern, and a set of variables describing these individual animal attributes was included. Principal component analysis as well as Spearman's rank correlations were conducted including a total of 265 variables. The attained plots describe several well-known associations between metabolic, immunological, and production traits. Main drivers of variance within the data set included milk production and efficiency as well as rumen fermentation and microbiome diversity attributes, whereas behavioral, metabolic, and immunological variables did not exhibit any strong interrelations with the other variables. The previously well-documented strong correlation of production traits with distinct prokaryote groups was confirmed. This mainly included a negative correlation of operational taxonomic units ascribed to the Prevotella genus with milk and fat yield and feed efficiency. A central role of the animals' feed intake behavior in this context could not be affirmed. Furthermore, different methodological and interpretability aspects concerning the microbiome analysis by 16S rRNA gene sequencing, such as the discrepancy between taxonomic classification and functional communality, as well as the comparability with other studies, are discussed. We concluded that, to further investigate the driving force that causes the difference between efficient and inefficient animals, studies including more sophisticated methods to describe phenotypical traits of the host (e.g., rumen physiology, metabolic and genetic aspects) as well as the rumen microbiome (e.g., metagenome, metatranscriptome, metaproteome, and metabolome analysis) are needed.

Influence of conjugated linoleic acids and vitamin E on biochemical, hematological, and immunological variables of dairy cows during the transition period.

The objective of this experiment was to determine the effects of conjugated linoleic acid (CLA) and vitamin E as well as their interaction on biochemical and hematological variables and on leukocyte populations and their functionality. We assigned 59 German Holstein cows between the 2nd and 9th lactation to 4 dietary groups in a 2 × 2 factorial design with the factors CLA and vitamin E. Six weeks before calving the cows had a BCS of 3.7 to provoke a higher risk of developing ketosis, which might impair their immune function. Blood samples for analyses were taken on d -42, -14, -7, -3, 1, 3, 7, 10, 14, 21, 28, 35, 42, 56, and 70 relative to parturition. Furthermore, peripheral blood mononuclear cells were cultured on d -42, -7, 1, 7, 14, 28, and 70 relative to calving. Most variables were characterized by a high variation between d 7 antepartum and d 7 postpartum. Treatments did not elicit any effect, with the exception of vitamin E, which increased serum urea concentrations and decreased monocyte percentages. Haptoglobin, aspartate-aminotransferase, red blood cell count, leukocyte percentage and populations, as well as peripheral blood mononuclear cells were influenced by parity. In conclusion, the impairment of immune function caused by calving was more severe in cows in ≥3rd parity than in younger cows. However, neither vitamin E nor CLA supplementation was successful to stabilize parity or parturition related variance in hematological and immunological traits.

Effects of energy supply and nicotinic acid supplementation on serum anti-oxidative capacity and on expression of oxidative stress-related genes in blood leucocytes of periparturient primi- and pluriparous dairy cows.

The periparturient period is accompanied by metabolic and oxidative stress. Niacin is known to decrease lipolysis but is also reported to have anti-oxidative effects. Therefore, we examined the effects of energy supply and a nicotinic acid (NA) supplementation on anti-oxidative serum parameters and on the expression of oxidative stress-related genes in blood leucocytes of periparturient dairy cows, differing in parity. Twenty-nine pluriparous and 18 primiparous cows were allocated to four different feeding groups 42 days before expected parturition until 100 days postpartum and fed a ration with either a low concentrate proportion of 30% (LC) or a high concentrate proportion of 60% (HC). After parturition, all animals received 30% concentrate which was increased to 50% either within 16 (LC group) or 24 days (HC group). Half of the animals per group were supplemented with 24 g NA per day from 42 days prepartum until 24 days postpartum. All investigated parameters varied significantly over time compared to parturition (p < .05). Ferric reducing ability (FRA) exhibited a nadir before parturition, and the antioxidant enzymes glutathione peroxidase (GPX) and superoxide dismutase (SOD) showed peak activities around parturition. Expression levels of GPX1, SOD2, xanthine dehydrogenase (XDH) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) peaked before calving. The concentrate level influenced GPX activity and mRNA abundance of SOD2, XDH and poly (ADP-ribose) polymerase 1 (PARP1). Pluriparous animals exhibited higher serum GPX activities, a more distinct nadir for FRA and higher expression levels for GPX1, SOD2 and XDH. Primiparous cows displayed higher serum SOD activities. NA supplementation increased serum SOD activity antepartum in LC animals. Parturition was characterised by an increased need for antioxidants and an increased expression of oxidative stress-related genes that clearly differed with parity and was influenced by energy supply while NA exerted only minor effects on the investigated parameters.

Impact of diet composition and temperature-humidity index on water and dry matter intake of high-yielding dairy cows.

The temperature-humidity index (THI) is widely used to characterize heat stress in dairy cattle. Diet composition is known to induce variation in metabolic-associated heat production. However, the relationships between THI and diet are poorly characterized with regard to performance and intake behaviour. Therefore, the objectives were to evaluate the impact of THI on water intake (WI), dry matter intake (DMI) and the frequency of drinking and feeding bouts in lactating dairy cows offered four dietary treatments: each contained 20% grass silage and additionally (i) 20% maize silage, 60% concentrate (M-HC); (ii) 60% maize silage, 20% concentrate (M-LC); (iii) 20% pressed beet pulp silage, 60% concentrate (BPS-HC); or (iv) 60% pressed beet pulp silage, 20% concentrate (BPS-LC) (DM basis). Individual WI and DMI were recorded from April to July 2013. Furthermore, dietary effects on milk production and reticular pH were estimated. Milk yield was lowest for M-LC, while energy-corrected milk was similar for all diets. Milk fat percentage was higher and milk protein amount lower for cows offered both LC diets. Reticular pH below 6.3, 6.0 and 5.8 lasted longest for BPS-LC. WI was higher for HC diets. However, the frequency of drinking bouts was not influenced by the ration. Lower DMI occurred for BPS-LC compared to M-LC. Frequency of feeding bouts was significantly higher for LC diets. THI was significantly related to WI, DMI as well as drinking and feeding bouts. Per increasing THI, WI increased slightly more for LC diets and DMI decreased more for HC diets. Frequency of drinking bouts increased slightly higher for BPS rations per rising THI, while the decrease in feeding bouts was highest for M-HC. In conclusion, TMR composition and moderate heat stress impacted WI and DMI of dairy cows, while both dietary energy density and ruminal filling might intensify the THI impact.

Influence of conjugated linoleic acids and vitamin E on milk fatty acid composition and concentrations of vitamin A and α-tocopherol in blood and milk of dairy cows.

The objective of this trial was to investigate the influences of conjugated linoleic acid (CLA) and vitamin E (Vit. E) and their interactions on fatty acid composition and vitamins in milk (α-tocopherol, retinol and ß-carotene) as well as on α-tocopherol in blood of pluriparous cows from week 6 ante partum until week 10 post-partum (p.p.). We assigned 59 pluriparous German Holstein cows to four treatment groups with the treatment factors CLA and Vit. E at two levels in a 2 × 2 factorial design. Milk fatty acid composition and milk vitamins were analysed on lactation days 7 and 28. α-tocopherol in blood serum was analysed on days -42, -7, 1, 7, 14, 28 and 70 relative to parturition. Milk concentration of α-tocopherol was influenced by Vit. E (p < .001) and CLA (p = .034). Percentage of cis-9, trans-11 CLA in total milk fat was influenced by treatment with CLA (p < .001), while for percentage of trans-10, cis-12 CLA an interaction between treatment and day (p = .019), driven by an increase in both CLA groups from day 7 to day 28, was found. Serum ratios of α-tocopherol to cholesterol were influenced by Vit. E (p < .001). Results suggest that treatment with CLA during late pregnancy and early lactation is suitable to enhance the proportion of trans-10, cis-12 CLA in milk and thereby influencing nutritional properties. As treatment with Vit. E did not have an impact on milk fatty acid composition, it might be possible to increase the antioxidative capacity of the dairy cow without affecting milk properties. Consequently, combined treatment with CLA and Vit. E might elicit synergistic effects on the cow and milk quality by increasing the proportion of CLA in milk fat as well as the excretion of Vit. E and the Vit. E levels in serum.

Relationships between body temperatures and inflammation indicators under physiological and pathophysiological conditions in pigs exposed to systemic lipopolysaccharide and dietary deoxynivalenol.

We studied the constancy of the relationship between rectal and intraabdominal temperature as well as their linkage to inflammatory markers (leucocyte counts, kynurenine-to-tryptophan ratio (Kyn-Trp ratio), tumour necrosis factor alpha (TNF-α) in healthy and in pigs exposed to lipopolysaccharide (LPS) and/or deoxynivalenol (DON). Barrows (n = 44) were fed 4 weeks either a DON-contaminated (4.59 mg DON/kg feed) or a control (CON) diet and equipped with an intraabdominal temperature logger and a multicatheter system (V.portae hepatis, V.lienalis, Vv.jugulares) facilitating infusion of 0.9% NaCl (CON) or LPS (7.5 µg/kg BW) and simultaneous blood sampling. Body temperatures were measured and blood samples taken every 15 min for leucocyte counts, TNF-α and Kyn-Trp ratio. Combination of diet and infusion created six groups: CON_CONjug .-CONpor. , CON_CONjug. -LPSpor. , CON_LPSjug. -CONpor. , DON_CONjug. -CONpor. , DON_CONjug. -LPSpor. , DON_LPSjug. -CONpor. . The relationship between both temperatures was not uniform for all conditions. Linear regression revealed that an intraabdominal increase per 1°C increase in rectal temperature was ~25% higher in all LPS-infused pigs compared to NaCl-infusion, albeit diet and site of LPS infusion modified the magnitude of this difference. Inflammatory markers were only strongly present under LPS influence and showed a significant relationship with body temperatures. For example, leucocyte counts in clinically inconspicuous animals were only significantly correlated to core temperature in DON-fed pigs, but in all LPS-infused groups, irrespective of diet and temperature method. In conclusion, the gradient between body core and rectal temperature is constant in clinically inconspicuous pigs, but not under various pathophysiological conditions. In the latter, measurement of inflammatory markers seems to be a useful completion.

Effect of increasing body condition on oxidative stress and mitochondrial biogenesis in subcutaneous adipose tissue depot of nonlactating dairy cows.

With the onset of lactation, dairy cows with a body condition score >3.5 are sensitive to oxidative stress and metabolic disorders. Adipose tissue (AT) can adapt to varying metabolic demands and energy requirements by the plasticity of its size during lactation. In AT, angiogenesis is necessary to guarantee sufficient oxygen and nutrient supply for adipocytes. Cellular energy metabolism is reflected mainly by mitochondria, which can be quantified by the mitochondrial DNA copy number per cell. In the present study, we aimed to investigate the effect of overconditioning on angiogenesis and mitochondrial biogenesis in AT of nonlactating cows, irrespective of the physiological influences of lactation and pregnancy. Eight nonpregnant, nonlactating cows received a ration of increasing energy density for 15 wk, during which body weight and body condition increased substantially. Subcutaneous AT was biopsied every 8 wk, and blood was sampled monthly. The blood concentrations of indicators of oxidative stress increased continuously throughout the experimental period, possibly damaging mitochondrial DNA. Concomitantly, HIF-1α, a major marker for hypoxia, increased until wk 8, indicating insufficient angiogenesis in the rapidly expanding AT. Based on the observation that the number of apoptotic cells decreased with increasing hypoxia, the increasing mitochondrial DNA copy numbers might compensate for the hypoxia, reinforcing the production of oxidative stressors. Key transcription factors of mitochondrial biogenesis were largely unaffected. Thus, increased oxidative stress does not impair mitochondrial DNA.

Effects of body condition, monensin, and essential oils on ruminal lipopolysaccharide concentration, inflammatory markers, and endoplasmatic reticulum stress of transition dairy cows.

Evidence exists that dairy cows experience inflammatory-like phenomena in the transition period. Rumen health and alterations in metabolic processes and gene networks in the liver as the central metabolic organ might be key factors for cows' health and productivity in early lactation. This study made use of an animal model to generate experimental groups with different manifestations of postpartal fat mobilization and ketogenesis. In total, 60 German Holstein cows were allocated 6 wk antepartum to 3 high-body condition score (BCS) groups (BCS 3.95) and 1 low-BCS group (LC; BCS 2.77). High-BCS cows were fed an antepartal forage-to-concentrate ratio of 40:60 on dry matter basis, in contrast to 80:20 in the LC group, and received a monensin controlled-release capsule (HC/MO), a blend of essential oils (HC/EO), or formed a control group (HC). We evaluated serum haptoglobin, kynurenine, tryptophan, ruminal lipopolysaccharide concentration and mRNA abundance of nuclear factor kappa B (NF-κB), nuclear factor E2-related factor 2 (Nrf2), and endoplasmatic reticulum stress-induced unfolded protein response (UPR) target genes in liver biopsy samples from d -42 until +56 relative to calving. Nearly all parameters were highly dependent on time, with greatest variation near calving. The ruminal lipopolysaccharide concentration and evaluated target genes were not generally influenced by antepartal BCS and feeding management. The kynurenine-to-tryptophan ratio was higher in LC than in HC/MO treatment on d 7. Ruminal lipopolysaccharide concentration was higher in HC/MO than in the HC group, but not increased in HC/EO group. Abundance of UPR target gene X-box binding protein 1 was higher in HC/MO than in HC/EO group on d 7. Hepatic mRNA abundance of Nrf2 target gene glutathione peroxidase 3 was higher, whereas expression of NF-κB target gene haptoglobin tended to be higher in LC than in HC/EO cows. The HC/MO cows showed the most prominent increase in the abundance of glutathione peroxidase 3 and haptoglobin after calving in comparison to antepartal values. Results indicate the presence of inflammatory-like phenomena near calving. Simultaneously, alterations in UPR and Nrf2 target genes with antioxidative properties and haptoglobin occurred, being most prominent in LC and HC/MO group.

Influence of conjugated linoleic acid and vitamin E on performance, energy metabolism, and change of fat depot mass in transitional dairy cows.

The objective of this experiment was to determine the effects of conjugated linoleic acid (CLA) and vitamin E as well as their interaction on performance variables and lipomobilization during late pregnancy and early lactation (wk 6 antepartum until wk 10 postpartum). For this purpose, 59 pluriparous German Holstein cows were assigned to 4 dietary groups in a 2 × 2 design with the factors CLA and vitamin E at 2 levels. For this trial, we selected cows with a high body condition score because they are more likely to mobilize fat and consequently are at a higher risk of developing ketosis. Furthermore, concentrate proportions were adjusted to provoke ketosis. Lactation performance variables were analyzed in 3 periods (d 42 antepartum until calving, 1 to 21 d in milk, 22 to 70 d in milk). Dry matter intake and net energy intake were reduced in animals receiving CLA. Milk fat content was reduced in the CLA group compared with the control group (4.83 vs. 5.46% in period 2; 3.36 vs. 4.57% in period 3). In the vitamin E and the CLA + vitamin E groups, reduction of milk fat content was observed in period 3 (3.76 vs. 4.57% compared with the control group). Milk yield was not affected by treatment. ß-Hydroxybutyrate concentrations and liver lipid contents were not influenced by CLA or vitamin E. Moreover, longitudinal changes of adipose tissue depot mass were not affected by dietary treatments. Results suggest that the effects CLA had on milk composition were compensated by an increased milk yield and a decreased dry matter intake. Reduced milk energy output in CLA-treated animals was compensated by a reduced dry matter intake. Therefore, the net energy balance was not affected by either treatment. Consequently, we found no group effect on the mobilization of adipose tissue.

Differential effects of monensin and a blend of essential oils on rumen microbiota composition of transition dairy cows.

In response to oral application, monensin alters the rumen microbiota, increasing ruminal propionate production and energy availability in the animal. Data from different studies indicate that the susceptibility of rumen bacteria to monensin is mainly cell-wall dependent but tracing its activity to specific microbial groups has been challenging. Several studies have shown a similar effect for essential oils but results are inconsistent. To investigate the influence of monensin and a blend of essential oils (BEO, containing thymol, guaiacol, eugenol, vanillin, salicylaldehyde, and limonene) on the rumen microbiome, rumen liquid samples were collected orally on d 56 postpartum from cows that had either received a monensin controlled-release capsule 3 wk antepartum, a diet containing a BEO from 3 wk antepartum onward, or a control diet (n = 12). The samples were analyzed for pH, volatile fatty acid, ammonia, and lipopolysaccharide concentrations and protozoal counts. A 16S rRNA gene fingerprinting analysis (PCR-single-strand conformation polymorphism) and sequencing revealed that the BEO treatment had no effect on the rumen microbiota, whereas monensin decreased bacterial diversity. Twenty-three bacterial species-level operational taxonomic units were identified for which monensin caused a significant decrease in their relative abundance, all belonging to the phyla Bacteroidetes (uncultured BS11 gut group and BS9 gut group) and Firmicutes (Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae). Ten bacterial operational taxonomic units belonging to the phyla Actinobacteria (Coriobacteriaceae), Bacteroidetes (Prevotella), Cyanobacteria (SHA-109), and Firmicutes (Lachnospiraceae and Ruminococcaceae) increased in relative abundance due to the monensin treatment. These results confirm the hypothesis that varying effects depending on cell-wall constitution and thickness might apply for monensin sensitivity rather than a clear-cut difference between gram-negative and gram-positive bacteria. No effect of monensin on the archaea population was observed, confirming the assumption that reported inhibition of methanogenesis is most likely caused through a decrease in substrate availability, rather than by a direct effect on the methanogens. The data support the hypothesis that the observed increase in ruminal molar propionate proportions due to monensin may be caused by a decrease in abundance of non-producers and moderate producers of propionate and an increase in abundance of succinate and propionate producers.