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.

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.

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.

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.

Effects of vitamin E and selenium supplementation on blood lipid peroxidation and cortisol concentration in dairy cows undergoing omentopexy.

Twenty dairy cows with left abomasal displacement were used to investigate the effects of vitamin E and selenium treatment on thiobarbituric acid reactive substances (TBARS) and blood cortisol in dairy cows stressed by omentopexy. The cows were randomly divided into two groups. Ten hours before surgery 6 g of DL-α-tocopheryl acetate (6 mg/kg) and 67 mg of natrium selenite (0.1 mg/kg) in volume of 40 ml (Vitaselen® ) were administered subcutaneously to 10 cows; the control animals (n = 10) received an equivalent volume of injectable water (40 ml). The injection of vitamin E and selenium produced a rapid rise (p < .05) in blood α-tocopherol and selenium concentrations. The serum vitamin E increased several times 10 hr after vitamin E and Se injection and raised continuously to the highest average concentration 21.6 mg/L at hr 24 after the surgery. The highest selenium concentration was seen 10 hr after selenium administration with holding the increased concentrations in comparison with initial ones during the whole study. Two-way ANOVA did not show significant treatment effect on plasma concentrations TBARS in the study. The plasma concentrations of thiobarbituric acid reactive substances reached the maximum value of 0.18 µmol/L in the control group 5 hr after the surgery. Twenty-four hours after the surgery, the TBARS values returned to the initial ones. Serum cortisol increased in both groups after surgery. The highest cortisol concentrations were reached at 1 hr after surgery in the experimental and control group (56.7 ± 28.8 and 65.3 ± 26.1 µg/L respectively). A return to the levels similar to the initial ones was recognized 24 hr after the surgery. The ANOVA revealed a significant effect of vitamin E and selenium injection on plasma cortisol (p < .05). In conclusion, we have demonstrated that abdominal surgery resulted in typical stress changes with no significant effects of a single vitamin E/Se injection on blood lipid peroxidation. In addition, a weaker cortisol response to the abdominal surgery was recognized in animals treated with vitamin E and selenium.

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.

Different milk feeding intensities during the first 4 weeks of rearing dairy calves: Part 2: Effects on the metabolic and endocrine status during calfhood and around the first lactation.

Feeding dairy calves at high intensity has been demonstrated to increase milk yield in later life. We investigated the effect of 3 different feeding regimens in the preweaning period on the metabolic and endocrine status during calfhood and in heifers at the onset of the first lactation. In trial 1, 57 German Holstein calves were allocated to 3 different feeding groups: milk replacer restricted to 6.78 kg/calf per day, 11.5% solids (MR-res, n = 20), milk replacer 13.8% solids, ad libitum (MR-ad lib, n = 17), and whole milk ad libitum (WM-ad lib, n = 20). All calves received ad libitum colostrum for 3 d postnatal (p.n.). From d 4 to 27, all calves were fed according to their respective feeding regimen, resulting in average intakes of 6.38, 9.25, and 9.47 kg/d in MR-res, MR-ad lib, and WM-ad lib, respectively. Thereafter, all calves were fed according to the MR-res regimen until weaning at d 55 (gradually until d 69 p.n.). Blood samples were collected on d 0 before colostrum intake and on d 1, 3, 11, 22, 34, 43, 52, 70, 90, and 108 p.n. Liver biopsies were taken on d 19 and 100, and on d 22, 52, and 108 p.n. intravenous glucose tolerance tests were performed. The male calves (n = 8 to 10 per group) underwent also an insulin tolerance test on d 24, 54, and 110 p.n. The females (n = 28) from trial 1 were further reared and bred as common practice, and were enrolled in trial 2 when beginning the last trimester of pregnancy. Blood samples were collected monthly antepartum starting 91 d before calving and weekly (0-70 d) postpartum. Trial 1 was subdivided into 4 phases (P): P0 (d 0-1), P1 (d 2-27), P2 (d 28-69), and P3 (d 70-110 p.n.). In trial 1, the leptin and adiponectin concentrations increased with colostrum intake. Differences in fatty acids, insulin, adiponectin, revised quantitative insulin sensitivity check index (RQUICKI), and variables from the glucose tolerance tests were largely limited to P1. The MR-res group had greater RQUICKI and fatty acid values, and lower insulin and, as a trend, adiponectin concentrations than in 1 or both ad lib groups. These differences were partly sustained in P2 (fatty acids, adiponectin, and RQUICKI) and in P3 (adiponectin). The hepatic mRNA abundance of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and pyruvatcarboxylase increased from d 19 to 100. None of the blood variables were different between the groups when tested in pregnancy and lactation. Our results do not support a sustained deflection of metabolic regulation by rearing at different feeding intensities; nevertheless, the differences observed during rearing might influence nutrient utilization in later life or the cellular development of organs, such as the mammary gland, and thereby affect milk yield. Further studies involving greater animal numbers and, thus, improved power will help to sort out the mechanisms of programming body function in later life via nutrition in early life.

The effect of conjugated linoleic acid supplements on oxidative and antioxidative status of dairy cows.

Dairy cows develop frequently negative energy balance around parturition and in early lactation, resulting in excessive mobilization of body fat and subsequently in increased risk of ketosis and other diseases. Dietary conjugated linoleic acid (CLA) supplements are used in dairy cows mainly for their depressing effect on milk fat content, but are also proposed to have antioxidative properties. As negative energy balance is associated with oxidative stress, which is also assumed to contribute to disease development, the present study was conducted to examine effects of CLA on oxidative and antioxidative status of lactating dairy cows. German Holstein cows (primiparous n=13, multiparous n=32) were divided into 3 dietary treatment groups receiving 100g/d of control fat supplement, containing 87% stearic acid (CON; n=14), 50g/d of control fat supplement and 50g/d of CLA supplement (CLA 50; n=15), or 100g/d of CLA supplement (CLA 100; n=16). The CLA supplement was lipid-encapsulated and contained 12% of trans-10,cis-12 CLA and cis-9,trans-11 CLA each. Supplementation took place between d1 and 182 postpartum; d 182 until 252 postpartum served as a depletion period. Blood was sampled at d -21, 1, 21, 70, 105, 140, 182, 224, and 252 relative to calving. The antioxidative status was determined using the ferric-reducing ability of plasma, α-tocopherol, α-tocopherol-to-cholesterol mass ratio, and retinol. For determination of oxidative status concentrations of hydroperoxides, thiobarbituric acid-reactive substances (TBARS), N'-formylkynurenine, and bityrosine were measured. Mixed models of fixed and random effects with repeated measures were used to evaluate period 1 (d -21 to 140) and 2 (d182-252) separately. Cows showed increased oxidative stress and lipid peroxidation during the periparturient period in terms of increased serum concentrations of hydroperoxides and TBARS, which decreased throughout lactation. During period 1, the supplemented cows had lower TBARS concentrations, which was not detectable in period 2. The other determined parameters were not affected by CLA supplementation. The obtained results show that dietary CLA supplementation in the chosen dosage, formulation, and application period had a marginal antioxidative effect in terms of lipid peroxidation in lactating dairy cows.

Longitudinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 1: The adipokines apelin and resistin and their relationship to receptors linked with lipolysis.

The transition from pregnancy to lactation is characterized by major changes in glucose and adipose tissue metabolism. Anti- and prolipolytic pathways mediated via the hydroxycarboxylic acid receptors 1 (HCAR1) and 2 (HCAR2) and tumor necrosis factor-α receptor 1 (TNFR1), as well as the adipokines apelin and resistin, are likely involved in regulating these processes. This study aimed to determine the mRNA abundance of the aforementioned receptors in both subcutaneous and visceral adipose tissue, to characterize the adipokine concentrations in serum, and to test the effects of feeding diets with either high or low portions of concentrate and a concomitant niacin supplementation from late gestation to early lactation. Twenty pluriparous German Holstein cows were all kept on the same silage-based diet until d 42 antepartum, when they were allocated to 2 feeding groups: until d 1 antepartum, 10 animals each were assigned to either a high-concentrate (60:40 concentrate-to-roughage ratio) or a low-concentrate diet (30:70). Both groups were further subdivided into a control and a niacin group, the latter receiving 24 g/d of nicotinic acid from d -42 until 24. From d 1 to 24 postpartum, the concentrate portion was increased from 30 to 50% for all cows. Biopsies of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) were taken at d -42, 1, 21, and 100 relative to parturition. Blood samples were drawn along with the biopsies and on d -14, 3, 7, 14, and 42. The concentrations of the adipokines apelin and resistin in serum were measured via ELISA. The mRNA of the 3 receptors in AT was quantified as well as the protein abundance of HCAR2 by Western blot. The feeding regimen did not affect the variables examined. The concentrations of apelin remained fairly constant during the observation period, whereas the resistin concentrations increased toward parturition and decreased to precalving levels within 1 wk after calving. The mRNA abundance of HCAR1, HCAR2, and TNFR1 changed in SCAT and RPAT during the considered time period. For the HCAR2 protein, time-dependent changes were restricted to SCAT. The mRNA abundance of all receptors was greater in RPAT than in SCAT. The tissue-specific correlations observed between the receptors point to a link between these factors and may indicate different regulatory roles in the respective tissues. This study provides insight into the complex metabolic adaptations during the transition period and supports a differential regulation of lipolysis among SCAT and RPAT in dairy cows.

Changes in lipid metabolism and ß-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation.

Dairy cattle will mobilize large amounts of body fat during early lactation as an effect of decreased lipogenesis and increased lipolysis. Regulation of lipid metabolism involves fatty acid synthesis from acetate and ß-adrenergic-stimulated phosphorylation of hormone-sensitive lipase (HSL) and perilipin in adipocytes. Although basic mechanisms of mobilizing fat storage in transition cows are understood, we lack a sufficiently detailed understanding to declare the exact regulatory network of these in a broad range of dairy cattle. The objective of the present study was to quantify 1) protein abundance of fatty acid synthase (FAS), 2) extent of phosphorylation of HSL and perilipin in vivo, and 3) ß-adrenergic stimulated lipolytic response of adipose tissues in vitro at different stages of the periparturient period. We fed 20 German Holstein cows an energy-dense or an energetically adequate diet prepartum and 0 or 24 g/d nicotinic acid (NA) supplementation. Biopsy samples of subcutaneous and retroperitoneal adipose tissue were obtained at d 42 prepartum (d -42) and at d 1, 21, and 100 postpartum (d +1, d +21, d +100, respectively). To assess ß-adrenergic response, tissue samples were incubated with 1 µ isoproterenol for 90 min at 37°C. The NEFA and glycerol release, as well as HSL and perilipin phosphorylation, was measured as indicators of in vitro stimulated lipolysis. In addition, protein expression of FAS and extent of HSL and perilipin phosphorylation were measured in fresh, nonincubated samples. There was no effect of dietary energy density or NA on the observed variables. The extent of HSL and perilipin phosphorylation under isoproterenol stimulation was strongly correlated with the release of NEFA and glycerol, consistent with the functional link between ß-adrenergic-stimulated protein phosphorylation and lipolysis. In the nonincubated samples, FAS protein expression was decreased at d +1 and d +21, whereas HSL and perilipin phosphorylation increased from d -42 to d +1 and remained at an increased level throughout the first 100 d of lactation. In vitro lipolytic response was significant in prepartum samples at times when in vivo lipolysis was only minimally activated by phosphorylation. These data extend our understanding of the complex nature of control of lipolysis and lipogenesis in dairy cows and could be useful to the ongoing development of systems biology models of metabolism to help improve our quantitative knowledge of the cow.

The rapid increase of circulating adiponectin in neonatal calves depends on colostrum intake.

Adiponectin, an adipokine, regulates metabolism and insulin sensitivity. Considering that the transplacental transfer of maternal proteins of high molecular weight is hindered in ruminants, this study tested the hypothesis that the blood concentration of adiponectin in neonatal calves largely reflects their endogenous synthesis whereby the intake of colostrum might modify the circulating concentrations. We thus characterized the adiponectin concentrations in neonatal and young calves that were fed either colostrum or formula. Three trials were performed: in trial 1, 20 calves were all fed colostrum for 3 d, and then formula until weaning. Blood samples were collected on d 0 (before colostrum feeding), and on d 1, 3, 11, 22, 34, 43, 52, 70, 90, and 108 postnatum. In trial 2, 14 calves were studied for the first 4 d of life. They were fed colostrum (n=7) or formula (n=7), and blood samples were taken right after birth and before each morning feeding on d 2, 3, and 4. In trial 3, calves born preterm (n=7) or at term received colostrum only at 24 h postnatum. Blood was sampled at birth, and before and 2 h after feeding. Additionally, allantoic fluid and blood from 4 Holstein cows undergoing cesarean section were sampled. Adiponectin was quantified by ELISA. In trial 1, the serum adiponectin concentrations recorded on d 3 were 4.7-fold higher than before colostrum intake. The distribution of the molecular weight forms of adiponectin differed before and after colostrum consumption. In trial 2, the colostrum group had consistently greater plasma adiponectin concentrations than the formula group after the first meal. In trial 3, the preterm calves tended to have lower concentrations of plasma adiponectin than the term calves at birth and before and 2 h after feeding. Furthermore, the adiponectin concentrations were substantially lower in allantoic fluid than in the sera from neonatal calves and from cows at parturition. Our results show that calves are born with very low blood concentrations of adiponectin and placental transfer of adiponectin to the bovine fetus is unlikely. In conclusion, colostrum intake is essential for the postnatal increase of circulating adiponectin in newborn calves.

Tocopherols and tocotrienols in serum and liver of dairy cows receiving conjugated linoleic acids or a control fat supplement during early lactation.

The fat-soluble vitamin E comprises the 8 structurally related compounds (congeners) α-, ß-, γ-, and δ-tocopherol (with a saturated side chain) and α-, ß-, γ-, and δ-tocotrienol (with a 3-fold unsaturated side chain). Little is known regarding the blood and liver concentrations of the 8 vitamin E congeners during the transition from pregnancy to lactation in dairy cows. We thus quantified tocopherols (T) and tocotrienols (T3) in serum and liver and hepatic expression of genes involved in vitamin E metabolism in pluriparous German Holstein cows during late gestation and early lactation and investigated whether dietary supplementation (from d 1 in milk) with conjugated linoleic acids (CLA; 100g/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 liver biopsies were collected on d -21, 1, 21, 70, and 105 (liver only) relative to calving. In both groups, the serum concentrations of αT, γT, ßT3, and δT3 increased from d -21 to d 21 and remained unchanged between d 21 and 70, but were unaffected by CLA. The concentrations of the different congeners of vitamin E in liver did not differ between the CTR and the CLA groups. In both groups, the concentrations of the vitamin E forms in liver changed during the course of the study. The hepatic mRNA abundance of genes controlling vitamin E status did not differ between groups, but α-tocopherol transfer protein and tocopherol-associated protein mRNA increased with time of lactation in both. In conclusion, the concentrations of vitamin E congeners and the expression of genes related to vitamin E status follow characteristic time-related changes during the transition from late gestation to early lactation but are unaffected by CLA supplementation at the dosage used.

Energy and metabolic sensing G protein-coupled receptors during lactation-induced changes in energy balance.

The free fatty acid receptor FFA1, FFA2, and FFA3 and hydroxy-carboxylic acid receptor (HCA2) are G protein-coupled receptors, acting as energy and metabolic sensors. Herein, we characterized the tissue-specific mRNA abundance of genes encoding for these receptors at different stages of lactation. In addition, potential effects of supplementation with or without conjugated linoleic acids (CLA) were tested. Tissues from pluriparous cows (subcutaneous adipose tissue [SAT] and liver) and from primiparous cows (3 SAT locations, 3 visceral adipose tissues, liver, mammary gland, and skeletal muscle) were used from 2 separate trials. In primiparous cows, the mRNA abundance of all receptors (FFA3 was not detectable by the applied protocol in muscle and udder) was lowest in muscle (P < 0.05). With the exception of FFA1, gene expression of the investigated receptors was higher in adipose tissue than in the non-adipose tissue. Expression of FFA1 in liver (P < 0.03), FFAR2 in SAT (P < 0.01), and HCA2 in SAT (P < 0.01) from pluriparous cows changed during the observation period (days 21 to 252 relative to parturition). The correlation between mRNA abundance of HCA2 and peroxisome proliferator-activated receptor gamma (PPARG) and likewise PPARG2 (P < 0.01) in SAT indicates a link between HCA2 and PPARG. Differences in receptor mRNA abundance between the CLA-fed and the control animals were scarce and limited to HCA2 and FFA1 in 1 and 2 time points, respectively (less hepatic HCA2mRNA in CLA-fed pluriparous cows and greater FFA1 mRNA abundance in 2 visceral adipose tissue depots in CLA-treated primiparous cows). In view of the metabolic changes occurring during the different phases of lactation, in particular, the altered concentrations of non-esterified fatty acids and ß-hydroxybutyrate acting as receptor ligands, the longitudinal tissue-specific characterization provided herein allows for a first insight into the regulation of these receptors at the gene expression level.

Lactation driven dynamics of adiponectin supply from different fat depots to circulation in cows.

Adipose tissue (AT) depots are heterogeneous in terms of morphology and adipocyte metabolism. Adiponectin, one of the most abundant adipokines, is known for its insulin sensitizing effects and its role in glucose and lipid metabolism. Little is known about the presence of adiponectin protein in visceral (vc) and subcutaneous (sc) AT depots. We assessed serum adiponectin and adiponectin protein concentrations and the molecular weight forms in vc (mesenterial, omental, and retroperitoneal) and sc (sternum, tail-head, and withers) AT of primiparous dairy cows during early lactation. Primiparous German Holstein cows (n = 25) were divided into a control (CON) and a conjugated linoleic acid (CLA) group. From day 1 of lactation until slaughter, CLA cows were fed 100 g of a CLA supplement/d (approximately 6% of cis-9, trans-11 and trans-10, cis-12 isomers each), whereas the CON cows received 100 g of a fatty acid mixture/d instead of CLA. Blood samples from all animals were collected from 3 wk before calving until slaughter on day 1 (n = 5, CON cows), 42 (n = 5 each of CON and CLA cows), and 105 (n = 5 each of CON and CLA cows) of lactation when samples from different AT depots were obtained. Adiponectin was measured in serum and tissue by ELISA. In all AT depots adiponectin concentrations were lowest on day 1 than on day 42 and day 105, and circulating adiponectin reached a nadir around parturition. Retroperitoneal AT had the lowest adiponectin concentrations; however, when taking total depot mass into consideration, the portion of circulating adiponectin was higher in vc than sc AT. Serum adiponectin was positively correlated with adiponectin protein concentrations but not with the mRNA abundance in all fat depots. The CLA supplementation did not affect adiponectin concentrations in AT depots. Furthermore, inverse associations between circulating adiponectin and measures of body condition (empty body weight, back fat thickness, and vc AT mass) were observed. In all AT depots at each time, adiponectin was present as high (approximately 300 kDa) and medium (approximately 150 kDa) molecular weight complexes similar to that of the blood serum. These data suggest differential contribution of AT depots to circulating adiponectin.

Effects of conjugated linoleic acids fed to dairy cows during early gestation on hematological, immunological, and metabolic characteristics of cows and their calves.

The aim of the present experiment was to test the stimulation ability of peripheral blood mononuclear cells (PBMC) expressed as stimulation index (SI) of newborn calves and of their dams fed a control fat supplement (CON, n=6) or 50 and 100g/d of a CLA-containing fat supplement (CLA50, n=5, and CLA100, n=6, respectively) during the preceding lactation period for 182 d after calving. The total intake of cis-9,trans-11 and trans-10,cis-12 CLA by groups CLA50 and CLA100 amounted to 4 and 8 g/d each, respectively. For this purpose, blood was collected immediately after parturition from calves before and after colostrum intake, and from cows after parturition and 21 d later. The SI was related to the fatty acid composition of erythrocyte and milk lipids and to various hematological and clinical-chemical parameters. Retrospective evaluation revealed that depletion time (i.e., the individual period elapsed between the day of terminating the feeding of the experimental diet in the preceding lactation period and the day of calving) ranged from 190 to 262 d, which corresponded to fetal exposure times of 19 to 102 d. The SI from cows increased significantly by 77 and 55%, within 21 d after calving according to the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and Alamar Blue assays, respectively. However, feeding of 50 g of the CLA product failed to demonstrate this increase in the MTT assay. Moreover, SI was significantly lower for calves whose dams belonged to the CLA50 group, whereas stimulation ability was comparable for the PBMC from calves whose mothers were treated with CON and CLA100. Plasma metabolites (total bilirubin, total cholesterol, glucose, nonesterified fatty acids, 3-ß-hydroxybutyrate, total protein, and albumin) and hematological parameters (hematocrit, white blood cell profile) were not significantly influenced by dietary treatments of the cows in the preceding lactation period. Although the fatty acid pattern of erythrocyte lipids of cows remained uninfluenced, that of calves showed alterations due to the feeding type of their dams. For example, C16:0 increased significantly from 14.4 to 16.9% of total fatty acid methyl esters, whereas cis-9,trans-11 CLA increased slightly from 0.11 to 0.15% at the same time in calves when their mothers were fed the CLA100 instead of the CON diet. Fatty acid profile of colostrum was significantly different from that of milk after 3 wk for most of the detected fatty acids, but was not influenced by diet type. In conclusion, feeding a CLA-containing fat supplement during the preceding lactation and gestation period exerted effects on the stimulation ability of PBMC from cows and calves for the subsequent parturition. However, CLA dose effects were inconsistent and require further investigation.

Bovine haptoglobin as an adipokine: serum concentrations and tissue expression in dairy cows receiving a conjugated linoleic acids supplement throughout lactation.

The present study aimed to characterize serum haptoglobin (Hp) concentrations throughout an entire lactation period in both primi- and multiparous cows and to compare them to the Hp mRNA expression in liver and - in view of Hp being potentially an adipokine - also in different subcutaneous (s.c.) and visceral fat depots. In addition, potential anti-inflammatory effects of long-term supplementation with conjugated linoleic acids (CLA) were evaluated by assessing Hp. Trial 1 comprised 33 cows and 16 Holstein heifers from day 21 ante partum until day 252 postpartum. The animals received 100 or 50 g/day CLA or a control fat supplement. Blood samples and biopsy (tail head fat and liver) samples were collected. Trial 2 included 25 Holstein heifers, 5 animals were slaughtered on the day of parturition, the remaining animals were allocated to either CLA (100 g/day, n=10) or control fat supplement (n=10) and slaughtered on days 42 and 105 postpartum, respectively. At slaughter, fat samples were collected from 3 different visceral depots, 3 s.c. depots and from liver tissue. Results indicated no effects of CLA on serum Hp and liver Hp mRNA for both trials and on Hp mRNA in biopsies from s.c. tail head fat. In omental and s.c. withers fat from trial 2, CLA reduced Hp mRNA on both day 42 and day 105. Hp mRNA was detectable in fat tissues from both trials with abundance values being significantly lower than in liver. The Hp mRNA abundance in the s.c. fat depots was generally higher than in the visceral depots. Haptoglobin mRNA abundance in the different tissues from trial 2 was correlated whereby all s.c. depots were interrelated. The evidence of Hp mRNA expression in adipose tissues and the presence of Hp-immune staining in histological fat sections confirm that Hp can be classified as a bovine adipokine. The lack of an evident relationship between circulating Hp concentrations and normal body fat portions in dairy cattle demonstrates that varying degrees of adiposity are not confounding factors when using Hp as inflammatory marker. The physiological changes in serum Hp concentration seem to be limited to parity and parturition. In view of the lack of effects of CLA on serum Hp concentrations, the observed reaction in two out of six different fat depots seems of marginal importance for the organisms as an entity.

Effect of trans-10, cis-12 conjugated linoleic acid on performance, adipose depot weights, and liver weight in early-lactation dairy cows.

In feeding practice, conjugated linoleic acid (CLA) supplements are used to decrease milk fat excretion in early-lactation dairy cows to save energy to counteract the physiological negative energy balance. The present study was conducted to examine the effects of CLA on energy metabolism, changes in liver weight, and the weight of different adipose depots during early lactation. Primiparous lactating German Holstein cows (n=25) were divided into 5 groups and each group contained 5 animals. The experiment started 21 d prepartum and continued until 105 d in milk (DIM). Cows were slaughtered at 1, 42, and 105 DIM. The experiment was divided into a prepartum period (21 d prepartum until calving), period 1 (1 until 42 DIM), and period 2 (>42 until 105 DIM). In the prepartum period, all animals were housed together and fed the same diet with no CLA supplementation. At 1 DIM, an initial group, with no CLA supplementation, was slaughtered. The 20 remaining cows were assigned to 2 diets. One group received 100g/d of a control fat supplement (CON; n=10) and the other group 100g/d of a CLA supplement (CLA; n=10) from 1 DIM until slaughter. Five cows of each feeding group were slaughtered after 42 DIM and the remaining animals after 105 DIM. The CLA supplement contained approximately 10% each of trans-10, cis-12 CLA and cis-9, trans-11 CLA. During the slaughter process the empty body weight was recorded and the omental, mesenteric, retroperitoneal, and s.c. adipose depots, as well as the liver, were dissected and weighed. The CLA treatment decreased milk fat content in period 1 (14.1%). In period 2, milk fat content (25.4%) and yield (17.1%) were lower in the CLA group. No effect of CLA on milk yield was observed. The net energy intake, milk energy output, and the calculated energy balance remained unchanged by CLA supplementation. No effect of CLA on the weights of liver, omental, mesenteric, or s.c. adipose depots was observed when related to empty body weight. Liver weight increased with DIM, whereas the retroperitoneal adipose depot weight decreased at the same time. Compared with the initial group, the retroperitoneal adipose depot weight for control animals slaughtered after 42 DIM was decreased (47.7%); however, for the CLA group slaughtered after 42 DIM, a trend to a lower retroperitoneal adipose depot weight (34.0%) was observed. This suggests a CLA-induced deceleration of mobilization of the retroperitoneal adipose depot during the first 42 DIM.

Physiological and conjugated linoleic acid-induced changes of adipocyte size in different fat depots of dairy cows during early lactation.

The aim of this study was to investigate the effects of lactation and conjugated linoleic acid (CLA) supplementation on adipocyte sizes of subcutaneous (s.c.) and visceral (VC) fat depots in primiparous dairy cows during the first 105 d in milk (DIM). German Holstein heifers (n=25) were divided into a control (CON) and a CLA group. From 1 DIM until sample collection, CLA cows were fed 100g of CLA supplement/d (about 6% of c9,t11 and t10,c12 isomers each), whereas the CON cows received 100g of fatty acid mixture/d instead of CLA. The CON cows (n=5 each) were slaughtered at 1, 42, and 105 DIM, and the CLA cows (n=5 each) were slaughtered at 42 and 105 DIM. Adipose tissues from 3s.c. depots (tailhead, withers, and sternum) and from 3 VC depots (omental, mesenteric, and retroperitoneal) were sampled. Hematoxylin-eosin staining was done to measure adipocyte area (µm(2)). Retroperitoneal adipocyte sizes were mostly larger than adipocytes from the other sites, independent of lactation time and treatment. Significant changes related to duration of lactation were limited to retroperitoneal fat: adipocyte sizes were significantly smaller at 105 DIM than at 1 DIM in CON cows. Adipocyte sizes were decreased in s.c. depots from the tailhead at 105 DIM and from the sternum at 42 DIM in CLA versus CON cows, whereas for VC depots, adipocyte sizes were decreased in mesenteric fat at 42 and 105 DIM, and in omental and retroperitoneal fat, at 105 DIM in CLA versus CON cows. Within the CLA group, adipocyte sizes were smaller in the s.c. depot from the tailhead at 105 DIM than at 42 DIM. Adipocyte sizes and depot weights were significantly correlated in s.c. depots (r=0.795) in the CLA group and in retroperitoneal fat both in the CON (r=0.698) and the CLA (r=0.723) group. In conclusion, CLA-induced decreases in adipocyte size indicate lipolytic or antilipogenic effects of CLA, or both effects, on adipose tissue in primiparous dairy cows.

Inter- and intra-individual differences in serum copper and zinc concentrations after feeding different amounts of copper and zinc over two lactations.

The influence of different copper and zinc contents in rations on blood serum concentrations was tested in a feeding trial at the Institute of Animal Nutrition of the Federal Research Institute for Animal Health (FLI). A total of 60 German Holstein breed cows (38 first lactating and 22 second lactating cows) were tested. All animals received a diet based on maize- and grass silage ad libitum. The animals were divided into two groups with 30 cows each. Group A received concentrates supplemented with copper and zinc as recommended, whereas Group B was offered a concentrate with roughly double the amount of copper and zinc. Blood samples were taken several times during the lactation. The concentrations of copper and zinc in the serum were measured and correlations between trace element intake and their serum concentrations were calculated. The mean dry matter intake was significantly higher for cows in the second lactation (19.1 kg/day) than for cows in the first lactation (16.4 kg/day), whereas the DM-intake was constant in the different feeding groups (17.3 and 17.4 kg/day). The correlation between feed intake in the first and the second lactation was r = 0.76. The copper concentration in the serum was not influenced by the different feedings (11.9 and 12.5 micromol/l), but high variations were found between the cows. The influence of the lactation number was not significant (12.1 and 12.4 micromol/l). In contrast to copper, the zinc concentration in the serum was significantly higher for Group B (14.0 micromol/l) than for Group A (12.2 micromol/l), respectively, for second lactating cows (13.8 micromol/l) in relation to 12.7 micromol/l for first lactating cows. The individual variation was also high as for copper. Neither the correlation between copper intake and copper serum concentration nor the correlation between zinc intake and zinc serum concentration was significant.

Prepartum nutrition alters fatty acid composition in plasma, adipose tissue, and liver lipids of periparturient dairy cows.

The fatty acyl profile of phospholipids (PL) determines the fluidity of cell membranes and affects cell function. The degree to which long-chain fatty acid (LCFA) composition of PL and triacylglycerols (TG) in liver and total lipids in adipose tissue can be altered by prepartum nutrition in peripartal dairy cows is unclear. Multiparous Holsteins (n = 25) were assigned to 1 of 4 prepartal diets: 1) CA, the control diet fed to meet 120% of energy requirements; 2) CR, a control diet fed to meet 80% of requirements; 3) S, a diet supplemented with mostly saturated free fatty acids (47% 16:0, 36% 18:0, 14% cis-18:1) and fed to meet 120% of requirements; or 4) U, a diet similar to S except that cows were abomasally infused with soybean oil so that the diet plus infused fat would meet 120% of requirements. Diets were fed for 40 d prepartum; all cows received a lactation diet postpartum. Groups CR and U had lower prepartum intakes of dry matter and net energy, but glucose concentrations in plasma were similar among treatments. Cows fed S, U, or CR had greater nonesterified fatty acids in plasma prepartum, but cows fed U had decreased beta-hydroxybutyrate postpartum. Postpartal concentrations of total lipids and glycogen in liver tissue were similar among treatments. Cows in group U had a greater percentage of 18:2 but less 16:0, 18:0, and 20:4 in plasma total lipids than cows fed S. Treatment U increased 18:2 and 18:3 and decreased 18:1 in subcutaneous adipose tissue at 1 d postpartum. Across diets, percentages of 16:0 and trans-18:1 were increased, and 18:0, 20:3, and 20:5 were decreased, in hepatic PL at d 1 postpartum. Significant treatment x time interactions indicated that treatment U increased 18:2 in hepatic PL at the expense of 18:1, 20:3, 20:4, 22:6, and 24:0 on d 1 postpartum, but changes were normalized by d 65 postpartum. The unsaturation index of hepatic PL was lower at d 1 than at d -45 or 65, which implies that hepatic membrane fluidity decreased around parturition. The unsaturation index at d 1 was greater for cows fed S than those fed CA or U. Percentages of 16:0, 18:1, and 22:0 were increased, and 18:0, 20:3, 20:4, 20:5, 24:0, and 26:0 were decreased, in hepatic TG at d 1. Prepartal feed restriction modestly affected tissue LCFA profiles. The LCFA profile of adipose tissue, liver PL, and liver TG can be altered by dietary LCFA supply prepartum; changes in liver are normalized by 65 d postpartum.