Effect of maternal supplementation with essential fatty acids and conjugated linoleic acid on metabolic and endocrine development in neonatal calves.

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

Modulation of colostrum composition and fatty acid status in neonatal calves by maternal supplementation with essential fatty acids and conjugated linoleic acid starting in late lactation.

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

Effects of colostrum instead of formula feeding for the first 2 days postnatum on whole-body energy metabolism and its endocrine control in neonatal calves.

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

Comparative analyses of estimated and calorimetrically determined energy balance in high-yielding dairy cows.

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

Indices of heart rate variability as potential early markers of metabolic stress and compromised regulatory capacity in dried-off high-yielding dairy cows.

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

Effects of feeding unlimited amounts of milk replacer for the first 5 weeks of age on rumen and small intestinal growth and development in dairy calves.

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

Evaluation of electrical broad bandwidth impedance spectroscopy as a tool for body composition measurement in cows in comparison with body measurements and the deuterium oxide dilution method.

Body fatness and degree of body fat mobilization in cows vary enormously during their reproduction cycle and influence energy partitioning and metabolic adaptation. The objective of the study was to test bioelectrical impedance spectroscopy (BIS) as a method for predicting fat depot mass (FDM), in living cows. The FDM is defined as the sum of subcutaneous, omental, mesenteric, retroperitoneal, and carcass fat mass. Bioelectrical impedance spectroscopy is compared with the prediction of FDM from the deuterium oxide (DO) dilution method and from body conformation measurements. Charolais × Holstein Friesian (HF; = 18; 30 d in milk) crossbred cows and 2 HF (lactating and nonlactating) cows were assessed by body conformation measurements, BIS, and the DO dilution method. The BCS of cows was a mean of 3.68 (SE 0.64). For the DO dilution method, a bolus of 0.23 g/kg BW DO (60 atom%) was intravenously injected and deuterium (D) enrichment was analyzed in plasma and whey by stabile isotope mass spectrometry, and total body water content was calculated. Impedance measurement was performed using a 4-electrode interface and time domain-based measurement system consisting of a voltage/current converter for applying current stimulus and an amplifier for monitoring voltage across the sensor electrodes. For the BIS, we used complex impedances over three frequency decades that delivers information on intra- and extracellular water and capacity of cell membranes. Impedance data (resistance of extra- and intracellular space, cell membrane capacity, and phase angle) were extracted 1) by simple curve fit to extract the resistance at direct current and high frequency and 2) by using an electrical equivalent circuit. Cows were slaughtered 7 d after BIS and D enrichment measurements and dissected for the measurement of FDM. Multiple linear regression analyses were performed to predict FDM based on data obtained from body conformation measurements, BIS, and D enrichment, and applied methods were evaluated by cross-validation. The FDM varied widely between cows and was correlated to D enrichment in plasma ( = 0.91, < 0.05). Prediction of FDM by body size measurements was less precise ( = 0.84), but FDM prediction was more accurate using D enrichment in plasma ( = 0.90) and BIS ( = 0.99) data. Therefore, both BIS and D enrichment analysis resulted in similarly good predictions of FDM in cows, and we conclude that BIS could have the potential to predict FDM in dairy cows from 40 to 380 kg.

Short communication: Free fatty acid receptors FFAR1 and FFAR2 during the peripartal period in liver of dairy cows grouped by their postpartum plasma ß-hydroxybutyrate concentrations.

Free fatty acid receptors (FFAR) play significant roles in various physiological processes, including energy metabolism, through interaction with their ligands, fatty acids. To determine whether the receptors FFAR1 and FFAR2 are involved in the regulation of liver metabolism during the peripartal period, we selected 13 German Holstein multiparous dairy cows and grouped them as high ß-hydroxybutyrate (H-BHB; n = 8) or low ß-hydroxybutyrate (L-BHB; n = 5) according to their individual maximum plasma BHB concentration observed within wk 2 or 3 postpartum (H-BHB: >1 mmol/L and L-BHB: <0.77 mmol/L). The selected cows had a milk yield of more than 10,000 kg/305 d during a previous lactation. The cows were fed a total mixed ration according to their requirements during the far-off dry period [5.9 MJ of net energy for lactation (NEL)/kg of dry matter (DM), crude protein (CP) 126 g/kg of DM], close-up dry period (6.5 MJ of NEL/kg of DM, CP 137 g/kg of DM), and lactation (7 MJ of NEL/kg of DM, CP 163 g/kg of DM). Blood samples were taken weekly, from d -34 to d 40 relative to parturition. Liver biopsies were taken on d -34, -17, 3, 18, and 30 relative to parturition and at slaughter (d 40). The protein abundance of FFAR1 was lower during the whole peripartal period in the H-BHB group. The abundance of FFAR2 increased over time and tended to be higher in H-BHB cows. The abundance of FFAR1 might be associated with imbalances of liver metabolism in peripartal dairy cows.

Variable liver fat concentration as a proxy for body fat mobilization postpartum has minor effects on insulin-induced changes in hepatic gene expression related to energy metabolism in dairy cows.

The liver plays a central role in adaptation for energy requirements around calving, and changes in the effects of insulin on hepatic energy metabolism contribute to metabolic adaptation in dairy cows. Hepatic insulin effects may depend on body fat mobilization. The objective of this study was to investigate the effects of insulin on the hepatic gene expression of enzymes involved in energy metabolism and factors related to nutrition partitioning in cows with high and low total liver fat concentration (LFC) after calving. Holstein cows were retrospectively grouped according to their LFC after calving as a proxy for body fat mobilization. Cows were classified as low (LLFC; LFC <24% fat/dry matter; n = 9) and high (HLFC; LFC >24.4% fat/dry matter; n = 10) fat-mobilizing after calving. Euglycemic-hyperinsulinemic clamps [6 mU/(kg × min) of insulin for 6 h] were performed in wk 5 antepartum (ap) and wk 3 postpartum (pp). Before and at the end of the euglycemic-hyperinsulinemic clamps, liver biopsies were taken to measure the mRNA abundance of enzymes involved in carbohydrate and lipid metabolism, expression related to the somatotropic axis, and adrenergic and glucocorticoid receptors. The mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK; PCK1), acyl-CoA-dehydrogenase very long chain (ACADVL), and hydroxyl-methyl-glutaryl-CoA-synthase 1 increased, but the mRNA abundance of solute carrier family 2 (SLC2A2 and SLC2A4), growth hormone receptor 1A (GHR1A), insulin-like growth factor 1 (IGF1), sterol regulatory element binding factor 1, adrenoceptor α 1A, and glucocorticoid receptor decreased from ap to pp. Insulin treatment was associated with decreased PCK1, mitochondrial PEPCK, glucose-6-phosphatase, propionyl-CoA-carboxylase α, carnitine-palmitoyl-transferase 1A, ACADVL, and insulin receptor mRNA, but increased IGF1 and SLC2A4 mRNA ap and pp and GHR1A mRNA pp. The mRNA abundance of SLC2A4 was greater, and the mRNA abundance of GHR1A and IGF1 tended to be lower in LLFC than in HLFC. Administration of insulin, albeit at a supraphysiological dose, was associated with inhibition of gene expression related to glucose production and ß-oxidation, but we observed variable effects in the degree of insulin depression of individual genes. Insulin status is important for regulation of nutrient partitioning, but different LFC pp had very little influence on changes in hepatic gene expression following administration of insulin.

Insulin-dependent glucose metabolism in dairy cows with variable fat mobilization around calving.

Dairy cows undergo significant metabolic and endocrine changes during the transition from pregnancy to lactation, and impaired insulin action influences nutrient partitioning toward the fetus and the mammary gland. Because impaired insulin action during transition is thought to be related to elevated body condition and body fat mobilization, we hypothesized that over-conditioned cows with excessive body fat mobilization around calving may have impaired insulin metabolism compared with cows with low fat mobilization. Nineteen dairy cows were grouped according to their average concentration of total liver fat (LFC) after calving in low [LLFC; LFC <24% total fat/dry matter (DM); n=9] and high (HLFC; LFC >24.4% total fat/DM; n=10) fat-mobilizing cows. Blood samples were taken from wk 7 antepartum (ap) to wk 5 postpartum (pp) to determine plasma concentrations of glucose, insulin, glucagon, and adiponectin. We applied euglycemic-hyperinsulinemic (EGHIC) and hyperglycemic clamps (HGC) in wk 5 ap and wk 3 pp to measure insulin responsiveness in peripheral tissue and pancreatic insulin secretion during the transition period. Before and during the pp EGHIC, [(13)C6] glucose was infused to determine the rate of glucose appearance (GlucRa) and glucose oxidation (GOx). Body condition, back fat thickness, and energy-corrected milk were greater, but energy balance was lower in HLFC than in LLFC. Plasma concentrations of glucose, insulin, glucagon, and adiponectin decreased at calving, and this was followed by an immediate increase of glucagon and adiponectin after calving. Insulin concentrations ap were higher in HLFC than in LLFC cows, but the EGHIC indicated no differences in peripheral insulin responsiveness among cows ap and pp. However, GlucRa and GOx:GlucRa during the pp EGHIC were greater in HLFC than in LLFC cows. During HGC, pancreatic insulin secretion was lower, but the glucose infusion rate was higher pp than ap in both groups. Plasma concentrations of nonesterified fatty acids decreased during HGC and EGHIC, but in both clamps, pp nonesterified fatty acid concentrations did not reach the ap levels. The study demonstrated a minor influence of different degrees of body fat mobilization on insulin metabolism in cows during the transition period. The distinct decrease in the glucose-dependent release of insulin pp is the most striking finding that explains the impaired insulin action after calving, but does not explain differences in body fat mobilization between HLFC and LLFC cows.

Short communication: Effects of oral flavonoid supplementation on the metabolic and antioxidative status of newborn dairy calves.

Scientific proof for flavonoids as a health tool in calf nutrition is inconsistent. We investigated the effects of the most abundant flavonoid, quercetin, and of a green tea extract (GTE) containing various catechins on the metabolic and antioxidative traits in dairy calves to clarify their potential health-promoting effects. Male newborn German Holstein calves (n=7 per group) received either no flavonoid (control group), 10mg of quercetin equivalents as quercetin aglycone or as rutin/kg of body weight (BW) per day, or 10mg/kg of BW per day of a GTE from d 2 to 26 of life. The supplements were provided with the morning and evening feeding. The calves were fed colostrum and milk replacer, and BW, feed intake, and health status were evaluated daily. Blood samples were collected from a jugular vein on d 1, 5, 12, 19, and 26 before the morning feeding to investigate the metabolic and antioxidative status of the calves. The growth performance and health status remained unchanged, but the GTE-fed calves had fewer loose feces than the controls. The plasma concentrations of quercetin changed over time and were higher in the rutin-fed group than in the control group, whereas the catechins were below the detection limit. The plasma Trolox equivalent antioxidative capacity and ferric reducing ability of plasma were measured as markers for plasma antioxidative capacity. The concentrations of Trolox equivalent antioxidative capacity increased, whereas ferric reducing ability of plasma decreased after the first day of life in all the groups. The oxidative stress markers in the plasma were measured as thiobarbituric acid reactive substances and F2-isoprostanes, but these did not indicate treatment or time effects. The plasma concentrations of total protein, albumin, urea, lactate, glucose, and nonesterified fatty acids and of insulin and cortisol varied over time, but no group differences were caused by the flavonoid supplementation. In summary, orally administered quercetin and catechins at the dosages used in the present study resulted in weak effects on health and no effects on the metabolic and antioxidative status of newborn dairy calves.

Bioavailability of the flavonol quercetin in neonatal calves after oral administration of quercetin aglycone or rutin.

Polyphenols, such as flavonoids, are secondary plant metabolites with potentially health-promoting properties. In newborn calves flavonoids may improve health status, but little is known about the systemically availability of flavonoids in calves to exert biological effects. The aim of this study was to investigate the oral bioavailability of the flavonol quercetin, applied either as quercetin aglycone (QA) or as its glucorhamnoside rutin (RU), in newborn dairy calves. Twenty-one male newborn German Holstein calves were fed equal amounts of colostrum and milk replacer according to body weight. On d 2 and 29 of life, 9 mg of quercetin equivalents/kg of body weight, either fed as QA or as RU, or no quercetin (control group) were fed together with the morning meal. Blood samples were taken before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 12, 24, and 48 h after feed intake. Quercetin and quercetin metabolites with an intact flavonol structure (isorhamnetin, tamarixetin, and kaempferol) were analyzed in blood plasma after treatment with glucuronidase or sulfatase by HPLC with fluorescence detection. Maximum individual plasma concentration was depicted from the concentration-time-curve on d 2 and 29, respectively. Additional blood samples were taken to measure basal plasma concentrations of total protein, albumin, urea, and lactate as well as pre- and postprandial plasma concentrations of glucose, nonesterified fatty acids, insulin, and cortisol. Plasma concentrations of quercetin and its metabolites were significantly higher on d 2 than on d 29 of life, and administration of QA resulted in higher plasma concentrations of quercetin and its metabolites than RU. The relative bioavailability of total flavonols (sum of quercetin and its metabolites isorhamnetin, tamarixetin, and kaempferol) from RU was 72.5% on d 2 and 49.6% on d 29 when compared with QA (100%). Calves fed QA reached maximum plasma concentrations of total flavonols much earlier than did RU-fed calves. Plasma metabolites and hormones were barely affected by QA and RU feeding in this experiment. Taken together, orally administrated QA resulted in a greater bioavailability of quercetin than RU on d 2 and 29, respectively, and quercetin bioavailability of quercetin and its metabolites differed markedly between calves aged 2 and 29 d.

Hepatic glucocorticoid and α1- and ß2-adrenergic receptors in calves change during neonatal maturation and are related to energy regulation.

Catecholamines and glucocorticoids are involved in fetal maturation of organ systems to prepare the fetus for extrauterine life. Calves, especially when born preterm, depend on function of the adrenergic system and the glucocorticoid axis to adapt energy metabolism for the neonatal period. We tested the hypothesis that hepatic glucocorticoid and α1- and ß2-adrenergic receptors in neonatal calves are involved in adaptation of energy metabolism around birth and that respective binding capacities depend on stage of maturation during the neonatal period. Calves (n=7 per group) were delivered by section preterm (PT, 9d before term) or were born at term (full-term, FT; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4d (FTC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT, FT) or on d 4 of life (FTC) to determine metabolic and endocrine changes. After slaughter at 26h after birth (PT, FT) or on d 4 of life (FTC), liver tissue was obtained to measure hepatic binding capacity of glucocorticoid and α1- and ß2-adrenergic receptors. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α1- and ß2-adrenergic receptors, respectively, and [(3)H]-dexamethasone for determination of glucocorticoid receptor in liver. Additional liver samples were taken to measure mRNA abundance of glucocorticoid and α1- and ß2-adrenergic receptors, of key enzymes and factors related to hepatic lipid metabolism, and of insulin-like growth factor 1 (IGF1). Plasma concentrations of ß-hydroxybutyrate and leptin changed with time, and leptin concentrations were affected by stage of maturation. The binding capacities for hepatic glucocorticoid and ß2-adrenergic receptors as well as gene expression of IGF1 were greater in FTC than in FT and PT, and binding affinity for ß2-adrenergic receptor was lowest in PT. The binding capacity of hepatic α1-adrenergic receptor was greatest in FTC and greater in FT than in PT. The binding capacities of glucocorticoid and α1-adrenergic receptors were mainly related to variables of glucose and lipid metabolism. In conclusion, our results indicate dependence of hepatic glucocorticoid and adrenergic receptors on stage of maturation in neonatal calves and emphasize the association of α1-adrenergic receptor and glucocorticoid receptor with neonatal glucose and lipid metabolism.

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and ß2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism.

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α1- and ß2-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2 h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α1- and ß2-AR and [(3)H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α1-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α1-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas ß2-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α1-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

Increased muscle fatty acid oxidation in dairy cows with intensive body fat mobilization during early lactation.

The beginning of lactation requires huge metabolic adaptations to meet increased energy demands for milk production of dairy cows. One of the adaptations is the mobilization of body reserves mainly from adipose tissue as reflected by increased plasma nonesterified fatty acid (NEFA) concentrations. The capacity of the liver for complete oxidation of NEFA is limited, leading to an increased formation of ketone bodies, reesterification, and accumulation of triglycerides in the liver. As the skeletal muscle also may oxidize fatty acids, it may help to decrease the fatty acid load on the liver. To test this hypothesis, 19 German Holstein cows were weekly blood sampled from 7 wk before until 5 wk after parturition to analyze plasma NEFA concentrations. Liver biopsies were obtained at d 3, 18, and 30 after parturition and, based on the mean liver fat content, cows were grouped to the 10 highest (HI) and 9 lowest (LO). In addition, muscle biopsies were obtained at d -17, 3, and 30 relative to parturition and used to quantify mRNA abundance of genes involved in fatty acid degradation. Plasma NEFA concentrations peaked after parturition and were 1.5-fold higher in HI than LO cows. Muscle carnitine palmitoyltransferase 1α and ß mRNA was upregulated in early lactation. The mRNA abundance of muscle peroxisome proliferator-activated receptor γ (PPARG) increased in early lactation and was higher in HI than in LO cows, whereas the abundance of PPARA continuously decreased after parturition. The mRNA abundance of muscle PPARD, uncoupling protein 3, and the ß-oxidative enzymes 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase, very long-chain acyl-CoA dehydrogenase, and 3-ketoacyl-CoA was greatest at d 3 after parturition, whereas the abundance of PPARγ coactivator 1α decreased after parturition. Our results indicate that around parturition, oxidation of fatty acids in skeletal muscle is highly activated, which may contribute to diminish the fatty acid load on the liver. The decline in muscle fatty acid oxidation within the first 4 wk of lactation accompanied with increased feed intake refer to greater supply of ruminally derived acetate, which as the preferred fuel of the muscle, saves long-chain fatty acids for milk fat production.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

[Quality of life of children and adolescents with psychiatric disorders. Results of the 1st multicenter study with an inventory to assess the quality of life in children and adolescents]. / Lebensqualität bei psychisch kranken Kindern und Jugendlichen. Ergebnisse der ersten multizentrischen Studie mit dem Inventar zur Erfassung der Lebensqualität bei Kindern und Jugendlichen (ILK).

OBJECTIVE: A newly developed, disorder-nonspecific instrument to measure the quality of life in children and adolescents (ILK, Mattejat et al., 1998) was tested with regard to its ability to differentiate between psychiatric in- and outpatient samples. METHODS: 626 children and adolescents from regionally different in- and outpatient clinics completed the ILK questionnaire. The data obtained were first described and then analyzed by means of logistic regression analysis. RESULTS: Whereas the descriptive analysis revealed that inpatients evaluated all areas of life more unfavorably than outpatients did, the logistic regression analysis modified these results. It became evident that some of the findings obtained were due to the effects of age and/or gender. However, given age and gender as covariates, there still remained some important life domains in which inpatients were more impaired than outpatients. CONCLUSIONS: The ILK turned out to be an efficient and economic instrument to measure the quality of life in children and adolescents depending on the severity of their disorder. Moreover, it is sensitive to effects of age and gender. As the ILK is able to reliably identify areas with a reduced quality of life, it offers valuable starting points for indications, therapy planning and initial therapeutic interventions.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans.

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

[An inventory for assessing the quality of life of children and adolescents--a pilot study]. / Das Inventar zur Erfassung der Lebensqualität bei Kindern und Jugendlichen (ILK)--Eine Pilotstudie.

Up to now, the quality of life of children and adolescents with psychiatric disorders has rarely been investigated. Not many suitable instruments are available. A new one is the "Inventory for the Assessment of the Quality of Live in Children and Adolescents" presented here. Objectives and underlying conceptual assumptions are discussed, followed by an explanation of the instrument itself (questionnaires, rating scales, etc.). Finally, experiences with the application of the instrument are reported together with initial empirical results from a clinical sample and a sample of high school students. These indicate that the instrument usefully assesses clinically relevant information about the quality of life of the patients. A broader empirical analysis of this method is being prepared.