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.

Glucose metabolism and the somatotropic axis in dairy cows after abomasal infusion of essential fatty acids together with conjugated linoleic acid during late gestation and early lactation.

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

Effects of a combined essential fatty acid and conjugated linoleic acid abomasal infusion on metabolic and endocrine traits, including the somatotropic axis, in dairy cows.

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

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.

Ad libitum milk replacer feeding, but not butyrate supplementation, affects growth performance as well as metabolic and endocrine traits in Holstein calves.

The enhanced growth performance of calves fed a higher plane of nutrition pre-weaning is well documented, and the effect of butyrate on the development of the gastrointestinal tract in calves has been evaluated. The aim of this study was to examine the synergistic effects of ad libitum milk replacer (MR) feeding and butyrate supplementation on growth performance and energy metabolism in calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (Res) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate supplementation. Colostrum and transition milk were fed in predefined amounts (Res or Adl) for the first 3 d postpartum. Ad libitum and restrictive MR feeding with or without butyrate was performed from d 4 until wk 8 of age. From wk 9 to 10, all calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate (CON), hay, and water were freely available. Intakes of MR and CON were measured daily. Calves were weighed at birth and weekly thereafter. Blood was drawn on d 1 before the first colostrum intake; on d 2, 4, and 7; and weekly thereafter until the end of the study to measure plasma concentrations of metabolites and hormones. Liver samples were taken at d 50 and at the end of the study to determine gene expression related to glucose metabolism. Milk, MR, and total nutrient intake were greater, but CON intake was lower in Adl than in Res calves, resulting in a greater body weight, but partially lower gain to feed ratio in Adl than in Res. Plasma concentrations of glucose and insulin were higher during the ad libitum milk-feeding period, whereas plasma ß-hydroxybutyrate was lower in Adl than in Res. Plasma concentrations of nonesterified fatty acids, lactate, total bilirubin, and cortisol were lower, but triglyceride and cholesterol concentrations were higher in Adl than in Res at specific time points. Feed intake, growth performance, and metabolic and endocrine changes were insignificantly affected by butyrate, and hepatic gene expression of enzymes related to endogenous glucose production was barely influenced by ad libitum MR feeding and butyrate supplementation. Intensive MR feeding indicated greater stimulation of growth and anabolic metabolism, but butyrate supplementation did not further improve postnatal growth or anabolic processes either in intensive or restrictive MR-fed calves.

Effects of general anesthesia with ketamine in combination with the neuroleptic sedatives xylazine or azaperone on plasma metabolites and hormones in pigs.

Physiological research with swine often includes sedation or general anesthesia (GA), which may influence the basal physiological responses of experimental animals and may have the potential to confound or interfere with the effects of experimental factors of interest. Using 6 adult female pigs, we investigated whether selected plasma metabolites and hormones are influenced by GA induced with ketamine (K) and 2 neuroleptic sedatives, namely azaperone (A) and xylazine (X). Fasted pigs rotationally received either no drug, a single intravenous administration of A or X, or A or X combined with ketamine (AK or XK, respectively), and plasma concentrations of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), glucagon, insulin, and cortisol were determined for a 5-h period following administration. Azaperone and X induced deep sedation, whereas AK and XK induced GA. Overall, the average plasma glucose concentrations were increased by A and X, with the latter exerting a stronger effect that was also associated with hypoinsulinemia ( < 0.05). Time-dependent effects indicated a more rapid increase in glucose concentration due to X or XK than AK. Plasma NEFA concentrations were elevated by A and AK and to a lesser extent by X and XK ( < 0.05). Plasma lactate and TG levels were elevated by A and AK and remained unaffected by X or XK. Plasma cortisol concentrations were elevated ( < 0.05) by X and XK and even more so with a single administration of A ( < 0.05), while the combined effect of A with ketamine resulted in the highest cortisol concentrations ( < 0.05). Our data suggest that the effects of azaperone are mediated by cortisol but less so for xylazine, which also indicates that azaperone elicits a stronger stress response in pigs. Xylazine probably induces long-lasting, fasting-state hyperglycemia through the stimulation of hepatic glucose production associated with hypoinsulinemia. A discriminant analysis based on the variation in all of the measured metabolites and hormones, collectively, indicated that ketamine induced no additional effect on the overall physiological response patterns than that of the individual sedatives. In conclusion, the neuroleptic sedatives azaperone, and to a lesser extent, xylazine, acutely affect the metabolism of pigs, so primary metabolic readouts obtained under these drugs may be confounded.

Effect of Follicle Size on In Vitro Maturation of Pre-Pubertal Porcine Cumulus Oocyte Complexes.

Very small follicles (<3.0 mm diameter) are over-represented on the surface of ovaries of non-cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre-pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5-4.0 mm) or large (4.5-6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5- to 6.0-mm follicles reach metaphase II (MII) compared with those from follicles with 2.5-4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß-oestradiol (E2 ) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P4 ) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP-mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.

Acute effects of general anesthesia with propofol, pentobarbital or isoflurane plus propofol on plasma metabolites and hormones in adult pigs.

Experimental setups for physiological research, in which acute operative interventions need to be performed, can require inclusion of general anesthesia (GA), which may interfere or confound with the effects of the experimental factors of interest on measured variables. It was recently shown that the most commonly used sedatives/anesthetics in pigs (e.g., ketamine, xylazine, azaperone) affect physiological responses and thus the primary metabolic readouts have the potential to be confounded. To extend the search for a physiologically-friendly anesthesia regime for such studies, we investigated effects of GA induced by propofol (Prop) or pentobarbital (Pent) or propofol plus isoflurane (Prop + Isof) on plasma concentrations of commonly measured metabolites and hormones. In 2 experimental runs, 6 female pigs fitted with jugular vein catheters were used. Fasted pigs received either no drug (CON) or anesthetized rotationally either with Prop, Pent or Prop + Isof on different days, separated with washout periods of sufficient length (2 to 3 d). Six-h profiles of glucose, lactate, non-esterified fatty acids (NEFA), triglycerides (TG), cholesterol, urea as well as hormones including glucagon, insulin and cortisol were determined. Concentrations of cholesterol, urea and glucagon remained unaffected by any of the treatments ( > 0.05). Pent tended to increase cortisol from 30 to 90 min after drug administration. Glucose and lactate concentrations were increased ( < 0.05) by Prop and Pent within the first hour of GA ( < 0.05). Propofol and Pent reduced NEFA concentrations, which were more pronounced during the last 2 h of the studied period. Triglyceride concentrations were increased by all 3 agents within the first 45 min with Prop containing treatments exerting a stronger effect than Pent. Our data suggest that GA with Prop and particularly with Pent adulterate plasma metabolite and hormone profiles of pigs acutely, and thus has the potential to confound the effects of experimental factors of interest. Although Prop + Isof anesthesia did not differ from the controls, providing a physiologically-friendly GA, both single and the isoflurane-combined treatment of Prop induced hypertriglyceridemia due to the lipid adjuvant of the Prop drugs. It is concluded that readouts obtained under GA may be influenced both by physiological adulterations as response to anesthesia as well as by artifacts due to accompanying ingredients of the drug formulations.

Postpartum levels of 8-iso-prostaglandin F2α in plasma and milk phospholipid fractions as biomarker of oxidative stress in first-lactating dairy cows.

F2-isoprostanes such as 8-iso-prostaglandin F2 (8-iso-PGF2α) are formed by free radical-catalyzed mechanisms from membrane phospholipids and from low density lipoproteins through peroxidation of arachidonic acid. Esterified 8-iso-PGF2α is cleaved by phospholipases, circulates in blood and is excreted as putatively harmful oxidatively modified lipid via the kidney into urine. In this study we demonstrate that 8-iso-PGF2α concentrations in plasma samples from heifers are higher (p<0.005) compared to those from first-lactating dairy cows at 71 days postpartum. Furthermore, plasma 8-iso-PGF2α concentrations vary with ovarian activity and differ in response to luteolytic initiation as well as activation of the hypothalamic-pituitary-gonadal axis between heifers and first-lactating cows. Sustainable concentrations of 8-iso-PGF2α (50-150 pg/ml) are detectable in the phospholipid fraction of milk, suggesting milk as an additional excretion route for 8-isoprostanes. Plasma levels largely paralleled levels in milk (p<0.001). Plasma phospholipid 8-iso-PGF2α concentrations in cyclic cows decreased (p<0.05) from day 38 to day 71 postpartum, whereas milk phospholipid 8-iso-PGF2α rather increased (p<0.05). Cyclic cows tend to have higher 8-isoprostane levels compared to acyclic animals. In contrast to lipohydroperoxides, concentration of 8-iso-PGF2α were not correlated with milk yield (p>0.05). Our data indicate 8-iso-PGF2α may be a novel biomarker of oxidative stress in dairy cow, which is detectable in blood as well as in milk.

Inactivation of the LOX-1 pathway promotes the Golgi apparatus during cell differentiation of mural granulosa cells.

In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX-1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)-dependent and ER-independent calcium pools. In the present study, we show that inhibition of LOX-1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium-dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose-dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum.

LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low-density lipoprotein (ox-LDL). Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a receptor of ox-LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)-dependent and ER-independent calcium pools. Inhibition of the LOX-1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX-1. Our data indicate that blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals.

Influence of 4-week intraduodenal supplementation of quercetin on performance, glucose metabolism, and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows.

Quercetin has been shown to be a potent antioxidant, acts hepatoprotectively, and affects glucose and lipid metabolism in monogastrics. If this is also true in ruminants, quercetin could be beneficial in periparturient high-yielding dairy cows by ameliorating the negative effects of free radical formation and reducing the severity of liver lipidosis and ketosis. In a first attempt to evaluate effects of a long-term quercetin treatment, we intraduodenally administered twice daily 18 mg of quercetin (Q)/kg of body weight to 5 late-lactation (215d in milk) dairy cows over a period of 28 d. Frequent blood samples were taken before and during administration to determine plasma concentrations of flavonols and metabolites. Before and after 1 and 4 wk of Q administration, glycogen and fat content as well as mRNA expression of selected genes were measured in liver biopsies. Furthermore, euglycemic, hyperinsulinemic, and hyperglycemic clamp studies were conducted before and after 2 wk of Q administration. During the experiment, dry matter intake and most other zootechnical data remained unchanged. Milk protein content was increased in wk 2 and 4 of Q administration compared with basal values, whereas fat and lactose contents of milk remained unchanged. Plasma nonesterified fatty acids, γ-glutamyl transferase, cholesterol, glutamate dehydrogenase, triglyceride, and albumin concentrations, as well as liver fat and glycogen concentrations, were not affected by Q supplementation. Plasma glucose and ß-hydroxybutyrate concentrations in plasma decreased and increased, respectively, under the influence of quercetin. During hyperglycemic clamp conditions, the relative increase of plasma insulin was higher after 2 wk of Q administration, and a tendency for an increased rQUICKI (revised quantitative insulin sensitivity check index) was observed. The relative mRNA expression levels of selected genes related to glucose metabolism, fat metabolism, and antioxidative status were not altered after 1 or 4 wk of Q supplementation. In conclusion, the effects on insulin release and sensitivity support the assumption that administration of Q could have positive effects on the metabolic adaption of high-yielding cows to early lactation. The increase of milk protein content in response to Q supplementation needs to be verified.

Initial characterization of an outbreed mouse model for male factor (in)fertility.

We analysed an outbreed mouse line which was selected for the phenotype 'high fertility' for 158 generations. During this selection period the mouse strain increased the number of offspring per litter from 10.4 to 17.1 and the total litter weight up to ~160%. In this study, we initially characterize the reproductive phenotype of high fertility males. Surprisingly, male bucks of the fertility line (FL1) show reduced percentage of motile and progressive motile spermatozoa; however, other sperm motility characteristics (e.g. velocity parameters) are improved compared with an unselected control line. Cytometrical investigation of the testicular cell-type composition indicated a significant increased concentration of diploid cells by a concomitant reduction in haploid cells in the testicular parenchyma of FL1. Furthermore, total testosterone concentrations in blood are dramatically increased in FL1 (>20 ng/mL). In line with increased testosterone levels, we observed increased expression rates of steroidogenic key enzymes Cyp11 and Cyp17 from FL1 testis samples. These data indicate that FL1 males have a manifest 'high fertility phenotype'. Diallelic crosses imply that male-only contribution largely determines the reproductive outcome in cross-breeding experiments. FL1 therefore is a promising model for future investigations on male factor (in)fertility. Our observation might also offer valuable cues for human reproductive medicine.

Maturity-onset diabetes of the young and hepatic adenomatosis - characterisation of a new mutation.

Hepatocyte nuclear factor 1 alpha (HNF1A) mutations cause maturity-onset diabetes of the young (MODY) type 3. Further extending the phenotypic spectrum, HNF1A mutations are associated with hepatic adenomatosis. A 20-year old lean, female patient with newly diagnosed diabetes mellitus was negative for diabetes-associated autoantibodies and had no relevant family history. Hepatic adenomatosis was diagnosed. Her HNF1A gene was examined and identified alterations further analysed.Sequencing of her HNF1A gene revealed a previously uncharacterised Q495X nonsense mutation, along with the known A98V polymorphism, both in the heterozygous state. The patient's father was also a carrier of both the mutation and the polymorphism. An oral glucose tolerance test (OGTT) revealed impaired glucose tolerance, whereas imaging of his liver was unremarkable. Wild type HNF1A and HNF1A carrying the Q495X mutation were co-transfected in reporter gene assays. The mutation causes a dominant-negative HNF1A protein variant which blocks HNF1A wild-type-mediated gene expression.The novel Q495X mutation is the likely cause of our patient's diabetes and hepatic adenomatosis. It may also cause her father's impaired glucose tolerance. More generally speaking, if non-autoimmune diabetes is suspected, examination of the liver may provide important diagnostic clues. Furthermore, patients with hepatic adenomatosis without known diabetes should be screened by OGTT. Relatives of patients with HNF1A mutations should also be screened by OGTT to detect potential early-stage diabetes.

Bone morphogenetic protein-6 (BMP-6): mRNA expression and effect on steroidogenesis during in vitro maturation of porcine cumulus oocyte complexes.

Oocyte secreted factors (OSFs) have emerged as important factors for follicular development. The present study investigated the effect of the potential OSF bone morphogenic protein (BMP)-6 on steroidogenesis in porcine cumulus oocyte complexes during in vitro maturation. Cumulus oocyte complexes (COCs), cumulus complexes (CCs) without oocytes and CCs with supplemented BMP-6 were cultured for 0, 5, 26 or 46 h. BMP-6 transcripts were detected in oocytes and cumulus cells at all time points. In both cell types the mRNA expression was most intense after 5h, and decreased during further maturation. After 26 and 46 h of culture, CCs secreted significantly less 17ß-estradiol than COCs. This effect was reversed by adding BMP-6 to CCs cultures. In addition, a down-regulation of Cyp19A1, the rate-limiting enzyme of 17ß-estradiol synthesis, was detected in CC cultures after 5h. As seen for 17ß-estradiol secretion, the addition of BMP-6 caused a significant increase in Cyp19A1 mRNA levels after 5, 26 and 46 h of culture. Progesterone secretion and transcripts of steroidogenic marker proteins StAR and 3ß-HSD were not affected considerably by oocyte removal or addition of BMP-6. Furthermore, BMP-6 did not affect the activity of the mitogen-activated protein kinase. The results indicated that BMP-6 is a potential OSF and is involved in the prevention of premature luteinisation in cumulus cells via enhancing 17ß-estradiol synthesis.

Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats.

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

Hormonal regulation of multiple promoters of the rat mitochondrial glycerol-3-phosphate dehydrogenase gene: identification of a complex hormone-response element in the ubiquitous promoter B.

Rat mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. Here, we demonstrate that thyroid hormone (3,5,3'-tri-iodo-L-thyronine) and steroid hormone but not the peroxisome proliferator clofibrate and retinoic acid stimulate the activation of the ubiquitous promoter B in a receptor-dependent manner, whereas the more tissue-restricted promoters A and C are not inducible by these hormones. Thyroid hormone action is mediated by a direct repeat +4 (DR+4) hormone-response element as identified by deletion and mutation analyses of promoter B in transient transfection analyses. The DR+4 element was able to bind to an in vitro translated thyroid hormone receptor in band-shift and supershift experiments. The hormone-response element comaps with a recognition site for the transcription factor Sp1, suggesting complex regulation of this sequence element. Mutation of this Sp1-recognition site reduces the basal promoter B activity dramatically in HepG2 and HEK293 cells in transient transfection and abolishes the binding of Sp1 in band-shift experiments. As demonstrated by Western-blot experiments, administration of tri-iodothyronine to euthyroid rats increases hepatic mGPDH protein concentrations in vivo. As it has recently been reported that human mGPDH promoter B is not regulated by tri-iodothyronine, this is the first example of a differentially tri-iodothyronine-regulated orthologous gene promoter in man and rat.

Two thyroid hormone-mediated gene expression patterns in vivo identified by cDNA expression arrays in rat.

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. Only a limited number of T3-target genes have been identified so far and their complex regulation pattern is poorly understood. We performed cDNA expression array hybridisation to identify T3-regulated genes and to investigate their expression pattern after various time points in vivo. Radioactively labelled cDNA was prepared from hepatic RNA of hypothyroid and hyperthyroid rats 6, 24 and 48 h after the administration of T3. Labelled cDNA probes were hybridised to rat Atlas Arrays. Twenty-three of 588 genes were shown to be differentially regulated, 18 of which were previously not known to be regulated by T3. The expression of 19 genes was verified by independent northern blot hybridisation. Two different expression time courses of T3 expression were observed. In a first expression profile ('early' expression) the transcription level of the target genes rises within 6 h, drops by 24 h and increases again within 48 h after the administration of T3. In a second expression profile ('late' expression) the mRNA level rose in the first 6 h and rose further by 48 h, indicating an additional regulation mechanism. Nuclear respiratory factor (NRF)-1 and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), but not NRF-2, were up-regulated within 6 h after T3 administration, suggesting NRF-1 and/or PGC-1 as key regulators for mediating the 'late' expression pattern.

Multiple promoters direct the tissue-specific expression of rat mitochondrial glycerol-3-phosphate dehydrogenase.

The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle which transfers reduction equivalents from the cytoplasm into the mitochondria. We analyzed the distribution of different exon 1-containing transcripts by RT-PCR in various tissues in vivo. Exon 1 a was predominantly expressed in brain, brown adipose tissue and pancreas, exon 1b was ubiquitously expressed, and exon 1c was exclusively expressed in testis. In transient transfection assays the ubiquitous promoter B showed a detectable activity, whereas promoters A and C were completely silent. A deletion mutational analysis located the basal promoter B activity to a 316 bp core sequence upstream of the transcription start site.

Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3'). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA two- to fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.