Absorption and intermediary metabolism of purines and pyrimidines in lactating dairy cows.

About 20 % of ruminal microbial N in dairy cows derives from purines and pyrimidines; however, their intermediary metabolism and contribution to the overall N metabolism has sparsely been described. In the present study, the postprandial patterns of net portal-drained viscera (PDV) and hepatic metabolism were assessed to evaluate purine and pyrimidine N in dairy cows. Blood was sampled simultaneously from four veins with eight hourly samples from four multi-catheterised Holstein cows. Quantification of twenty purines and pyrimidines was performed with HPLC-MS/MS, and net fluxes were estimated across the PDV, hepatic tissue and total splanchnic tissue (TSP). Concentration differences between veins of fifteen purine and pyrimidine nucleosides (NS), bases (BS) and degradation products (DP) were different from zero (P≤ 0·05), resulting in the net PDV releases of purine NS (0·33-1·3 mmol/h), purine BS (0·0023-0·018 mmol/h), purine DP (7·0-7·8 mmol/h), pyrimidine NS (0·30-2·8 mmol/h) and pyrimidine DP (0·047-0·77 mmol/h). The hepatic removal of purine and pyrimidine was almost equivalent to the net PDV release, resulting in no net TSP release. One exception was uric acid (7·9 mmol/h) from which a large net TSP release originated from the degradation of purine NS and BS. A small net TSP release of the pyrimidine DP ß-alanine and ß-aminoisobutyric acid (-0·032 to 0·37 mmol/h) demonstrated an outlet of N into the circulating N pool. No effect of time relative to feeding was observed (P>0·05). These data indicate that considerable amounts of N are lost in the dairy cow due to prominent intermediary degradation of purines, but that pyrimidine N is reusable to a larger extent.

Whey and casein labeled with L-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion.

Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1.

Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough.

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short-chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl-CoA carboxylase, methylmalonyl-CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6-bisphosphatase and glucose 6-phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high-producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist.

BACKGROUND: The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia. METHODS: First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion. RESULTS: Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ΔT (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ΔT > -3°C for more than 12 hours. CONCLUSIONS: Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.

Isolation of the cDNA encoding the acid labile subunit (ALS) of the 150 kDa IGF-binding protein complex in cattle and ALS regulation during the transition from pregnancy to lactation.

During the transition from pregnancy to lactation, dairy cows experience a 70% reduction in plasma IGF-I. This reduction has been attributed to decreased hepatic IGF-I production. IGF-I circulates predominantly in multi-protein complexes consisting of one molecule each of IGF-I, IGF binding protein-3 and the acid labile subunit (ALS). Recent studies in the mouse have shown that absence of ALS results in accelerated turnover and severely depressed concentration of plasma IGF-I. These observations suggest that reduced plasma ALS could be a second factor contributing to the fall of plasma IGF-I in peri-parturient cows. This possibility has not been studied due to the lack of bovine ALS reagents. To address this, we isolated the bovine ALS cDNA and used its sequence to develop a ribonuclease protection assay (RPA) and a bovine ALS antiserum. Using the RPA, ALS mRNA abundance was approximately fivefold higher in liver than in lung, small intestine, adipose tissue, kidney and heart, but was absent in muscle and brain. The antiserum detected the highest ALS levels in plasma followed by ovarian follicular fluid, lymph and colostrum. A portion of colostrum and follicular fluid ALS appears to be synthesized locally as ALS mRNA was found in mammary epithelial cells and ovarian follicular cells. Finally, we measured plasma ALS in dairy cows during the peri-parturient period (days -35 and +56 relative to parturition on day 0). Plasma ALS dropped by 50% between late pregnancy and the first day of lactation and returned to prepartum levels by day +56. To determine whether this reflected a change in hepatic expression, ALS mRNA was measured in liver biopsies collected on days -35, +3 and +56. ALS mRNA expression was significantly lower on day +3 than on day -35, but recovered completely by day +56. Finally, we examined the ability of GH to increase plasma ALS abundance at selected times before and after parturition (weeks -5, -2, +1 and +5). GH increased plasma ALS at weeks -5, -2 and +5, but not at week +1. Identical effects of GH were seen when the response considered was plasma IGF-I. We conclude that the decline in plasma ALS after parturition is a consequence of hepatic GH resistance and contributes to the associated reduction of plasma IGF-I.

Indicators of induced subacute ruminal acidosis (SARA) in Danish Holstein cows.

BACKGROUND: The prevalence of subacute ruminal acidosis (SARA) in dairy cows is high with large impact on economy and welfare. Its current field diagnosis is based on point ruminal pH measurements by oral probe or rumenocentesis. These techniques are invasive and inaccurate, and better markers for the diagnosis of SARA are needed. The goal of this study was to evaluate clinical signs of SARA and to investigate the use of blood, faecal and urinary parameters as indicators of SARA. Six lactating, rumen cannulated, Danish Holstein cows were used in a cross-over study with three periods. The first and second periods included two cows on control diet and two cows on nutritional SARA challenge. The third period only included two cows on SARA challenge. Control diet was a conventional total mixed ration [45.5% dry matter (DM), 17.8% crude protein, 43.8% neutral detergent fibre, and 22.5% acid detergent fibre (DM basis)]. SARA challenge was conducted by substituting control diet with grain pellets (50% wheat/barley) over 3 days to reach 40% grain in the diet. Ruminal pH was measured continuously. Blood samples were collected once daily at 7 h after feeding. Samples of faeces and urine were collected at feeding, and at 7 and 12 h after feeding. Blood samples were analysed for pCO2, pO2, pH, electrolytes, lactate, glucose, packed cell volume (PCV), and total plasma protein concentration. Milk composition, ruminal VFA, and pH of faeces and urine were measured. RESULTS: SARA was associated with decreased (P < 0.05) minimum ruminal, faecal and urinary pH. Daily times and areas of ruminal pH below 5.8, and 5.6 were increased to levels representative for SARA. Significant differences were detected in milk composition and ruminal VFAs. Blood calcium concentration was decreased (P < 0.05), and pCO2 tended to be increased (P = 0.10). Significant differences were not detected in other parameters. CONCLUSIONS: SARA challenge was associated with changes in faecal and urinary pH, blood calcium concentration and pCO2. These may be helpful as indicators of SARA. However changes were small, and diurnal variations were present. None of these parameters are able to stand alone as indicators of SARA.

High environmental temperature increases glucose requirement in the developing chicken embryo.

Environmental conditions during the perinatal period influence metabolic and developmental processes in mammals and avian species, which could impact pre- and postnatal survival and development. The current study investigated the effect of eggshell temperature (EST) on glucose metabolism in broiler chicken embryos. Broiler eggs were incubated at a high (38.9°C) or normal (37.8°C) EST from day 10.5 of incubation onward and were injected with a bolus of [U-(13)C]glucose in the chorio-allantoic fluid at day 17.5 of incubation. After [U-(13)C]glucose administration, (13)C enrichment was determined in intermediate pools and end-products of glucose metabolism. Oxidation of labeled glucose occurred for approximately 3 days after injection. Glucose oxidation was higher in the high than in the normal EST treatment from day 17.6 until 17.8 of incubation. The overall recovery of (13)CO2 tended to be 4.7% higher in the high than in the normal EST treatment. An increase in EST (38.9°C vs 37.8°C) increased (13)C enrichment in plasma lactate at day 17.8 of incubation and (13)C in hepatic glycogen at day 18.8 of incubation. Furthermore, high compared to normal EST resulted in a lower yolk-free body mass at day 20.9 (-2.74 g) and 21.7 (-3.81 g) of incubation, a lower hepatic glycogen concentration at day 18.2 (-4.37 mg/g) and 18.8 (-4.59 mg/g) of incubation, and a higher plasma uric acid concentration (+2.8 mg/mL/+43%) at day 21.6 of incubation. These results indicate that the glucose oxidation pattern is relatively slow, but the intensity increased consistently with an increase in developmental stage of the embryo. High environmental temperatures in the perinatal period of chicken embryos increased glucose oxidation and decreased hepatic glycogen prior to the hatching process. This may limit glucose availability for successful hatching and could impact body development, probably by increased gluconeogenesis from glucogenic amino acids to allow anaerobic glycolysis.