Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Dairy Science
Non Technical Summary
Nitrogen is an expensive nutrient and environmentally harmful waste product. Under current nutritional practices, 75% of the nitrogen consumed by a lactating dairy cow is excreted in manure. To feed the growing world population, while maintaining fresh water and air quality, dairy systems need to drastically increase nitrogen efficiency. Our long-term goal is to develop dietary strategies that increase productivity of dietary protein and reduce N excretion in manure.The majority of nitrogen losses occur in the liver when amino acids (AA) are converted to urea. The overall hypothesis is that stimulation of hepatic anabolic activity increases splanchnic extraction of AA but reduces urea production. Using dietary approaches and a splanchnic multi-catheterized dairy cow model we aim to:Determine the effect of amino acid supplementation on splanchnic AA extraction and metabolism.Analyze the effect of dietary energy sources on splanchnic AA fluxes.Investigate cellular mechanisms of insulin and AA regulation of hepatic metabolism.Update a lactating dairy cow model of hepatic AA metabolism.This project contributes to more efficient milk production, reduced environmental impact of U.S. dairy systems, and satisfaction of humanity's need for high-quality food. Our overall research program on liver and mammary metabolism could lead to an increase in nitrogen efficiency of 50% (to 37.5% nitrogen efficiency), which considering the 9.4 million dairy cows in the U.S., would translate into more than one million acres of protein production land released and 100 tons less nitrogen excreted in dairy cattle manure yearly.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
The long-term goal of this proposal is to identify dietary strategies that maximize AA utilization for milk protein synthesis and minimize splanchnic AA catabolism and N excretion. Within this proposal, we aim to understand AA and energy regulation of pDVC and hepatic AA extraction and metabolism. We hypothesize that mTORC1 mediate effects on splanchnic extraction of AA increase reversible splanchnic use of N (e.g. protein synthesis) and reduce irreversible N conversion to urea for excretion.The specific objectives are:Determine the effect of G1AA and BCAA on splanchnic individual AA fluxes, hepatic metabolism and urea output, and milk components yield in dairy cows.Analyze the effect of dietary energy sources on BCAA regulation of splanchnic individual AA fluxes, hepatic metabolism, and milk components yield in dairy cows.Determine the role of insulin on G1AA and BCAA stimulation of hepatic mTORC1 activity in lactating dairy cows.Incorporate nutritional regulatory mechanisms into a mathematical model of hepatic AA metabolism in the lactating dairy cow.
Project Methods
Objective 1:Treatments will consist of abomasal infusion of water (negative control), His and Met (1:1 mass ratio) or the BCAAs Ile and Leu (0.6:1 mass ratio) to reduce the MP deficiency by 50% (12.5% deficiency), or the combination of both EAA groups to meet MP requirements. Ruminally cannulated multiparous cows will be fixed with indwelling catheters in a jejunal branch of the mesenteric artery, a duodenal branch of the mesenteric vein, a small branch of the right ruminal vein, portal vein, and a small branch of the hepatic vein, at least 4 weeks before the beginning of the experiment as previously described (Huntington et al., 1989).Cows will adapt to the experimental diet for 2 weeks before starting infusion. Infusion periods will be 10 days long with 6 days wash out period in between. Diets will be restricted to 95% of the intake during the adaptation period. During AA infusion, diets will be fed in 6 equal meals and every 2 hr for the last 24 hr. Feed consumption will be recorded at every meal. Milk production will be measured in every milking from infusion day 6 to 10, and milk composition will be analyzed in composed samples from those 8 milkings. Body weight will be recorded at the beginning and end of each period for two consecutive days.On the last day of AA infusion pAH will be continuously infused into the mesenteric and ruminal vein for 4 hr at a rate of 6 g/h (12 g/h total) to determine blood flow (Larsen et al., 2015), and arterial, portal and hepatic vein samples will be collected every 30 min. Hematocrit will be determined immediately after sampling in arterial samples by centrifugation in capillary tubes at room temperature (20ºC). Blood samples will be immediately cooled on ice. Glucose, lactate, BHBA, urea-N and ammonia-N will be analyzed from deproteinized whole blood samples. Plasma will be isolated by centrifugation and pAH, individual AA, VFA, NEFA, triglyceride, insulin, albumin and total protein will be analyzed. For AA analysis, plasma samples will be gravimetrically combined with13C universally labeled internal AA standards for LC-MS analysis.Objective 2:Peak lactation dairy cows will be fed isoenergetic high starch or high fiber diets to meet NEL requirements, with adequate levels of RDP and limited RUP to generate a 25% MP deficiency (NRC, 2001). Under each dietary treatment, water or the BCAAs Ile and Leu (0.6:1 mass ratio) will be abomasally infused to reduce the MP deficiency by 2 thirds. The effect of BCAA infusion on visceral and hepatic individual AA clearance rate constants will be compared under the two dietary energy sources in a 2 by 2 factorial arrangement of treatments.Ruminally cannulated multiparous cows will be fixed with indwelling catheters as described above. Infusion periods will be 10 days long plus 6 days for diet adaptation or infusion wash out, and 10 days for infusion of each group of EAA. Diets will be restricted to 95% of the intake during the adaptation period for the lowest intake treatment. During AA infusion, diets will be fed in 6 equal meals and every 2 hr for the last 24 hr. Feed consumption will be recorded at every meal. Milk production will be measured in every milking from infusion day 7 to 10, and milk composition will be analyzed in composed samples from those 8 milkings. Body weight will be recorded at the beginning and end of each period for two consecutive days.Infusions and sample collection and analysis will be as described in Objective 1. Dietary energy source and EAA infusion effects and interactions on dependent variables will be analyzed with a mixed model in R, with random effects of cow and period and fixed effects of energy, BCAA and the interaction within cow and period. Repeated measures within infusion periods will be considered for not composed samples.Objective 3:An independent study to this proposal will be performed to determine the effect of insulin on G1AA and BCAA stimulation of mammary AA extraction and milk protein synthesis in lactating dairy cows. Either water, the G1AA His and Met, or the BCAA Ile and Leu will be infused for 10 days. On day 7 of AA infusion, cows will be subjected to insulinemic-euglycemic clamp, where insulin will be continuously infused to rise plasma insulin concentration, and glucose will be infused at the required rate to maintain euglycemia. Liver biopsies will be collected on day 6 (pre-clamp) and day 10 (clamped) of abomasal infusions to determine the effect of insulin on G1AA and BCAA stimulation of mTORC1 signaling, mRNA expression of metabolic enzymes, and tissue free AA concentrations.Liver tissue samples will be flush frozen in liquid N and stored at -80ºC for analysis. Frozen tissue will be lysed with 1.0 mm glass beads in a Mini-Beadbeater-24. For LC-MS analysis of tissue lysates will be deproteinized in perchloric acid, filtered (0.2 μm) and 2 μL will be injected into a quadrupole LC-MS for analysis. Standard curves will be generated for each AA. For qPCR analysis of mRNA expression, tissues will be homogenized in TRI Reagent (Sigma) and RNA will be extracted following provider instructions. Complementary DNA (cDNA) will be synthesized from RNA (1μg) by Superscript III reverse transcriptase (Invitrogen) in a thermocycler. Primers for target genes including in the mTORC1 pathway (AKT1, TSC1, TSC2, MTOR, RPS6KB1, EIF4BP1), ribosomal RNA genes (18S and 28S), and AA transporters (SLC1A1, 1A5, 3A2, 7A1, 7A5, and 36A1) will be designed with the IDT primer design tool, and real time PCR will be performed using SYBR Green dye-based PCR amplification in a BioRad Real Time PCR machine. Protein expression and phosphorylation of proteins in the mTOR pathway including Akt, mTOR, S6K, 4E-BP1 and rpS6 will be determined by western blotting as previously described (Arriola Apelo et al., 2014b).Objective 4: The current hepatic model (Hanigan et al., 2004a) will be evaluated by residual analysis with literature and experimental data. If as expected, model reparameterization is required, up to three possible mathematical functions will be considered based on error pattern (e.g. linear, quadratic, and exponential). Revised model parameterization will be performed by the maximum likelihood method using the Nelder-Mead algorithm (Press, 2007). Prediction errors will be randomly sampled to generate a population of parameter estimates by non-parametric bootstraping (Efron and Tibshirani, 1993). Means, standard errors, and 95% confidence intervals of that population of parameters will be used as descriptors of model parameters. A multivariate analysis based on AIC and residual analysis will be performed using mean parameter values to compare the original and revised model. Global sensitivity analysis of the final model will be performed for input variable and individual function parameters (Saltelli et al., 1999).