An in vitro method for assessment of amino acid bidirectional transport and intracellular metabolic fluxes in mammary epithelial cells.

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15N-labeled AA for 24 h followed by removal of the 15N-labeled medium and incubation with medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15N and 13C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.

Convergent responses of fish belonging to different feeding guilds to sewage pollution

This study aimed to evaluate if the presence of pollutants promotes changes in feeding habits of fish species from different trophic guilds: the detritivorous species, Hypostomus francisci, and the piscivorous, Hoplias intermedius. Both species were sampled at 12 sites (with different degrees of pollution) in the Rio das Velhas basin, which is heavily polluted by domestic and industrial sewage from the Metropolitan Region of Belo Horizonte (MRBH). Stable isotope analyses of carbon (δ13C) and nitrogen (δ15N) of fish tissue and the main food resources were performed. Fishes from both trophic guilds altered their diets in degraded environments, but the detritivorous species showed greater trophic plasticity. The isotopic niche of both trophic guilds was broadest in unpolluted sites and more δ15N enriched in polluted regions. The detritivorous species presented high niche-breadth in unpolluted sites, probably due to the greater variety of resources consumed. In addition, the δ15N of the detritivorous was more enriched than the piscivorous species in polluted sites. In conclusion, fishes from both trophic guilds presented similar isotopic responses to environmental pollution. However, the detritivorous species was more sensitive to these alterations and therefore, is likely a better indicator of environmental condition than the piscivorous.(AU)

Este estudo teve como objetivo avaliar se a presença de poluentes promove mudanças nos hábitos alimentares de espécies de peixes de diferentes guildas tróficas: a espécie detritívora, Hypostomus francisci, e a piscívora, Hoplias intermedius. Ambas espécies foram amostradas em 12 locais (com diferentes níveis de poluição) na bacia do Rio das Velhas, que é altamente poluída por esgoto doméstico e industrial da Região Metropolitana de Belo Horizonte (RMBH). Foram realizadas análises de isótopos estáveis de carbono (δ13C) e nitrogênio (δ15N) dos tecidos dos peixes e dos principais recursos alimentares. Espécies de ambas guildas tróficas alteraram suas dietas em ambientes degradados, mas a espécie detritívora apresentou maior plasticidade trófica. O nicho isotópico de ambas as espécies foi mais amplo em locais menos perturbados e mais enriquecido em δ15N em regiões poluídas. A espécie detritívora apresentou grande amplitude em seu nicho isotópico em locais menos perturbados, provavelmente devido à maior variedade de recursos consumidos. Além disso, o δ15N da espécie detritívora foi mais enriquecido que a espécie piscívora em locais poluídos. Em conclusão, ambas as espécies apresentaram respostas isotópicas semelhantes à poluição ambiental. No entanto, a espécie detritívora foi mais sensível a essas alterações e, portanto, é provavelmente uma melhor indicadora de condição ambiental do que a espécie piscívora.(AU)

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system.

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH4 production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH4 and CO2 by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, 13C-labeled H13COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both 13CH4 and 13CO2 production quadratically increased (P<0.01) with the addition of H13COOH. The total 13C (13CH4 and 13CO2) CR was also quadratically increased (P<0.01) when H13COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH3-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH4 and CO2 by rumen microorganisms.

Methionine to cystine ratio in the total sulfur amino acid requirements and sulfur amino acid metabolism using labelled amino acid approach for broilers.

BACKGROUND: Assuming that part of Methionine (Met) is converted into Cystine (Cys), but ignoring the rates with which such phenomenon occurs may lead to an excessive supply of Met in poultry diets. Such inconvenient could be easily avoided with the knowledge of the ideal Met:Cys/Total sulfur amino acids (TSAA) ratio and the rates of Met conversion into Cys. RESULTS: Met sources did not affect performance. Met:Cys/TSAA ideal ratio was determined using curvilinear-plateau regression model. Both optimum body weight gain and feed conversion ratio were estimated in 1007 g/day and 1.49, respectively, at 52% Met/TSAA ratio. Feed intake was not affected by Met:Cys/TSAA ratios. In the labelled amino acid assay, the rates with which Met was converted into Cys ranged from 27 to 43% in response to changes in Met:Cys/TSAA ratios, being higher at 56:44. CONCLUSION: Based on performance outcomes, the minimum concentration of Met relative to Cys in diets for broilers from 14 to 28 d of age based on a TSAA basis, is 52% (52:48 Met:Cys/TSAA). The outcomes from labelled amino acid assay indicate that highest the Met supply in diets, the highest is its conversion into Cys.

Effect of dietary nutrients on ileal endogenous losses of threonine, cysteine, methionine, lysine, leucine and protein in broiler chicks.

An isotope dose technique was utilized (i) to determine endogenous amino acid (AA) and protein losses and (ii) to propose adjusted values for AA requirements. The endogenous flow rate was calculated from the pool of enrichment in plasma AA, assuming similitude to enrichment of endogenous AA. In experiment 1, chicks were orally administered D4-lysine at 2% of estimated lysine intake from 16 to 24 days to find the isotopic steady state of the atom percent excess (APE) of lysine for plasma and jejunal and ileal digesta. The APE of D4-lysine in plasma, jejunal digesta and ileal digesta reached the isotopic steady state at 5.5, 3.4 and 2.0 days, respectively, by using the broken-line model. It was assumed that the isotopic steady state at 5 days identified for D4-lysine is also representative for the 15N-labeled AA. In experiment 2, chicks were fed diets from 1 to 21 days with increasing levels of fat (6%, 8%, 12%, 13% extract ether), protein (26%, 28.5%, 31% CP) or fiber (14%, 16%, 18% NDF) by adding poultry fat, soybean meal, blended animal protein or barley. Chicks were orally administered 15N-threonine, 15N-cysteine, 15N-methionine, 15N-lysine and 15N-leucine at 2% of estimated daily intake for 5 days from 17 to 21 days of age. Dietary nutrients influenced endogenous losses (EL), where dietary fat stimulated EL of lysine (P=0.06), leucine and protein (P=0.07); dietary protein enhanced EL of leucine and protein; and finally the dietary fiber increased EL of leucine. Dietary nutrients also affected apparent ileal digestibility (AID). Dietary fat increased AID of cysteine but decreased AID of lysine. Dietary protein reduced AID of protein, threonine, lysine and leucine, and similarly dietary fiber decreased AID of protein, threonine, methionine, lysine and leucine. In contrast, dietary fat or protein did not affect real ileal digestibility (RID) of protein and AA except threonine and leucine. The dietary fiber reduced the RID of protein, threonine and leucine. This indicate that variations of some endogenous AA and protein losses due to dietary nutrients almost eliminates the effects of RID, and thus the EL coming from the body should be utilized to adjust the AA requirement instead of changing the true digestible nutrients of ingredients. The present data suggest that 5 days' feeding labeled AA was enough to reach the isotopic steady state and AA requirements should be adjusted when additional dietary protein, fat or fiber is fed.

Isotopic Evidence of a Wide Spectrum of Feeding Strategies in Southern Hemisphere Humpback Whale Baleen Records.

Our current understanding of Southern hemisphere humpback whale (Megaptera novaeangliae) ecology assumes high-fidelity feeding on Antarctic krill in Antarctic waters during summer, followed by fasting during their annual migration to and from equatorial breeding grounds. An increase in the number of reported departures from this feeding/fasting model suggests that the current model may be oversimplified or, alternatively, undergoing contemporary change. Information about the feeding and fasting cycles of the two Australian breeding populations of humpback whales were obtained through stable isotope analysis of baleen plates from stranded adult individuals. Comparison of isotope profiles showed that individuals from the West Australian breeding population strongly adhered to the classical feeding model. By contrast, East Australian population individuals demonstrated greater heterogeneity in their feeding. On a spectrum from exclusive Antarctic feeding to exclusive feeding in temperate waters, three different strategies were assigned and discussed: classical feeders, supplemental feeders, and temperate zone feeders. Diversity in the inter-annual feeding strategies of humpback whales demonstrates the feeding plasticity of the species, but could also be indicative of changing dynamics within the Antarctic sea-ice ecosystem. This study presents the first investigation of trophodynamics in Southern hemisphere humpback whales derived from baleen plates, and further provides the first estimates of baleen plate elongation rates in the species.

The oral (13)C-bicarbonate technique for estimation of energy expenditure in dogs: validation against indirect calorimetry.

To get more knowledge about the energy requirements of dogs and to formulate appropriate feeding guidelines, it is essential to determine their energy expenditure (EE) in a reliable and feasible way. In this study, the non-invasive oral stable isotope (13)C-bicarbonate technique (o(13)CBT) was validated against indirect calorimetry (IC) for the determination of CO2-production and EE in dogs. Eleven privately owned dogs were simultaneously measured with IC and the o(13)CBT after being fasted overnight. All dogs were measured twice on two separate days. For calculation, measurements were divided into two groups depending on dogs' behaviour during the measurement. Dogs of Group 1 (n = 17) were resting calmly in the chamber and dogs of Group 2 (n = 5) were more active. Mean heart rate was significantly higher in Group 2 (102 beats per minute [bpm]) than in Group 1 (77 bpm) (p < 0.001). Within groups, the CO2-production and EE [kJ d(-1) kg BW(-0.75)] estimated by the o(13)CBT or IC did not differ significantly (Group 1: [Formula: see text] = 368; EEIC = 363; Group 2: [Formula: see text] = 701; EEIC = 718). However, the estimated (13)C recovery factor (RF) for the estimation of CO2-production was significantly different between Groups 1 and 2 (0.72 and 0.94, respectively, p < 0.001). The respiratory quotient (RQ), which is needed for the estimation of EE, did not differ between groups. This study shows that the non-invasive o(13)CBT can be used for accurate estimation of the CO2-production rate and EE in resting dogs. A value of 0.77 can be applied as an estimate of the RQ in fasted dogs and 0.72 as an appropriate estimate for RF when dogs are resting calmly during the measurements.

Stable isotope-labelled feed nutrients to assess nutrient-specific feed passage kinetics in ruminants.

Knowledge of digesta passage kinetics in ruminants is essential to predict nutrient supply to the animal in relation to optimal animal performance, environmental pollution and animal health. Fractional passage rates (FPR) of feed are widely used in modern feed evaluation systems and mechanistic rumen models, but data on nutrient-specific FPR are scarce. Such models generally rely on conventional external marker techniques, which do not always describe digesta passage kinetics in a satisfactory manner. Here the use of stable isotope-labelled dietary nutrients as a promising novel tool to assess nutrient-specific passage kinetics is discussed. Some major limitations of this technique include a potential marker migration, a poor isotope distribution in the labelled feed and a differential disappearance rate of isotopes upon microbial fermentation in non-steady state conditions. Such limitations can often be circumvented by using intrinsically stable isotope-labelled plant material. Data are limited but indicate that external particulate markers overestimate rumen FPR of plant fibre compared with the internal stable isotope markers. Stable isotopes undergo the same digestive mechanism as the labelled feed components and are thus of particular interest to specifically measure passage kinetics of digestible dietary nutrients.

Trophic ecology of Arapaima in Guyana: giant omnivores in Neotropical floodplains

Using stable nitrogen and carbon isotope signatures, we investigated the trophic ecology and identified potential prey fish groups supporting the giant Arapaima within floodplain lakes of the Essequibo River basin in southwestern Guyana. Morphological descriptions of feeding structures and digestive tract are presented together with preliminary data on Arapaima diets. Stable isotope results suggest that algivorous/detritivorous and omnivorous fishes contributed most to Arapaima biomass, and generally, that was consistent with what is known about Arapaima diets. Stable nitrogen isotope ratios for piscivorous fishes in these lakes were higher than nitrogen isotope ratios for Arapaima, indicating that piscivorous fishes are unlikely to constitute a major source of energy for Arapaima. This population of Arapaima has an intestine averaging 1.45 times total body length, relatively small teeth, and numerous, closely-spaced gill rakers. These morphological features, together with isotope data, support our inference that Arapaima are secondary consumers and may be better characterized as omnivores and not top predators.

Utilizando firmas de isotopos estables de nitrógeno y carbón, investigamos la ecología trófica e identificamos los grupos de peces que potencialmente mantienen a la Arapaima en los lagos inundables de la cuenca del río Essequibo, al suroeste de Guyana. Presentamos descripciones morfológicas de las estructuras alimentarias y tracto digestivo de la Arapaima, conjuntamente a datos preliminares de sus dietas. Los isotopos estables sugieren que peces algívoros/detritívoros y peces omnívoros son los principales contribuyentes de la biomasa de la Arapaima, y estos resultados son compatibles con lo que se conoce actualmente de la dieta de la Arapaima. A diferencia, las proporciones del isotopo estable de nitrógeno para peces piscívoros en estos lagos resultaron más altas que los valores obtenidos para el isotopo estable de nitrógeno en la Arapaima. Esto indica que es improbable que sean peces piscívoros los que constituyan la fuente energética principal de la Arapaima. La población de Arapaima estudiada presenta un intestino que promedia 1,45 veces la longitud total del cuerpo, dientes relativamente pequeños, y agallas con branquiespinas numerosas y cercanamente espaciadas. Estas características morfológicas, conjuntamente a los datos obtenidos a través del uso de isotopos estables apoyan nuestra inferencia que la Arapaima es un consumidor secundario y que puede ser caracterizada como un pez omnívoro y no como un depredador mayor.

Host behaviour and physiology underpin individual variation in avian influenza virus infection in migratory Bewick's swans.

Individual variation in infection modulates both the dynamics of pathogens and their impact on host populations. It is therefore crucial to identify differential patterns of infection and understand the mechanisms responsible. Yet our understanding of infection heterogeneity in wildlife is limited, even for important zoonotic host-pathogen systems, owing to the intractability of host status prior to infection. Using novel applications of stable isotope ecology and eco-immunology, we distinguish antecedent behavioural and physiological traits associated with avian influenza virus (AIV) infection in free-living Bewick's swans (Cygnus columbianus bewickii). Swans infected with AIV exhibited higher serum δ13C (-25.3±0.4) than their non-infected counterparts (-26.3±0.2). Thus, individuals preferentially foraging in aquatic rather than terrestrial habitats experienced a higher risk of infection, suggesting that the abiotic requirements of AIV give rise to heterogeneity in pathogen exposure. Juveniles were more likely to be infected (30.8% compared with 11.3% for adults), shed approximately 15-fold higher quantity of virus and exhibited a lower specific immune response than adults. Together, these results demonstrate the potential for heterogeneity in infection to have a profound influence on the dynamics of pathogens, with concomitant impacts on host habitat selection and fitness.

Resolving the trophic relations of cryptic species: an example using stable isotope analysis of dolphin teeth.

Understanding the foraging ecology and diet of animals can play a crucial role in conservation of a species. This is particularly true where species are cryptic and coexist in environments where observing feeding behaviour directly is difficult. Here we present the first information on the foraging ecology of a recently identified species of dolphin (Southern Australian bottlenose dolphin (SABD)) and comparisons to the common bottlenose dolphin (CBD) in Victoria, Australia, using stable isotope analysis of teeth. Stable isotope signatures differed significantly between SABD and CBD for both δ(13)C (-14.4‰ vs. -15.5‰ respectively) and δ(15)N (15.9‰ vs. 15.0‰ respectively), suggesting that the two species forage in different areas and consume different prey. This finding supports genetic and morphological data indicating that SABD are distinct from CBD. In Victoria, the SABD is divided into two distinct populations, one in the large drowned river system of Port Phillip Bay and the other in a series of coastal lakes and lagoons called the Gippsland Lakes. Within the SABD species, population differences were apparent. The Port Phillip Bay population displayed a significantly higher δ(15)N than the Gippsland Lakes population (17.0‰ vs. 15.5‰), suggesting that the Port Phillip Bay population may feed at a higher trophic level--a result which is supported by analysis of local food chains. Important future work is required to further understand the foraging ecology and diet of this newly described, endemic, and potentially endangered species of dolphin.

In vivo investigations of neurotensin receptors in adipocytes, hepatocytes and enterocytes of rat.

INTRODUCTION: Atherosclerotic vascular disease is currently the biggest threat and the highest cause of death worldwide, approaching almost 60%.The development of atherosclerosis is affected by ecological factors associated with industry and pollution of the environment. Neurotensin (NT) is a peptide acting via 3 kinds of neurotensin receptors (NTR) localized in target tissues. In several studies, the presence of its receptors has been shown in chicken liver, and the influence of NT on the metabolism of this organ was confirmed (glycogenolysis stimulation through sympathetic nervous system, enterohepatic circulation of bile acids, metabolism of lipoproteins). MATERIALS AND METHODS: Healthy male WISTAR rats weighing 300}30 grams, were used for the experiments. The animals were divided into 4 groups: 1) control group, to which 0.9% NaCl was administrated (i.p.); 2) the second group was given levocabastine 1mg/kg i.p.; 3) the third group received SR 48692 0.4 mg/; 4) the fourth group was given NT analog [D-Trp 11]-neurotensin 15 nM/kg. Plasmatic membranes of liver, small intestine and adipose tissue were prepared according to the method of Havrankova. Analysis of results obtained in the investigation of NT receptors was performed using the Scatchard method from LIGAND-Pc, v. 3.1 software. RESULTS: The investigation of antigenity of I125NT showed proper antigen-antibody reaction. No binding of the I125NT with plasmatic membranes of adipocytes or enterocytes was observed. Unspecific binding of I125NT with 10 µmol/L of free NT was observed in the plasmatic membranes of hepatocytes. CONCLUSION: The presence of NT receptors only in the membranes of hepatocytes may suggest their role in the regulation of lipid metabolism via receptor ­ ligand way.

In vitro evaluation of feline leukocytes radiolabeled in whole blood with 99mTc stannous colloid.

Technetium-99m stannous colloid (9mTcSnC) has been used to radiolabel human leukocytes to investigate various inflammatory disorders. We investigated the in vitro behavior of feline leukocytes labeled in whole blood with 99mTcSnC. Heparinized blood samples were collected from healthy cats and divided into control and test aliquots. The latter were labeled with 99mTcSnC using a standard procedure. Leukocyte viability was determined for each sample using a trypan blue exclusion test. Labeling efficiency was determined for test aliquots. Test aliquots were layered onto Histopaque-1077 and centrifuged before measurement of radioactivity of the blood components. Leukocytes from radiolabeled and control samples were washed and incubated with opsonized zymosan particles to allow assessment of phagocytic function. Aliquots were taken from radiolabeled feline leukocyte samples at 1, 3, 4, and 7 h postlabelling. After centrifugation of each aliquot, radioactivity of the supernatant and pellet was measured and the labeling retention determined. Leukocyte viability in both radiolabeled and control samples was > 98%. The labeling efficiency was 95.2 +/- 0.14%. The distribution of radioactivity in feline blood was found to be 3.4 +/- 0.18% in plasma, 39.0 +/- 0.37% in erythrocytes, and 57.6 +/- 0.38% in leukocytes. Labeled feline leukocytes had phagocytic activity of 90.9 +/- 0.18% (control 91.3 +/- 0.15%). The radiolabeled leukocytes retained 93.4 +/- 0.19% of the radioactivity up to 7h postlabeling. 99TcSnC efficiently labeled feline leukocytes with no effect on viability and minimal effect on phagocytic function. The percentage retention of radioactivity by the leukocytes was still high at 7h postlabeling.

Transgenerational marking of marine fish larvae: stable-isotope retention, physiological effects and health issues.

This study examined the toxicological and physiological responses of a commercially important coral-reef grouper, Plectropomus leopardus (Serranidae), to injection of enriched stable-isotope barium chloride (BaCl(2)) solution. Thirty adult P. leopardus were subject to one of two (138)BaCl(2) injection treatment groups (corresponding to dosage rates of 2 and 4 mg (138)Ba kg(-1) body mass), and a control group in which fish were injected with 0.9% sodium chloride (NaCl) solution. Fish from each group were sampled at post-injection intervals of 48 h and 1, 3, 5 and 8 weeks, at which time blood and tissue samples were removed from each fish. Residual concentrations of Ba and (138)Ba:(137)Ba ratios were measured in muscle, gonad, liver and bone tissues of each experimental fish. Elevated Ba concentrations were detected in all treatment fish tissue samples within 48 h post injection. Residual Ba concentrations decreased throughout the remainder of the 8 week experimental period in all tissues except bone. The BaCl(2) injection had no significant effects on measured whole blood variables or on the plasma concentrations of steroid hormones. Enriched Ba stable isotopes can therefore be used at low dosages to mark larvae of commercially important marine fishes, without adverse effects on the health of the fishes or on humans who may consume them.

Application of stable isotopes and mass isotopomer distribution analysis to the study of intermediary metabolism of nutrients.

Genomic investigations in animals have begun to reveal the metabolic and physiological functions of genes and protein products. However, a thorough understanding of the genomic roadmaps will require investigative approaches that yield qualitative and quantitative information on the activities, fluxes, and connectivity of pathways involved in nutrient use in farm animals; that is, the metabolic phenotype. Recently, the commercial availability of stable isotope (13C, 15N, 2H)-labeled compounds and highly accurate mass spectrometers has made it possible to probe the details of metabolic pathways involved in macronutrient use. For years, the biological sciences have exploited uniformly 13C-labeled substrates (e.g., glucose, amino acids, nucleic acids) and 13C-mass isotopomer distribution (MID) in their metabolic investigations, whereas their use in the animal sciences is very limited. When [U-13C] substrates are fed, infused, or added to cell incubations, the 13C-skeletons distribute throughout metabolic networks. 13C-Mass isotopomer distribution in intermediates and end products of the pathways provides a signature of the fluxes and activities of pathway enzymes traveled by the precursor molecule. This paper highlights aspects of animal nutrition and metabolism in which [U-13C] substrates and MID can be applied to investigations of amino acid, carbohydrate, and fat metabolism. We will focus on [U-13C] glucose as a tracer in chickens, fish, sheep, and cell cultures to investigate the interconnectivity of the pathways of macronutrient and nucleic acid metabolism, and provide demonstration of the central position of the Krebs cycle in preserving metabolic flexibility via anaplerotic and cataplerotic sequences. Exploitation of this approach in animal sciences offers endless opportunities to provide missing details of the biochemical networks of nutrient use that may prove to be strictly under genomic control.

Measuring splanchnic amino acid metabolism in vivo using stable isotopic tracers.

The splanchnic bed comprises the liver and the portal-drained viscera (PDV). The PDV, which include the stomach, intestines, pancreas, and spleen, represent 4 to 6% of BW, yet they account for 20 to 35% of whole-body protein turnover and energy expenditure. Because the PDV are the first to be exposed to the diet, their nutrient needs are met first. Consequently, the extraction of dietary nutrients, especially AA, by the intestine will have a critical influence on their availability to peripheral tissues and therefore, on whole body requirements. Moreover, the systemic availability of dietary AA is a key determinant of lean body growth rate. A complicating factor in the measurement of intestinal nutrient use is that the intestinal epithelial cells receive nutrients from 2 sources: the diet and the arterial circulation. However, combining measurements of the net portal balance with those of isotopic enrichments from enterally and intravenously administered stable isotope-labeled AA provides an in vivo model that can be used to determine the proportion of AA extracted by the intestine from either source. Using this technique in fed animals demonstrated that the PDV contribute significantly to the use of essential (>60% of threonine) and nonessential (>90% of glutamate) AA provided by the diet. The relative use by the PDV of individual AA from the diet and arterial inputs varies widely, and dietary AA are the preferred fuel over dietary glucose. Stable isotope-labeled AA also enable the determination of the metabolic fate of individual AA. Using this technique, studies have shown that an insufficient protein supply or the mode of feeding affects AA use by the PDV, and consequently, may affect whole-body growth.

Measuring synthesis rates of nitrogen-containing polymers by using stable isotope tracers.

The major N-containing polymer compounds in the body include protein, RNA, and DNA. The endogenous gastrointestinal secretions as well as the portal-drained visceral and peripheral immune responses are basic physiological functions. Elevated endogenous secretions and immune activities, as affected by developmental stages, diets, and management factors, decrease the availability of dietary nutrients for peripheral muscle synthesis and deposition. Measurements of in vivo protein, RNA, and DNA synthesis rates associated with the viscera, peripheral immune cells, and skeletal muscles should, in principle, be the sensitive biochemical and cellular endpoints for studying factors affecting nonruminant nutrition, metabolism, and growth. The selection of stable isotope tracers for precursors, routes of tracer delivery, and mass spectrometric analyses of tracer enrichments are the major methodological considerations. To measure in vivo protein, RNA, and DNA synthesis rates, oral feeding with heavy water (2H2O), and continuous infusion of [U-13C]glucose and [15N]Gly intravenously for labeling the sugar moieties ribose and deoxyribose and de novo purine base synthesis have been established. Flooding doses of tracer Phe, for example, L-[ring-2H5]Phe, via the i.p. route are reliable and cost-effective for measuring in vivo protein synthesis rates, especially for the viscera in small nonruminants. Therefore, measurements of the major N-containing polymer synthesis rates in the viscera, the peripheral immune cells, and muscles through oral feeding with 2H2O and/or i.p. flooding doses of Phe tracers are the emerging tools for studying nonruminant nutrition, metabolism, and growth under research and field test conditions.

Immune response to Sarcocystis neurona infection in naturally infected horses with equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is one of the most common neurologic diseases of horses in the United States. The primary etiologic agent is Sarcocystis neurona. Currently, there is limited knowledge regarding the protective or pathophysiologic immune response to S. neurona infection or the subsequent development of EPM. The objectives of this study were to determine whether S. neurona infected horses with clinical signs of EPM had altered or suppressed immune responses compared to neurologically normal horses and if blood sample storage would influence these findings. Twenty clinically normal horses and 22 horses with EPM, diagnosed by the presence of S. neurona specific antibodies in the serum and/or cerebrospinal (CSF) and clinical signs, were evaluated for differences in the immune cell subsets and function. Our results demonstrated that naturally infected horses had significantly (P<0.05) higher percentages of CD4 T-lymphocytes and neutrophils (PMN) in separated peripheral blood leukocytes than clinically normal horses. Leukocytes from naturally infected EPM horses had significantly lower proliferation responses, as measured by thymidine incorporation, to a non-antigen specific mitogen than did clinically normal horses (P<0.05). Currently, studies are in progress to determine the role of CD4 T cells in disease and protection against S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. Finally, overnight storage of blood samples appears to alter T lymphocyte phenotypes and viability among leukocytes.

Estimation of endogenous phosphorus loss in growing and finishing pigs fed semi-purified diets.

Thirty-six barrows were used in a series of 3 P-balance experiments in which growing and finishing pigs were fed highly digestible, semi-purified diets at or below the dietary available P requirement to estimate the effect of BW on endogenous P loss. Experiments 1, 2, and 3 were conducted with pigs averaging 27, 59, and 98 kg of BW, respectively. In each experiment, pigs were placed in metabolism crates and allotted by weight and litter to 3 dietary treatments. The basal diet consisted of sucrose, dextrose, cornstarch, and casein fortified with minerals (except P) and vitamins. Diets 1, 2, and 3 in Exp. 1 were the basal diet with 0, 0.078, or 0.157% added P, respectively, from monosodium phosphate. In Exp. 2 and 3, diets 1, 2, and 3 were the basal diet with 0, 0.067, and 0.134% added P, respectively, from monosodium phosphate. Within replicate, pigs were fed equal amounts of feed twice daily. Pigs were adjusted to treatments for 7 d before a 6-d, marker-to-marker collection of feces and urine. Phosphorus intakes for pigs fed the 3 diets ranged from 1.73 to 3.91 g/d in Exp. 1, from 2.18 to 5.32 g/d in Exp. 2, and from 1.96 to 6.26 g/d in Exp. 3. Fecal P excretion and P absorption increased linearly (P < 0.05) with increasing P intake. In the 3 experiments, urinary P excretion (g/d) was low for pigs fed diet 1 (0.010, 0.011, 0.019) and diet 2 (0.013, 0.058, 0.084) and was low for pigs fed diet 3 in Exp. 1 (0.037); however, urinary P was greater in pigs fed diet 3 in Exp. 2 and 3 (0.550 and 0.486, respectively). When P absorption (Y, g/d) was regressed on P intake (X, g/d) in Exp. 1, 2, and 3, the relationships were linear (P < 0.01): Y = -0.110 + 0.971X (R2 = 0.999), Y = -0.156 + 0.939X (R2 = 0.998), and Y = -0.226 + 0.8919X (R2 = 0.982), respectively. Thus, our estimates of endogenous P loss at zero P intake were 110, 156, and 226 mg/d for 27-, 59-, and 98-kg pigs, respectively. When these Y-intercepts were regressed on BW, the relationship was Y = 63.06 + 1.632X (R2 = 0.996), where Y = endogenous P loss in mg/d and X = BW in kg. Based on these data, we estimate the endogenous P loss of pigs fed highly digestible, semi-purified diets to increase by approximately 1.632 mg for each 1-kg increase in BW from 25 to 100 kg.