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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.

The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 1.11.1.7) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml).

Comparison of a radioactive and non-radioactive method for sequencing foot and mouth disease virus isolates.

The authors compare the radioactive method of detecting foot and mouth disease virus sequence products with a non-radioactive, silver stain sequencing method. The latter was found to compare favourably to the radioactive technique for detecting such products. The silver stain sequencing method was simple and did not require expensive specialised equipment. This new approach will be particularly useful in developing countries, since the method does not depend on the availability of fresh radioactive isotopes and is also safer and considerably cheaper.

A review of the use of intrinsically 14C and rare earth-labeled neutral detergent fiber to estimate particle digestion and passage.

Methodology that allows simultaneous measurement of dynamic events affecting NDF digestion in passage from the rumen should improve our understanding of factors influencing intake and digestion. Ideally, particle flow is measured with a marker indelibly attached to or intrinsically part of the feed. If flow measurements are to reflect physiological conditions, marked and unmarked feed must be digested and passed with identical fractional rates. Application of 14C-labeled plant fiber to the study of ruminal dynamics has been slow because of expense and difficulty in producing 14C-labeled plant material. Recently, alfalfa was intrinsically labeled with 14C under field conditions to produce plant material similar in composition to unlabeled material. Carbon-14 specific activity was similar in all particle sizes, and in indigestible and digestible NDF. Greater concentrations of ytterbium (Yb) were associated with smaller vs larger particles. Larger differences in turnover rates among animals than differences attributable to treatments force comparisons of markers to be made within animal or with in vitro systems. The uncertainty about how extrinsic markers respond under various environments resulting from interaction of feed properties and gut function, and the high error inherent in measuring dynamic systems, raise serious questions on the interpretability of results. Advantages of 14C-labeled NDF over other markers include simultaneous measurements of particle breakdown, digestion and passage rates as well as the potential to study microbial attachment, VFA, CO2 and CH4 production and rate of incorporation of labeled metabolites into tissues.

Technical note: labeling of forages with 13C for nutrition and metabolism studies.

Alfalfa was labeled in the field with 99 atom % 13CO2 and cut either the same day (C1) or 30 d after labeling (C30). The C1 alfalfa contained 84% of the 13C label in cell contents, whereas C30 alfalfa contained 47% of the 13C label in cell contents. In two separate trials, C1 and C30 alfalfa were dosed to two or four Suffolk ewes fed natural abundance alfalfa diets. Carbon isotope ratios (13C/12C, expressed as delta 13C/1000 [parts per thousand] vs Pee Dee Belemnite standard) were determined for breath, feces, blood, and blood serum from ewes fed C1 alfalfa and blood and feces from ewes fed C30 alfalfa. In the C1 trial, carbon isotope ratios of respired CO2 peaked 4 h after feeding, then declined to baseline levels by 40 h after the dose. Fecal samples increased in 13C only slightly from 12 to 40 h after the meal. Blood serum values increased by approximately .5/1000 from 0 to 4 h after the dose and remained relatively constant thereafter. In both trials, carbon isotope values from whole blood were constant. In the C30 trial, fecal samples peaked in carbon isotope value approximately 30 to 36 h after dosing, then declined; the time of this peak corresponded closely to that from a concurrent study that used a pulse dose of Yb-labeled alfalfa hay. Thus, when incorporated into cell wall material, the excretion pattern of 13C in feces was similar to that of Yb-labeled hay, but little 13C enrichment in feces was found when 13C was primarily in cell contents of the labeled forage.(ABSTRACT TRUNCATED AT 250 WORDS)

A modification to the isotope-dilution technique for estimating milk intake of pigs using pig serum.

The aim of this study was to compare the measurement of deuterium oxide (D2O) directly in pig serum with that in sublimed whole blood. This was to assess whether excluding vacuum sublimation before analysis would cause any significant loss of accuracy in estimates of pig milk intake. Water and serum standards were made in deionized water and serum, respectively, and were assayed with samples under the same conditions on a fixed-filter, infrared spectrophotometer. The mean concentration of D2O in sublimed samples was 2,244 microg/mL of body water, and the mean concentration of D2O in serum samples was 2,184 microg/mL of body water. The mean ratio of D2O concentration in deionized water to the D2O concentration in serum was 1.0275, which was used as a correction factor to convert serum D2O concentration to D2O concentrations in body water. Using this method, the mean concentration of D2O in all serum samples was identical to that in sublimed samples (i.e., 2,244 microg/mL of body water). Mean milk intake of pigs based on sublimed samples was 1,006 g/d and that based on serum samples was 1,012 g/d. This confirms that milk intake determined from measurement of D2O directly in pig serum is sufficiently precise.

Modified in vitro method to label equine red blood cells with technetium 99m in concentrated whole blood.

An in vitro method to label equine RBC with technetium 99m was modified to achieve quantitative labeling of cells in concentrated whole blood. After a blood sample was incubated with a reducing agent (stannous citrate), an oxidizing reagent (NaOCl) and a chelating agent (EDTA) were added to inactivate residual Sn2+ in the plasma. This step prevented premature reduction of pertechnetate in plasma. Labeling of RBC from 9 healthy horses, using a standard whole blood protocol, resulted in only moderate labeling efficiency (44 to 85%) and indicated a linear relationship between labeling efficiency and PCV. Effects of increased incubation time, increased incubation temperature, prelabeling sedimentation, and double addition of NaOCl/EDTA were investigated in whole blood from 10 healthy horses. Labeling efficiency was improved by each independent factor and by combination of factors. Highest labeling efficiencies (96 to 97%) were achieved when blood samples were sedimented for 20 minutes before being labeled, regardless of incubation time or incubation temperature. Morphologic features of RBC were unaffected by labeling procedures. In vivo whole blood clearance time for labeled cells was determined in 5 healthy horses. Sedimented blood samples were labeled, using a standard 15-minute incubation time at 20 to 22 C. Mean clearance half-time for 5 horses was approximately 20 hours. More than 95% of 99mTc activity was associated with the cells during the 24 hours after reinjection.

Radiolabeling of equine platelets in plasma with 111In-(2-mercaptopyridine-N-oxide) and their in vivo survival.

A method is presented for the in vitro isolation and radiolabeling of equine platelets with the isotope indium 111 (111In: half-life = 2.8 days, gamma = 173 keV, 89%; 247 keV, 94%). The technique described involves complexing 111In with the lipid-soluble chelating agent, 2-mercaptopyridine-N-oxide (merc), in an aqueous medium. 111In-merc platelet-labeling efficiencies in autologous plasma pretreated with or without ferric citrate reagent were 82 +/- 7% and 24 +/- 12%, respectively. Mean intravascular survivals of 111In-merc-radiolabeled platelets in 8 healthy horses according to simple linear, exponential, mean, weighted-mean residual sum of squares analysis, and multiple-hit model were 5.5 +/- 0.49, 3.5 +/- 0.53, 4.5 +/- 0.18, 4.3 +/- 0.65, and 3.6 +/- 0.97 days, respectively.

In vitro comparison of equine granulocytes labeled with 99mTc-hexamethylpropyleneamine oxime or 111In-oxine.

Isolated equine granulocytes (WBC), radiolabeled with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) or 111In-oxine, were evaluated in vitro for their labeling characteristics, viability, and phagocytic function over a 6-hour postlabeling period. Mean +/- SD labeling efficiency for 111In-oxine-WBC was 62.2 +/- 15.3%, which was significantly (P less than 0.001) higher than that for 99mTc-HMPAO-WBC (32.0 +/- 17.0%). In vitro elution of radiolabel from cells was significantly (P less than 0.02) greater for 99mTc-HMPAO-WBC at 0.5, 2, and 4 hours, but was not significantly different from elution of radiolabel for 111In-oxine-WBC at 6 hours. Viability, assessed by trypan blue dye exclusion, for 99mTc-HMPAO-WBC, 111In-oxine-WBC, and nonlabeled control WBC ranged from 97 to 100%, and was not significantly different among groups. Cell function was assessed by use of a phagocytosis assay and was reported as phagocytic index. The phagocytic index ranged from 0.86 to 0.96 for 99mTc-HMPAO-WBC, and from 0.76 to 0.97 for 111In-oxine-WBC. The phagocytic index was not significantly different at 0.5, 2, or 4 hours, but was significantly (P = 0.038) greater at 6 hours for 99mTc-HMPAO-WBC. Because of the superior imaging characteristics of 99mTc-HMPAO-WBC and equal or better labeling characteristics than those for 111In-oxine at 6 hours, 99mTc-HMPAO-WBC appear to be a good alternative to 111In-oxine-WBC.

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.