Weight-loss in obese dogs promotes important shifts in fecal microbiota profile to the extent of resembling microbiota of lean dogs

Background Among the undesirable changes associated with obesity, one possibility recently raised is dysbiosis of the intestinal microbiota. Studies have shown changes in microbiota in obese rats and humans, but there are still few studies that characterize and compare the fecal microbiota of lean, obese and dogs after weight loss. Thus, this study aimed to evaluate the effects of a weight loss program (WLP) in fecal microbiota of dogs in addition to comparing them with those of lean dogs. Twenty female dogs of different breeds, aged between 1 and 9 years were selected. They were equally divided into two groups: Obese group (OG), with body condition score (BCS) 8 or 9/9, and body fat percentage greater than 30%, determined by the deuterium isotope dilution method, and lean group (LG) with BCS 5/9, and maximum body fat of 15%. Weight loss group (WLG) was composed by OG after loss of 20% of their current body weight. Fecal samples were collected from the three experimental groups. Total DNA was extracted from the feces and these were sequenced by the Illumina methodology. The observed abundances were evaluated using a generalized linear model, considering binomial distribution and using the logit link function in SAS (p < 0.05). Results The WLP modulated the microorganisms of the gastrointestinal tract, so that, WLG and LG had microbial composition with greater biodiversity than OG, and intestinal uniformity of the microbiota (Pielou’s evenness index) was higher in OG than WLG dogs (P = 0.0493) and LG (P = 0.0101). In addition, WLG had values of relative frequency more similar to LG than to OG. Conclusion The fecal microbiota of the studied groups differs from each other. The weight loss program can help to reverse the changes observed in obese dogs.

Influence of Vitamin A Status on the Choice of Sampling Time for Application of the Retinol Isotope Dilution Method in Theoretical Children

ABSTRACT Background Vitamin A status may influence the choice of a blood sampling time for applying the retinol isotope dilution (RID) equation to predict vitamin A total body stores (TBS) in children. Objectives We aimed to identify time(s) after administration of labeled vitamin A that provide accurate estimates of TBS in theoretical children with low or high TBS. Methods We postulated 2- to 5-y-old children (12/group) with low (&lt;200 μmol) or high TBS (≥700 μmol) and used compartmental analysis to simulate individual subject values for the RID equation TBS = FaS/SAp (Fa, fraction of dose in stores; S, retinol specific activity in plasma/in stores; SAp, retinol specific activity in plasma). Using individual SAp and group geometric mean FaS values from 1–28 d, we calculated individual and group mean TBS and compared them to assigned values. Results Mean TBS was accurately predicted for both groups at all times. For individuals, predicted and assigned TBS were closest when the CV% for FaS was low [12–14%; 4–13 d (low), 12–28 d (high)]. The mean percentage error for TBS was &lt;10% from 2–19 d (low) and 7–28 d (high). Predicted TBS was within 25% of assigned TBS for ≥80% of children from 3–23 d (low) and 9–28 d (high). Within groups, RID tended to overestimate lower TBS and underestimate higher TBS. Conclusions Using a good estimate for FaS, accurate RID predictions of TBS for individuals will be obtained at many times. If vitamin A status is low, results indicate that early sampling (e.g., 4–13 d) is optimal; if vitamin A status is high, sampling at 12–28 d is indicated. When vitamin A status is unknown, sampling at 14 d is recommended, or a super-subject design can be used to obtain the group mean FaS at various times for RID prediction of TBS in individuals.

Does the Amount of Stable Isotope Dose Influence Retinol Kinetic Responses and Predictions of Vitamin A Total Body Stores by the Retinol Isotope Dilution Method in Theoretical Children and Adults?

Background To minimize both cost and perturbations to the vitamin A system, investigators limit the amount of stable isotope administered when estimating vitamin A total body stores (TBS) by retinol isotope dilution (RID). Objectives We hypothesized that reasonable increases in the mass of stable isotope administered to theoretical subjects would have only transient impacts on vitamin A kinetics and minimal effects on RID-predicted TBS. Methods We adapted previously-used theoretical subjects (3 children, 3 adults) with low, moderate, or high assigned TBS and applied compartmental analysis to solve a steady state model for tracer and tracee using assigned values for retinol kinetic parameters and plasma retinol. To follow retinol trafficking when increasing amounts of stable isotope were administered [1.39-7 (children) and 2.8-14 µmol retinol (adults)], we added assumptions to an established compartmental model so that plasma retinol homeostasis was maintained. Using model-simulated data, we plotted retinol kinetics versus time and applied the RID equation TBS = FaS/SAp [Fa, fraction of dose in stores; S, retinol specific activity (SA) in plasma/SA in stores; SAp, SA in plasma] to calculate vitamin A stores. Results The model predicted that increasing the stable isotope dose caused transient early increases in hepatocyte total retinol; increases in plasma tracer were accompanied by decreases in tracee to maintain plasma retinol homeostasis. Despite changes in kinetic responses, RID accurately predicted assigned TBS (98-105%) at all loads for all theoretical subjects from 1-28 d postdosing. Conclusions Results indicate that, compared with doses of 1.4–3.5 µmol used in recent RID field studies, doubling the stable isotope dose should not affect accuracy of TBS predictions, thus allowing for experiments of longer duration when including a super-subject design (Ford et al., J Nutr 2020;150:411–8) and/or studying retinol kinetics.

A Dilute-and-Shoot UHPLC-MS/MS Isotope Dilution Method for Simultaneous Determination and Confirmation of Eleven Mycotoxins in Dried Distillers Grains with Solubles

Background Natural contamination of mycotoxins in dried distiller’s grains with solubles (DDGS) as a mainstream animal feed ingredient poses risk to animal health. Objective A regulatory method was needed for the agency to simultaneously detect eleven mycotoxins of high regulatory priority in DDGS. Methods Ten grams of DDGS sample were extracted twice with acetonitrile/water under mildly acidic condition. Two aliquots from the combined crude extract were taken and processed separately: (1) diluted 400-fold with solvent for analysis of deoxynivalenol and fumonisins B1 and B2; (2) pH adjusted to 7.5, then diluted 15.7-fold for analysis of aflatoxins B1, B2, G1, G2, ochratoxin A, zearalenone, and T-2 and HT-2 toxins. Uniformly-labelled 13C-isotopologues of these mycotoxins were added as internal standards to the diluted extracts for quantitative analysis by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). Results. The linear quantitation ranges (µg/kg) were: aflatoxin B1, B2, G1, and G2, 1.57 to 105; zearalenone, 16.3 to 1090; T-2 toxin, 3.14 to 208; HT-2 toxin, 48.2 to 3220; ochratoxin A, 0.47 to 31.4; deoxynivalenol, 240 to 16000; fumonisin B1 and B2, 320 to 21200. Accuracies for these analytes at each of three fortification levels range from 70.7% to 100%, with corresponding relative standard deviations between 1.4% to 10.5%. True recoveries were all higher than 83%. Conclusions This method was successfully validated to meet the agency’s performance guidelines for regulatory methods. Highlights This method is easy, quick and robust to simultaneously quantify and confirm presence of eleven regulated mycotoxins in DDGS.

A stable isotope dilution method for a highly accurate analysis of karrikins

Background Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. Results We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). Conclusions Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.

Species Interactions and Nitrogen Use during Early Intercropping of Intermediate Wheatgrass with a White Clover Service Crop

Perennial grain crops intercropped with legumes are expected to use nitrogen (N) resources efficiently. A pot experiment using the 15N isotope dilution method demonstrated interspecific competition and use of N from the soil and N2 fixation in intermediate wheatgrass (Thinopyrum intermedium (Host) Barkworth & D.R. Dewey, IWG) and white clover (Trifolium repens L., WC) intercrops at five species-relative frequencies and four levels of inorganic N fertilizer in a replacement series design. The proportion of N in WC derived from the atmosphere increased from 39.7% in a sole crop to 70.9% when intercropped with IWG, and 10.1% N in IWG transferred from WC. Intermediate wheatgrass showed high fitness with maintained high total dry matter production at low relative frequencies. Decreasing IWG-relative frequency only increased dry matter and N accumulation of WC, resulting in increased amounts of N2 fixed. Increased levels of N fertilization increased the proportion of N acquired from the fertilizer in IWG and WC but decreased the N fixed by WC and N absorbed by IWG from the soil. Our study indicates that WC supply sufficient fixed N2 for IWG intercrop biomass yields under appropriate levels of soil N fertility and species-relative frequencies.

The Dynamic of Nitrogen Uptake from Different Sources by Pea (Pisum sativum L.)

Nitrogen uptake from various sources by plants capable of biological reduction of N2 in symbiotic systems with root nodule bacteria is influenced by many factors. The aim of the study was to examine the influence of the development stage and variety of pea (Pisum sativum L.) cultivated in years with different temperature and precipitation conditions on the dynamics of nitrogen uptake from the atmosphere (Ndfa), fertilizer (Ndff), and soil (Ndfs). Pre-sowing nitrogen fertilization with the 15N isotope and the isotope dilution method were used in the research. The highest rate of Ndfa uptake was noted between the three-internode stage and the stage of the first visible flower buds outside the leaves, while Ndff and Ndfs uptake was highest between the 4-leaf stage and the 3-internode stage. The lowest rate of Ndfa uptake was noted from sowing to the four-leaf stage, while Ndff and Ndfs uptake was lowest between the stage when 50% of pods were of typical length and full maturity. Nitrogen uptake from all sources was similar for all pea cultivars, but significantly depended on the variable temperature and precipitation conditions (years of the study).

Quantification of Biologically Fixed Nitrogen by White Lupin (Lupins albus L.) and Its Subsequent Uptake by Winter Wheat Using the 15N Isotope Dilution Method

A field experiment was carried out in 2016–2018 in a white lupin (Lupinus albus L.)-winter wheat (Triticum aestivum cv. ‘Bogatka’) crop rotation. The aim of this study was to determine the amount of nitrogen (N) that was biologically fixed by the white lupin crop in the first year of the rotation and to estimate how much of this N was then taken up from the lupin residues by winter wheat in the second and third years of the rotation. Biologically fixed N was determined by the isotope-dilution method (ID15N) by applying 30 kg N ha−1 of 15N-labeled fertilizer (15NH4)2SO4 (containing 20.1 at.% 15N) to the white lupin and the reference plant spring wheat. The yields of white lupin seeds and crop residues were 3.92 t ha−1 and 4.30 t ha−1, respectively. The total amount of N in the white lupin biomass was 243.2 kg ha−1, which included 209.3 kg ha−1 in the seeds and 33.9 kg ha−1 in the residues. The 15N-labeled residue of white lupin was cut and ploughed into soil. Our results indicate that 111.2 kg N ha−1 was fixed from the atmosphere by the lupin plants, with 93.7 kg ha−1 found in the seeds and 17.5 kg ha−1 in the residues. In the second and third years of the rotation when winter wheat was cultivated, the plots were divided into two groups of subplots (1) without N-fertilization (control) and (2) with an application of 100 kg N ha−1. In the first year of winter wheat cultivation, 20.0% and 21.0% of N from the crop residues was taken up by the control and N-fertilization plots, respectively, while in the second year, uptake was lower at 7.12% and 9.27% in the control and N-fertilized plots, respectively.