Fecal Collection Method Research Articles

Controlling Preanalytical Process in High-Coverage Quantitative Metabolomics: Spot-Sample Collection for Mouse Urine and Fecal Metabolome Profiling.

Compared to conventional MS and NMR techniques, high-performance chemical isotope labeling (CIL) LC-MS provides accurate relative quantification of many more metabolites in biological samples. However, to apply this technique for urine and fecal metabolomics studies of animal models, the entire workflow, including the preanalytical process, needs to be strictly controlled to avoid or minimize quantitative errors. In this study, we report our investigation of the effects of mouse urine and fecal sample collection methods on CIL LC-MS metabolome analysis. Metabolic-cage collection and spot-sample collection of urine and feces were compared in a mouse model of CCl4-induced liver disease. 13C-/12C-dansylation LC-MS was used for quantitative profiling of the amine-/phenol-submetabolome changes. A total of 5026, 4963, 4238, and 4600 peak pairs or metabolites were detected from spot urine, spot feces, cage-collected urine, and cage-collected feces, respectively. It was found that samples collected using metabolic cages, widely used in low coverage metabolomics, could be contaminated with food as well as cross-specimen (urine in feces or feces in urine) to the extent that metabolomic comparison of different groups of mice could be seriously compromised in high-coverage metabolomics. In contrast, spot urine and spot feces could be collected without contamination and should be used in CIL LC-MS metabolomics.

Evaluation of titanium dioxide and chromic oxide as digestibility markers in ponies fed alfalfa hay in relation to marker dosing frequency

In equines, Cr2O3 is widely accepted as an indigestible marker, but there are health concerns regarding the carcinogenic properties of Cr2O3. Recently, TiO2 has been suggested to be an alternative digestibility marker in equines. However, a comparison between Cr2O3 and TiO2 has not been made in equines. Six Welsh pony geldings (initial BW: 254±3 kg; 7 years of age) fed chopped alfalfa hay were used to evaluate the use of TiO2 (Ti) and Cr2O3 (Cr) as markers for calculating apparent digestibility and to investigate the effect of frequency of marker administration on the measurement of digestibility values. Diets contained 4.65 kg dry matter (DM) chopped alfalfa hay supplemented with minerals, vitamins, TiO2 (3.3 g Ti/day) and Cr2O3 (3.2 g Cr/day). Ponies were dosed with either 3.3 g Ti and 3.2 g Cr once daily (DF1) or with 1.65 g Ti and 1.60 g Cr twice daily (DF2). After adaptation to the diets and procedures for 14 days, voluntary voided faeces were collected quantitatively over 7 days and analysed for moisture, ash, Ti and Cr. Apparent total tract DM digestibility (DMD) and organic matter digestibility (OMD) were calculated using the total faecal collection (TFC) and marker method (Ti and Cr). The overall mean cumulative faecal recovery of Cr and Ti (as % of intake) were 102.0% and 96.6%, respectively. Mean daily faecal recoveries of Cr as well as of Ti were not different (P=0.323; P=0.808, respectively) between treatments. Overall daily faecal recovery of Cr differed (P=0.019) from 100% when the marker was dosed once daily, whereas overall daily faecal recovery was similar to 100% for both administration frequencies when Ti was used as a marker. For both markers, the coefficient of variation of the mean faecal marker recovery between horses was lower when the markers were administrated twice per day. Across treatments, cumulative DMD and OMD estimated with Ti were similar (P=0.345; P=0.418, respectively) compared with those values determined by TFC method. When Cr was used, the calculated cumulative DMD tended (P=0.097) to be greater compared with those estimated with TFC, and cumulative OMD values were overestimated (P=0.013). Orally supplemented Ti recovery in the faeces of ponies fed chopped alfalfa hay with Ti administered once or twice daily was close to 100%, making it the preferred marker for digestibility trials in equines.

METABOLIZABLE ENERGY OF RICE BRAN, COTTONSEED MEAL AND WHEAT BRAN FOR SLOW-GROWING BROILERS AT TWO AGES

Abstract The aim of this study was to evaluate bromatological composition and metabolizable energy values of cottonseed meal, rice meal, and wheat bran when provided to slow growth rate broilers at different ages. Energy values of 15- to 25- and 35- to 45-day-old broilers were determined by the total feces collection method using two digestibility bioassays. The birds were distributed in a completely randomized experimental design to evaluate the age effect, with six replicates of 10 chickens each, for each ingredient. Values of apparent metabolizable energy adjusted by nitrogen balance (kcal/kg), for the first and second ages were: 1,255 and 1,564 for cottonseed meal, 3,124 and 3,301 for rice meal, and, 2,211 and 2,383 for wheat bran, respectively. The ingredients showed some variations regarding chemical composition and energy values, which were higher (P < 0.05) in older broiler chickens.

Digestible energy of a corn-soybean meal-based diet supplemented with xylanase for nursery pigs in metabolism crates and floor pens

Two metabolism trials were conducted in the current study to determine the digestible energy (DE) of a corn-soybean meal-based diet supplemented with xylanase. In trial 1, 32 PIC L1050 barrows (initial body weight (BW) 8.6 ± 0.4 kg) were used. All the barrows were housed individually in metabolism crates (2.0 × 0.8 m2). Daily feed allowance was set at about 3.5% of the average BW of pigs. In trial 2, 32 PIC L1050 × L337 barrows (initial BW 8.7 ± 1.0 kg) were used. Sixteen barrows were kept individually in floor pens (3.0 × 1.8 m2), and the other 16 in metabolism crates (2.0 × 0.8 m2). All the pigs in both pens and crates had free access to feed and water. In trial 1, there were 4 dietary treatments in a 2 by 2 factorial arrangement by combining 2 concentrations of xylanase (0 and 0.2 g/kg feed) with 2 particle sizes of corn: 249 ± 1.7 and 427 ± 2.3 µm. In trial 2, there were 2 concentrations of xylanase (0 and 0.2 g/kg feed) in combination with 2 types of housing conditions (crates and floor pens). In both trials, there were 8 replicates for each treatment and 1 pig in each crate/pen. The results of trial 1 showed that there was no interaction between the particle size of corn and the supplementation of xylanase. Based on both total fecal collection and index methods, the supplementation of xylanase increased dietary DE by 27–29 kcal compared with the unsupplemented treatment (P > 0.05). There was no interaction between the fecal collection method and the supplementation of xylanase. The digestibility of dry matter and gross energy, and DE based on index method were less than those based on total fecal collection method (P < 0.001). In trial 2, there was no interaction between the housing conditions and the supplementation of xylanase. The pigs in the floor pens had a greater average daily feed intake (ADFI; P = 0.021) and a greater average final BW (P = 0.022), but tended to have a less digestibility of DM (P = 0.093). The supplementation of xylanase only tended to improve the ADFI irrespective of the housing conditions (P = 0.055). In conclusion, the supplementation of xylanase released 27–29 kcal DE of a corn-soybean meal-based diet with pigs housed in metabolism crates or floor pens. The supplementation of xylanase could improve the fecal digestibility of DM and increase the feed intake of pigs when allowed a free access to feed.

Reliability of a participant-friendly fecal collection method for microbiome analyses: a step towards large sample size investigation

BackgroundThe effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods.ResultsFourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods.ConclusionOur results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.

Digestibilidad aparente de dietas con harina de semillas de maracuyá sobre el desempeño productivo del pez nativo vieja azul (Aequidens rivulatus) en la etapa de cría

El objetivo de este estudio fue valorar el efecto de dietas a base de torta de maracuyá sobre el rendimiento productivo, calculando el incremento de peso (IP), incremento de peso relativo (IPR), tasa de crecimiento (TC), tasa de crecimiento incremental (TCI), conversión alimenticia (CA), digestibilidad aparente de materia seca (MS), proteína (PB) y energía bruta (EB), así como, el costo de alimentación y la mortalidad del pez nativo vieja azul en la etapa de cría. Se determinaron: el peso final (PF), IP, IPR, TC, TCI, CA, los coeficientes de digestibilidad MS, PB y EB, costos de alimentación y mortalidad. No hay diferencias (P&gt;0,05) hasta un nivel del 3% de torta de maracuyá, si hubo diferencias en los valores 6 y 9% en: PF, IP, IPR, TC y TCI. La CA fue eficiente hasta el 6% de maracuyá. A medida que se incrementó el maracuyá el costo disminuyó. El reemplazo parcial de la harina de pescado hasta un 6% del maracuyá en las dietas, no afectó el desempeño productivo en el PF, IP, IPR, TC, TCI y CA. Se utilizó el método de la recolección total de heces en cada uno de los tratamientos y repeticiones, mediante el sistema Guelph modificado, con este método directo se obtuvieron los coeficientes de digestibilidad aparente de MS, PB y EB, encontrándose diferencias estadísticas entre (T1) en relación a T2, T3 y T4, concuerdan de manera general con los descritos en diferentes investigaciones con tilapia y cachama para MS, PB y EB que fueron similares

Dietas para ovinos contendo capim elefante maduro e raspa de mandioca: estimativa de digestibilidade aparente por meio de indicadores

Objetivou-se a avaliar a estimativa da digestibilidade aparente (DA) em dietas para ovinos por meio dos indicadores internos MSi (matéria seca indigestível), FDNi (fibra em detergente neutro indigestível) e FDAi (fibra em detergente ácido indigestível) e o indicador externo LIPE®. Utilizou-se oito ovinos machos Santa Inês, castrados, fistulados no rúmen, com peso médio corporal de 50 kg. Os animais receberam dietas a base de raspa de mandioca e do capim elefante maduro (120 dias). Os indicadores internos (MSi, FDNi e FDAi) foram estimados após 288 horas de incubação in situ das amostras de alimentos e fezes. O indicador externo LIPE® foi fornecido na forma de cápsulas na dosagem de 250 mg/animal/dia. Os resultados de DA estimados pelo indicador externo LIPE® diferiram estatisticamente (P0,05) entre o método da coleta total de fezes e os indicadores internos FDNi e FDAi. Os indicadores internos FDNi e FDAi podem ser utilizados em estudos de digestibilidade em ovinos, pois, permitem estimar de forma semelhante ao método da coleta total de fezes.

Long-term persistence of horse fecal DNA in the environment makes equids particularly good candidates for noninvasive sampling.

Fecal DNA collected noninvasively can provide valuable information about genetic and ecological characteristics. This approach has rarely been used for equids, despite the need for conservation of endangered species and management of abundant feral populations. We examined factors affecting the efficacy of using equid fecal samples for conservation genetics. First, we evaluated two fecal collection methods (paper bag vs. ethanol). Then, we investigated how time since deposition and month of collection impacted microsatellite amplification success and genotyping errors. Between May and November 2014, we collected feral horse fecal samples of known age each month in a feral horse Herd Management Area in western Colorado and documented deterioration in the field with photographs. Samples collected and dried in paper bags had significantly higher amplification rates than those collected and stored in ethanol. There was little difference in the number of loci that amplified per sample between fresh fecal piles and those that had been exposed to the environment for up to 2 months (in samples collected in paper bags). After 2 months of exposure, amplification success declined. When comparing fresh (0–2 months) and old (3–6 months) fecal piles, samples from fresh piles had more matching genotypes across samples, better amplification success and less allelic dropout. Samples defecated during the summer and collected within 2 months of deposition had highest number of genotypes matching among samples, and lowest rates of amplification failure and allelic dropout. Due to the digestive system and amount of fecal material produced by equids, as well as their occurrence in arid ecosystems, we suggest that they are particularly good candidates for noninvasive sampling using fecal DNA.

Influence of marker particle size on nutrient digestibility measurements and particle movement through the digestive system of shrimp

A range of inert digestibility markers, such as yttrium(Y) and ytterbium (Yb) oxides, are used to indirectly calculate the digestibility of nutrients in aquafeeds. Gross over-estimations of nutrient digestibility can be encountered if several assumptions are not met, including that nutrients move through the digestive system at the same rate as the markers. The present study sought to assess the effect of particle size on apparent nutrient digestibility, and traced the movement of several inert metal markers (Y, Yb and gold) of various sizes (>3 μm, 400 nm, 100 nm, 50 nm and 5 nm) through the shrimp digestive tract. The first of two experiments assessed the effect of particle size on nutrient digestibility using traditional faecal collection methods, while the second traced particle movement within the stomach, hepatopancreas and intestine at various times (1, 2, 3, 5, 10 and 18 h) after a set ration. This work showed that particle size had a significant effect on nutrient digestibility, and that 50 nm and 5 nm gold particles had relative particle retention coefficients of 37.3 ± 2.7% and 48.9 ± 1.8%, respectively. Gold particles were detected in the hepatopancreas of animals fed 5 nm gold for >18 h, indicating that small particles remain within the animal for long periods of time. This is despite that the shrimp digestion was extremely rapid, with evacuation rate of the digestive tract calculated at 22.1% h−1. Large Y and Yb particles (>3 μm) found within the animal 1-hour after feeding showed that the amount of true feed intake was lower than measured feed intake by 15–25%, and provided an estimate of manipulative losses during feeding. Overall, this study showed that the shrimp filter press was highly efficient at excluding small indigestible particles, to below 400 nm, and that extremely high rates of nutrient digestion were maintained despite rapid movement through the digestive system. This work may in part account for the disparity between high levels of apparent nutrient digestibility and low nutrient utilisation efficiency measured in shrimp.

Effect of exercise on apparent total tract digestibility of nutrients and faecal recovery of ADL and TiO2 in ponies

Exercise and physical training are known to affect gastrointestinal function and digestibility in horses and can lead to inaccurate estimates of nutrient and energy digestibility when markers are used. The effect of exercise on apparent nutrient digestibility and faecal recoveries of ADL and TiO2 was studied in six Welsh pony geldings subjected to either a low- (LI) or high-intensity (HI) exercise regime according to a cross-over design. Ponies performing LI exercise were walked once per day for 45 min in a horse walker (5 km/h) for 47 consecutive days. Ponies submitted to HI exercise were gradually trained for the same 47 days according a standardized protocol. Throughout the experiment, the ponies received a fixed level of feed and the daily rations consisted of 4.7 kg DM of grass hay and 0.95 kg DM of concentrate. The diet was supplemented with minerals, vitamins and TiO2 (3.0 g Ti/day). Total tract digestibility of DM, organic matter (OM), CP, crude fat, NDF, ADF, starch, sugar and energy was determined with the total faeces collection (TFC) method. In addition, DM and OM digestibility was estimated using internal ADL and the externally supplemented Ti as markers. Urine was collected on the final 2 days of each experimental period. Exercise did not affect apparent digestibility of CP, crude fat, starch and sugar. Digestibility of DM (DMD), OM (OMD), ADF and NDF tended to be lower and DE was decreased when ponies received the HI exercise regime. For all treatments combined, mean faecal recoveries of ADL and Ti were 87.8±1.7% and 99.3±1.7%, respectively. Ti was not detected in the urine, indicating that intestinal integrity was maintained with exercise. Dry matter digestibility estimated with the TFC, ADL and Ti for ponies subjected to LI exercise were 66.3%, 60.3% and 64.8%, respectively, while DMD for HI ponies were 64.2%, 60.3% and 65.2%, respectively. In conclusion, physical exercise has an influence on the GE digestibility of the feed in ponies provided with equivalent levels of feed intake. In addition, the two markers used for estimating apparent DMD and OMD indicate that externally supplemented Ti is a suitable marker to determine digestibility of nutrients in horses performing exercise unlike dietary ADL.

Comparison of Feeding Value of Some Popular Tree Fodders for Goats in the Mid-hill Region of Nepal

Large numbers of species of tree fodder are being used as green roughage source for goats in several farming systems. A study was carried out at Goat Research Station, Bandipur, Nepal in order to compare the feeding value of commonly used tree fodder leaves in mid-hills of Nepal. Altogether five treatments; tree fodder leaves of Ficus semicordata, Shorea robusta, Ficus glaberrima and Ficus lacor and mixed fodder as the conventional practices of the farmers were evaluated in Randomized Complete Block Design (RCBD) with three replications. Total of 30 male goats were used for the experiment in five groups, 6 for each treatment. The subsequent experiment was carried out to determine the nutrient digestibility of the tree fodder leaves by using fecal collection method. Nutrient composition, voluntary intake and nutrient digestibility of the fodder leaves and growth performance of goats were monitored. Crude protein (CP), total ash (TA), organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), hemicelluloses and cellulose proportion were better for F. glaberrima and F. lacor leaves, whereas the digestibility of those nutrients were better for F. semicordata leaves and moderate for F. glaberrima and S. robusta. Voluntary fodder dry matter intake was highest for F. glaberrima and least for mixed fodders. Growth performance of goats was higher for F. lacor and F. glaberrima than other tree fodder leaves and mixed fodders. The leaves of F. lacor and F. glaberrima had shown better feeding value than the conventional practices of farmers, mixed fodders. The leaves of S. robusta and F. semicordata were observed similar with the mixed fodders for their feeding value.Int. J. Appl. Sci. Biotechnol. Vol 5(4): 442-448

Rapid regrowth and detection of microbial contaminants in equine fecal microbiome samples.

Advances have been made to standardize 16S rRNA gene amplicon based studies for inter-study comparisons, yet there are many opportunities for systematic error that may render these comparisons improper and misleading. The fecal microbiome of horses has been examined previously, however, no universal horse fecal collection method and sample processing procedure has been established. This study was initialized in large part to ensure that samples collected by different individuals from different geographical areas (i.e., crowdsourced) were not contaminated due to less than optimal sampling or holding conditions. In this study, we examined the effect of sampling the surface of fecal pellets compared to homogenized fecal pellets, and also the effect of time of sampling after defecation on ‘bloom’ taxa (bloom taxa refers to microbial taxa that can grow rapidly in horse feces post-defecation) using v4 16S rRNA amplicon libraries. A total of 1,440,171 sequences were recovered from 65 horse fecal samples yielding a total of 3,422 OTUs at 97% similarity. Sampling from either surface or homogenized feces had no effect on diversity and little effect on microbial composition. Sampling at various time points (0, 2, 4, 6, 12 h) had a significant effect on both diversity and community composition of fecal samples. Alpha diversity (Shannon index) initially increased with time as regrowth taxa were detected in the amplicon libraries, but by 12 h the diversity sharply decreased as the community composition became dominated by a few bloom families, including Bacillaceae, Planococcaeae, and Enterococcaceae, and other families to a lesser extent. The results show that immediate sampling of horse feces must be done in order to ensure accurate representation of horse fecal samples. Also, several of the bloom taxa found in this study are known to occur in human and cattle feces post defecation. The dominance of these taxa in feces shortly after defecation suggests that the feces is an important habitat for these organisms, and horse fecal samples that were improperly stored can be identified by presence of bloom taxa.

Dietary maifanite supplementation did not affect the apparent total tract digestibility of calcium and phosphorus in growing pigs.

ObjectiveThis study was conducted to determine the effects of dietary maifanite supplementation and fecal collection method on the apparent total tract digestibility (ATTD) of calcium (Ca) and phosphorus (P) and blood parameters in growing pigs.MethodsThirty-six growing barrows (Duroc×Landrace×Yorkshire; 27.0±2.6 kg) were allotted to six dietary treatments with 6 pigs per treatment according to body weight in a completely randomized design. The experimental treatments were: i) Low Ca+cornstarch (2.25%), ii) Low Ca+maifanite (2.25%), iii) Medium Ca+cornstarch (1.42%), iv) Medium Ca+maifanite (1.42%), v) High Ca+cornstarch (0.64%), and vi) High Ca+maifanite (0.64%). Feces were collected by the total collection (TC) and indicator method (IM). At the beginning and the end of the experiment, blood samples were collected from each pig.ResultsFor the TC method, there were no difference in Ca intake, fecal Ca output, Ca retention and the ATTD of Ca between cornstarch and maifanite diets at the same dietary Ca level. However, urinary Ca excretion was lower (p = 0.01) in pigs fed low Ca diets without maifanite supplementation compared with other dietary treatments. Dietary maifanite supplementation had no effect on the P metabolism in growing pigs. For the IM method, there was no difference in Ca digestibility between cornstarch and maifanite diets at the same dietary Ca level. The ATTD of P was greater (p<0.01) in pigs fed the high Ca diet with maifanite supplementation compared with the high Ca diet with cornstarch treatment. Dietary inclusion of maifanite had no effect on blood parameters in growing pigs.ConclusionDietary maifanite supplementation had no effect on the ATTD of Ca and P and serum parameters in growing pigs. The IM resulted in lower digestibility values than the TC method.

Digestibility markers of sheep diets containing hydrolyzed sugarcane-top hay

SUMMARY This experiment evaluated the correlation between the estimates of fecal digestibility and dry matter digestibility (DMD) and nutrients obtained using internal (indigestibles dry materiDM, neutral detergent fiber-iNDF, acid detergent fiber-iADF and acid detergent ligniniADL) and external (chromium oxide-Cr2O3, titanium dioxide-TiO2, and purified and enriched lignin from eucalyptus-LIPE®) markers and the values obtained by the total feces collection method. Twenty uncastrated male sheep, with 29.64±5.53 kg average live weight; approximately 12 months old, were kept in metabolic cages and fed diets containing untreated sugarcane-top hay or sugarcane-top hay hydrolyzed with 3 and 6% urea or 1.5 and 3% calcium oxide (CaO). There was a positive correlation (P&lt;.05) between the fecal production estimated by the markers and via total feces collection, with coefficients higher than 90% for iDM, Cr2O3, and TiO2. The DMD and nutrients estimated with the use of markers was positively correlated (P&lt;.05) with that obtained by total feces collection. Coefficients of correlation for digestibility obtained by total collection were higher than those obtained with Cr2O3, but close to those obtained using TiO2. The coefficient of correlation between the digestibility of protein and fiber fraction estimated with iDM and iNDF and that obtained by total collection was higher than 70%. Internal marker iDM and external markers Cr2O3 and TiO2 are effective in estimating fecal production and DMD and nutrients.

Effect of enzyme supplements on macronutrient digestibility by healthy adult dogs.

Some enzyme supplement products claim benefits for healthy dogs to compensate for alleged suboptimal production of endogenous enzymes and the loss of enzymes in commercial pet foods secondary to processing. The objective of the current study was to determine macronutrient and energy digestibility by healthy adult dogs fed a commercial maintenance diet with or without supplementation with plant- and animal-origin enzyme products at the dosage recommended by their respective manufacturers. A group of fourteen healthy neutered adult Beagle dogs (average age 8 years) was divided into two equal groups and fed the basal diet alone and then with either the plant- or animal-origin enzyme supplement in three consecutive 10-d periods; the treatment groups received the opposite enzyme supplement in the third period. Digestibility in each period was performed by the total faecal collection method. Serum trypsin-like immunoreactivity (TLI) was measured at the end of each trial. Data were analysed by repeated measures and the α level of significance was set at 0·05. There were no differences in energy and nutrient digestibility between enzyme treatments. When comparing basal with enzyme supplementation, fat digestibility was higher for the basal diet compared with the animal-origin enzyme treatment, which could be a period effect and was not biologically significant (94·7 v. 93·5%). Serum TLI was not affected by supplementation with either enzyme product. Exogenous enzyme supplementation did not significantly increase digestibility of a typical commercial dry diet in healthy adult dogs and routine use of such products is not recommended.

Highlights of This Issue

To identify common genetic variants linked to aggressive prostate cancer, Koboldt and colleagues used whole-exome sequencing followed by targeted sequencing in aggressive prostate cancer patients and disease-free controls in both African and European Americans. In each population, the authors tested rare variants for association using two gene-based aggregation tests. TET2 in African Americans was associated with aggressive disease, with 24.4% of cases harboring a rare deleterious variant, compared to 9.6% of controls. Eight additional genes were identified with suggestive evidence of association as well as rare truncation variants. These findings suggest that rare variants influence risk of clinically relevant prostate cancer and if validated could serve to identify men for screening, prophylaxis, and treatment.The effects of use of different types of hormone therapy (HT) on breast cancer risk according to prognostic factors are largely unknown. Román and colleagues linked data from the Norwegian Prescription Database and the Cancer Registry of Norway to estimate the rate ratios and 95% confidence intervals for breast cancer in relation to HT. Users of the most common estrogen and progestin combinations were at a 4- to 5-fold elevated risk of grade I tumors, and estradiol–NETA users were also at a 2- to 3-fold increased risk of medium differentiated (grade II) tumors. The HT preparations most commonly used in the Nordic countries are associated with both breast cancers with good and less favorable prognosis characteristics.Health insurance may lengthen or inhibit time to follow-up after positive screening mammography. Durham and colleagues assessed the association between insurance status and time to initial diagnostic follow-up after a positive screening mammogram. Among 43,026 women included in the study, 73% were &lt;65 years and 27% were 65+. Median time until initial diagnostic follow-up was similar by age group and insurance status. In the adjusted model for women &lt;65, uninsured women experienced a longer time to initiation of diagnostic follow-up versus women with private insurance. There was also an increased odds of these uninsured women not meeting the CDC guideline for follow-up within 60 days. Among women ages 65+, women with private insurance experienced a faster time to follow-up compared to women with Medicare and private insurance.The gut metabolome may be associated with incidence and progression of measuring metabolite levels in the feces. But questions remain with regards to the effects of fecal sample collection methods on metabolomic measures. Loftfield and colleagues collected fecal samples from 18 volunteers using four methods: no solution, 95% ethanol, fecal occult blood test (FOBT) cards, and fecal immunochemical test (FIT). One set of samples was frozen after collection and a second set was frozen after 96 hours at room temperature. Intraclass correlation coefficients (ICC) estimating technical reproducibility were high for replicate samples for each collection method, and ICCs estimating stability at room temperature were high for 95% ethanol and FOBT but not FIT. Future epidemiologic studies should collect feces using 95% ethanol or FOBT if interested in studying fecal metabolomics.

The genotoxic and antigenotoxic properties of citrus phenolic limonene on mammalian cell lines

This study aimed to investigate the effects of a natural saline pasture on digestibility, intake, growth, carcass traits and meat quality of lambs. Forty weaned female Queue Fine de l′Ouest lambs (25.4 ± 4.33 kg) were allotted into two groups. The first group raised indoor (I) with oat straw ad libitum and the second grazed natural saline pasture (SP). Both groups were supplemented the same concentrate with 300 and 500 g for SP and I respectively, to have the same average daily gain (ADG). On the 49th day, digestibility and intake were determined on 8 lambs randomly selected from each group, using total fecal collection and Acid Insoluble Ash (AIA) marker method. After 83 days, the 16 lambs were slaughtered at 34 kg. Dry matter (DM), organic matter (OM), crude protein (CP) digestibility and metabolizable energy (ME) were higher (P < 0.001) for SP. Digestible crude protein (DCP)/ME ratio was higher (P < 0.05) for SP group. DM intake (P < 0.05) was lower for SP, while digestible crude protein intake (DCPI) and ME intake (MEI) were similar between the two groups. Water intake was higher for SP (P < 0.001). Final weight and ADG were similar between groups. Carcass dressing and high-priced joints percentages were higher for SP group. Lambs from the SP group had a higher percentage of muscle (P < 0.001) and a lower fat proportion (P < 0.01). No effect of feeding system on carcass linear measurements and on compactness indexes (P > 0.05) was noted. Kidney fat decreased in favor of SP group (P < 0.01). There were no differences for fat color, whereas meat of I group had a higher lightness and yellowness than that of SP one. Meat of SP group had a higher DM, protein level (P < 0.05) and lower intramuscular fat (P < 0.01). It has higher content of Fe and Zn (P < 0.01). It could be concluded rearing lambs on saline pasture, with a moderate concentrate supplementation, allowed acceptable growth performances, leaner carcass, and higher nutritional meat quality lambs.

Apparent digestibility of pacu (Piaractus mesopotamicus) diets containing different levels of phosphorus

&lt;p&gt;The present study aimed to evaluate pacu (&lt;em&gt;Piaractus mesopotamicus&lt;/em&gt;) diets and determine the apparent digestibility at different levels of phosphorus using the feces collection method with an intestinal dissection marker. A total of 320 pacu with average weights of 768.5±36.34 g were used, and they were distributed in four 5 m³ net cages. The fish were fed isonitrogenous (27%), isoenergetic (3000 kcal/kg) and isocalcium (1.50%) diets with total phosphorus levels of 0.6, 0.8, 1.0 and 1.2%. The fish were incised with lateral openings to allow for feces removal from the rectum, and the fish were then sent for carcass chemical composition analysis. Significant differences were observed (P&amp;lt;0.01) between treatments for the apparent diet digestibility, apparent phosphorous digestibility, available phosphorus and residual phosphorus. There was a linear decrease in apparent diet digestibility and apparent phosphorus digestibility with increasing levels of phosphorous in the diet, and the highest values were observed for the diet with 0.6% total phosphorus. For available phosphorus and residual phosphorus, there was a linear increase based on total phosphorus levels in the diet. No significant differences were observed for the chemical composition of the pacu carcasses. Therefore, the greatest digestibility of total phosphorus in the diet obtained through dissection was observed in the diet containing 0.6% total phosphorus, and the different levels of phosphorus did not influence the chemical composition of the pacu carcasses. &lt;/p&gt;

Apparent digestibility of pacu (Piaractus mesopotamicus) diets containing different levels of phosphorus

The present study aimed to evaluate pacu (Piaractus mesopotamicus) diets and determine the apparent digestibility at different levels of phosphorus using the feces collection method with an intestinal dissection marker. A total of 320 pacu with average weights of 768.5±36.34 g were used, and they were distributed in four 5 m³ net cages. The fish were fed isonitrogenous (27%), isoenergetic (3000 kcal/kg) and isocalcium (1.50%) diets with total phosphorus levels of 0.6, 0.8, 1.0 and 1.2%. The fish were incised with lateral openings to allow for feces removal from the rectum, and the fish were then sent for carcass chemical composition analysis. Significant differences were observed (P&lt;0.01) between treatments for the apparent diet digestibility, apparent phosphorous digestibility, available phosphorus and residual phosphorus. There was a linear decrease in apparent diet digestibility and apparent phosphorus digestibility with increasing levels of phosphorous in the diet, and the highest values were observed for the diet with 0.6% total phosphorus. For available phosphorus and residual phosphorus, there was a linear increase based on total phosphorus levels in the diet. No significant differences were observed for the chemical composition of the pacu carcasses. Therefore, the greatest digestibility of total phosphorus in the diet obtained through dissection was observed in the diet containing 0.6% total phosphorus, and the different levels of phosphorus did not influence the chemical composition of the pacu carcasses.

Fecal collection methods for the determination of protein digestibility in bullfrogs

Adequate methods for the determination of protein digestibility in bullfrogs are important for the understanding of nutrient utilization. Therefore, this study evaluated two methods of feces collection: intestinal dissection and fecal decantation, using cylindric-conical tanks. Frogs were fed with a commercial diet (45% crude protein) which was ground and supplemented with 0.5% chromium oxide III. The frogs were fasted 48h before force-feeding (5% of the animal's live weight). For the decantation method, the animals were sacrificed 36 h after force-feeding and feces were collected directly from the large intestine. For the sedimentation method, feces were collected when they appeared in the tubes attached to the front end of the cylindric tanks. No significant difference (P&gt;0.05) in the apparent digestibility coefficients of crude protein for dietary was observed between the methods tested (74.0% and 76.4% for the dissection and decantation methods, respectively). In conclusion, both methods can be used for the determination of protein digestibility of bullfrog feeds