Equine Feces Research Articles

Diagnosis, risk factors analysis and first molecular characterization of Cryptosporidium spp. in horses from Rio de Janeiro, Brazil

An analysis was made of the frequency of Cryptosporidium spp. in fecal samples from horses raised on farms in the Teresópolis city, state of Rio de Janeiro, Brazil, and the risk factors that favored this infection. Between 2019 and 2020, 314 samples of equine feces were collected, 287 of which came from English Thoroughbred horses and 27 from ponies. Information on the horses and their management were retrieved from a stud book and forms filled out by trainers. The fecal samples were subjected to macroscopic analysis, modified Sheather's and Lutz parasitological techniques, safranin staining, and to enzyme-linked immunosorbent assay (ELISA) for the detection of coproantigens. All the samples that tested positive by these techniques underwent partial sequence analysis of the 18S rRNA gene to characterize the protozoan species. Cryptosporidium spp. was identified in 35 (11.1%) of the samples, 34 from English Thoroughbred horses and one from a pony. Based on a logistic regression model, it was found that the presence of dogs and small ruminants on the farms, and drinking water from a spring, were significantly associated with the animals' infection by the protozoan (p<0.05). Eight of the English Thoroughbred horse samples underwent molecular characterization, which revealed the presence of Cryptosporidium felis in one sample and Cryptosporidium parvum in seven. The seven samples containing C. parvum were subjected to gp60 gene analysis, based on which nucleotide sequences typical of the IIa family were identified, which are usually transmitted from animals to humans. In addition, the genotype IIaA15G2R1, which is considered to have the highest profile of zoonotic transmissibility, was identified in one Thoroughbred horse. This is the first study conducted in the state of Rio de Janeiro that molecularly characterized Cryptosporidium spp. in horses, and the first on the American continent to detect C. felis in the feces of these animals.

Impact of Storage Conditions on Equine Fecal Inoculum for Estimating In Vitro Digestibility

Simple SummarySample handling and storage may affect the fermentative capacity of fecal inoculum. The need to collect rectal grabs from individuals can be a limiting factor in utilizing fecal inoculum from very young or feral animals. This study evaluated the effect of storage conditions of equine feces on the viability of microbial inoculum used for in vitro equine digestibility trials. Pooled fecal material was stored anaerobically at 39 °C for 15 min (control), while aerobic samples were stored at 22 °C for 6 h (SC1), 3 °C for 6 h (SC2), and −18 °C for 24 h (SC3). Results supported that fecal material stored aerobically for six hours at 22 °C provided similar digestibility estimates compared to the control, while dry matter digestibility decreased by 3.86% at SC2 and by 4.08% at SC3.This study evaluated the effect of storage conditions of equine fecal material on the viability of microbial inoculum used for in vitro equine digestibility trials. Pooled fecal material from three mature Quarter Horse geldings was stored at 39 °C anaerobically for 15 min (control), while aerobic samples were stored at 22 °C for 6 h (SC1), 3 °C for 6 h (SC2), and −18 °C for 24 h (SC3). Following storage, the feces were utilized to prepare microbial inoculum for the digestion of six different forages using the Daisy II Incubator. After incubation, DM, NDF, and ADF compositions were determined and used to calculate DMD, NDFD, and ADFD. Analysis using the OLS regression model for differences in DMD, NDFD, and ADFD across the storage conditions found significant interactions between the forage sample and the storage condition (p < 0.05). The results between the control and SC1, SC2, and SC3 were not different (p < 0.8). Fecal material stored aerobically for six hours at 22 °C provided similar digestibility estimates compared to the control, while DMD decreased by 3.86% in SC2 and by 4.08% in SC3.

The Effect of Ryegrass Silage Feeding on Equine Fecal Microbiota and Blood Metabolite Profile.

Silage is fed to horses in China and other areas in the world, however, knowledge about the impact of feeding silage on horse health is still limited. In the current study, 12 horses were assigned into two groups and fed ryegrass silage and ryegrass hay, respectively, for 8 weeks. High-throughput sequencing was applied to analyze fecal microbiota, while liquid chromatography–tandem mass spectrometry (LC–MS/MS) based metabolomics technique was used for blood metabolite profile to investigate the influence of feeding ryegrass silage (group S) compared to feeding ryegrass hay (group H) on equine intestinal and systemic health. Horses in group S had significantly different fecal microbiota and blood metabolomes from horses in group H. The results showed that Verrucomicrobia was significantly less abundant which plays important role in maintaining the mucus layer of the hindgut. Rikenellaceae and Christensenellaceae were markedly more abundant in group S and Rikenellaceae may be associated with some gut diseases and obesity. The metabolomics analysis demonstrated that ryegrass silage feeding significantly affected lipid metabolism and insulin resistance in horses, which might be associated with metabolic dysfunction. Furthermore, Pearson’s correlation analysis revealed some correlations between bacterial taxa and blood metabolites, which added more evidence to diet-fecal microbiota-health relationship. Overall, ryegrass silage feeding impacted systemic metabolic pathways in horses, especially lipid metabolism. This study provides evidence of effects of feeding ryegrass silage on horses, which may affect fat metabolism and potentially increase risk of insulin resistance. Further investigation will be promoted to provide insight into the relationship of a silage-based diet and equine health.

The Equine Faecal Microbiota of Healthy Horses and Ponies in The Netherlands: Impact of Host and Environmental Factors.

Simple SummarySeveral studies have described the bacterial composition in the intestines of horses, and several factors of influence have been detected. Variation in the results between studies, however, is substantial. Therefore, the current study aimed to study the bacterial composition in the faeces of healthy horses and ponies kept under standard housing and management condition in The Netherlands. Seventy-nine horses and ponies originating from two farms were included. Several factors, such as location, age, the season of sampling, horse type (horses vs. ponies) and pasture access significantly affected the bacterial composition. The current study provides important baseline information on variation in the bacterial composition in healthy horses and ponies under standard housing and management conditions. The aforementioned factors identified in this study to affect the bacterial population of the gut should be considered in future studies regarding the bacterial population of the equine gut.Several studies have described the faecal microbiota of horses and the factors that influence its composition, but the variation in results is substantial. This study aimed to investigate the microbiota composition in healthy equids in The Netherlands under standard housing and management conditions and to evaluate the effect of age, gender, horse type, diet, pasture access, the season of sampling and location on it. Spontaneously produced faecal samples were collected from the stall floor of 79 healthy horses and ponies at two farms. The validity of this sampling technique was evaluated in a small pilot study including five ponies showing that the microbiota composition of faecal samples collected up to 6 h after spontaneous defaecation was similar to that of the samples collected rectally. After DNA extraction, Illumina Miseq 16S rRNA sequencing was performed to determine microbiota composition. The effect of host and environmental factors on microbiota composition were determined using several techniques (NMDS, PERMANOVA, DESeq2). Bacteroidetes was the largest phylum found in the faecal microbiota (50.1%), followed by Firmicutes (28.4%). Alpha-diversity and richness decreased significantly with increasing age. Location, age, season, horse type and pasture access had a significant effect on beta-diversity. The current study provides important baseline information on variation in faecal microbiota in healthy horses and ponies under standard housing and management conditions. These results indicate that faecal microbiota composition is affected by several horse-related and environment-related factors, and these factors should be considered in future studies of the equine faecal microbiota.

Methodical investigation of mineral and trace element concentrations in equine feces with special consideration of the sampling location

Objective measurements of the mineral supply in horses are rarely performed. As a result, incorrect elements or an improper amount of elements are provided. The analysis of feces could represent a novel method to evaluate the nutritive supply. The prerequisite is a knowledge of methodological factors influencing the mineral concentration in the fecal samples. Within the scope of this investigation, the effects of different kinds of mineral supply and the influence of the sampling location on the concentration of minerals in equine feces samples were analyzed. Additionally, the methodical error of the measurement procedure was estimated. Six minerals and 22 trace elements in 30 fecal samples of 5 warmbloods, 3 haflinger horses and 2 ponies with differing mineral supplementation (none, calculated amount, mineral lick mass for free intake) were investigated. The samples derived from 3 locations (cranial rectum, ampulla recti and dung heaps) were dried and analyzed using ICP-OES and ICP-MS. Besides a descriptive data analysis, the effects of the fixed factors sampling location and mineral supplementation as well as repeatability and the relative error of the method were assessed. The element concentrations were not influenced by the sampling location, while they varied significantly depending on the type of mineral supplementation. The repeatability of analysis was high with an average measure of certainty of 0.949 (0.894-0.978) and an average median relative error of 0.18 (0.01-0.86). The study outlines a methodological framework for the determination of mineral and trace element concentrations in equine feces. The location of fecal sampling did not affect the results, therefore, preference may be given to noninvasive sample collection from a fecal pile. Furthermore, as the types of mineral supplementation result in significantly differing fecal element concentrations, varying concentrations in feces allow for conclusions concerning the amount of mineral intake of the individual horse.

Effects of Pasture Grass, Silage, and Hay Diet on Equine Fecal Microbiota.

Simple SummaryThe intestinal microbial community in horses is very complex and interacts closely with diets. Apart from traditional forage diets, such as hay and pasture grass, silage is used to feed horses in China and other areas of the world for economic reasons or convenience of storage. Additionally, silage is also used for its convenience of harvesting and its nutrient components, including lactic acid and volatile fatty acids. In this study, we detected the characteristic composition of a fecal microbial community in horses that were fed silage with the use of a relatively new sequencing technique; we compared this result with that from horses that were fed hay and pasture grass. This study revealed some characteristic findings on the fecal microbial composition in horses that were given each of type of diet and showed significant differences between the groups. Our results provided novel data about the fecal microbial composition in horses on the silage diet. We hope that these could help balance the intestinal microbiota in horses that are mainly fed silage in combination with other types of forages in order to maintain intestinal health.Diet is an important factor affecting intestinal microbiota in horses. Fecal microbiota is commonly used as a substitute for studying hindgut microbiota when investigating the relationship between intestinal microbial changes and host health. So far, no study has compared the difference between the fecal microbiota found in horses that are fed pasture grass, silage, and hay. The present study aims to characterize the fecal microbiota in horses that were exclusively on one of the three forage diets, and to analyze the potential impact of these forages, especially silage, on horse intestinal health. There were 36 horses randomly assigned to each of the three groups; each group was fed only one type of forage for 8 weeks. High throughput sequencing was applied to analyze the bacterial taxa in fecal samples collected from the horses at the end of the feeding trial. The Lachnospiraceae family was statistically more abundant in horses fed with hay, while it was the least abundant in horses fed with silage. The Streptococcaceae spp., considered a core microbial component in equine intestinal microbiota, were present in significantly lower quantities in feces from horses that were fed pasture grass as compared to those from horses fed hay or silage. The novel data may help promote the balancing of horse intestinal microbiota and the maintenance of intestinal health in horses.

Sequential Modulation of the Equine Fecal Microbiota and Fibrolytic Capacity Following Two Consecutive Abrupt Dietary Changes and Bacterial Supplementation.

Simple SummaryThe equine hindgut is colonized by microorganisms, some of which are involved in fiber digestion and are crucial for the horse’s nutrition and health. These key microorganisms are very sensitive to dietary changes, which have been identified as a risk factor for colics. This study assessed the stressful effect of two consecutive abrupt dietary changes on the diversity, the composition, and the activity of fecal microorganisms focusing on fibrolytic bacteria. Twelve horses were subjected to an abrupt change from forage to a concentrate-rich diet, followed by a second change from a concentrate to forage-rich diet 5 days later. Half of the horses were given a supplement of living bacteria as a probiotic. Two days after the sudden change from forage to concentrate diet, the proportions and types of microorganisms were altered drastically, as was their capacity to degrade fibers. After this dietary stress, it took 3–4 weeks of a high-fiber diet to recover the basal state. Supplementation with probiotics promoted an earlier recovery of fibrolytic bacteria after the dietary stress. Abrupt dietary changes should be limited in horse management to protect the hindgut microorganisms and their capacity to use forage fibers, and consequently to limit the development of colic.In horses, abrupt changes from high-fiber (HF) to high-starch (HS) diets can affect the cecal and colonic microbiota. This study investigated modifications and recovery of fecal microbiota after two consecutive abrupt dietary changes. Twelve horses fed HF for 2 weeks were changed to HS for 5 days then returned to HF for 7 weeks. Six received lactic acid bacteria supplementation. Bacterial population diversity, structure, and activity, especially fibrolysis, were assessed to obtain an overview of alteration in hindgut microbiota. Two days after the abrupt change from HF to HS, the findings in feces were consistent with those previously reported in the cecum and colon, with a decrease in fibrolytic activity and an increase in amylolytic activity. Fecal parameters stabilized at their basal level 3–4 weeks after the return to HF. A bloom of cellulolytic bacteria and lower pH were observed after 1.5 weeks, suggesting a higher level of fiber degradation. In supplemented horses the relative abundance of potentially fibrolytic genera was enhanced 2 days after HS and 2 days to 2–3 weeks after the return to HF. Fecal analysis could be a promising technique for monitoring hindgut microbial variations accompanying dietary changes.

Antimicrobial Resistance and Extended-Spectrum Beta-Lactamase (ESBL) Genes in E. coli Isolated from Equine Fecal Samples in Turkey

The extensive use of antibacterial agents used for treatment in human and veterinary clinics to increase yield in livestock and aquaculture in developing countries causes the detection and spread of multidrug-resistant (MDR) strains in different sources. The presence of Escherichia coli strains is very common in racehorses and it's a serious problem on horse farms. Extended-spectrum beta-lactamase (ESBL) production is an important source of MDR development in Gram-negative strains such as E.coli. This study aimed to detect the presence of ESBL genes in E. coli strains isolated from horse farms in eastern Turkey.A total of 200 equine fecal samples were collected from 16 horse farms (70 Thoroughbred and 130 Arabian horses) in the Adana and Şanlıurfa provinces of Turkey. The presence of ESBL genes, such as blaCTX-M, blaTEM, blaSHV, blaOXA-48, and blaKPC in the E. coli strains was investigated using real-time PCR.According to the real-time PCR results, 107 (53.5%) out of 200 E. coli strains were positive for at least one gene. BlaCTX-M, blaSHV, and blaTEM were detected in 11, 6, and 4 strains respectively. blaCTX-M+blaSHV coexisted in 16 strains, blaCTX-M+blaTEM in 11 strains, blaSHV+blaOXA-48 in 8 strains, blaTEM+blaSHV in 8 strains, blaCTX+blaSHV+ blaOXA-48 coexistent in 3 strains, blaCTX-M+blaTEM+blaSHV+blaOXA-48 in 7 strains, blaCTX-M+ blaTEM+blaSHV+blaKPC in 2 strains, and finally, blaCTX-M+blaTEM+blaSHV+blaOXA-48+blaKPC in 3 strains.In a summary, ESBL-producing E. coli strains were frequently seen in the racehorses from eastern Turkey. The excessive misuse of antibiotics has led to the evolution of MDR strains such as ESBL-producing E. coli that cause serious health problems and are difficult to treat. Routine molecular epidemiology studies are warranted to closely monitor these resistant strains.

168 The application of near infrared spectroscopy to determine concentrations of organic constituents in equine faeces

168 The application of near infrared spectroscopy to determine concentrations of organic constituents in equine faeces

Long-term storage of feces at -80 °C versus -20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome.

The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.

FACTORS INFLUENCING THE INCIDENCE OF EIMERIA LEUCKARTI IN HORSES

Cross sectional study was planned to figure out the prevalence rate of Eimeria (E.) leuckarti in horses and factors influencing its occurrence in district TT Singh, Punjab, Pakistan. From April 2009 to March 2010, equine feces were collected. Two stage cluster method was adopted for random selection of horses. Total of 484 fecal samples were collected from whole district and collected samples were subjected to quantitative floatation method. It was found that 50.41% of sampled horses were positive for E. leuckarti with prevalence reaching to its peak in August (OR=1.156; χ2=20.055) while lowest occurence was recorded in April, May and June, coinciding with the period of lowest humidity and precipitation of the year in Pakistan. It can be inferred from these observations that wet season seems to be favorable for propagation of this disease. Foals (62.94%; OR=0.422; χ2=20.825) were found to be more susceptible (P&lt;0.05) to E. leuckarti in comparison to adults (41.81%) while mares with prevalence of 56.48% (OR=0.512; χ2=13.265) were found to be more susceptible (P&lt;0.05) as compared to and male horses (35.04%). Horses kept on non-cemented floor, mix farming with other animals, given food on ground and watered in ponds/common places were found to have higher rates of prevalence of E. leuckarti (P&lt;0.05) in comparison to those kept on cemented/ partially cemented floor, separate farming, food in trough and tap watered, respectively. It is inferred from findings of this survey that disease can be controlled by improving management systems.

Feasibility of biogas as a green energy source in the central part of South Sulawesi

The goal of this work is the feasibility of biogas as a green energy source produced from organic matter from ruminants, equine and farming activities in the central part of South Sulawesi. The study was conducted with survey and laboratory analysis to calculate the number of ruminants, corn, and rice stalk using supporting data from the South Sulawesi Statistics office. Amounts of 84,567, 112,915, and 3,579 ruminants have been raised in Maros, Gowa, and Makassar, respectively. While 6,757, 9,529, and 50 monogastric are raised in Maros, Gowa, and Makassar. Corn and rice stalks were produced 465,878.06 tons year-1 in Gowa and Maros. Every cattle and equine can produce feces up to 25 kg day-1 and 30 kg day-1, daily feces production equals 2,316,885; 3,108,745; and 90,975 kg day-1 in Maros, Gowa, and Makassar, respectively. These waste products can be converted to biogas as source of green energy with a total yield of 667,752,675,489.20 x 103 cc. Its production ratio is 1 kg dry matter: 1,051.3-liter biogas for beef feces and 1 kg dry matter: 3,980-liter biogas for equine feces and corn and rice stalks can be applied as organic fertilizer as a significant factor in anaerobic fermentation of biogas production.

Effect of Gender Bias on Equine Fecal Microbiota

Many studies on human intestinal microbiota indicate that gender difference is one of the key factors influencing microbial community composition. To date, the degree of influence that gender has on equid intestinal microbiota has not been reported. Thus, microbiota was analyzed in feces of seven female Przewalski’s horses (FRPHs) and seven male Przewalski’s horses (MRPHs) in this study, determining which microbiota characteristics respond to gender biases. The microbial community composition and structure were explored by 16S rRNA sequencing, followed by diversity analysis and difference analysis. Female Przewalski’s horses showed higher Shannon diversity than MRPHs, no difference in Simpson diversity, and displayed difference in beta diversity. Although gender had little effect on the overall microbiota, it significantly changed the dominant microbial community in each classification level. Male Przewalski’s horses contained significantly higher amounts of microorganisms related with diseases, including spirochetes (phylum), deltaproteobacteria (class), fibrobacteria (class), spirochaetia (class), desulfovibrionales (order), fibrobacterales (order), spirochaetales (order), and spirochaetaceae (family). Female Przewalski’s horses showed less than MRPHs in the top 10 genera. To our knowledge, this study is the first to document the gender-related intestinal microbiota profile in equines and discovered notable differences between the gender, which suggests that gender should be considered as a biological variable in future microbiota studies.

Novel Equine Faecal Egg Diagnostics: Validation of the FECPAKG2.

Faecal egg counts (FECs) are the standard method of diagnosing the level of parasitic helminth egg shedding in horses and other grazing animals. Testing before treatment is an important factor in slowing the appearance of anthelmintic resistance in nematode parasites. The FECPAKG2, optimised for livestock, is reported to allow owners to perform FECs on their own animals without the need for a separate microscope or any specialist knowledge by tapping into remote expertise. However, the performance of the FECPAKG2 has yet to be assessed for equids. Therefore, a comparison of the FECPAKG2 (G2) method with an accepted equine FEC method (FECPAKG1(G1)) was performed, using faecal samples from 57 horses in Wales and 22 horses in New Zealand. There was a significant correlation between the FECs obtained by the two methods (p < 0.001) and no effect of the country of origin on the data (p = 0.157). The mean percentage accuracy compared to the control values (mean G2 count as a percentage of the mean G1 count, ±SStandard Error (SE)) was 101 ± 4%. There was no significant interaction between the method applied and the country of origin of the data (p = 0.814). The relative accuracy of the G2 method compared to the control method (FECPAKG1) was not affected by the level of infection (p = 0.124) and it was concluded that the FECPAKG2 method is a suitable method of performing FECs in horses. It is anticipated that the user-friendliness of the method will increase the uptake of FECs amongst horse owners, either by the direct use of the technology or through their veterinary practice, likely slowing the development of anthelmintic resistance.

Models Based on the Mitscherlich Equation for Describing Typical and Atypical Gas Production Profiles Obtained from In Vitro Digestibility Studies Using Equine Faecal Inoculum

Simple SummaryFeedstuff evaluation through animal trials is time consuming and expensive. An alternative, the gas production method, measures the amount of fermentation gas produced from incubating feedstuffs with microbes from ruminal fluid or faecal samples. Models can be applied to gas production profiles to determine extent of feedstuff degradation either in the rumen or in the hindgut. Typical gas production profiles show a monotonically increasing monophasic pattern. However, atypical gas production profiles exist whereby at least two consecutive phases of gas production are present; these profiles are much less well described. Two models are proposed to fit these biphasic profiles, a sum of two Mitscherlich equations, and sum of Mitscherlich + linear equations. Additionally, two models that describe typical monophasic gas production curves, the simple Mitscherlich and the generalised Mitscherlich (root-t) model, were assessed for comparison. Models were fitted to 25 gas production profiles resulting from incubating feedstuffs with faecal inocula from equines. Of these 25 profiles, 17 displayed atypical biphasic patterns, and 8 displayed typical monophasic patterns. The two biphasic models were found to describe both the atypical and typical gas production profiles accurately. These models allow for the evaluation of feedstuffs using cost- and time-efficient methods.Two models are proposed to describe atypical biphasic gas production profiles obtained from in vitro digestibility studies. The models are extensions of the standard Mitscherlich equation, comprising either two Mitscherlich terms or one Mitscherlich and one linear term. Two models that describe typical monophasic gas production curves, the standard Mitscherlich and the France model [a generalised Mitscherlich (root-t) equation], were assessed for comparison. Models were fitted to 25 gas production profiles resulting from incubating feedstuffs with faecal inocula from equines. Seventeen profiles displayed atypical biphasic patterns while the other eight displayed typical monophasic patterns. Models were evaluated using statistical measures of goodness-of-fit and by analysis of residuals. Good agreement was found between observed atypical profiles values and fitted values obtained with the two biphasic models, and both can revert to a simple Mitscherlich allowing them to describe typical monophasic profiles. The models contain kinetic fermentation parameters that can be used in conjunction with substrate degradability information and digesta passage rate to calculate extent of substrate degradation in the rumen or hindgut. Thus, models link the in vitro gas production technique to nutrient supply in the animal by providing information relating to digestion and nutritive value of feedstuffs.

Dietary Trace Mineral Level and Source Affect Fecal Bacterial Mineral Incorporation and Mineral Leaching Potential of Equine Feces

Minerals excreted in feces have the potential to leach or runoff to water-ways, negatively impacting water quality. This study examined the effect of dietary trace mineral levels, and their source, on the leaching potential of minerals from equine feces. Nine horses were used in a replicated 3 × 3 Latin Square, with three dietary treatments provided as pellets: no added trace minerals (CON), added inorganic trace minerals (ING), and added organic trace minerals (ORG). Supplemental trace minerals included Co, Cu, Mn, and Zn. Horses were allowed ad libitum access to forage and fed their treatment pellets for 16 days prior to fecal sample collection. Estimated dietary mineral intake exceeded requirements for supplemented minerals. Regardless of the source, adding dietary trace minerals increased the fecal leaching potential of Cu, Zn, and P (p &lt; 0.05). More Co leached from ORG compared to ING, while Zn leached in greater amounts from ING compared to ORG (p &lt; 0.05). Fecal bacterial Zn content was greater (p &lt; 0.05) for ORG compared to ING. Negative correlations were observed between bacterial mineral content and leaching for several minerals. Supplementing trace minerals in forms that increase microbial incorporation may provide a strategy to control fecal mineral leaching.

Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control.

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.

The recovery of added nematode eggs from horse and sheep faeces by three methods

BackgroundNematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces were added to egg free ovine and equine faeces to give counts of 10, 50, 200 and 500 eggs per gram (EPG) of faeces.ResultsThe Cornell-Wisconsin significantly underestimated egg counts and McMaster showed a low analytical sensitivity, revealing 100% of sensitivity only for concentrations greater than 200 EPG. EPG values detected by Mini-FLOTAC did not differ significantly from expected counts at any level of egg density.ConclusionsMini-FLOTAC combined to Fill-FLOTAC which provides an accurate method of weighing without need for a balance and filtering out debris, could be used for FEC on the farm as well as in the laboratory.

A comparison of sample preparation methods for extracting volatile organic compounds (VOCs) from equine faeces using HS-SPME

IntroductionDisturbance to the hindgut microbiota can be detrimental to equine health. Metabolomics provides a robust approach to studying the functional aspect of hindgut microorganisms. Sample preparation is an important step towards achieving optimal results in the later stages of analysis. The preparation of samples is unique depending on the technique employed and the sample matrix to be analysed. Gas chromatography mass spectrometry (GCMS) is one of the most widely used platforms for the study of metabolomics and until now an optimised method has not been developed for equine faeces.ObjectivesTo compare a sample preparation method for extracting volatile organic compounds (VOCs) from equine faeces.MethodsVolatile organic compounds were determined by headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GCMS). Factors investigated were the mass of equine faeces, type of SPME fibre coating, vial volume and storage conditions.ResultsThe resultant method was unique to those developed for other species. Aliquots of 1000 or 2000 mg in 10 ml or 20 ml SPME headspace were optimal. From those tested, the extraction of VOCs should ideally be performed using a divinylbenzene-carboxen-polydimethysiloxane (DVB-CAR-PDMS) SPME fibre. Storage of faeces for up to 12 months at − 80 °C shared a greater percentage of VOCs with a fresh sample than the equivalent stored at − 20 °C.ConclusionsAn optimised method for extracting VOCs from equine faeces using HS-SPME-GCMS has been developed and will act as a standard to enable comparisons between studies. This work has also highlighted storage conditions as an important factor to consider in experimental design for faecal metabolomics studies.

Impact of diet on bacterial lipopolysaccharides in equine feces and blood

Feeding horses with high-starch diet can lead to alteration of their hindgut and fecal bacteria composition, with a potential shift in gram negative and positive bacteria. Gram-negative bacteria contain LPS in their outer membrane, which can translocate into bloodstream following hindgut microbial disturbances. The aim of our study was to evaluate if diet composition (high-fiber or high-starch diets) can modulate the LPS concentration in equine feces and plasma using an innovative measurement method. Six horses were used in a longitudinal study composed of three consecutive periods. During the first period (H1; 3 weeks), they were fed 100% hay. After a gradual transition, they received during the second period (HB; 4 weeks) a diet composed of 57% hay and 43% barley. During the third period (H2; 6 weeks), they returned to the 100% hay diet without transition. Colonic, fecal and blood samples were collected on days 10 and 20 in H1 and HB periods and on days 10, 20 and 40 in H2 period. Total anaerobic, amylolytic and lactate-utilizing bacteria were enumerated in colon and feces using culture technics. Fecal and plasma LPS concentration was determined by direct quantitation of 3HM by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using a method described recently in human for plasma samples (Pais de Barros et al., 2015), and by adapting this method for fecal samples. The fecal LPS concentrations increased significantly when horses changed from hay diet to hay/barley diet and significantly decreased when horses returned to the hay diet (values comparable to those found during the first hay diet). Similar variations were observed on the bacteria concentrations in colon and feces of horses. The LPS concentration did not vary in plasma samples when the hay/barley diet was fed. It was assumed that dietary stress was not important enough to disrupted the hindgut mucosa and allow LPS translocation into plasma. These data showed that fecal LPS concentrations are very sensitive to diet changes, and could reflect alteration in the hindgut bacteria composition occurring under the dietary change. The measure of LPS through the 3HM raised the question of the accurate representation of LPS. In conclusion, quantification of 3HM could be a simple method to follow the evolution of bacterial composition in the feces, and rapidly state on the balance or imbalance of the bacteria. Further researches have to be conducted to use this parameter as an indicator of bacterial disturbances.