Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome.

BACKGROUND: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. METHODS: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0-D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14-D21), during 5 days of oral TMS treatment (D22-D26) and after discharge from the hospital up to 6 months later (D27-D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. RESULTS: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3")-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later. CONCLUSIONS: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment. Video Abstract.

Differential effect of two dietary protein sources on time course response of muscle anabolic signaling pathways in normal and insulin dysregulated horses.

The objective of the study was to characterize the temporal changes of phosphorylation patterns of mTOR signaling proteins in response to two dietary protein sources in insulin dysregulated (ID, n = 8) and non-ID (n = 8) horses. Horses were individually housed and fed timothy grass hay and 2 daily concentrate meals so that protein was the first limiting nutrient and the total diet provided 120% of daily DE requirements for maintenance. On sample days, horses randomly received 0.25 g CP/kg BW of a pelleted alfalfa (AP) or commercial protein supplement (PS). Blood samples were collected before and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, and 480 min post feeding and analyzed for plasma glucose, insulin and amino acid (AA) concentrations. Gluteus Medius muscle samples were obtained before and 90, 180, and 300 min after feeding and analyzed for relative abundance of phosphorylated mTOR pathway components using western immunoblot analysis. There was no effect of protein source on postprandial glucose and insulin responses (P ≥ 0.14) but consumption of PS elicited a 2 times larger AUC for essential AA (EAA), greater peak concentrations of EAA and a shorter time to reach peak EAA concentrations compared to AP. Abundance of phosphorylated mTOR (P = 0.08) and rpS6 (P = 0.10) tended to be ~1.5-fold greater after consumption of PS at 90 min compared to AP. Dephosphorylation patterns differed between protein sources and was slower for AP compared to PS. ID horses had a 2 times greater (P = 0.009) AUC and 3 times higher postprandial peak concentrations (P < 0.0001) for insulin compared to non-ID horses after consumption of both treatment pellets, but EAA responses were similar between groups (P = 0.53). Insulin status did not affect rpS6 or mTOR phosphorylation after consumption of either protein source (P ≥ 0.35), but phosphorylated rpS6 abundance was twice as high in ID compared to non-ID horses (P = 0.007). These results suggest that the consumption of higher quality protein sources may result in greater postprandial activation of the mTOR pathway compared to equal amounts of a forage-based protein source. Moreover, ID does not impair postprandial activation of mTOR and rpS6 proteins in horses following a protein-rich meal.

Free Faecal Water: Analysis of Horse Faecal Microbiota and the Impact of Faecal Microbial Transplantation on Symptom Severity.

Free faecal water (FFW) in equines results in pollution of the hindquarters and tail and can also involve clinical signs. Though the cause of FFW is unknown, it was hypothesized that it may involve the gut microbiota. This hypothesis was addressed as follows. First, the faecal prokaryotic community composition of horses suffering from FFW relative to healthy controls (n = 10) was compared. Second, FFW horses were treated with a standardised faecal microbiota transplantation (FMT) protocol (n = 10), followed by assessment of FFW symptom severity and faecal prokaryotic community composition over a follow-up period of 168 days. No significant differences were found in the faecal microbiota composition of FFW horses compared to healthy controls (p > 0.05). Relative to before FMT, FFW symptom severity decreased in affected horses 14 days after FMT (p = 0.02) and remained decreased for the remainder of the study (p < 0.02). However, individual animal responses to FMT varied. FMT had no effect on FFW horse faecal prokaryotic community composition in terms of alpha or beta diversity. Alpha diversity of the donor inocula used in the FMT was always lower than that of the faecal microbiota of the FFW treated horses (p < 0.001). In conclusion, whilst findings indicate FFW horses do not have an altered hindgut microbiota, some horses that received FMT had a temporary alleviation of FFW symptom severity without causing changes in the faecal microbiota. Future studies using controls are now needed to confirm the effectiveness of FMT to treat FFW.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes.

Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes (IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN), DNMTs (DNMT1 and DNMT3B), glucose (SLC2A1), fructose (SLC2A5) and amino acid (SLC7A2) transporters following ET from an HE to a control mare. Expression of amino acid transporters (SLC1A5 and SLC7A1) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Effect of a long-term high-energy diet on cardiovascular parameters in Shetland pony mares.

BACKGROUND: Changes in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown. OBJECTIVES: Determine the order in which EMS-associated changes in cardiovascular parameters arise. ANIMALS: Twenty Shetland pony mares. METHODS: High-energy (HE) diet mares were fed 200% of net energy requirements for 1 (n = 3) or 2 (n = 7) consecutive diet-years, with 17 weeks of hay-only between years. Noninvasive BP measurements and echocardiograms were performed during both years. Resting 24-hour ECGs and measurements of autonomic tone (splenic volume and packed cell volume [PCV]) were performed at the end of diet-year 1. Results were compared to control mares receiving a maintenance diet for 1 (n = 7) or 2 (n = 3) consecutive years. RESULTS: In year 1, HE mares had significantly higher values than control mares for mean relative left ventricular wall thickness (P = .001). After 2 diet-years, mean systolic (P = .003), diastolic (P < .001) and mean arterial BP (P = .001), heart rate (HR; P < .001), and mean left ventricular wall thickness (P = .001) also were significantly increased in HE compared to control mares. No pathological arrhythmias or differences in splenic volume or PCV were detected. CONCLUSIONS AND CLINICAL IMPORTANCE: Ingesting a HE diet first induced minor changes in BP, and progressed to left-sided cardiac hypertrophy in Shetland pony mares. These findings are of interest given the increasing incidence of obesity in horses.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

Pathways regulating equine skeletal muscle protein synthesis respond in a dose-dependent manner to graded levels of protein intake.

Activation of the mechanistic target of rapamycin (mTOR)-controlled anabolic signaling pathways in skeletal muscle of rodents and humans is responsive to the level of dietary protein supply, with maximal activation and rates of protein synthesis achieved with 0.2 to 0.4 g protein/kg body weight (BW). In horses, few data are available on the required level of dietary protein to maximize protein synthesis for maintenance and growth of skeletal muscle. To evaluate the effect of dietary protein level on muscle mTOR pathway activation, five mares received different amounts of a protein supplement that provided 0, 0.06, 0.125, 0.25, or 0.5 g of crude protein (CP)/kg BW per meal in a 5 × 5 Latin square design. On each sample day, horses were fasted overnight and were fed only their protein meal the following morning. A preprandial (0 min) and postprandial (90 min) blood sample was collected and a gluteus medius muscle sample was obtained 90 min after feeding the protein meal. Blood samples were analyzed for glucose, insulin, and amino acid concentrations. Activation of mTOR pathway components (mTOR and ribosomal protein S6 [rpS6]) in the muscle samples was measured by Western immunoblot analysis. Postprandial plasma glucose (P = 0.007) and insulin (P = 0.09) showed a quadratic increase, while total essential amino acid (P < 0.0001) concentrations increased linearly with the graded intake of the protein supplement. Activation of mTOR (P = 0.02) and its downstream target, rpS6 (P = 0.0008), increased quadratically and linearly in relation to the level of protein intake, respectively. Comparisons of individual doses showed no differences (P > 0.05) between the 0.25 and 0.5 g of protein intake for either mTOR or rpS6 activation, indicating that protein synthesis may have reached near maximal capacity around 0.25 g CP/kg BW. This is the first study to show that the activation of muscle protein synthetic pathways in horses is dose-dependent on the level of protein intake. Consumption of a moderate dose of high-quality protein resulted in near maximal muscle mTOR pathway activation in mature, sedentary horses.

Effect of long-term overfeeding of a high-energy diet on glucose tolerance in Shetland pony mares.

BACKGROUND: Overfeeding is associated with obesity and insulin dysregulation (ID), which are both risk factors for equine metabolic syndrome. How chronic overfeeding affects development of these factors is poorly understood. OBJECTIVES: To examine the influence of long-term high-energy diet provision on body condition and ID. ANIMALS: Eleven Shetland pony mares. METHODS: In a 3-phase study, the high-energy group (n = 7) was fed 200% of net energy (NE) requirements (hay; concentrate: 36% sugar and starch, 13% fat) for 24 weeks, followed by 17 weeks hay-only feeding before resuming the high-energy diet (n = 4) for an additional 29 weeks. Mares were weighed weekly. Oral glucose tolerance tests were performed 3 to 4 times per dietary period. Results were compared with those of a control group (phase 1, n = 4; phases 2 and 3, n = 6) that received 100% NE requirements, using a general linear mixed model with post hoc Bonferroni testing. RESULTS: The mean body weight of the high-energy group increased by 27% per high-energy feeding period. During both feeding periods, area under the curve (AUC) for plasma glucose concentration decreased (P < .01), whereas AUC for plasma insulin concentration increased. Mean basal plasma glucose concentration and peak plasma insulin concentrations were higher (P < .05) in the high-energy group than in the control group. CONCLUSION AND CLINICAL IMPORTANCE: Feeding a high-energy diet to healthy nonobese Shetland pony mares led to more efficient glucose metabolism within 5 weeks, followed by significant hyperinsulinemia and obesity. Hyperinsulinemic status was reversed during 17 weeks of hay-only feeding, regardless of body condition, but returned rapidly after restarting the high-energy diet.

Evaluation of equine rectal inoculum as representative of the microbial activities within the horse hindgut using a fully automated in vitro gas production technique system.

The in vitro gas production technique (IVGPT) has been a valuable tool in ruminant nutrition research for decades and has more recently been used in horse nutrition studies to investigate fermentation activities of the equine hindgut though primarily using feces as inoculum. This study was conducted to evaluate the use of equine rectal content in the IVGPT system as a viable inoculum that can be considered representative of the activities throughout the equine hindgut. Additionally, the study was conducted to measure the effects on fermentation kinetics and end-product production using inoculum from horses fed supplemental levels of coated sodium butyrate in an IVGPT system. Eight warmblood horses were fed a diet consisting of haylage (1% DM intake based on ideal body weight [BW]) and a mash concentrate formulated to provide 2.5 g nonstructural carbohydrate (NSC)/kg BW per meal. The diet was intended to create a NSC challenge to the microbial populations of the hindgut. The horses were randomly assigned to treatment or control group and after a 1-wk diet-adaptation period, the treatment group received 0.4 g/kg BW per day of a coated sodium butyrate supplement, while the control group received a placebo (coating only). After a 3-wk treatment period, the animals were sacrificed and digesta from the cecum, left ventral colon, right dorsal colon, and the rectum were collected within 30 min postmortem and used as inocula for the IVGPT trial. Haylage and concentrates fed to the test animals were also used as substrates in vitro. Sodium butyrate supplementation was not significant for gas production parameters or VFA measured suggesting no effect of sodium butyrate supplementation on the extent or kinetics of gas production or microbial end-product production (P ≥ 0.073). Differences in inocula were significant for organic matter corrected cumulative gas production (P = 0.0001), asymptotic gas production of the second phase (A2) (P < 0.0001); and maximal rate of OM degradation of the second phase (Rmax2) (P = 0.002). Inocula had a significant effect on total VFA (P = 0.0002), butyrate (Bu) (P = 0.015), branched chain fatty acids (P < 0.0001), pH (P < 0.0001), and ammonia (NH3) (P = 0.0024). In conclusion, based on observed results from this study, total tract digestibility may be overestimated if using rectal content inoculum to evaluate forage-based feeds in an IVGPT system.