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Additional file 5. Differentially abundant ASVs grouped by family at different time points during the study (before vs. after transportation; before vs. after hospitalisation for one week without antimicrobial treatment; before vs. after five days of treatment with TMS; the start vs. the end of the study).

Additional file 2. Relative abundance of specified phyla over time at the level of the individual ponies.

Additional file 4. Relative abundance of bacterial families of the Bacteroidetes and Firmicutes phylum in faecal samples collected from Welsh ponies at the farm, during hospitalisation without treatment, during hospitalisation and treatment with TMS and after discharge from the hospital with six months follow-up.

Simple Summary In pregnant individuals, maternal overnutrition is associated with disturbances in the expression of specific genes and nutrient transporters in the early embryo, 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 several genes, nutrient transporters and DNA methyltransferases (DNMTs; play an important role in regulating gene expression) were determined in extra-embryonic membranes after recovery on day 28 of gestation. The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after embryo removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven embryos were assessed. Maternal overfeeding resulted in elevated expression of several genes, DNMTs and nutrient transporters following embryo transfer from an HE to a control mare. The expression of two amino acid transporters was also elevated in the endometrium after embryo transfer from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in day-seven embryos, or glucose uptake by membranes of day-28 embryos. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life. Abstract 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.

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

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.

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

Correct and well-balanced nutrition of the horse will improve wellbeing, health and performance. Formulation of well-balanced diets depends on accurate determination of the energy value of any feedstuff, as well as of the total diet. The energetic value of feeds is closely related to their organic matter digestibility (OMD) and this assumption is the underlying basis of feed evaluation systems. Accurate information on OMD of feeds for animals is obtained by conducting in vivo digestibility experiments. In a digestibility trial, the feed under study is given to the animal in known amounts and faecal output is measured. Then, OMD is estimated by subtracting the total amount of organic matter (OM) contained in the faeces from the total amount of OM contained in the feed intake. Therefore, accurate determination of feed intake and total collection of faeces are necessary to determine reliable in vivo digestibility of nutrients in equines. The gold standard for the determination of OMD of feedstuffs is the total collection of faeces, which is time consuming, laborious, requires quantitative separation of urine and faeces and cannot be used with grazing or working horses. Moreover, animals are hindered to express their species-specific behavior due to the collection procedure. Refinement of digestibility experiments, by means of shortening of length of confinement and alternative procedures for total faeces collection are preferred for obtaining equine-specific OMD values. The marker method is suggested as an alternative procedure for indirect quantitation of nutrient digestibility in horses. This method eliminates quantitative collection of the faeces and would enable periodic collection of a representative sample of faeces or even allow grab sampling. The availability of an appropriate marker would minimize interference with animal behavior patterns and might be a potential strategy in order to make a reliable estimate faecal excretion or nutrient digestibility. In equine digestibility studies, acid-insoluble ash (AIA), acid detergent lignin (ADL) as internal markers and chromic oxide (Cr2O3) as external marker have been widely used in both grazing and stabled horses. However, these markers do not seem to reflect the digestibility of the feed accurately. Moreover, the use of Cr2O3 as a feed additive is prohibited due to its potential carcinogenicity. Titanium dioxide (TiO2) is presented as a promising alternative to estimate faecal output or nutrient digestibility in different animal species. Although, in horses TiO2 has not been evaluated or validated as a marker yet. The studies reported in this thesis indicate that TiO2 can be used as a digestibility marker as it provides an accurate estimate of faecal excretion and subsequently OMD in equines. In addition, titanium dioxide appeared to be more accurate in its prediction than Cr2O3 and is not systematically affected by the type of forages, the forage-to-concentrate ratio or exercise. The application of TiO2 as digestibility marker, as described in this thesis, can lead to a standardized procedure for obtaining accurate equine-specific values on OMD. This facilitates future exchange of digestibility values of feedstuffs between countries and/or feed evaluation systems.