Baselining physiological parameters in three muscles across three equine breeds. What can we learn from the horse?

Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species. Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism. Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters. Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds. Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.

Dynamics of training and acute exercise-induced shifts in muscular glucose transporter (GLUT) 4, 8, and 12 expression in locomotion versus posture muscles in healthy horses.

Important changes in glucose transporter (GLUT) expression should be expected if the glucose influx plays a pivotal role in fuelling or connecting metabolic pathways that are upregulated in response to exercise. The aim was to assess GLUT4, 8, and 12 dynamics in response to training and acute exercise. Methods: Sixteen untrained Standardbred mares (3-4 year) performed an incremental SET at the start and end of 8 weeks harness training. M. pectoralis (PM) and M. vastus lateralis (VL) muscle biopsies were taken before and after each SET, allowing for comparing rest and acute samples in untrained (UT) and trained (T) condition using Western Blot for GLUT quantification and Image Pro v.10 for Blot analysis. Data were normalized against GAPDH. Basal GLUT-levels of PM versus VL were analysed with the Wilcoxon matched-pairs signed rank test. The effect of acute exercise or training was assessed using the Friedman test with a post hoc Dunn's. Results: Basal GLUT4 and GLUT12 protein expression were significantly higher in the VL compared to the PM (PGLUT4 = 0.031 and PGLUT12 = 0.002). Training had no effect on basal GLUT4 expression, neither in the VL (p > 0.9999), nor the PM (p > 0.9999). However, acute exercise in trained condition significantly decreased GLUT4 expression in the VL (p = 0.0148). Neither training nor acute exercise significantly changed total GLUT8 protein expression. Training significantly decreased total GLUT12 protein expression in rest biopsies, only visible in the VL (p = 0.0359). This decrease was even more prominent in the VL after acute exercise in trained condition (PVL = 0.0025). Conclusion: The important changes seen in GLUT12 expression downregulation, both in response to training and acute exercise in the horse, the downregulation of GLUT4 expression after acute exercise in trained condition and the lack of differential shifts in GLUT8 expression in any of the studied conditions, questions the importance of glucose as substrate to fuel training and exercise in healthy horses. These findings encourage to further explore alternative fuels for their involvement in equine muscular energetics.

Relationship between the Cycle Threshold Value (Ct) of a Salmonella spp. qPCR Performed on Feces and Clinical Signs and Outcome in Horses.

The objective of this retrospective study was to evaluate the clinical significance of fecal quantitative real-time polymerase chain reaction (qPCR) Salmonella results when taking the cycle threshold values (Ct) into account. The study included 120 Salmonella qPCR-positive fecal samples obtained from 88 hospitalized horses over a 2-year period. The mean Ct of the qPCR test was evaluated in regard to (1) clinical outcome and (2) systemic inflammatory response syndrome (SIRS) status (no SIRS, moderate SIRS, or severe SIRS) of the sampled horses. An ROC analysis was performed to establish the optimal cut-off Ct values associated with severe SIRS. The mean ± SD Ct value was significantly lower in samples (1) from horses with a fatal issue (27.87 ± 5.15 cycles) than in surviving horses (31.75 ± 3.60 cycles), and (2) from horses with severe SIRS (27.87 ± 2.78 cycles) than from horses with no (32.51 ± 3.59 cycles) or moderate (31.54 ± 3.02 cycles) SIRS. In the ROC analysis, the optimal cut-off value of Ct associated with a severe SIRS was 30.40 cycles, with an AUC value of 0.84 [95% confidence interval 0.76-0.91] and an OR of 0.64 [0.51-0.79]. Results suggest that including the Ct value in the interpretation of fecal qPCR results could improve the diagnostic value of this test for clinical salmonellosis in horses.

Profiling the Aerobic Window of Horses in Response to Training by Means of a Modified Lactate Minimum Speed Test: Flatten the Curve.

There is a great need for objective external training load prescription and performance capacity evaluation in equestrian disciplines. Therefore, reliable standardised exercise tests (SETs) are needed. Classic SETs require maximum intensities with associated risks to deduce training loads from pre-described cut-off values. The lactate minimum speed (LMS) test could be a valuable alternative. Our aim was to compare new performance parameters of a modified LMS-test with those of an incremental SET, to assess the effect of training on LMS-test parameters and curve-shape, and to identify the optimal mathematical approach for LMS-curve parameters. Six untrained standardbred mares (3-4 years) performed a SET and LMS-test at the start and end of the 8-week harness training. The SET-protocol contains 5 increments (4 km/h; 3 min/step). The LMS-test started with a 3-min trot at 36-40 km/h [until blood lactate (BL) > 5 mmol/L] followed by 8 incremental steps (2 km/h; 3 min/step). The maximum lactate steady state estimation (MLSS) entailed >10 km run at the LMS and 110% LMS. The GPS, heartrate (Polar®), and blood lactate (BL) were monitored and plotted. Curve-parameters (R core team, 3.6.0) were (SET) VLa1.5/2/4 and (LMS-test) area under the curve (AUC>/ 0.80), Bland-Altman method, and ordinary least products (OLP) regression analyses were determined for test-correlation and concordance. Training induced a significant increase in VLa1.5/2/4. The width of the AW increased significantly while the AUCLMS and LMS decreased post-training (flattening U-curve). The LMS BL steady-state is reached earlier and maintained longer after training. BLmax was significantly lower for LMS vs. SET. The 40° angular method is the optimal approach. The correlation between LMS and VMLSS was significantly better compared to the SET. The VLa4 is unreliable for equine aerobic capacity assessment. The LMS-test allows more reliable individual performance capacity assessment at lower speed and BL compared to SETs. The LMS-test protocol can be further adapted, especially post-training; however, inducing modest hyperlactatemia prior to the incremental LMS-stages and omitting inclusion of a per-test recovery contributes to its robustness. This LMS-test is a promising tool for the development of tailored training programmes based on the AW, respecting animal welfare.

Comparison of Shifts in Skeletal Muscle Plasticity Parameters in Horses in Three Different Muscles, in Answer to 8 Weeks of Harness Training.

Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all muscle plasticity parameters show the same expression time profile across muscles. This knowledge is important to underpin results of metabolomic studies. Twelve non-competing Standardbred mares were subjected to standardized harness training. Muscle biopsies were taken on a non-training day before and after 8 weeks. Shifts in muscle fiber type composition and muscle fiber cross-sectional area (CSA) were compared in the m. pectoralis, the m. vastus lateralis, and the m. semitendinosus. In the m. vastus lateralis, which showed most pronounced training-induced plasticity, two additional muscle plasticity parameters (capillarization and mitochondrial density) were assessed. In the m. semitendinosus, additionally the mean minimum Feret's diameter was assessed. There was a significant difference in baseline profiles. The m. semitendinosus contained less type I and more type IIX fibers compatible with the most pronounced anaerobic profile. Though no baseline fiber type-specific and overall mean CSA differences could be detected, there was a clear post-training decrease in fiber type specific CSA, most pronounced for the m. vastus lateralis, and this was accompanied by a clear increase in capillary supply. No shifts in mitochondrial density were detected. The m. semitendinosus showed a decrease in fiber type specific CSA of type IIAX fibers and a decrease of type I fiber Feret's diameter as well as mean minimum Feret's diameter. The training-induced increased capillary supply in conjunction with a significant decrease in muscle fiber CSA suggests that the muscular machinery models itself toward an optimal smaller individual muscle fiber structure to receive and process fuels that can be swiftly delivered by the circulatory system. These results are interesting in view of the recently identified important fuel candidates such as branched-chain amino acids, aromatic amino acids, and gut microbiome-related xenobiotics, which need a rapid gut-muscle gateway to reach these fibers and are less challenging for the mitochondrial system. More research is needed with that respect. Results also show important differences between muscle groups with respect to baseline and training-specific modulation.

Effects of Aleurone Supplementation on Glucose-Insulin Metabolism and Gut Microbiome in Untrained Healthy Horses.

Aleurone, a layer of the bran fraction, is deemed to be responsible for the positive health effects associated with the consumption of whole-grain products. Studies on rodents, pigs, and humans report beneficial effects of aleurone in five main areas: the reduction of oxidative stress, immunomodulatory effects, modulation of energy management, digestive health, and the storage of vitamins and minerals. Our study is the first aleurone supplementation study performed in horses. The aim of this study was to investigate the effect of an increase in the dose levels of aleurone on the postprandial glucose-insulin metabolism and the gut microbiome in untrained healthy horses. Seven adult Standardbred horses were supplemented with four different dose levels of aleurone (50, 100, 200, and 400 g/day for 1 week) by using a Latin square model with a 1-week wash out in between doses. On day 7 of each supplementation week, postprandial blood glucose-insulin was measured and fecal samples were collected. 16S ribosomal RNA (rRNA) gene sequencing was performed and QIIME2 software was used for microbiome analysis. Microbial community function was assessed by using the predictive metagenome analysis tool Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and using the Metacyc database of metabolic pathways. The relative abundancies of a pathway were analyzed by using analysis of composition of microbiomes (ANCOM) in R. There was a significant dose-dependent increase in the postprandial time to peak of glucose (p = 0.030), a significant delay in the time to peak of insulin (p = 0.025), and a significant decrease in both the insulin peak level (p = 0.049) and insulin area under the curve (AUC) (p = 0.019) with increasing dose levels of aleurone, with a consideration of 200 g being the lowest significant dose. Alpha diversity and beta diversity of the fecal microbiome showed no significant changes. Aleurone significantly decreased the relative abundance of the genera Roseburia, Shuttleworthia, Anaerostipes, Faecalibacter, and Succinovibrionaceae. The most pronounced changes in the relative abundance at phyla level were seen in Firmicutes and Verrucomicrobia (downregulation) and Bacteroidetes and Spirochaetes (upregulation). The PICRUSt analysis shows that aleurone induces a downregulation of the degradation of L-glutamate and taurine and an upregulation of the three consecutive pathways of the phospholipid membrane synthesis of the Archaea domain. The results of this study suggest a multimodal effect of aleurone on glucose-insulin metabolism, which is most likely to be caused by its effect on feed texture and subsequent digestive processing; and a synergistic effect of individual aleurone components on the glucose-insulin metabolism and microbiome composition and function.

State-of-the-Art Diagnostic Methods to Diagnose Equine Spinal Disorders, With Special Reference to Transcranial Magnetic Stimulation and Transcranial Electrical Stimulation.

Spinal cord disorders are a common problem in equine medicine. However, finding the site of the lesion is challenging for veterinarians because of a lack of sensitive diagnostic methods that can assess neuronal functional integrity in horses. Although medical imaging is frequently applied to help diagnose corticospinal disorders, this approach does not reveal functional information. For the latter, transcranial magnetic stimulation (TMS) and more recently transcranial electrical stimulation (TES) can be useful. These are brain stimulation techniques that create either magnetic or electrical fields passing through the motor cortex, inducing muscular responses, which can be recorded either intramuscularly or extramuscularly by needle or surface electrodes. This permits the evaluation of the functional integrity of the spinal motor tracts and the nerve conduction pathways. The interest in TES in human medicine emerged these last years because unlike TMS, TES tends to bypass the motor cortex of the brain and predominantly relies on direct activation of corticospinal and extrapyramidal axons. Results from human medicine have indicated that TMS and TES recordings are mildly if not at all affected by sedation. Therefore, this technique can be reliably used in human patients under either sedation or full anesthesia to assess functional integrity of the corticospinal and adjunct motor tracts. This opens important new avenues in equine medicine.