Nicotinamide N-methyltransferase inhibition mimics and boosts exercise-mediated improvements in muscle function in aged mice.

Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies.

Sex Differences in Quadriceps Atrophy After Anterior Cruciate Ligament Tear.

BACKGROUND: Female athletes lag behind their male counterparts in recovery from anterior cruciate ligament (ACL) injury. Quadriceps muscle size and strength are crucial factors for regaining function after ACL injury, but little is known about how these metrics vary due to biological sex. HYPOTHESIS: Female patients have reduced vastus lateralis fiber cross-sectional area (CSA) and lower quadriceps strength after ACL injury than male patients. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 4. METHODS: A total of 60 participants with recent ACL tear were evaluated for vastus lateralis muscle fiber CSA, isometric quadriceps peak torque, and quadriceps rate of torque development. Linear mixed models were fit to determine differences across sex and limb for each variable of interest. RESULTS: The female group averaged almost 20% atrophy between limbs (P < 0.01), while the male group averaged just under 4% (P = 0.05). Strength deficits between limbs were comparable between female and male groups. CONCLUSION: Immediately after ACL injury, female patients have greater between-limb differences in muscle fiber CSA but between-limb strength deficits comparable with those of male patients. CLINICAL RELEVANCE: These results indicate that the underpinnings of strength loss differ based on biological sex, and thus individual patients could benefit from a sex-specific treatment approach to ACL injury.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage.

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/-). MnSOD+/- mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

Vitamin D status associates with skeletal muscle loss after anterior cruciate ligament reconstruction.

BACKGROUNDAlthough 25-hydroxyvitamin D [25(OH)D] concentrations of 30 ng/mL or higher are known to reduce injury risk and boost strength, the influence on anterior cruciate ligament reconstruction (ACLR) outcomes remains unexamined. This study aimed to define the vitamin D signaling response to ACLR, assess the relationship between vitamin D status and muscle fiber cross-sectional area (CSA) and bone density outcomes, and discover vitamin D receptor (VDR) targets after ACLR.METHODSTwenty-one young, healthy, physically active participants with recent ACL tears were enrolled (17.8 ± 3.2 years, BMI 26.0 ± 3.5 kg/m2). Data were collected through blood samples, vastus lateralis biopsies, dual energy x-ray bone density measurements, and isokinetic dynamometer measures at baseline, 1 week, 4 months, and 6 months after ACLR. The biopsies facilitated CSA, Western blotting, RNA-seq, and VDR ChIP-seq analyses.RESULTSACLR surgery led to decreased circulating bioactive vitamin D and increased VDR and activating enzyme expression in skeletal muscle 1 week after ACLR. Participants with less than 30 ng/mL 25(OH)D levels (n = 13) displayed more significant quadriceps fiber CSA loss 1 week and 4 months after ACLR than those with 30 ng/mL or higher (n = 8; P < 0.01 for post hoc comparisons; P = 0.041 for time × vitamin D status interaction). RNA-seq and ChIP-seq data integration revealed genes associated with energy metabolism and skeletal muscle recovery, potentially mediating the impact of vitamin D status on ACLR recovery. No difference in bone mineral density losses between groups was observed.CONCLUSIONCorrecting vitamin D status prior to ACLR may aid in preserving skeletal muscle during recovery.FUNDINGNIH grants R01AR072061, R01AR071398-04S1, and K99AR081367.

Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear-Resultant Quadriceps Atrophy.

BACKGROUND: Anterior cruciate ligament (ACL) tear (ACLT) leads to protracted quadriceps muscle atrophy. Protein turnover largely dictates muscle size and is highly responsive to injury and loading. Regulation of quadriceps molecular protein synthetic machinery after ACLT has largely been unexplored, limiting development of targeted therapies. PURPOSE: To define the effect of ACLT on (1) the activation of protein synthetic and catabolic signaling within quadriceps biopsy specimens from human participants and (2) the time course of alterations to protein synthesis and its molecular regulation in a mouse ACL injury model. STUDY DESIGN: Descriptive laboratory study. METHODS: Muscle biopsy specimens were obtained from the ACL-injured and noninjured vastus lateralis of young adult humans after an overnight fast (N = 21; mean ± SD, 19 ± 5 years). Mice had their limbs assigned to ACLT or control, and whole quadriceps were collected 6 hours or 1, 3, or 7 days after injury with puromycin injected before tissue collection for assessment of relative protein synthesis. Muscle fiber size and expression and phosphorylation of protein anabolic and catabolic signaling proteins were assessed at the protein and transcript levels (RNA sequencing). RESULTS: Human quadriceps showed reduced phosphorylation of ribosomal protein S6 (-41%) in the ACL-injured limb (P = .008), in addition to elevated phosphorylation of eukaryotic initiation factor 2α (+98%; P = .006), indicative of depressed protein anabolic signaling in the injured limb. No differences in E3 ubiquitin ligase expression were noted. Protein synthesis was lower at 1 day (P = .01 vs control limb) and 3 days (P = .002 vs control limb) after ACLT in mice. Pathway analyses revealed shared molecular alterations between human and mouse quadriceps after ACLT. CONCLUSION: (1) Global protein synthesis and anabolic signaling deficits occur in the quadriceps in response to ACL injury, without notable changes in measured markers of muscle protein catabolism. (2) Importantly, these deficits occur before the onset of significant atrophy, underscoring the need for early intervention. CLINICAL RELEVANCE: These findings suggest that blunted protein anabolism as opposed to increased catabolism likely mediates quadriceps atrophy after ACL injury. Thus, future interventions should aim to restore muscle protein anabolism rapidly after ACLT.

Muscle-Specific Cellular and Molecular Adaptations to Late-Life Voluntary Concurrent Exercise.

Murine exercise models can provide information on factors that influence muscle adaptability with aging, but few translatable solutions exist. Progressive weighted wheel running (PoWeR) is a simple, voluntary, low-cost, high-volume endurance/resistance exercise approach for training young mice. In the current investigation, aged mice (22-mo-old) underwent a modified version of PoWeR for 8 wk. Muscle functional, cellular, biochemical, transcriptional, and myonuclear DNA methylation analyses provide an encompassing picture of how muscle from aged mice responds to high-volume combined training. Mice run 6-8 km/d, and relative to sedentary mice, PoWeR increases plantarflexor muscle strength. The oxidative soleus of aged mice responds to PoWeR similarly to young mice in every parameter measured in previous work; this includes muscle mass, glycolytic-to-oxidative fiber type transitioning, fiber size, satellite cell frequency, and myonuclear number. The oxidative/glycolytic plantaris adapts according to fiber type, but with modest overall changes in muscle mass. Capillarity increases markedly with PoWeR in both muscles, which may be permissive for adaptability in advanced age. Comparison to published PoWeR RNA-sequencing data in young mice identified conserved regulators of adaptability across age and muscles; this includes Aldh1l1 which associates with muscle vasculature. Agrn and Samd1 gene expression is upregulated after PoWeR simultaneous with a hypomethylated promoter CpG in myonuclear DNA, which could have implications for innervation and capillarization. A promoter CpG in Rbm10 is hypomethylated by late-life exercise in myonuclei, consistent with findings in muscle tissue. PoWeR and the data herein are a resource for uncovering cellular and molecular regulators of muscle adaptation with aging.

Elevated dietary selenium rescues mitochondrial capacity impairment induced by decreased vitamin E intake in young exercising horses.

Maintenance of mitochondrial health, which is supported in part by dietary antioxidants such as selenium (Se) and vitamin E (vitE), is pertinent to optimizing athletic performance. Deficiencies in Se and vitE negatively impact muscle health but mitochondrial adaptations to various levels of dietary Se and vitE are poorly understood. Young Quarter Horses (mean ± SD: 17.6 ± 0.9 mo) undergoing submaximal exercise training were used to test the hypothesis that a proprietary antioxidant blend containing elevated Se yeast (EconomasE, Alltech, Inc., Nicholasville, KY) would improve mitochondrial characteristics compared to Se at current requirements, even with reduced vitE intake. Horses were balanced by age, sex, body weight (BW), and farm of origin and randomly assigned to one of three custom-formulated concentrates fed at 1% BW (dry matter, DM basis) for 12 wk: 1) 100 IU vitE/kg DM and 0.1 mg Se/kg DM (CON, n = 6); 2) no added vitE plus EconomasE to provide 0.1 mg Se/kg DM (ESe1, n = 6); or 3) no added vitE plus EconomasE to provide 0.3 mg Se/kg DM (ESe3, n = 6). Samples collected at week 0 and 12 were analyzed for serum Se and middle gluteal glutathione peroxidase (GPx) and mitochondrial enzyme activities by kinetic colorimetry and mitochondrial capacities by high-resolution respirometry. Data were analyzed using mixed linear models in SAS v9.4 with repeated measures (time) and fixed effects of time, diet, and time × diet; horse(diet) served as a random effect. Serum Se tended to increase in all horses by week 12 (P = 0.08) but was unaffected by diet. Muscle GPx activity remained similar among all horses throughout the duration of the study. Mitochondrial volume density (citrate synthase [CS] activity), integrative function (cytochrome c oxidase [CCO] activity per mg protein), and integrative (per mg tissue) oxidative (P) and electron transfer (E) capacities increased from week 0 to 12 in all horses (P ≤ 0.01). Intrinsic (relative to CS) CCO activity decreased in all horses (P = 0.001), while intrinsic P and E capacities decreased only in ESe1 horses from week 0 to 12 (P ≤ 0.002). These results suggest that feeding EconomasE to provide 0.3 mg Se/kg DM may prevent adverse effects of removing 100 IU dietary vitE/kg DM on mitochondria in young horses. More research is needed to determine optimal dietary Se and vitE levels in performance horses to maximize mitochondrial energy production.

Mitochondria, colloquially referred to as the powerhouses of the cell, are essential for sustained energy production, which is particularly important for athletic performance. During exercise, reactive oxygen species (ROS) are produced as a normal byproduct of muscle contraction. ROS act as critical signaling molecules and are essential to stimulate adaptation to exercise and other stressors. However, if excess ROS are produced and not sequestered by antioxidants, they may damage cellular components such as lipids, proteins, and DNA. Selenium (Se) and vitamin E (vitE) are two primary dietary antioxidants that aid in quenching excess ROS. To evaluate the impact of Se and vitE on mitochondria, three diets differing in Se and vitE levels were provided to lightly exercising young horses for 12 wk. Skeletal muscle mitochondrial capacity was negatively impacted by the reduction of dietary vitE, which was rescued with elevated dietary Se. The results highlight the importance of determining optimal levels of minerals and vitamins in performance horse diets to ensure proper energy production during exercise.

A glitch in the matrix: the pivotal role for extracellular matrix remodeling during muscle hypertrophy.

Multinuclear muscle fibers are the most voluminous cells in skeletal muscle and the primary drivers of growth in response to loading. Outside the muscle fiber, however, is a diversity of mononuclear cell types that reside in the extracellular matrix (ECM). These muscle-resident cells are exercise-responsive and produce the scaffolding for successful myofibrillar growth. Without proper remodeling and maintenance of this ECM scaffolding, the ability to mount an appropriate response to resistance training in adult muscles is severely hindered. Complex cellular choreography takes place in muscles following a loading stimulus. These interactions have been recently revealed by single-cell explorations into muscle adaptation with loading. The intricate ballet of ECM remodeling involves collagen production from fibrogenic cells and ECM modifying signals initiated by satellite cells, immune cells, and the muscle fibers themselves. The acellular collagen-rich ECM is also a mechanical signal-transducer and rich repository of growth factors that may directly influence muscle fiber hypertrophy once liberated. Collectively, high levels of collagen expression, deposition, and turnover characterize a well-trained muscle phenotype. The purpose of this review is to highlight the most recent evidence for how the ECM and its cellular components affect loading-induced muscle hypertrophy. We also address how the muscle fiber may directly take part in ECM remodeling, and whether ECM dynamics are rate limiting for muscle fiber growth.

Late-life exercise mitigates skeletal muscle epigenetic aging.

There are functional benefits to exercise in muscle, even when performed late in life, but the contributions of epigenetic factors to late-life exercise adaptation are poorly defined. Using reduced representation bisulfite sequencing (RRBS), ribosomal DNA (rDNA) and mitochondrial-specific examination of methylation, targeted high-resolution methylation analysis, and DNAge™ epigenetic aging clock analysis with a translatable model of voluntary murine endurance/resistance exercise training (progressive weighted wheel running, PoWeR), we provide evidence that exercise may mitigate epigenetic aging in skeletal muscle. Late-life PoWeR from 22-24 months of age modestly but significantly attenuates an age-associated shift toward promoter hypermethylation. The epigenetic age of muscle from old mice that PoWeR-trained for eight weeks was approximately eight weeks younger than 24-month-old sedentary counterparts, which represents ~8% of the expected murine lifespan. These data provide a molecular basis for exercise as a therapy to attenuate skeletal muscle aging.

Vitamin D Promotes Skeletal Muscle Regeneration and Mitochondrial Health.

Vitamin D is an essential nutrient for the maintenance of skeletal muscle and bone health. The vitamin D receptor (VDR) is present in muscle, as is CYP27B1, the enzyme that hydroxylates 25(OH)D to its active form, 1,25(OH)D. Furthermore, mounting evidence suggests that vitamin D may play an important role during muscle damage and regeneration. Muscle damage is characterized by compromised muscle fiber architecture, disruption of contractile protein integrity, and mitochondrial dysfunction. Muscle regeneration is a complex process that involves restoration of mitochondrial function and activation of satellite cells (SC), the resident skeletal muscle stem cells. VDR expression is strongly upregulated following injury, particularly in central nuclei and SCs in animal models of muscle injury. Mechanistic studies provide some insight into the possible role of vitamin D activity in injured muscle. In vitro and in vivo rodent studies show that vitamin D mitigates reactive oxygen species (ROS) production, augments antioxidant capacity, and prevents oxidative stress, a common antagonist in muscle damage. Additionally, VDR knockdown results in decreased mitochondrial oxidative capacity and ATP production, suggesting that vitamin D is crucial for mitochondrial oxidative phosphorylation capacity; an important driver of muscle regeneration. Vitamin D regulation of mitochondrial health may also have implications for SC activity and self-renewal capacity, which could further affect muscle regeneration. However, the optimal timing, form and dose of vitamin D, as well as the mechanism by which vitamin D contributes to maintenance and restoration of muscle strength following injury, have not been determined. More research is needed to determine mechanistic action of 1,25(OH)D on mitochondria and SCs, as well as how this action manifests following muscle injury in vivo. Moreover, standardization in vitamin D sufficiency cut-points, time-course study of the efficacy of vitamin D administration, and comparison of multiple analogs of vitamin D are necessary to elucidate the potential of vitamin D as a significant contributor to muscle regeneration following injury. Here we will review the contribution of vitamin D to skeletal muscle regeneration following injury.

Complexed trace mineral supplementation alters antioxidant activities and expression in response to trailer stress in yearling horses in training.

To test the hypothesis that complexed trace mineral supplementation would increase antioxidant capacity and decrease muscle oxidative stress and damage in young horses entering an exercise training program, Quarter Horses (mean [Formula: see text] SD; 9.7 ± 0.7 mo) balanced by age, sex, and BW were assigned to receive complexed (CTM; n = 8) or inorganic (INORG; n = 8) trace minerals at -12 week relative to this study. Blood and muscle samples were collected before (week 0) and after 12 week of light exercise training surrounding a 1.5-h trailer stressor. Muscle glutathione peroxidase (GPx) activity was higher for CTM than INORG horses (P ≤ 0.0003) throughout the study. Following both trailer stressors, serum creatine kinase increased (P < 0.0001) and remained elevated through 24 h post-trailering (P < 0.0001). At week 0, muscle malondialdehyde, expression of superoxide dismutase 2, and whole blood GPx activity increased (P [Formula: see text] 0.003) following trailering but trailering did not affect these measures at week 12. Young horses supplemented with CTM had higher muscle GPx activity than horses receiving INORG, but CTM did not affect damage markers following a stressor. Dietary CTM may be useful for improving antioxidant capacity during exercise training in young equine athletes.

Dietary conjugated linoleic acid supplementation alters skeletal muscle mitochondria and antioxidant status in young horses.

Conjugated linoleic acid (CLA) improves oxidative stress and mitochondrial biogenesis in various species but has not been thoroughly investigated in horses. We collected blood and muscle samples from lightly exercising horses before and 6 and 12 wk after receiving either soybean oil (CON; n = 5) or CLA (CLA; n = 5) supplementation. Samples were analyzed for markers of mitochondrial characteristics, antioxidant status, oxidative stress, and muscle damage. Data were analyzed using a linear model with repeated measures. In the triceps brachii (TB), citrate synthase (CS) activity was higher in CON than CLA horses (P = 0.003) but was unaffected by diet in the gluteus medius (GM). Integrative (relative to mg protein) cytochrome c oxidase (CCO) activity was higher in TB than the GM (P < 0.0001), while intrinsic (relative to CS) CCO was lower in the TB than the GM (P = 0.02) and tended to be lower in CON than CLA horses (P = 0.06). Neither CS nor integrative CCO activities were affected by time. In the GM, superoxide dismutase activity tended to increase in CON through week 12 (P = 0.10). Over both muscle groups, glutathione peroxidase activity tended to be higher in CON compared with CLA at week 12 (P = 0.06). Malondialdehyde was higher in the TB than the GM (P = 0.0004) but was unaffected by diet, while serum creatine kinase activity tended to be lower in CLA than CON horses (P = 0.07). These results suggest that CLA supplementation may lead to mitochondrial adaptations and prevent myofiber perturbation in skeletal muscle of young, lightly exercised horses.

Skeletal Muscle Adaptations to Exercise Training in Young and Aged Horses.

In aged humans, low-intensity exercise increases mitochondrial density, function and oxidative capacity, decreases the prevalence of hybrid fibers, and increases lean muscle mass, but these adaptations have not been studied in aged horses. Effects of age and exercise training on muscle fiber type and size, satellite cell abundance, and mitochondrial volume density (citrate synthase activity; CS), function (cytochrome c oxidase activity; CCO), and integrative (per mg tissue) and intrinsic (per unit CS) oxidative capacities were evaluated in skeletal muscle from aged (n = 9; 22 ± 5 yr) and yearling (n = 8; 9.7 ± 0.7 mo) horses. Muscle was collected from the gluteus medius (GM) and triceps brachii at wk 0, 8, and 12 of exercise training. Data were analyzed using linear models with age, training, muscle, and all interactions as fixed effects. At wk 0, aged horses exhibited a lower percentage of type IIx (p = 0.0006) and greater percentage of hybrid IIa/x fibers (p = 0.002) in the GM, less satellite cells per type II fiber (p = 0.03), lesser integrative and intrinsic (p ≤ 0.04) CCO activities, lesser integrative oxidative phosphorylation capacity with complex I (PCI; p = 0.02) and maximal electron transfer system capacity (ECI+II; p = 0.06), and greater intrinsic PCI, ECI+II, and electron transfer system capacity with complex II (ECII; p ≤ 0.05) than young horses. The percentage of type IIx fibers increased (p < 0.0001) and of type IIa/x fibers decreased (p = 0.001) in the GM, and the number of satellite cells per type II fiber increased (p = 0.0006) in aged horses following exercise training. Conversely, the percentage of type IIa/x fibers increased (p ≤ 0.01) and of type IIx fibers decreased (p ≤ 0.002) in young horses. Integrative maximal oxidative capacity (p ≤ 0.02), ECI+II (p ≤ 0.07), and ECII (p = 0.0003) increased for both age groups from wk 0 to 12. Following exercise training, aged horses had a greater percentage of IIx (p ≤ 0.002) and lesser percentage of IIa/x fibers (p ≤ 0.07), and more satellite cells per type II fiber (p = 0.08) than young horses, but sustained lesser integrative and intrinsic CCO activities (p ≤ 0.04) and greater intrinsic PCI, ECI+II, and ECII (p ≤ 0.05). Exercise improved mitochondrial measures in young and aged horses; however, aged horses showed impaired mitochondrial function and differences in adaptation to exercise training.

Temperament influences mitochondrial capacity in skeletal muscle from 8 through 18 mo of age in Brahman heifers.

Temperamental cattle tend to yield carcasses of poorer quality, and Brahman cattle are reportedly more temperamental than non-indicus cattle breeds. A potential link between temperament and product quality may be mitochondrial activity. We hypothesized that mitochondrial measures would be greater in temperamental compared with calm heifers and that the relationships between temperament and mitochondria would persist as heifers age. Serum cortisol and skeletal muscle (longissimus thoracis [LT] and trapezius [TRAP]) mitochondrial profiles and antioxidant activities were quantified from the same calm (n = 6) and temperamental (n = 6) Brahman heifers at 8, 12, and 18 mo of age. Data were analyzed using a mixed model ANOVA in SAS (9.4) with repeated measures. Serum cortisol was greater in temperamental compared with calm heifers throughout the study (P = 0.02). Mitochondrial volume density (citrate synthase [CS] activity) increased over time (P < 0.0001) but was similar between temperament and muscle groups. Mitochondrial function (cytochrome c oxidase activity) was greatest in the temperamental LT at 8 mo of age (P ≤ 0.0006), greatest in the temperamental TRAP at 18 mo of age (P ≤ 0.003), and did not differ by temperament at 12 mo of age. Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI+II), noncoupled electron transfer system capacity (ECI+II), and E with functional complex II only (ECII) were greater in the TRAP than LT for calm heifers at all ages (P ≤ 0.002), but were similar between muscle groups in temperamental heifers. Overall, calm heifers tended to have greater intrinsic (relative to CS activity) PCI and flux control of PCI+II (P ≤ 0.1) than temperamental heifers, indicating greater utilization of complex I paired with greater coupling efficiency in calm heifers. Within the LT, integrative PCI+II was greater (P = 0.05) and ECI+II tended to be greater (P = 0.06) in temperamental compared with calm heifers. From 8- to 18-mo old, glutathione peroxidase (GPx) activity decreased (P < 0.0001) and superoxide dismutase activity increased (P = 0.02), and both were similar between muscle groups. The activity of GPx was greater in temperamental compared with calm heifers at 8 (P = 0.004) but not at 12 or 18 mo of age. These results detail divergent skeletal muscle mitochondrial characteristics of live Brahman heifers according to temperament, which should be further investigated as a potential link between temperament and product quality.

RAPID COMMUNICATION: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses1.

Responses of equine skeletal muscle characteristics to growth and training have been shown to differ between breeds. These differential responses may arise in part because muscle fiber type and mitochondrial density differ between breeds, even in untrained racing-bred horses. However, it is not known when these breed-specific differences manifest. To test the hypothesis that weanling Standardbreds (SB) and Thoroughbreds (TB) would have higher mitochondrial measures than Quarter Horses (QH), gluteus medius samples were collected from SB (mean ± SD; 6.2 ± 1.0 mo; n = 10), TB (6.1 ± 0.5 mo; n = 12), and QH (7.4 ± 0.6 mo; n = 10). Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were assessed as markers of mitochondrial density and function, respectively. Mitochondrial oxidative (P) and electron transport system (E) capacities were assessed by high-resolution respirometry (HRR). Data for CCO and HRR are expressed as integrated (per mg protein and per mg tissue wet weight, respectively) and intrinsic (per unit CS). Data were analyzed using PROC MIXED in SAS v 9.4 with breed as a fixed effect. Mitochondrial density (CS) was higher for SB and TB than QH (P ≤ 0.0007). Mitochondrial function (integrated and intrinsic CCO) was higher in TB and QH than SB (P ≤ 0.01). Integrated CCO was also higher in TB than QH (P < 0.0001). However, SB had higher integrated maximum P (PCI+II) and E (ECI+II) than QH (P ≤ 0.02) and greater integrated and intrinsic complex II-supported E (ECII) than both QH and TB (P ≤ 0.02), whereas TB exhibited higher integrated P with complex I substrates (PCI) than SB and QH (P ≤ 0.003) and higher integrated PCI+II and ECI+II than QH (P ≤ 0.02). In agreement, TB and QH had higher contribution of complex I (CI) to max E than SB (P ≤ 0.001), whereas SB had higher contribution of CII than QH and TB (P ≤ 0.002). Despite having higher mitochondrial density than QH and TB, SB showed lower CCO activity and differences in contribution of complexes to oxidative and electron transport system capacities. Breed differences in mitochondrial parameters are present early in life and should be considered when developing feeding, training, medication, and management practices.

Rapid Communication: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses.

Responses of equine skeletal muscle characteristics to growth and training have been shown to differ between breeds. These differential responses may arise in part because muscle fiber type and mitochondrial density differ between breeds, even in untrained racing-bred horses. However, it is not known when these breed-specific differences manifest. To test the hypothesis that weanling Standardbreds (SB) and Thoroughbreds (TB) would have higher mitochondrial measures than Quarter Horses (QH), gluteus medius samples were collected from SB (mean ± SD; 6.2 ± 1.0 mo; n = 10), TB (6.1 ± 0.5 mo; n = 12), and QH (7.4 ± 0.6 mo; n = 10). Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were assessed as markers of mitochondrial density and function, respectively. Mitochondrial oxidative (P) and electron transport system (E) capacities were assessed by high-resolution respirometry (HRR). Data for CCO and HRR are expressed as integrated (per mg protein and per mg tissue wet weight, respectively) and intrinsic (per unit CS). Data were analyzed using PROC MIXED in SAS v 9.4 with breed as a fixed effect. Mitochondrial density (CS) was higher for SB and TB than QH (P ≤ 0.0007). Mitochondrial function (integrated and intrinsic CCO) was higher in TB and QH than SB (P ≤ 0.01). Integrated CCO was also higher in TB than QH (P < 0.0001). However, SB had higher integrated maximum P (PCI+II) and E (ECI+II) than QH (P ≤ 0.02) and greater integrated and intrinsic complex II-supported E (ECII) than both QH and TB (P ≤ 0.02), while TB exhibited higher integrated P with complex I substrates (PCI) than SB and QH (P ≤ 0.003) and higher integrated PCI+II and ECI+II than QH (P ≤ 0.02). In agreement, TB and QH had higher contribution of complex I (CI) to max E than SB (P ≤ 0.001), while SB had higher contribution of CII than QH and TB (P ≤ 0.002). Despite having higher mitochondrial density than QH and TB, SB showed lower CCO activity and differences in contribution of complexes to oxidative and electron transport system capacities. Breed differences in mitochondrial parameters are present early in life and should be considered when developing feeding, training, medication, and management practices.

Evaluating equine feeding behavior utilizing GrowSafe Systems: a pilot study.

Equine research and management is limited to single-housing systems if individual animal intake is to be precisely recorded. Even then, dry forage intake is difficult to quantify accurately due to stomping or mixing hay with fecal matter and bedding. In cattle management, GrowSafe Systems (GrowSafe) is a commonly used tool to closely monitor individual animal feeding data using radio frequency identification (RFID) tag technology. Animals are equipped with a unique RFID tag that is read by the feed bunks each time the animal lowers its head into the bunk to consume feed. The objectives of this pilot study were 1) to test the feasibility of use of the GrowSafe system with horses by measuring intake of dry hay and 2) to characterize feeding behaviors of horses in an individually housed (without competition) or group-housed (with competition) setting. To test the hypothesis that horses would consume more hay when individually (NOCOMP) compared to group-housed (COMP), 10 mature Quarter Horses (14 ± 1.5 yr) were placed in one of four pens containing GrowSafe feed bunks in a 4-wk crossover design consisting of two 2-wk treatment periods. Pen 1 contained five horses with access to two GrowSafe bunks (Period 1: n = 4 mares, n = 1 gelding; Period 2: n = 5 geldings); pens 2, 3, and 4 contained one horse each with access to one bunk. Horses were individually fed 0.25% body weight (BW; dry matter [DM] basis) of a commercial concentrate once per day and were allowed Coastal bermudagrass hay in the GrowSafe bunks ad libitum. Although five horses were used in the group-housed (COMP) pen to more closely mimic a true group environment, only data from horses that experienced both housing systems (n = 3 mares and n = 3 geldings) were used for statistical analyses. Hourly (P = 0.008) and daily (P = 0.003) durations of hay feeding were higher for NOCOMP compared to COMP horses, and total daily intake (g DM/kg BW) of NOCOMP horses tended to be greater (P = 0.09) than COMP horses. Conversely, eating rate (g DM/kg BW/min) was greater (P = 0.04) for COMP compared to NOCOMP mares but was unaffected by housing in geldings. The GrowSafe system may provide an opportunity for efficient and effective monitoring of individual horse feed intake and feeding behavior in group-housing situations in horses.

Erratum: Evaluating equine feeding behavior utilizing GrowSafe Systems: a pilot study.

[This corrects the article DOI: 10.1093/tas/txz002.].