1 Repeated muscle collections induce few physiological changes in mature sedentary horses

Abstract

Repeated skeletal muscle collections are often used to determine tissue-specific responses to interventions or stressors but little is known about the physiological impacts of the collections themselves. Twenty-six mature sedentary horses (8 geldings, 18 mares; mean ± SD 9.5 ± 3.5y) were used to test the hypothesis that repeated muscle collections would not impact mitochondrial function or markers of inflammation and muscle damage. Horses were stratified by age and sex and randomly assigned to have gluteus medius samples collected at: 1) 0 and 24 h (n = 7); 2) 0 and 6 h (n = 6); 3) 0, 6, and 12 h (n = 7); or 4) 0, 6, 12, and 24 h (n = 6). Blood was collected from all horses every 6 h for 72 h total, starting 24 h before the first muscle collection. Muscle mitochondrial oxidative phosphorylation and electron transfer (E) capacities were determined via high-resolution respirometry, and mitochondrial volume density and function by citrate synthase (CS) and cytochrome c oxidase activities. Muscle and plasma concentrations of interleukin (IL)- 4, IL-8, IL-10, and tumor necrosis factor (TNF)α were quantified, as well as serum amyloid A (SAA) and cortisol concentrations and creatine kinase (CK) activity. Data were analyzed using linear models in SAS with time as a repeated measure, horse as the subject, time as a fixed effect, and sex when significant. For blood parameters, additional fixed effects includedcollection group and the time × group interaction. Integrative (per mg tissue) E supported by complex II decreased from 0 to 6 h (P = 0.004) but returned to baseline by 12 h post-initial muscle collection. Intrinsic (per unit CS) Leak was greater at 6 than at 0 and 12 h (P < 0.03) but did not differ from 24 h. Activity of CS was greater at 0 than 12 and 24 h (P ≤ 0.02). Regardless of group, serum CK activity was similar from −24 through 0 h but increased in all horses at 6 h and remained elevated through 48 h, peaking at 24 and 30 h post-initial muscle collection (P < 0.05). Geldings had greater serum CK activity and plasma IL-8 concentrations than mares (P ≤ 0.006). Serum cortisol concentrations displayed a circadian pattern throughout the 72-h collection period (P < 0.0001) but were not impacted by tissue collection group. Overall, horses in group 2 (0 and 6 h) had greater SAA concentrations than all other groups (P < 0.006). No other variable was impacted by repeated tissue collections. In unstressed, mature horses, repeated gluteal muscle collections did not profoundly impact local and systemic markers of mitochondrial function, select cytokines, and stress indicators; however, serum CK should be interpreted with caution in studies employing repeated tissue collections.