Magnetic resonance imaging shows spinal curvature in Chinook salmon (Oncorhynchus tshawytscha) is associated with chronic inflammation of peri-vertebral soft tissues.

Chinook salmon (Oncorhynchus tshawytscha) farmed in New Zealand are known to develop abnormal spinal curvature late in seawater production. Its cause is presently unknown, but there is evidence to suggest a neuromuscular pathology. Using magnetic resonance imaging (MRI), we evaluated the relationship between soft tissue pathology and spinal curvature in farmed Chinook salmon. Regions of interest (ROIs) presenting as pathologic MRI signal hyper-intensity were identified from scans of 24 harvest-sized individuals: 13 with radiographically-detectable spinal curvature and 11 without. ROIs were excised from individuals using anatomical landmarks as reference points and histologically analysed. Pathologic MRI signal was observed more frequently in individuals with radiographic curvature (92%, n = 12) than those without (18%, n = 2), was localized to the peri-vertebral connective tissues and musculature, and presented as three forms: inflammation, fibrosis, or both. These pathologies are consistent with a chronic inflammatory process, such as that observed during recovery from a soft tissue injury, and suggest spinal curvature in farmed Chinook salmon may be associated with damage to and/or compromised integrity of the peri-vertebral soft tissues. Future research to ascertain the contributing factors is required.

Voluntary physical activity in early life attenuates markers of fatty liver disease in adult male rats fed a high-fat diet.

Paediatric fatty liver disease (FLD) can develop into steatohepatitis, cirrhosis and hepatocellular carcinoma in adulthood. We assessed if early life physical exercise reduced the effects of high-fat (HF) diet-induced steatosis. Male HF-fed rats with access to a running wheel from weaning until day (D)60 (early exercise) or from D67 to D120 (late exercise) were compared with control HF- or chow-fed groups with no wheel. At D63 and D120, liver histopathology (Kleiner score), type I collagen and plasma enzymes were assessed. At D63, early life activity significantly reduced histopathology scores (total, portal inflammation, steatosis, ballooning, but not lobular inflammation or fibrosis) and the number of rats affected. At D120, HF control scores were higher than in chow-fed controls, but the effect of activity was selective: early exercise reduced portal inflammation, steatosis, ballooning and fibrosis, but late activity affected only portal inflammation and ballooning. The chow-fed portal inflammation score was significantly less than all HF groups, but lobular inflammation was lower in the HF control group only. The fibrosis score in the HF early exercise and control chow group were lower than in the late exercise and sedentary HF groups, indicating that early life exercise was more effective than when activity was introduced later in life. Plasma biomarkers showed minor between-group differences. The retained effect on liver histopathology rat at D120 after only early life exposure activity suggests that timing of introduction of exercise is critical in reducing FLD scores and prevalence in children, young adults and possibly into adulthood.

Toxicity of oxidized fish oil in pregnancy: a dose-response study in rats.

Fish oil (FO) supplements are consumed during pregnancy to increase dietary omega-3. However, FO is often oxidized past recommended limits. In rats, a large dose of highly oxidized FO substantially increased newborn mortality, but the effects of human-relevant doses of less oxidized oil are unknown. A dose-response study in rats was conducted to estimate the safe level of oxidation during pregnancy. Sprague-Dawley rat dams were mated, then individually housed and provided with a gel treatment on each day of pregnancy. Treatment groups differed only in the FO content of the gel; control (no oil), PV5, PV10, and PV40 [0.05 mL of FO oxidized to a peroxide value (PV) of 5, 10, or 40 meq/kg], or PV40(1 mL) (1 mL of PV40). A subset of dams was culled on gestational day 20 to enable sampling, and the remainder were allowed to give birth. Newborn mortality was recorded. Offspring were sampled on postnatal days 2 and 21, and dams on day 21. There were no signs of unwellness during pregnancy. However, there was markedly increased neonatal mortality affecting the PV40(1 mL) (12.8%) and PV40 (6.3%) groups, but not the control, PV5, or PV10 groups (1%-1.4%). Dietary-oxidized FO altered the expression of placental genes involved in antioxidant pathways and the production of free radicals. Highly oxidized FO was toxic in rat pregnancy leading to a marked increase in mortality even at a human-relevant dose. We observed no toxic effects of FOs with PV ≤10 meq/kg, suggesting that this is an appropriate maximum limit.

Microstructural changes in cartilage and bone related to repetitive overloading in an equine athlete model.

The palmar aspect of the third metacarpal (MC3) condyle of equine athletes is known to be subjected to repetitive overloading that can lead to the accumulation of joint tissue damage, degeneration, and stress fractures, some of which result in catastrophic failure. However, there is still a need to understand at a detailed microstructural level how this damage progresses in the context of the wider joint tissue complex, i.e. the articular surface, the hyaline and calcified cartilage, and the subchondral bone. MC3 bones from non-fractured joints were obtained from the right forelimbs of 16 Thoroughbred racehorses varying in age between 3 and 8 years, with documented histories of active race training. Detailed microstructural analysis of two clinically important sites, the parasagittal grooves and the mid-condylar regions, identified extensive levels of microdamage in the calcified cartilage and subchondral bone concealed beneath outwardly intact hyaline cartilage. The study shows a progression in microdamage severity, commencing with mild hard-tissue microcracking in younger animals and escalating to severe subchondral bone collapse and lesion formation in the hyaline cartilage with increasing age and thus athletic activity. The presence of a clearly distinguishable fibrous tissue layer at the articular surface immediately above sites of severe subchondral collapse suggested a limited reparative response in the hyaline cartilage.

Innovative approach to investigating the microstructure of calcified tissues using specular reflectance Fourier transform-infrared microspectroscopy and discriminant analysis.

Although bone fracture has become a serious global health issue, current clinical assessments of fracture risk based on bone mineral density are unable to accurately predict whether an individual is likely to suffer a fracture. There is increasing recognition that the chemical structure and composition, or microstructure, of mineralized tissues has an important role to play in determining the fracture resistance of bone. The objective of this preliminary study was to evaluate the use of specular reflectance Fourier transform infrared (SR FT-IR) microspectroscopy in conjunction with discriminant analysis as an innovative technique for providing future insights into the origins of orthopedic abnormalities. The impetus for this approach was that SR FT-IR microspectroscopy would offer several advantages over conventional transmission methods. Bone samples were obtained from young racehorses at known fracture predilection sites and spectra were successfully obtained from calcified cartilage and subchondral bone for the first time. By applying discriminant analysis to the spectral data set in biologically relevant regions, microstructural differences between groups of individuals were found to be related to features associated with both the mineral and organic components of the bone. The preliminary findings also suggest that differences in bone microstructure may exist between healthy individuals of the same age, raising important questions around the normal limits of individual variation and whether individuals may be predisposed to later fracture as a result of detrimental microstructural changes during early growth and development.

Fish oil supplementation of rats fed a high fat diet during pregnancy improves offspring insulin sensitivity.

Introduction: In rats, a maternal high-fat diet (HFD) leads to adverse metabolic changes in the adult offspring, similar to the children of mothers with obesity during pregnancy. Supplementation with a high dose of fish oil (FO) to pregnant rats fed a HFD has been shown to prevent the development of insulin resistance in adult offspring. However, the effects of supplementation at a translationally relevant dose remain unknown. Aim: To determine whether supplementation with a human-relevant dose of FO to pregnant rats can prevent the long-term adverse metabolic and cardiovascular effects of a maternal HFD on adult offspring. Methods: Female rats (N = 100, 90 days of age) were assigned to HFD (45% kcal from fat) or control diet (CD) for 14 days prior to mating and throughout pregnancy and lactation. Following mating, dams received a gel containing 0.05 ml of FO (human equivalent 2-3 ml) or a control gel on each day of pregnancy. This produced 4 groups, CD with control gel, CD with FO gel, HFD with control gel and HFD with FO gel. Plasma and tissue samples were collected at day 20 of pregnancy and postnatal day 2, 21, and 100. Adult offspring were assessed for insulin sensitivity, blood pressure, DXA scan, and 2D echocardiography. Results: There was an interaction between maternal diet and FO supplementation on insulin sensitivity (p = 0.005) and cardiac function (p < 0.01). A maternal HFD resulted in impaired insulin sensitivity in the adult offspring (p = 0.005 males, p = 0.001 females). FO supplementation in the context of a maternal HFD prevented the reduction in insulin sensitivity in offspring (p = 0.05 males, p = 0.0001 females). However, in dams consuming CD, FO supplementation led to impaired insulin sensitivity (p = 0.02 males, p = 0.001 females), greater body weight and reduced cardiac ejection fraction. Conclusion: The effects of a human-relevant dose of maternal FO on offspring outcomes were dependent on the maternal diet, so that FO was beneficial to the offspring if the mother consumed a HFD, but deleterious if the mother consumed a control diet. This study suggests that supplementation with FO should be targeted to women expected to have abnormalities of metabolism such as those with overweight and obesity.

Voluntary exercise in pregnant rats positively influences fetal growth without initiating a maternal physiological stress response.

The effects of increased physical activity during pregnancy on the health of the offspring in later life are unknown. Research in this field requires an animal model of exercise during pregnancy that is sufficiently strenuous to cause an effect but does not elicit a stress response. Previously, we demonstrated that two models of voluntary exercise in the nonpregnant rat, tower climbing and rising to an erect bipedal stance (squat), cause bone modeling without elevating the stress hormone corticosterone. In this study, these same models were applied to pregnant rats. Gravid Wistar rats were randomly divided into three groups: control, tower climbing, and squat exercise. The rats exercised throughout pregnancy and were killed at day 19. Maternal stress was assessed by fecal corticosterone measurement. Maternal bone and soft tissue responses to exercise were assessed by peripheral quantitative computed tomography and dual-energy X-ray absorptiometry. Maternal weight gain during the first 19 days of pregnancy was less in exercised than in nonexercised pregnant control rats. Fecal corticosterone levels did not differ between the three maternal groups. The fetuses responded to maternal exercise in a uterine position-dependent manner. Mid-uterine horn fetuses from the squat exercise group were heavier (P < 0.0001) and longer (P < 0.0001) and had a greater placental weight (P = 0.001) than those from control rats. Fetuses from tower-climbing dams were longer (P < 0.0001) and had heavier placentas (P = 0.01) than those from control rats, but fetal weight did not differ from controls. These models of voluntary exercise in the rat may be useful for future studies of the effects of exercise during pregnancy on the developmental origins of health and disease.

Short-term voluntary exercise in the rat causes bone modeling without initiating a physiological stress response.

Recent research has revealed a neuroendocrine connection between the skeleton and metabolism. Exercise alters both bone modeling and energy balance and may be useful in further developing our understanding of this complex interplay. However, research in this field requires an animal model of exercise that does not cause a physiological stress response in the exercised subjects. In this study, we develop a model of short-term voluntary exercise in the female rat that causes bone modeling without causing stress. Rats were randomly assigned to one of three age-matched groups: control, tower climbing, and squat exercise (rising to an erect bipedal stance). Exercise for 21 days resulted in bone modeling as assessed by peripheral quantitative computed tomography. Fecal corticosterone output was used to assess physiological stress at three time points during the study (preexercise, early exercise, and late in the exercise period). There were no differences in fecal corticosterone levels between groups or time points. This model of voluntary exercise in the rat will be useful for future studies of the influence of exercise on the relationship between skeletal and metabolic health and may be appropriate for investigation of the developmental origins of those effects.

Acute whole-body vibration elicits post-activation potentiation.

Whole-body vibration (WBV) leads to a rapid increase in intra-muscular temperature and enhances muscle power. The power-enhancing effects by WBV can, at least in part, be explained by intra-muscular temperature. However, this does not exclude possible neural effects of WBV occurring at the spinal level. The aim of this study was to examine if muscle twitch and patellar reflex properties were simultaneously potentiated from an acute bout of WBV in a static squat position. Six male and six female athletes performed three interventions for 5 min, static squat with WBV (WBV+, 26 Hz), static squat without WBV (WBV-) and stationary cycling (CYCL, 70 W). Transcutaneous muscle stimulation consisting of a single 200 micros pulse and three patellar tendon taps were administered prior to and then 90 s, 5, 10 min post-intervention. Ninety-seconds after WBV+ muscle twitch peak force (PF) and rate of force development (RFD) were significantly higher (P < 0.01) compared to WBV- and CYCL. However the patellar tendon reflex was not potentiated. An acute continuous bout of WBV caused a post-activation potentiation (PAP) of muscle twitch potentiation (TP) compared to WBV- and CYCL indicating that a greater myogenic response was evident compared to a neural-mediated effect of a reflex potentiation (RP).

Effects of early exercise on metacarpophalangeal joints in horses.

OBJECTIVE: To determine the effects of exercise at an early age on tissues in the metacarpophalangeal joints of horses. ANIMALS: Twelve 18-month-old horses. PROCEDURES: All horses were pasture reared, but 6 horses had additional exercise starting at 3 weeks of age until 18 months of age. At that time, computed tomography, articular cartilage metabolism evaluation, and histologic assessments of synovial membrane, articular cartilage, and subchondral bone were performed. RESULTS: Exercised horses had fewer gross lesions, less articular cartilage matrix staining in the dorsal aspect of the condyle, greater bone fraction in the dorsolateral aspect of the condyle, and higher bone formation rate, compared with nonexercised horses. CONCLUSIONS AND CLINICAL RELEVANCE: Exercise at a young age may be protective to joints, although more research is needed to characterize changes in articular cartilage matrix. Results suggested that exercise can be safely imposed at an early age.

Changes in collagen fibril network organization and proteoglycan distribution in equine articular cartilage during maturation and growth.

The aim of this study was to record growth-related changes in collagen network organization and proteoglycan distribution in intermittently peak-loaded and continuously lower-level-loaded articular cartilage. Cartilage from the proximal phalangeal bone of the equine metacarpophalangeal joint at birth, at 5, 11 and 18 months, and at 6-10 years of age was collected from two sites. Site 1, at the joint margin, is unloaded at slow gaits but is subjected to high-intensity loading during athletic activity; site 2 is a continuously but less intensively loaded site in the centre of the joint. The degree of collagen parallelism was determined with quantitative polarized light microscopy and the parallelism index for collagen fibrils was computed from the cartilage surface to the osteochondral junction. Concurrent changes in the proteoglycan distribution were quantified with digital densitometry. We found that the parallelism index increased significantly with age (up to 90%). At birth, site 2 exhibited a more organized collagen network than site 1. In adult horses this situation was reversed. The superficial and intermediate zones exhibited the greatest reorganization of collagen. Site 1 had a higher proteoglycan content than site 2 at birth but here too the situation was reversed in adult horses. We conclude that large changes in joint loading during growth and maturation in the period from birth to adulthood profoundly affect the architecture of the collagen network in equine cartilage. In addition, the distribution and content of proteoglycans are modified significantly by altered joint use. Intermittent peak-loading with shear seems to induce higher collagen parallelism and a lower proteoglycan content in cartilage than more constant weight-bearing. Therefore, we hypothesize that the formation of mature articular cartilage with a highly parallel collagen network and relatively low proteoglycan content in the peak-loaded area of a joint is needed to withstand intermittent stress and shear, whereas a constantly weight-bearing joint area benefits from lower collagen parallelism and a higher proteoglycan content.

The pathobiology of exercise-induced superficial digital flexor tendon injury in Thoroughbred racehorses.

Despite the high incidence of superficial digital flexor tendon (SDFT) injury in racehorses, the pathobiology of the condition is not clearly defined. The SDFT improves locomotor efficiency by storing elastic energy, but as a result it has low mechanical safety margins. As with the Achilles tendon in humans, rupture during athletic activity often follows accumulation of exercise and age-induced degenerative change that is not repaired by tenocytes. There is limited understanding of tenocyte biology and pathology, including responses to high mechanical strains and core temperatures during exercise. Unfortunately, much of the current information on SDFT pathology is derived from studies of collagenase-induced injury, which is a controversial model. Following rupture the overlapping phases of reactive inflammation, proliferation, remodelling and maturation do not necessarily reconstitute normal structure and function, resulting in long-term persistence of scar tissue and high re-injury rates. Tissue engineering approaches are likely to be applicable to SDFT lesions, but will require significant advances in cell biology research.

Influence of early conditioning exercise on the development of gross cartilage defects and swelling behavior of cartilage extracellular matrix in the equine midcarpal joint.

OBJECTIVE: To investigate the influence of early conditioning exercise on the development of gross cartilage defects and swelling behavior of cartilage extracellular matrix (ECM) in the midcarpal joint of horses. ANIMALS: 12 Thoroughbreds. PROCEDURES: 6 horses underwent early conditioning exercise from birth to 18 months of age (CONDEX group), and 6 horses were used as control animals (PASTEX group). The horses were euthanized at 18 months of age, and the midcarpal joints were harvested. Gross defects of the cartilage surface were classified and mapped. Opposing surfaces of the third and radial carpal bones were used to quantify swelling behavior of the cartilage ECM. RESULTS: A wide range of gross defects was detected in the cartilage on the opposing surfaces of the bones of the midcarpal joint; however, there was no significant difference between the CONDEX and PASTEX groups. Similarly, no significant difference in swelling behavior of the cartilage ECM was evident between the CONDEX and PASTEX groups. CONCLUSIONS AND CLINICAL RELEVANCE: In the study reported here, we did not detect negative influences of early conditioning exercise on the prevalence of gross defects in cartilage of the midcarpal joint or the quality of the cartilage ECM as defined by swelling behavior. These results suggested that early conditioning exercise may be used without negative consequences for the midcarpal joint and the cartilage ECM of the third and radial carpal bones.

High resolution microscopic survey of third metacarpal articular calcified cartilage and subchondral bone in the juvenile horse: possible implications in chondro-osseous disease.

The aim was to survey articular calcified cartilage (ACC) and subchondral bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of young horses with minimal or no signs of musculo-skeletal abnormality. Back-scattered electron scanning electron microscopy (BSE SEM) was conducted on polymethyl methacrylate-embedded mediolateral slices and macerated wedges of the right distal Mc3 from seven each of trained and untrained 2-year-old Thoroughbred horses. Furrows or grooves visible to the naked eye in the mineralizing front (MF) of ACC are the commonest "lesion" and are most common in the palmar portions of the medial and lateral condylar grooves. Cracks running predominantly in the parasagittal plane that infill with hypercalcified matrix are found in the same domain. Common to all these defects are deficiencies or absence of the ACC MF. Other anomalies include local excrescences or depressed areas of the MF. More important condylar lesions show displaced fragmented hypermineralized ACC with underlying excess resorption in the bone domain, leaving a thin ACC layer with cavernous space beneath it. The fragments may dislodge and displace to the joint space. Obvious although small lesions are present in horses that have undertaken little or no training. The nature and sites of the lesions indicate that they are possibly the earliest morphological evidence of changes that may lead to specific joint abnormalities. The lesions appear unlikely to be solely due to functional traumatic forces, and developmental influences are likely to be important in their initiation.

Effects of exercise on chondrocyte viability and subchondral bone sclerosis in the distal third metacarpal and metatarsal bones of young horses.

The objective was to determine the effects of early exercise on the articular cartilage and subchondral bone at specific sites of the distal third metacarpal and metatarsal bones of 12 young Thoroughbred horses allowed free choice exercise at pasture. Six of the horses had additional controlled exercise 5 days per week from mean age of 21+/-20 days of age (range: 3-83 days) until 17.1 months of age. Confocal laser scanning microscopy was used to quantify viable and non-viable chondrocytes. Proteoglycan scoring and modified Mankin scoring was performed and subchondral bone mineral density measured by computed tomography. The number of viable chondrocytes was significantly greater in the conditioned group, which also had a higher Safranin O/Fast Green (SOFG) score than did the group which could exercise only at pasture. There was no difference in mean bone mineral density between groups, nor was there relationship between subchondral bone mineral density and chondrocyte viability. The apparent beneficial effects of early conditioning exercise may support the use of exercise to optimise development of articular cartilage in young individuals.

Future tools for early diagnosis and monitoring of musculoskeletal injury: biomarkers and CT.

This article provides an overview of two relatively new techniques that can be used for the early detection of musculoskeletal injury: biochemical markers and CT. The emphasis in the biomarker section is on the early detection of joint disorders because these are most important clinically and most of the research has been conducted in this area. In the section on CT, bone is the target tissue.

Statistical modeling of the equine third metacarpal bone incorporating morphology and bone mineral density.

The objective of this study was to describe the three-dimensional shape and subchondral bone mineral density (BMD) variation of the equine distal third metacarpal bone (MC3) using a statistical shape model. The association between form and function builds upon previous two-dimensional observations of MC3 epiphyseal structure. It was expected that the main source of variation would be an increase in overall MC3 bone size, correlated to an increase in subchondral BMD. Geometry and bone mineral density was obtained from CT image data of 40 healthy Thoroughbred horses. This was used to create a statistical shape model, in which the first ten components described 75% of the variation in geometry and BMD. The first principal component described an increase in overall size of the MC3 distal epiphysis, coupled with higher BMD on the disto-palmar and dorso-proximal surfaces. The second component was qualitatively described as an increased convexity of the sagittal ridge at the dorsal junction of the epiphysis and the metaphysis, coupled to increased BMD in that region. The third component showed an increase in lateral condylar surface area relative to medial condylar area. As the condyle reduced in relative surface area, the BMD at both dorsal condyles increased. The statistical shape analysis produced a compact description of 3-D shape and sub-chondral bone mineral density variation for the third metacarpal bone. This study uniquely illustrates the shape variations in a sample population of MC3 bones, and the corresponding changes in subchondral BMD.

Mechanisms of bone response to injury.

Bone, despite its relatively inert appearance, is a tissue that is capable of adapting to its environment. Wolff's law, first described in the 19th century, describes the ability of bone to change structure depending on the mechanical forces applied to it. The mechanostat model extended this principle and suggested that the amount of strain a bone detects depends on bone strength and the amount of muscle force applied to the bone. Experimental studies have found that low-magnitude, high-frequency mechanical loading is considered to be the most effective at increasing bone formation. The osteocyte is considered to be the master regulator of the bone response to mechanical loading. Deformation of bone matrix by mechanical loading is thought to result in interstitial fluid flow within the lacunar-canalicular system, which may activate osteocyte mechanosensors, leading to changes in osteocyte gene expression and ultimately increased bone formation and decreased bone resorption. However, repetitive strain applied to bone can result in microcracks, which may propagate and coalesce, and if not repaired predispose to catastrophic fracture. Osteocytes are a key component in this process, whereby apoptotic osteocytes in an area of microdamage promote targeted remodeling of the damaged bone. If fractures do occur, fracture repair can be divided into 2 types: primary and secondary healing. Secondary fracture repair is the most common and is a multistage process consisting of hematoma formation and acute inflammation, callus formation, and finally remodeling, whereby bone may return to its original form.

Neonatal leptin treatment reverses the bone-suppressive effects of maternal undernutrition in adult rat offspring.

Alterations in the early life environment, including maternal undernutrition (UN) during pregnancy, can lead to increased risk of metabolic and cardiovascular disorders in offspring. Leptin treatment of neonates born to UN rats reverses the programmed metabolic phenotype, but the possible benefits of this treatment on bone tissue have not been defined. We describe for the first time the effects of neonatal leptin treatment on bone in adult offspring following maternal UN. Offspring from either UN or ad libitum-fed (AD) rats were treated with either saline or leptin (2.5 µg/ g.d on postnatal days (D)3-13) and were fed either a chow or high fat (HF) diet from weaning until study completion at D170. Analysis of micro-tomographic data of the left femur showed highly significant effects of UN on cortical and trabecular bone tissue indices, contributing to inferior microstructure and bone strength, almost all of which were reversed by early leptin life treatment. The HF fat diet negatively affected trabecular bone tissue, but the effects of only trabecular separation and number were reversed by leptin treatment. The negative effects of maternal UN on skeletal health in adult offspring might be prevented or attenuated by various interventions including leptin. Establishment of a minimal efficacious leptin dose warrants further study.

A Memory of Early Life Physical Activity Is Retained in Bone Marrow of Male Rats Fed a High-Fat Diet.

Studies have reported opposing effects of high-fat (HF) diet and mechanical stimulation on lineage commitment of the bone marrow stem cells. Yet, how bone marrow modulates its gene expression in response to the combined effects of mechanical loading and a HF diet has not been addressed. We investigated whether early-life (before onset of sexual maturity at 6 weeks of age) voluntary physical activity can modulate the effects of a HF diet on male Sprague Dawley rats. In the bone marrow, early-life HF diet resulted in adipocyte hypertrophy and a pro-inflammatory and pro-adipogenic gene expression profile. The bone marrow of the rats that undertook wheel exercise while on a HF diet retained a memory of the early-life exercise. This memory lasted at least 60 days after the cessation of the voluntary exercise. Our results are consistent with the marrow adipose tissue having a unique response to HF feeding in the presence or absence of exercise.