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1.  Reduced high intensity training distance had no effect on VLa4 but attenuated heart rate response in 2-3-year-old Standardbred horses 
Background
Training of Standardbred race horses aims to improve cardiovascular and metabolic functions but studies on the effects of different training strategies from breaking till racing are lacking. Sixteen horses with the goal to race as 3-year-olds were studied from breaking (1-year-olds) to December as 3-year-olds. Horses were allocated to either a control (C) or reduced (R) training program from 2 years of age. The aim was to evaluate the effect of reducing the distance of high intensity exercise by 30% with respect to velocity at lactate concentration 4 mmol/l (VLa4), blood lactate and cardiovascular response. All training sessions were documented and heart rate (HR) was recorded. A standardized exercise test of 1,600 m was performed 10 times and a VLa4 test was performed five times.
Results
C horses initially exercised for a longer time with a HR >180 beats per minute compared to R horses (P < 0.05) but after 6–9 months, time with HR >180 bpm decreased in C and were similar in the two groups (P > 0.05). Over the 2-year period, recovery HR after the 1,600 m-test decreased in both groups but was within 2 months lower in C than in R (P < 0.05). C horses also had lower resting HR as 3-year-olds (P < 0.01) than R horses. In C, post exercise hematocrit was higher than in R (P < 0.05). There was a tendency (P < 0.1) towards a larger aortic diameter in C as 3-year-olds (C: 1.75 ± 0.05, R: 1.70 ± 0.05 cm/100 kg BW). Left ventricle diameter and blood volume (in December as 2-year-olds) did not differ between groups. There were no differences between groups in post exercise blood lactate concentration or in VLa4. Both groups were equally successful in reaching the goal of participation in races.
Conclusions
Horses subjected to a reduced distance of high intensity training from the age of 2 showed an attenuated heart rate response, but were able to maintain the same VLa4 and race participation as horses subjected to longer training distances.
doi:10.1186/s13028-015-0107-1
PMCID: PMC4389305  PMID: 25884463
Anaerobic threshold; Cardiovascular response; Hematocrit; Lactate; Heart rate
2.  Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses 
PLoS ONE  2013;8(6):e66897.
Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life.
doi:10.1371/journal.pone.0066897
PMCID: PMC3686748  PMID: 23840549
3.  Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent 
BMC Veterinary Research  2015;11:140.
Background
Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.
Results
In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG).
Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment.
In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups.
In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.
Conclusion
Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10.
The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0452-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12917-015-0452-3
PMCID: PMC4476236  PMID: 26100265
Equine melanoma; Grey horse; MIDGE vector; Cytokines; CpG; IL-12; IL-18; Transfection reagent; DNA vaccine; Cationic lipid
4.  Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses 
BMC Physiology  2009;9:12.
Background
The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.
In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).
Results
Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.
Conclusion
These results suggest that MMP-1 and IL-8 are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.
doi:10.1186/1472-6793-9-12
PMCID: PMC2705340  PMID: 19552796
5.  Exercise Training and Beta-Alanine-Induced Muscle Carnosine Loading 
Purpose
Beta-alanine (BA) supplementation has been shown to augment muscle carnosine concentration, thereby promoting high-intensity (HI) exercise performance. Trained muscles of athletes have a higher increase in carnosine concentration after BA supplementation compared to untrained muscles, but it remains to be determined whether this is due to an accumulation of acute exercise effects or to chronic adaptations from prior training. The aim of the present study was to investigate whether high-volume (HV) and/or HI exercise can improve BA-induced carnosine loading in untrained subjects.
Methods
All participants (n = 28) were supplemented with 6.4 g/day of BA for 23 days. The subjects were allocated to a control group, HV, or HI training group. During the BA supplementation period, the training groups performed nine exercise sessions, consisting of either 75–90 min continuous cycling at 35–45% Wmax (HV) or 3 to 5 repeats of 30 s cycling at 165% Wmax with 4 min recovery (HI). Carnosine content was measured in soleus and gastrocnemius medialis by proton magnetic resonance spectroscopy.
Results
There was no difference in absolute increase in carnosine content between the groups in soleus and gastrocnemius muscle. For the average muscle carnosine content, a higher absolute increase was found in HV (+2.95 mM; P = 0.046) and HI (+3.26 mM; P = 0.028) group compared to the control group (+1.91 mM). However, there was no additional difference between the HV and HI training group.
Conclusion
HV and HI exercise training showed no significant difference on BA-induced muscle carnosine loading in soleus and gastrocnemius muscle. It can be suggested that there can be a small cumulative effect of exercise on BA supplementation efficiency, although differences did not reach significance on individual muscle level.
doi:10.3389/fnut.2015.00013
PMCID: PMC4429226  PMID: 25988141
beta-alanine; muscle contractions; sport supplements; carnosine loading; training
6.  Higher Cardiorespiratory Fitness in Older Trained Women is Due to Preserved Stroke Volume 
Previous literature has shown that sedentary older women rely on peripheral adaptations to improve cardiorespiratory fitness with endurance training i.e. they show minimal increases in central parameters (cardiac output, Q) in response to endurance training. The purpose of this study therefore was to determine whether endurance trained older women were able to preserve maximal exercise Q and were characterized by a high stroke volume (SV) when compared to physically inactive older women. Trained (n = 7) and untrained (n = 1 0) women attended two maximal and one submaximal laboratory session. Breath-by-breath analysis was conducted using mass spectrometry and Q was assessed using acetylene open circuit inert gas wash-in. Multivariate analysis of variance and paired samples t-tests were used to determine between and within group differences. Trained women had a significantly higher VO2max (37.5 vs. 24.1 ml-1·kg·min-1) compared to untrained women. There were no differences for peripheral oxygen extraction (VO2/Q) at either submaximal or maximal work rates; however trained women had a significantly higher SV at maximal (119.3 vs. 94.6 ml) exercise compared to untrained women. In both trained and untrained women, SV did not rise significantly between submaximal and maximal exercise. Conclusion: Highly fit, endurance trained older women are able to preserve central parameters of VO2max. Peripheral oxygen extraction is similar between older trained and untrained women.
Key points
Older women are able to preserve stroke volume with high volumes of endurance training.
Stroke volume of endurance trained older women does not continue to rise until maximal exercise, as is the case in young endurance trained adults.
Peripheral oxygen extraction does not differ between endurance trained and physically inactive older women.
PMCID: PMC3763323  PMID: 24150087
cardiac output; aging; exercise; running; VO2max
7.  Physical Fitness and Mitochondrial Respiratory Capacity in Horse Skeletal Muscle 
PLoS ONE  2012;7(4):e34890.
Background
Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.
Methodology/Principal Findings
We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.
Oxidative phosphorylation (OXPHOS) capacity (pmol O2•s−1•mg−1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness.
Conclusions/Significance
The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.
doi:10.1371/journal.pone.0034890
PMCID: PMC3329552  PMID: 22529950
8.  A prospective study on a cohort of horses and ponies selected for participation in the European Eventing Championship: reasons for withdrawal and predictive value of fitness tests 
Background
Eventing is generally recognized as a challenging equestrian discipline and wastage figures for this discipline are relatively high. There is a need for information that provides insight into the causes of wastage and withdrawal from competition, for animal welfare and economic reasons. The aim of the present investigation was to conduct a prospective study following the entire national selection of event horses (n = 20) and ponies (n = 9) in the Netherlands that prepared for the European Championship in 2010 (ponies) and 2011 (horses), noting causes of withdrawal and monitoring fitness using standardized exercise tests (SETs), with heart rate (HR; beats/min), speed (V; m/s) and plasma lactate concentrations (LA; mmol/L) as measured parameters.
Results
In SET-I, performed at the beginning of the season, horses (n = 17) had a mean VLA4 (V at LA 4 mmol/L) of 10.3 ± 0.4 m/s with a mean V200 (V at 200 beats/min) of 11.4 ± 0.8 m/s and ponies (n = 9) a mean VLA4 of 7.8 ± 0.9 m/s and V200 of 9.6 ± 0.7 m/s. Before SET-II, performed six weeks before the European Championship, 16/20 horses and 6/9 ponies were withdrawn. The most common reason for withdrawal was locomotor injury (9/16 horses, 4/6 ponies; P < 0.001 and P = 0.011, respectively). Other reasons included an animal ‘not meeting the competition criteria’ (4/16 horses, 2/6 ponies) and being sold (3/16 horses). Animals were divided on the basis of VLA4 and recovery-HR during SET-I into good and average performers. Average performers were significantly more likely to be injured (50.0%) than good performers (0%, P = 0.05). In a subpopulation of ten horses, in which all condition training sessions were evaluated for HR and speed, HRpeak was significantly lower in horses that stayed sound (186 ± 9 beats/min) compared with horses withdrawn from training and competition because of injury (201 ± 5 beats/min; P = 0.016).
Conclusions
Of the national selection, 45% of all animals were unavailable for the European Championship because of locomotor injuries. Field tests were useful in assessing the potential injury risk, as individuals with better fitness indices (good performers) were less likely to become injured than average performers. Furthermore, monitoring of training sessions showed predictive value for future injuries, as horses withdrawn because of injury later on showed already higher peak HRs during condition training than horses that stayed sound. Therefore the increase in peak HR seemed to precede visible lameness in a horse.
doi:10.1186/1746-6148-9-182
PMCID: PMC3848563  PMID: 24034152
Horse; Eventing; Exercise; Fitness; Monitoring; Training
9.  Carbohydrate restricted recovery from long term endurance exercise does not affect gene responses involved in mitochondrial biogenesis in highly trained athletes 
Physiological Reports  2015;3(2):e12184.
The aim was to determine if the metabolic adaptations, particularly PGC-1α and downstream metabolic genes were affected by restricting CHO following an endurance exercise bout in trained endurance athletes. A second aim was to compare baseline expression level of these genes to untrained. Elite endurance athletes (VO2max 66 ± 2 mL·kg−1·min−1, n = 15) completed 4 h cycling at ∼56% VO2max. During the first 4 h recovery subjects were provided with either CHO or only H2O and thereafter both groups received CHO. Muscle biopsies were collected before, after, and 4 and 24 h after exercise. Also, resting biopsies were collected from untrained subjects (n = 8). Exercise decreased glycogen by 67.7 ± 4.0% (from 699 ± 26.1 to 239 ± 29.5 mmol·kg−1·dw−1) with no difference between groups. Whereas 4 h of recovery with CHO partly replenished glycogen, the H2O group remained at post exercise level; nevertheless, the gene expression was not different between groups. Glycogen and most gene expression levels returned to baseline by 24 h in both CHO and H2O. Baseline mRNA expression of NRF-1, COX-IV, GLUT4 and PPAR-α gene targets were higher in trained compared to untrained. Additionally, the proportion of type I muscle fibers positively correlated with baseline mRNA for PGC-1α, TFAM, NRF-1, COX-IV, PPAR-α, and GLUT4 for both trained and untrained. CHO restriction during recovery from glycogen depleting exercise does not improve the mRNA response of markers of mitochondrial biogenesis. Further, baseline gene expression of key metabolic pathways is higher in trained than untrained.
doi:10.14814/phy2.12184
PMCID: PMC4393183  PMID: 25677542
baseline mRNA expression; glycogen depletion; PGC-1α; type 1 fibers
10.  Interday variation and effect of transportation on indirect blood pressure measurements, plasma endothelin-1 and serum cortisol in Standardbred and Icelandic horses 
Background
Systemic hypertension is a prominent feature in humans with metabolic syndrome (MS) and this is partly caused by an enhanced endothelin-1 (ET-1) mediated vasoconstriction. There are indications that systemic hypertension might be a feature in equine metabolic syndrome (EMS) but if ET-1 is involved in the development of hypertension in horses is not known. Increased levels of cortisol have also been found in humans with MS but there are no reports of this in horses. Before blood pressure, plasma ET-1 and serum cortisol can be evaluated in horses with EMS, it is necessary to investigate the interday variation of these parameters on clinically healthy horses. The aims of the present study were therefore to evaluate the interday variation and influence of transportation on systemic blood pressure, plasma ET-1 and serum cortisol in healthy Standardbred and Icelandic horses, and to detect potential breed differences.
Methods
Nine horses of each breed were included in the study. Blood pressure was measured and blood samples were collected between 6 and 9 am on two separate days. Eight of the horses (four of each breed) were transported to a new stable were they stayed overnight. The next morning, the sampling procedure was repeated.
Results
The interday variation was higher for plasma ET-1 (37%) than for indirect pressure measurements (8-21%) and serum cortisol (18%). There were no differences in systemic blood pressure between the two breeds. The Icelandic horses had significantly lower serum cortisol and significantly higher plasma ET-1 concentrations compared to the Standardbred horses. Plasma ET-1 was significantly elevated after transportation, but systemic blood pressure and serum cortisol did not differ from the values obtained in the home environment.
Conclusions
Indirect blood pressure, plasma ET-1 and serum cortisol are of interest as markers for cardiovascular dysfunction in horses with EMS. The elevated plasma ET-1 concentrations recorded after transportation was likely caused by a stress response. This needs to be considered when evaluating plasma ET-1 in horses after transportation. The differences detected in plasma ET-1 and serum cortisol between the two breeds might be related to differences in genetic setup, training status as well as management conditions.
doi:10.1186/1751-0147-54-37
PMCID: PMC3503793  PMID: 22682151
Horse; Plasma endothelin-1; Cortisol; Blood pressure; Transportation
11.  Effects of exercise on soluble transferrin receptor and other variables of the iron status 
Background: Soluble transferrin receptor (sTfr) is a new marker of iron status and erythropoietic activity. It has been included in multivariable blood testing models for the detection of performance enhancing erythropoietin misuse in sport.
Objective: To evaluate the effect of different types and volumes of physical activity on sTfr concentration, variables of iron status (ferritin, transferrin, iron, and protein), and haematological indices.
Methods: Thirty nine subjects were divided into three groups: 1, untrained (n = 12); 2, moderately trained (n = 14); 3, highly trained (n = 13, seven men, six women). Groups 1 and 2 carried out two exercise tests: an incremental running test until exhaustion (test A) and a 45 minute constant speed running test at 70% VO2MAX (test B). Group 3 performed three days (women) or four days (men) of prolonged aerobic cycling exercise. The above variables together with haemoglobin and packed cell volume were analysed in venous blood samples before and after exercise. Changes in blood and plasma volume were estimated.
Results: sTfr levels were slightly increased in trained and untrained subjects immediately after test A. Test B and aerobic exercise had no significant effect on sTfr. Ferritin levels were increased after the laboratory tests for trained and untrained subjects and after prolonged aerobic exercise in male cyclists. Transferrin was increased significantly in trained and untrained subjects after both laboratory tests, but remained unchanged after prolonged exercise. Plasma and blood volumes were decreased after the laboratory tests but increased after aerobic exercise. No differences in the variables were observed between trained and untrained subjects with respect to response to exercise.
Conclusion: The changes in sTfr and the variables of iron status can be mainly attributed to exercise induced changes in volume. Taking these limitations into account, sTfr can be recommended as a marker of iron deficiency in athletes.
doi:10.1136/bjsm.36.3.195
PMCID: PMC1724494  PMID: 12055114
12.  Heart Rate Recovery After Exercise and Neural Regulation of Heart Rate Variability in 30-40 Year Old Female Marathon Runners 
The aim of the present study was to examine the effects of endurance training on heart rate (HR) recovery after exercise and cardiac autonomic nervous system (ANS) modulation in female marathon runners by comparing with untrained controls. Six female marathon runners (M group) aged 32-40 years and eight age-matched untrained females (C group) performed a maximum-effort treadmill running exercise. Maximal oxygen uptake (VO2max) was measured during the exercise with a gas analyzer connected to subjects through a face mask. Heart rate, blood pressure and blood lactate were measured before and after the exercise. Rating of perceived exertion (RPE) to the exercise was obtained immediately after the exercise. Holter ECG was recorded and analyzed with power spectral analysis of heart rate variability (HRV) to investigate the cardiac ANS modulation. The M group had significantly higher VO2max, faster HR recovery after exercise, higher Mean RR, SDRR, HF power and lower LF/HF ratio at rest compared with the C group. The M group also presented greater percent decrease of blood pressure after exercise, although their blood pressure after exercise was higher than the C group. It is suggested that endurance training induced significant alterations in cardiac ANS modulation at rest and significant acceleration of HR recovery after exercise in female marathon runners. Faster HR recovery after exercise in the female marathon runners should result from their higher levels of HRV, higher aerobic capacity and exaggerated blood pressure response to exercise compared with untrained controls.
Key PointsThe effects of endurance training on HR recovery after exercise and cardiac ANS modulation were investigated in female marathon runners by comparing with untrained controls.Time and frequency domain analysis of HRV was used to investigate cardiac ANS modulation.As compared with untrained controls, the female marathon runners showed faster HR recovery after exercise, which should result from their higher levels of HRV, higher aerobic capacity and exaggerated blood pressure response to exercise.
PMCID: PMC3880089  PMID: 24431956
Heart rate recovery; heart rate variability; female marathon runner
13.  Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins. 
Journal of Virology  1994;68(3):1459-1467.
Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.
Images
PMCID: PMC236601  PMID: 8107209
14.  Red blood cell variables in highly trained pubescent athletes: a comparative analysis 
Background—A suboptimal haematological status has often been recorded in athletes involved in intensive physical activity. There have even been reports of "sports anaemia" associated with intensive physical exercise. However, studies on the effect of different types of exercise practiced over a long period of time on the red blood cell variables in pubescent athletes are very few.
Aim—To assess the basic red blood cell variables in highly trained pubescent athletes from different sports and to compare the results with those for a control untrained group. Sex related differences in these variables were also assessed.
Methods—876 highly trained athletes (559 boys and 317 girls) were included in the study. Their mean (SEM) age, weight, and duration of training were: 14.01 (0.06) years, 56.24 (0.52) kg, and 3.52 (0.07) years respectively. The control group consisted of 357 untrained subjects (171 boys and 186 girls) with mean (SEM) age and weight of 14.58 (0.09) years and 57.75 (0.67) kg. The group of athletes was divided into seven subgroups according to the sport practiced: athletics (105), swimming (107), rowing (230), wrestling (225), weight lifting (47), various team sports (92), and other sports (67). Venous blood samples were drawn from the cubital vein, and the red blood cell count, packed cell volume, haemoglobin concentration, and mean corpuscular volume were measured. Statistical indices were computed for each group and for each variable, and analysis of variance factorial analysis was performed to evaluate the statistical significance of the differences detected.
Results—The highly trained group was found to have lower red blood cell count, packed cell volume, and haemoglobin concentration (p<0.001) than the control untrained group (4.61 (0.01) x 1012/1 v 4.75 (0.02) x 1012/l, 0.389 (0.001) v 0.404 (0.002) l/l, and 133.01 (0.38) v 139.9 (0.62) g/l respectively). These variables were lower for the boys of the trained group than for the boys of the control group (p<0.001), and similarly for the girls (p<0.001). The lowest red blood cell count, packed cell volume, and haemoglobin concentration were measured in blood samples from the boys of the swimming subgroup (4.54 (0.06) x 1012/l, 0.386 (0.006) l/l, and 129.38 (1.80) g/1 respectively) and the rowing subgroup (4.66 (0.03) x 1012/l, 0.400 (0.003) l/l, and 136.21 (0.94) respectively). The same distribution was found for the girls: lowest in the rowing subgroup (4.32 (0.04) x 1012/1, 0.314 (0.003) l/l, and 124.27 (0.93) g/1) and the swimming subgroup (4.40 (0.05) x 1012/l, 0.375 (0.005) l/l, and 125.90 (1.30) g/1). No differences were found in the mean corpuscular volume.
Conclusions—Continuous (more than one year) high intensity sports training (twice a day/five days a week) results in a decrease in the basic red blood cell variables in pubescent boys and girls, this being most pronounced in the submaximal sports.
Key Words: erythrocytes; haemoglobin; packed cell volume; mean corpuscular volume; pubescence; training
doi:10.1136/bjsm.34.3.200
PMCID: PMC1763253  PMID: 10854020
15.  Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq 
Molecules and Cells  2015;38(3):210-220.
Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.
doi:10.14348/molcells.2015.2138
PMCID: PMC4363720  PMID: 25666347
DNA methylation; exercise; MeDIP-Seq; thoroughbred horse; transposable elements
16.  Effect of Energy Expenditure and Training Status on Leptin Response to Sub-Maximal Cycling 
We examined the leptin response and related hormones during and after two sub-maximal exercise protocols in trained and untrained subjects. During this study, plasma concentrations of leptin [Lep], insulin [I], cortisol [C], growth hormone [GH], glucose [G] and lactate [La] were measured. 7 elite volleyball trained players (TR) and 7 untrained (UTR) subjects (percent body fat: 13.2 ± 1.8 versus 15.7 ± 1.0, p < 0.01, respectively) were examined after short and prolonged sub-maximal cycling exercise protocols (SP and PP). Venous blood samples were collected before each protocol, during, at the end, and after 2 and 24 h of recovery. SP and PP energy expenditures ranged from 470 ± 60 to 740 ± 90 kcal for TR and from 450 ± 60 to 710 ± 90 kcal for UTR, respectively. [Lep] was related to body fat percentage and body fat mass in TR (r = 0. 84, p < 0.05 and r = 0.93, p < 0.01) and in UTR (r = 0.89, p < 0.01 and r = 0.92, p < 0. 01, respectively). [Lep] did not change significantly during both protocols for both groups but was lower (p < 0.05) in all sampling in TR when compared to UTR. Plasma [I] decreased (p < 0.01) and [GH] increased (p < 0.01) significantly during both SP and PP and these hormones remained lower (I: p < 0.01) and higher (GH: p < 0.01) than pre-exercise levels after a 2-h recovery period, returning to base-line at 24-h recovery. Plasma [La] increased (p < 0.01) during both protocols for TR and UTR. There was no significant change in [C] and [G] during and after both protocols for all subjects. It is concluded that 1) leptin is not sensitive to acute short or prolonged sub-maximal exercises (with energy expenditure under 800 kcal) in volleyball/ anaerobically trained athletes as in untrained subjects, 2) volleyball athletes showed significantly lower resting and exercise leptin response with respect to untrained subjects and 3) it appears that in these anaerobically trained athletes leptin response to exercise is more sensitive to the level of energy expenditure than hormonal or metabolic modifications induced by acute exercise.
Key pointsTrials concerning acute exercise and leptin indicated discrepant results.Acute exercise with energy expenditure higher than 800 kcal can decrease leptinemia.Elite volleyball players presented decreased leptin levels than untrained subjects.
PMCID: PMC3761489  PMID: 24149525
Hormones; anaerobic training; acute exercise; body fat.
17.  Pronounced Muscle Deoxygenation During Supramaximal Exercise Under Simulated Hypoxia in Sprint Athletes 
The purpose of this study was to determine whether acute hypoxia alters the deoxygenation level in vastus lateralis muscle during a 30 s Wingate test, and to compare the muscle deoxygenation level between sprint athletes and untrained men. Nine male track sprinters (athletic group, VO2max 62.5 ± 4.1 ml/kg/min) and 9 healthy untrained men (untrained group, VO2max 49.9 ± 5.2 ml·kg-1·min-1) performed a 30 s Wingate test under simulated hypoxic (FIO2 = 0.164 and PIO2 = 114 mmHg) and normoxic conditions. During the exercise, changes in oxygenated hemoglobin (OxyHb) in the vastus lateralis were measured using near infrared continuous wave spectroscopy. Decline in OxyHb, that is muscle deoxygenation, was expressed as percent change from baseline. Percutaneous arterial oxygen saturation (SpO2), oxygen uptake (VO2), and ventilation (VE) were measured continuously. In both groups, there was significantly greater muscle deoxygenation, lower SpO2, lower peakVO2, and higher peakVE during supramaximal exercise under hypoxia than under normoxia, but no differences in peak and mean power output during the exercise. Under hypoxia, the athletic group experienced significantly greater muscle deoxygenation, lower SpO2, greater decrement in peakVO2 and increment in peakVE during the exercise than the untrained group. When the athletic and untrained groups were pooled, the increment of muscle deoxygenation was strongly correlated with lowest SpO2 in the 30 s Wingate test under hypoxia. These results suggest that acute exposure to hypoxia causes a greater degree of peripheral muscle deoxygenation during supramaximal exercise, especially in sprint athletes, and this physiological response would be explained mainly by lower arterial oxygen saturation.
Key pointsThe deoxygenation trends in the vastus lateralis muscle during 30 s Wingate test in track sprinters and untrained men under simulated hypoxic and normoxic conditions was investigated using near infrared spectroscopy.Acute hypoxia caused a greater degree of peripheral muscle deoxygenation than normoxia, whereas there were no changes in performance such as power output during 30 s Wingate test.Sprint athletes show a greater degree of peripheral muscle deoxygenation during 30 s Wingate test in hypoxia when compared with untrained subjects.A larger difference in muscle deoxygenation between hypoxia and normoxia is accompanied by lowest SpO2 at the 30 s Wingate test in hypoxia.
PMCID: PMC3761931  PMID: 24149959
NIRcws; muscle deoxygenation; hypoxic; 30s Wingate test; athletes
18.  Serum amyloid A (SAA) concentration after training sessions in Arabian race and endurance horses 
Background
Serum amyloid A (SAA) is the major acute phase protein in horses. Its concentration increases in various pathologies but also in response to prolonged, strenuous effort. The purpose of this study was to establish whether routine race and endurance training produces changes in the SAA level in Arabian horses. Additionally, the differences between SAA response in experienced endurance horses and endurance horses that were beginning their career were investigated.
Results
There were no changes in SAA concentrations after race training and endurance training in experienced horses. In horses that were beginning their endurance training, exercise produced an increase in SAA level as compared with rest level.
Conclusion
In Arabians, the SAA concentration seems to be a good indicator of endurance training but is useless in race training. The routine training of experienced horses, which were prepared for long distance rides, did not promote any changes in the SAA level. In contrast, a significant increase in the SAA concentration was observed in horses that were beginning their endurance training and were only prepared for moderate distance rides and underwent the same effort. Further research is needed to elucidate whether this difference reflects too heavy training or adaptation to an increasing workload. Additionally, the adaptation to long distance rides in Arabians may include a reduced acute phase response.
doi:10.1186/1746-6148-9-91
PMCID: PMC3655847  PMID: 23634727
Serum amyloid A; Race horses; Endurance horses; Training
19.  Short-Term Intensified Cycle Training Alters Acute and Chronic Responses of PGC1α and Cytochrome C Oxidase IV to Exercise in Human Skeletal Muscle 
PLoS ONE  2012;7(12):e53080.
Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training. Nine untrained, healthy participants (mean±SD; VO2peak 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO2peak). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90–100% VO2peak) and six prolonged moderate-intensity sessions (45–90 min; 75% VO2peak). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training. These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.
doi:10.1371/journal.pone.0053080
PMCID: PMC3532354  PMID: 23285255
20.  Effects of Prolonged Exercise on Oxidative Stress and Antioxidant Defense in Endurance Horse 
Increased oxidative stress during prolonged endurance exercise may end up with muscle damage, fatigue and decreased physical performance. We have recently shown that acute exercise at moderate intensity induced lipid peroxidation, protein oxidation and oxygen radical absorbance capacity (ORAC) in trained trotters. The aim of this study was to measure the changes in oxidative stress and antioxidant defense following an 80-km ride in the blood of endurance horses. Blood samples were collected before and immediately after the ride. Unlike to our previous studies performed on trotters, in endurance horses there were no measurable changes in antioxidants or oxidative stress marker lipid hydroperoxides (LPO) after prolonged exercise. ORAC, vitamin E and lipid hydroperoxide (LPO) concentration or glutathione related enzyme activities were not altered due to the 80-km ride. However, the base line levels of oxidative stress marker were higher in endurance horses compared to trotters. A positive correlation between the pre-ride LPO concentration and erythrocyte glutathione peroxidase (GPx) activity after the ride was observed, which may indicate a protective response of glutathione peroxidase against exercise-induced oxidative stress. Our results suggest that endurance horses have higher oxidative stress levels compared to trotters and a single 80-km ride probably did not suffice to induce oxidative stress and to activate antioxidant defense mechanisms.
Key PointsReactive oxygen species (ROS) at lower concentrations have physiological role in the signal transduction and in the regulation of cellular functions. However, the overproduction of ROS results in oxidative stress, an imbalance favoring pro-oxidants over antioxidants.Increased oxidative stress which occurred during prolonged and strenuous physical exercise may end up with muscle damage, fatigue and decreased performance.Prolonged exercise at moderate intensity does not induce oxidative stress in endurance horses.Endurance horses have higher oxidative stress at rest compared to trotters which were trained for short bouts of exercise.
PMCID: PMC3899657  PMID: 24501555
Horse; endurance; oxidative stress; antioxidants; ORAC
21.  Lifelong endurance training attenuates age-related genotoxic stress in human skeletal muscle 
Background
The aim of the present study was to determine the influence of age and habitual activity level, at rest and following a single bout of high-intensity exercise, on the levels of three proteins poly(ADP-ribose) polymerase-1 (PARP-1), cleaved-PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), involved in the DNA repair and cell death responses to stress and genotoxic insults. Muscle biopsies were obtained from the vastus lateralis of young trained (22 ± 3 years, n = 6), young untrained (24 ± 4 years, n = 6), old trained (64 ± 3 years, n = 6) and old untrained (65 ± 6 years, n = 6) healthy males before, immediately after and three days following a high-intensity interval exercise bout.
Results
PARP-1, which catalyzes poly(ADP-ribosyl)ation of proteins and DNA in response to a range of intrinsic and extrinsic stresses, was increased at baseline in old trained and old untrained compared with young trained and young untrained participants (P ≤ 0.05). Following exercise, PARP-1 levels remained unchanged in young trained participants, in contrast to old trained and old untrained where levels decreased and young untrained where levels increased (P ≤ 0.05). Interestingly, baseline levels of the cleaved PARP-1, a marker of apoptosis, and PARG, responsible for polymer degradation, were both significantly elevated in old untrained compared with old trained, young trained and young untrained (P ≤ 0.05). Despite this baseline difference in PARG, there was no change in any group following exercise. There was a non-significant statistical trend (P = 0.072) towards increased cleaved-PARP-1 expression post-exercise in younger but not old persons, regardless of training status.
Conclusions
Collectively, these results show that exercise slows the progression towards a chronically stressed state but has no impact on the age-related attenuated response to acute exercise. Our findings provide valuable insight into how habitual exercise training could protect skeletal muscle from chronic damage to macromolecules and may reduce sarcopenia in older people.
doi:10.1186/2046-2395-2-11
PMCID: PMC3922955  PMID: 24472304
PARP-1; Cleaved PARP-1; PARG; Apoptosis; DNA repair; Exercise; Training; Ageing
22.  Differences in Muscle Fiber Recruitment Patterns between Continuous and Interval Exercises 
Journal of equine science  2011;21(4):59-65.
We evaluated differences in muscle fiber recruitment patterns between continuous and interval training to develop an optimal training program for Thoroughbred horses. Five well trained female thoroughbred horses (3–4 years old) were used. The horses performed two different exercises on a 10% inclined treadmill: 90%VO2 max for 4 min (continuous) and 90% VO2 max for 2 min × 2 times with 10-min interval (interval). Muscle samples were obtained from the middle gluteal muscle before and immediately after the exercises. Four muscle fiber types (type I, IIA, IIA/X, and IIX) were immunohistochemically identified, and the optical density of periodic acid Schiff staining (OD-PAS) in each fiber type and glycogen content of the muscle sample were determined by quantitative histochemical and biochemical procedures, respectively. No significant differences were found in the OD-PASs and glycogen contents between the continuous and interval exercises, but the decreases in OD-PAS of fast-twitch muscle fibers were obvious after interval as compared to continuous exercise. Interval exercise may be a more effective training stimulus for the glycolytic capacity of fast-twitch muscle fiber. The data about muscle fiber recruitment can provide significant insights into the optimal training program not only for thoroughbred horses, but also for human athletes.
doi:10.1294/jes.21.59
PMCID: PMC4013969  PMID: 24833978
glycogen; interval exercise; muscle fiber; Thoroughbred
23.  Oxidative stress responses to a graded maximal exercise test in older adults following explosive-type resistance training☆ 
Redox Biology  2013;2:65-72.
We recently demonstrated that low frequency, moderate intensity, explosive-type resistance training (EMRT) is highly beneficial in elderly subjects towards muscle strength and power, with a systemic adaptive response of anti-oxidant and stress-induced markers. In the present study, we aimed to evaluate the impact of EMRT on oxidative stress biomarkers induced in old people (70–75 years) by a single bout of acute, intense exercise. Sixteen subjects randomly assigned to either a control, not exercising group (n=8) or a trained group performing EMRT protocol for 12-weeks (n=8), were submitted to a graded maximal exercise stress test (GXT) at baseline and after the 12-weeks of EMRT protocol, with blood samples collected before, immediately after, 1 and 24 h post-GXT test. Blood glutathione (GSH, GSSG, GSH/GSSG), plasma malonaldehyde (MDA), protein carbonyls and creatine kinase (CK) levels, as well as PBMCs cellular damage (Comet assay, apoptosis) and stress–protein response (Hsp70 and Hsp27 expression) were evaluated. The use of multiple biomarkers allowed us to confirm that EMRT per se neither affected redox homeostasis nor induced any cellular and oxidative damage. Following the GXT, the EMRT group displayed a higher GSH/GSSG ratio and a less pronounced increase in MDA, protein carbonyls and CK levels compared to control group. Moreover, we found that Hsp70 and Hsp27 proteins were induced after GXT only in EMRT group, while any significant modification within 24 h was detected in untrained group. Apoptosis rates and DNA damage did not show any significant variation in relation to EMRT and/or GXT.
In conclusion, the adherence to an EMRT protocol is able to induce a cellular adaptation allowing healthy elderly trained subjects to cope with the oxidative stress induced by an acute exercise more effectively than the aged-matched sedentary subjects.
Graphical abstract
Highlights
•A low frequency, moderate intensity, explosive-type resistance training (EMRT) does not affect redox homeostasis at rest.•EMRT improves the general adaptive response to oxidative stress induced by graded maximal effort.•EMRT is effective intervention for improving the overall health of the older people.
doi:10.1016/j.redox.2013.12.004
PMCID: PMC4297938  PMID: 25460722
Explosive-type moderate intensity resistance training (EMRT); Graded maximal exercise test (GXT); Oxidative stress; HSPs; Apoptosis; Elderly
24.  Effect of regular aerobic exercise with ozone exposure on peripheral leukocyte populations in Wistar male rats 
BACKGROUND:
The immune system in endurance athletes may be at risk for deleterious effects of gasous pollutants such as ambient ozone. Therefore, this study was performed to assess the effect of regular aerobic exercise with ozone exposure on peripheral leukocytes populations in male Wistar rats.
METHODS:
Twenty eight 8 weeks old rats were selected and randomly divided into four groups of ozone-unexposed and untrained (control or group 1, n = 6), ozone-exposed and untrained (group 2, n = 6), ozone-unexposed and trained (group 3, n = 8), ozone-exposed and trained (group 4, n = 8). All animals in groups 3 and 4 were regularly running (20 m/min, 30 min/day) on a treadmill for 7 weeks (5 day/week). After the last ozone exposure [0.3 ppm, 30 min per sessions], blood samples were obtained from the cardiac puncture and hematological parameters as well as blood lactate were measured using automatic analyzers. Data were expressed as means (± SD) and analyzed by ANOVA and Pearson's correlation tests at p < 0.05.
RESULTS:
All the hematological parameters differences (except RBC and hemoglobin rate) were significantly higher in the trained groups (p < 0.001). However, ozone-induced leukocytosis in the trained (but not in the sedentary) rats was statistically higher than in the counterpart groups.
CONCLUSIONS:
Repeated acute ozone exposure has more additive effect on peripheral leukocyte counts in active animals. But, more researches are needed to identify effects of ozone exposure on other components of the immune system in athletes and non-athletes.
PMCID: PMC3129096  PMID: 21772896
Moderate Aerobic Exercise; Ozone Exposure; Leukocytosis; Wistar Rats
25.  Wrestlers’ immune cells produce higher interleukin-6 and lower interleukin-12 and interleukin-13 in response to in vitro mitogen activation 
Objective(s):
Although recent investigations have shown chronic inflammation and inflammation-associated diseases might be ameliorated by exercise; little is known about the relation between exercise training with anti/pro-inflammatory cytokines.
Materials and Methods:
This cross sectional study was conducted to compare interleukin-4 (IL-4), IL-6, IL-10, IL-12, IL-13, interferon gamma (IFN-γ ) levels in serum, and their in vitro production by whole blood (WB) cells and by peripheral blood mononuclear cells (PBMCs) in response to mitogens lipopolysaccharide and phytohemagglutinin. Twelve elite wrestlers with history of three times per week exercise training for about 9.5 years, and thirteen healthy silent controls were recruited. To analysis the cytokines by enzyme linked immunosorbent assay (ELISA), the blood samples were taken 24 hr after the last training session from the wrestlers.
Results:
Serum analysis for IL-4, IL-6, IL-10, IL-12, IL-13 and IFN-γ indicated no statistical difference between the two groups. Meanwhile, 48 hr in vitro activation of WB and PBMCs by the mitogens revealed that IL-6 production was elevated in both WB and PBMCs. Whereas, IL-12 and IL-13 were decreased in supernatant of PBMCs and WB cells cultures, respectively.
Conclusion:
It seems that wrestling cause immune system cells to produce anti-inflammatory myokine IL-6 and decrease production of pro-inflammatory cytokine IL-12 and IL-13.
PMCID: PMC4328102  PMID: 25691935
Cytokines; Wrestling; Interleukins; Mitogens; PBMC; Whole blood culture

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