A three-criteria performance score for rats exercising on a running treadmill.

In this study, we propose a novel three-criteria performance score to semiquantitatively classify the running style, the degree of involvement and compliance and the validity of electric shock count for rats exercising on a treadmill. Each score criterion has several style-marks that are based on the observational registry of male Sprague-Dawley rats running for 4-7 weeks. Each mark was given a score value that was averaged throughout a session-registry and resulting in a session score for each criterion, ranging from "0" score for a hypothetical "worst runner", to score "1" for a hypothetical "perfect runner" rat. We found significant differences throughout a training program, thus providing evidence of sufficient sensitivity of this score to reflect the individual evolution of performance improvement in exercise capacity due to training. We hypothesize that this score could be correlated with other physiological or metabolic parameters, thus refining research results and further helping researchers to reduce the number of experimental subjects.

Contractile Activity Is Necessary to Trigger Intermittent Hypobaric Hypoxia-Induced Fiber Size and Vascular Adaptations in Skeletal Muscle.

Altitude training has become increasingly popular in recent decades. Its central and peripheral effects are well-described; however, few studies have analyzed the effects of intermittent hypobaric hypoxia (IHH) alone on skeletal muscle morphofunctionality. Here, we studied the effects of IHH on different myofiber morphofunctional parameters, investigating whether contractile activity is required to elicit hypoxia-induced adaptations in trained rats. Eighteen male Sprague-Dawley rats were trained 1 month and then divided into three groups: (1) rats in normobaria (trained normobaric inactive, TNI); (2) rats subjected daily to a 4-h exposure to hypobaric hypoxia equivalent to 4,000 m (trained hypobaric inactive, THI); and (3) rats subjected daily to a 4-h exposure to hypobaric hypoxia just before performing light exercise (trained hypobaric active, THA). After 2 weeks, the tibialis anterior muscle (TA) was excised. Muscle cross-sections were stained for: (1) succinate dehydrogenase to identify oxidative metabolism; (2) myosin-ATPase to identify slow- and fast-twitch fibers; and (3) endothelial-ATPase to stain capillaries. Fibers were classified as slow oxidative (SO), fast oxidative glycolytic (FOG), fast intermediate glycolytic (FIG) or fast glycolytic (FG) and the following parameters were measured: fiber cross-sectional area (FCSA), number of capillaries per fiber (NCF), NCF per 1,000 µm2 of FCSA (CCA), fiber and capillary density (FD and CD), and the ratio between CD and FD (C/F). THI rats did not exhibit significant changes in most of the parameters, while THA animals showed reduced fiber size. Compared to TNI rats, FOG fibers from the lateral/medial fields, as well as FIG and FG fibers from the lateral region, had smaller FCSA in THA rats. Moreover, THA rats had increased NCF in FG fibers from all fields, in medial and posterior FIG fibers and in posterior FOG fibers. All fiber types from the three analyzed regions (except the posterior FG fibers) displayed a significantly increased CCA ratio compared to TNI rats. Global capillarisation was also increased in lateral and medial fields. Our results show that IHH alone does not induce alterations in the TA muscle. The inclusion of exercise immediately after the tested hypoxic conditions is enough to trigger a morphofunctional response that improves muscle capillarisation.

Modulation of mitochondrial biomarkers by intermittent hypobaric hypoxia and aerobic exercise after eccentric exercise in trained rats.

Unaccustomed eccentric contractions induce muscle damage, calcium homeostasis disruption, and mitochondrial alterations. Since exercise and hypoxia are known to modulate mitochondrial function, we aimed to analyze the effects on eccentric exercise-induced muscle damage (EEIMD) in trained rats using 2 recovery protocols based on: (i) intermittent hypobaric hypoxia (IHH) and (ii) IHH followed by exercise. The expression of biomarkers related to mitochondrial biogenesis, dynamics, oxidative stress, and bioenergetics was evaluated. Soleus muscles were excised before (CTRL) and 1, 3, 7, and 14 days after an EEIMD protocol. The following treatments were applied 1 day after the EEIMD: passive normobaric recovery (PNR), 4 h daily exposure to passive IHH at 4000 m (PHR) or IHH exposure followed by aerobic exercise (AHR). Citrate synthase activity was reduced at 7 and 14 days after application of the EEIMD protocol. However, this reduction was attenuated in AHR rats at day 14. PGC-1α and Sirt3 and TOM20 levels had decreased after 1 and 3 days, but the AHR group exhibited increased expression of these proteins, as well as of Tfam, by the end of the protocol. Mfn2 greatly reduced during the first 72 h, but returned to basal levels passively. At day 14, AHR rats had higher levels of Mfn2, OPA1, and Drp1 than PNR animals. Both groups exposed to IHH showed a lower p66shc(ser36)/p66shc ratio than PNR animals, as well as higher complex IV subunit I and ANT levels. These results suggest that IHH positively modulates key mitochondrial aspects after EEIMD, especially when combined with aerobic exercise.

Effects of intermittent hypoxia and light aerobic exercise on circulating stem cells and side population, after strenuous eccentric exercise in trained rats.

Our goal was to address if intermittent hypobaric hypoxia (IHH) exposure can help to increase the number of peripheral blood circulating progenitor cells and side population (SP) stem cells, in order to establish the usefulness of this intervention for skeletal muscle repair, because these cells play a role in tissue regeneration. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with 1, 3, 7 and 14 days stages of damage recovery of trained rats that had suffered skeletal muscle injury. Three experimental groups were studied: rats with passive recovery (CTRL); rats exposed to IHH after muscle damage (HYP); and, trained rats that, in addition to IHH, performed light aerobic exercise sessions (EHYP). We observed an increase in hematopoietic stem cells (HSCs) (mean = 0.153% of cells) and endothelial progenitor cells (EPCs) (mean = 0.0020% of cells) in EHYP on day 7. Also these cells showed characteristics of more primitive progenitors in comparison to the other experimental groups (mean = 0.107% of cells), as deduced by retention of the promising fluorescent probe Vybrant Dye Cycle Violet. We concluded that intermittent exposure to hypobaric hypoxia in combination with light aerobic exercise increased the number of HSCs and EPCs on the 7th day in EHYP group, although the exercise-induced stimulus showed a reverse effect on SP kinetics.

Effect of intermittent hypoxia and exercise on blood rheology and oxygen transport in trained rats.

Intermittent hypobaric hypoxia (IHH) exposure, accompanied or not with active recovery, can help to skeletal muscle repair. However, the erythropoietic response elicited can disturb blood rheology and thus alter the oxygen delivery to tissues. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with early (1-3 days) and late (7-14 days) stages of damage recovery in three groups of trained rats that had suffered skeletal muscle injury: Control, passive recovery rats; HYP, rats exposed to IHH after muscle damage; and EHYP, trained rats that performed light aerobic exercise sessions in addition to IHH. Hematocrit, RBC count and hemoglobin were only elevated in the late stage of recovery in HYP (13%; 14% and 8%) and EHYP (18%; 13% and 15%) groups. Blood viscosity increased about double for EHYP rats. It is concluded that intermittent exposure to hypobaric hypoxia in combination with light aerobic exercise in normoxia has an erythropoietic effect, but also provides advantageous hemorheological conditions for the perfusion of damaged muscle.

Analysis of fluoroquinolones in animal feeds by liquid chromatography with fluorescence detection.

An analytical method for the analysis of six fluoroquinolones (FQs) in animal feeds was developed. The sample treatment consists of a simple and rapid extraction of the analytes by manual shaking with an acetonitrile-water mixture containing hydrochloric acid without further sample cleanup. Matrix effects were minimized by diluting the extract with water. Determination was carried out by liquid chromatography using fluorimetric detection. The method was validated in-house in four different feed matrices (poultry, cow, pig, and lamb feed). Mean recoveries ranging from 80 to 105%, with relative standard deviations below 12%, were achieved from spiked animal feed samples on the 0.2-2.0 µg/g level. No relevant differences were observed between the studied feeds, this ensuring that the method was reliable for a wide variety of feed matrices. Decision limit and detection capability values are below 0.08 and 0.13 mg/kg, respectively, for most FQs. The results obtained demonstrate the feasibility of the analytical method developed for a routine use to control the illegal use of these substances in feeding stuffs.