Animals and environmental conditions

Thirty male Wistar rats (Rattus norvegicus albinus) were housed from 45 days of age in polypropylene cages (40 cm length, 30 cm width, 15 cm height, with five animals per cage) with food and water provided ad libitum. Environmental conditions were maintained throughout the experiment, including temperature (22 ± 2 °C), relative humidity (45-55%), noise (< 85 decibels), and a 12-hour light/dark cycle, with lights off at 06:00 h (zeitgeber time 12). Incandescent lamps (565-590 nm; 60 lx, measured with a lux meter) were used during the 12-hour light cycle. Reflectors were covered with a red filter (ROSCO brand, model # fire19; >600 nm; <15 lx; [17] to conduct experimental interventions with the rats during the dark cycle. The experimental procedures were conducted in accordance with Ethical Principles in Animal Research and approved by the Animal Ethics Committee of an institution affiliated with one of the authors. All procedures followed the ARRIVE guidelines 2.0.

Experimental design

After aquatic adaptation and the endurance swimming test, all animals performed the endurance swimming exercise. They were then randomly allocated into six groups (n = five per group), as follows: Exercised (E) and treated with melatonin (M, 10 mg.kg-¹), euthanized 1 h (EM1), 3 h (EM3), or 24 h (EM24) post-exercise; or Exercised and treated with vehicle solution (Ex, 0.9% NaCl and ethanol < 0.1%), euthanized 1 h (Ex1), 3 h (Ex3), or 24 h (Ex24) after the endurance swimming exercise (Fig. 1). No formal allocation concealment was applied during group assignment and data analyses were performed without blinding to group allocation. The sample size was calculated using GPower 3.1 software. No inclusion/exclusion criteria were established.

Fig. 1

Experimental design of the study illustrating the steps from beginning to end of the experiment. ZT: zeitgeber time; iMAC: intensity of maximum aerobic capacity; BM: body mass; Ex1: Animals treated with vehicle solution and euthanized 1 h after exercise; Ex3: Animals treated with vehicle solution and euthanized 3 h after exercise; Ex24: Animals treated with vehicle solution and euthanized 24 h after exercise; EM1: Animals treated with melatonin and euthanized 1 h after exercise; EM3: Animals treated with melatonin and euthanized 3 h after exercise; EM24: Animals treated with melatonin and euthanized 24 h after exercise

Exercise protocol

At 80 to 89 days of age, the rats underwent a structured adaptation period to the aquatic environment and swimming protocol. They were exposed to water for 5 to 20 min per session, with progressively increasing depth (10-80 cm) and load (0 or 4% of body mass), in cylindrical, opaque tanks measuring 100 cm in height, 80 cm in water depth, and 30 cm in diameter. At 90 days of age, each animal performed an incremental test (IT) to determine the intensity corresponding to maximal aerobic capacity (iMAC). The IT involved progressive increases in load to identify the point at which a disproportionate rise in blood lactate concentration occurred, following the method described by Beck et al. [18]. The protocol consisted of 5-min stages with overloads ranging from 3.0 to 7.0% of body mass (% bm), increasing by 0.5% intervals, with the weights secured to the animals’ chests using elastic straps. After each stage, blood samples (25 µL) were collected from the distal portion of the tail to determine lactate concentration. The relationship between exercise intensity and blood lactate levels was plotted and the iMAC was determined by interpolation on the Y-axis. This test served to establish the intensity for the subsequent exercise session. At 100 days of age, all animals (body mass: 396.07 ± 4.40 g, with comparable body mass between the melatonin and vehicle groups, indicating appropriate standardization of the experimental conditions) performed a continuous 60-min swimming session at 90% of iMAC (zeitgeber time 12), the swimming endurance test, under the supervision of the same researcher (Fig. 1).

Determination of blood lactate concentration

During the swimming endurance test, blood samples (25 µL) were collected from the animals’ tails using heparinized and calibrated glass capillaries. The samples were placed into plastic microtubes (1.5 mL) containing 400 µL of trichloroacetic acid (4%), mixed, and stored at 4 °C. After shaking and centrifugation (3000 rpm for 3 min), 50 µL of the supernatant was extracted and transferred to a 96-well microplate where 250 µL of reactive solution (glycine/EDTA and hydrazine hydrate stock), NAD (β-nicotinamide adenine dinucleotide), and LDH (L-lactic dehydrogenase, bovine heart), prepared for immediate use, were added. The samples were subjected to spectrophotometric measurements (Spectramax i3, Molecular Devices; San José, CA, USA) at 340 nm to compare sample values to a standard curve constructed from a serial dilution of 1-15 mmol.L-¹ of L-lactate [19].

Melatonin administration

Melatonin (Sigma Aldrich Chemical Corporation; St. Louis, MO, USA; M-5250, > 98%) was dissolved in ethanol (< 0.1%) and diluted in saline (0.9% NaCl) for administration at a dose of 10 mg.kg-¹ [5]. The preparation was carried out just prior to use and was stored in an amber bottle wrapped in aluminum foil. Control animals received an equal volume of the vehicle alone (0.9% NaCl and ethanol < 0.1%). Administration was intraperitoneal and occurred immediately after the endurance swimming exercise.

Euthanasia, obtention, and processing of biological samples

The animals were euthanized 1, 3, or 24 h after the end of the experimental procedures by decapitation, in accordance with the guidelines of the American Veterinary Medical Association [20]. After euthanasia, an aliquot of approximately 2.0 mL of blood was obtained and allowed to rest for 20 min (4 °C) before subsequent centrifugation (15 min, 3000 rpm, 10 °C). These serum samples were stored in different aliquots to avoid freeze-thaw cycles and were kept at -20 °C for further analysis. Then, skeletal muscle tissues (soleus, gluteus maximus, red and white portions of gastrocnemius) and liver were collected, immediately frozen in liquid nitrogen, and stored at -80 °C for further analyses.

Blood glucose analysis

To determine blood glucose levels, 3 µL of serum was mixed with the kit reagent (300 µL; LaborLab; Guarulhos, SP, Brazil) and incubated for 25 min (25 °C). The kit reagent is composed of GOD (≥ 15 kU.L-1), POD (≥ 2 kU.L-1), 4-AAT (0.5 mmol.L-1), phosphates (pH = 7.5, 250 mmol.L-1), and phenol (5 mmol.L-1). The glucose absorbance was determined using a spectrophotometer (SpectraMax i3, Molecular Devices; San José, CA, USA) at 505 nm according to the kit’s guidelines. Blood samples were collected into standard serum tubes and immediately kept on ice (4 °C). To minimize glucose degradation, samples were allowed to rest for 20 min before centrifugation at 10 °C (3000 rpm, 15 min). After centrifugation, serum was aliquoted and stored at -20 °C until analysis, avoiding freeze-thaw cycles. Each group had five animals for the present analysis (Fig. 2).

Blood triglyceride analysis

To determine blood triglyceride levels, 3 µL of serum was mixed with the kit reagent (300 µL; LaborLab; Guarulhos, SP, Brazil) and incubated for 20 min (25 °C). The kit reagent is composed of buffer (pH = 6.8, 50 mmol.L-1), chlorophenol (2 mmol.L-1), lipoprotein lipase (≥ 800 U.L-1), GK (≥ 500 U.L- =1), GPO (≥ 1500 U.L-1), POD (≥ 900 U.L-1), ATP (2 mmol.L-1), and 4-AF (0.4 mmol.L-1). The triglyceride absorbance was determined using a spectrophotometer (SpectraMax i3, Molecular Devices; San José, CA, USA) at 505 nm according to the kit’s guidelines. Each group had five animals for the present analysis (Fig. 2).

Determination of muscle glycogen content

The glycogen content in skeletal muscle (gluteus maximus, red and white portions of the gastrocnemius) and the liver was determined as described by Dubois et al. [21]. Both skeletal muscle (250 mg) and liver (500 mg) were initially immersed in potassium hydroxide (30%; Êxodo Científica; Sumaré, SP, Brazil) and then mixed with a saturated sodium sulfate solution (20 µL; Dinâmica Química Contemporânea Ltda; Indaiatuba, SP, Brazil) and ethanol (70%) for glycogen precipitation. The samples were homogenized with phenol (10 µL; Êxodo Científica; Sumaré, SP, Brazil) and sulfuric acid (2 mL; Dinâmica Química Contemporânea Ltda; Indaiatuba, SP, Brazil), and heated in a water bath for 5 min (85 °C). Finally, the absorbance was measured using a spectrophotometer (Hach Company, Loveland, CO, USA; 490 nm), and the glycogen content was calculated using a calibration glucose curve. Each group had five animals for the present analysis (Fig. 2).

Determination of muscle triglyceride content

To determine triglyceride content, liver and skeletal muscle samples (soleus, gluteus maximus, red and white portions of the gastrocnemius) (100-200 mg) were macerated using liquid nitrogen and then transferred to plastic tubes containing Triton X-100 (1%) at the ratio of 200 mg of tissue per 1 mL of Triton. Subsequently, the samples were homogenized overnight at 4 °C using magnetic stir bars (5 × 3 mm). After homogenization, the samples were centrifuged at 4000 rpm for 10 min, and 10 µL of the supernatant was pipetted into a 96-well microplate and mixed with the kit reagent (200 µL; LaborLab; Guarulhos, SP, Brazil). The mixture was then incubated for 20 min at 25 °C. Triglyceride absorbance was measured using a spectrophotometer (SpectraMax i3, Molecular Devices; San José, CA, USA) at 505 nm, according to the kit’s guidelines. Each group had five animals for the present analysis (Fig. 2).

Acylcarnitine extraction and quantification

Acylcarnitine determination was conducted using flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS), following the established methodology of Sarafian et al. [23], with tissue-specific adaptations previously validated in our laboratory [22]. The complete analytical workflow, from tissue extraction through data acquisition and quantification, is schematically represented in Fig. 2. Tissue samples (50-150 mg) were thawed at room temperature and extracted with isopropanol pre-cooled to 4 °C in a ratio of 1:10 (mg tissue:µL solvent). This ratio proved essential for efficient protein precipitation while retaining lipid intermediates intact. After initial vortexing (30 s), samples underwent three freeze-thaw cycles in liquid nitrogen (1 min intervals), each followed by thorough vortexing to ensure complete cell membrane disruption in muscle and hepatic tissues. Sonication was performed for 10 min at 4 °C (40-45 kHz ultrasonic frequency), followed by a final 30 s vortex. After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant (400 µL) was carefully transferred to a clean tube, avoiding the protein pellet, and evaporated to dryness under a gentle stream of nitrogen gas. The dried extract was reconstituted in 250 µL of mobile phase solution (acetonitrile: water, 82:18 v/v, acidified with formic acid 0.1%) containing the internal standard L-Leucine-5,5,5-d₃ at 0.73 µg.mL-¹ to correct for potential ionization suppression artifacts.

Data acquisition was conducted on a Waters Xevo TQD triple quadrupole mass spectrometer interfaced with a Shimadzu LC-20AD pump system, utilizing procedures adapted from Moura [24]. Rather than employing liquid chromatography with a packed column, flow injection analysis (FIA) was selected to enable rapid analysis (4 min per sample) without compromising specificity. The mobile phase flow was programmed as a gradient from 0.01 to 0.50 mL.min-¹. Positive electrospray ionization (ESI+) was used to generate molecular ions. Acylcarnitine species (C0-C18) were quantified via multiple reaction monitoring (MRM), with each analyte monitored using precursor-to-product ion transitions specific to the carnitine moiety (m/z 85.0), as detailed in Supplementary Table S1. As depicted in Fig. 2, this analytical pipeline integrated four critical stages, as follows: metabolite extraction under controlled temperature conditions, high-throughput FIA-MS/MS data acquisition, MRM-based quantification targeting the diagnostic carnitine fragment, and compound identification via TargetLynx software. Quantification was performed by measuring the peak area ratio of analyte to internal standard and referencing this value against a 7-point calibration curve spanning 0.1-100 µmol.L-¹ (R² consistently > 0.99). Tissue concentrations were normalized to wet tissue weight and reported in µmol.g-¹. Quality control (QC) samples were prepared from pooled tissue extracts and interspersed throughout each analytical batch (every 10 injections) to monitor instrumental performance. Results were accepted only when QC values fell within ± 15% of the expected concentration, ensuring data integrity throughout the run.

Fig. 2

Schematization of the analysis methods of the present study. Blood glucose analysis using Spectrophotometer; Blood triglyceride analysis; Determination of muscle glycogen content; and Determination of muscle triglyceride content using spectrophotometer; Determination of acylcarnitine using tandem mass-spectrometry analysis. Analytical workflow for acylcarnitine profiling in muscle and liver tissues. The complete pipeline comprised four sequential stages, as follows: (i) Tissue extraction through mechanical (freeze-thaw cycles in liquid nitrogen, 1 min per cycle) and ultrasonic (40-45 kHz, 4 °C) disruption followed by centrifugation to obtain lipid-rich supernatants; (ii) Flow injection analysis (FIA) coupled to triple quadrupole mass spectrometry (Waters Xevo TQD; Shimadzu LC-20AD pump) enabling rapid sample throughput (4 min per injection) without compromising specificity; (iii) Multiple reaction monitoring (MRM) quantification of acylcarnitine species (C0-C18) via optimized precursor-to-product ion transitions terminating at the diagnostic carnitine head-group fragment (m/z 85.0); and (iv) Peak area integration and compound identification using TargetLynx software with reference to MRM parameters, detailed in Supplementary Table S1. Both endurance exercise Melatonin (EM) and Exercise control (Ex) groups underwent identical processing protocols to ensure analytical reproducibility and minimize bias

Statistical analysis

The data are presented as mean ± standard error of the mean. Normality was verified using the Shapiro-Wilk test (p > 0.05). Data normality was assessed using the Shapiro-Wilk test. Variables that did not meet normality assumptions were transformed using an inverse (1/x) transformation, after which parametric statistical analyses were applied. A two-way ANOVA followed by a Newman-Keuls post hoc test was applied to analyze the effects of melatonin (two levels: with or without melatonin) and the effect of time (three levels: 1, 3, and 24 h). Effect size analysis (E.S) [25] was used as a complementary test. The thresholds for small, moderate, and large effects were 0.20, 0.50, and 0.80, respectively. All large effect sizes were included in the figures. A significance level of 5% and Statistica 7.0 (StatSoft, Inc.; Tulsa, OK, USA) were used for all analyses. In addition, the Integrated Biomarkers Responses (IBR) was used according to the instructions of Beliaeff & Burgeot [26] to observe all amino acids in a single analysis, through the area calculation of a star plot for each acylcarnitine, generating the IBR-value. The vehicle group was always the control of the melatonin group within each time point. There was no exclusion of data in the present analyses. Given the number of dependent variables analyzed, statistical tests were conducted in an exploratory manner and p-values were not adjusted for multiple comparisons.