The plants to produce EOLS and EOCC were cultivated in Petrolina, PE, Brazil. The collections of the fresh leaf samples were carried out in autumn 2018. The extraction of essential oils was carried out by steam distillation using a Clevenger-type apparatus (British Pharmacopoeia 2007) for 2 h in the Natural Products Laboratory of the Instituto Federal do Sertão Pernambucano, Petrolina, PE, Brazil. The determination of their chemical compounds was carried out by gas chromatography/mass spectrometry (GC-MS) in the Multi-User Characterization and Analysis Laboratory of the Universidade Federal da Paraíba, João Pessoa, PB, Brazil.
The GC-MS analysis was performed using a gas chromatograph coupled to a mass selective detector (Shimadzu GCMS-QP2010 Ultra – Barueri, SP, Brazil). The capillary column was DB-5MS (Agilent technologies; 30 m x 0.25 mm x 0.25 µm). Helium (99.99%) was used as the gas carrier. The conditions used were as follows: a constant flow of 1.1 mL min−1; injection volume of 1.0 µL with injector split ratio of 1:10 (250°C); electron impact mode at 70 eV; ion source temperature at 280°C; and transfer line temperature at 260°C. The oven temperature was programmed to 60°C, with a progressive increase of 3°C min−1 until it reached 240°C. A mixture of linear hydrocarbons (C8H18 – C20H42) was injected under the same experimental conditions.
The identification of the essential oils constituents was realized by comparing retention indices in the equipment database (NIST, 2008) and Kovats retention index. The essential oils used in this study were the same as those used by Felix e Silva et al. (in press). The major components found for EOLS were carvacrol (44.50%), p−cymene (14.06%), and thymol (7.99%); for EOCC, they were α−citral (73.56%), myrcene (12.65%), and geraniol (3.51%).
Location and animals
Two different sizes of fish were tested, termed here as juveniles I (0.82 ± 0.02 g; 3.24 ± 0.05 cm) and juveniles II (2.40 ± 0.08 g; 4.78 ± 0.05 cm). Initially, we evaluated sedation, anesthesia induction, and anesthetic recovery times with EOLS or EOCC in juveniles I and II (n total = 144). Afterwards, a second experiment was performed to evaluate the effects of these essential oils on the transportation of juveniles II (n total = 48). Finally, a third experiment, evaluating VR, was conducted with juveniles II (n = 40). For the second and third experiments, only juveniles II were selected, because juveniles I were smaller, severely impairing the analysis of blood glucose, muscle glycogen, gill histology, and VR.
The experiments were carried out in the Laboratory of Study and Physiology of Aquatic Fauna of the Universidade Federal da Bahia (UFBA) under registration no. 03-2019 at Ethical Committee of the Biology Institute of the UFBA. A total of 232 juveniles from a commercial fish farm (Lauro de Freitas, BA, Brazil) were used.
Before testing, the fish were kept in two 500-L tanks with constant aeration and mechanical and biological filters. Juveniles were fed four times a day (commercial diet, 50% crude protein, Sera Discus Color Red, Germany) for an acclimation period of 10 days until apparent satiety. Feeding was suspended 24 h before the beginning of the experiments.
Induction and anesthetic recovery
The procedure involved transferring juveniles (I and II) to aquaria containing 2 L of water and EOLS or EOCC at concentrations of 10, 25, 50, 75, 100, 150, 200, and 250 mg L−1 diluted 1:10 with absolute ethanol (1:10). Densities for EOLS and EOCC were, respectively, 0.93 and 0.90 g cm−3. A control group was transferred to aquaria containing only ethanol (2,250 mg L−1) at a concentration equivalent to the dilution used for 250 mg L−1 of both essential oils. A second control group with fish kept in aquariums containing only water was also performed.
In each treatment, eight juveniles I and eight juveniles II were used. For sedation (partial loss of balance and erratic swimming) and anesthesia (complete loss of balance and cessation of swimming) evaluation, two fish were simultaneously placed in each aquarium (adapted from Oliveira et al. 2019a). The fish were evaluated for up to 30 min, and each fish was used only once. Subsequently, the juveniles were transferred to a 5-L aquarium containing anesthetic-free dechlorinated water to measure the post-anesthetic recovery time (swimming, equilibrium and, behaviors similar to those of the fish kept in the maintenance tanks) (adapted from Sena et al. 2016). Survival was monitored up to 72 h after anesthetic induction.
Another 48 freshwater angelfish juveniles II were used, and five different treatments (n = 8 fish per treatment) were evaluated: fish transported without essential oil (control) or with the addition of 10 and 15 mg L−1 of EOLS or EOCC. Duration of transport was 8 h. In a pilot experiment, concentrations of 10, 15, 20 and, 25 mg L−1 in plastic bags were used, and 20 and 25 mg L−1 of both essential oils showed potential to induce fish to anesthesia after 8 h of transport. Therefore, it was decided to use only the concentrations of 10 and 15 mg L−1 in this experiment, which triggered sedation, but not anesthesia, in this same transport time. Another eight juveniles were not subjected to transport and designated ‘before transport’.
The fish were placed in 2-L plastic bags (20 x 40 cm) closed with elastic straps containing 0.5 L of water and the remainder was filled with pure oxygen. The plastic bags were stored in plastic boxes for transportation in a closed car under ambient temperature (30 ºC), simulating the routine transport of ornamental fish.
The physical and chemical parameters of the water were measured before (0 h) and after transportation (8 h), in quadruplicate. Dissolved oxygen and temperature were measured with an oxygen meter (YSI 200A, Yellow Springs, USA) and pH meter (HmPA-210P, Piracicaba, Brazil). The hardness, carbon dioxide, chlorine and nitrite were measured with a commercial kit (JBL GmbH and Co. KG, Neuhofen, Germain) and total ammonia with another commercial kit (Alfatecnoquímica, Florianópolis, Brazil). Un-ionized ammonia levels were calculated according to a conversion table specific to freshwater.
Blood, muscle, and gill samples were collected from eight fish per treatment before (0 h) and after transportation (8 h). Blood was collected by venocaudal puncture using heparinized syringes to determine blood glucose levels, which were analyzed using microfilm strips and a digital glucometer (Accu-Chek, Mannheim, Germany) immediately after blood collection. Then, juveniles II were euthanized with a lethal dose of benzocaine hydrochloride (250 mg L−1) (CONCEA, 2013) to collect gills and muscle.
Muscle glycogen was determined according to Bidinotto et al. (1997). The muscle samples (100 mg) were collected and preserved at -80 ºC until analysis. These samples were homogenized in buffer (10 mM phosphate/20 mM tris-pH 7.0) using a mechanical homogenizer (Marconi MA039, Piracicaba, SP, Brazil) before centrifugation at 600 x g for 3 min (4°C). The reading in a spectrophotometer (UV-Vis, Quimis®, Diadema, Brazil) was done at 480 nm.
Gills destined for histopathologic evaluation were fixed in 10% neutral buffered formalin for 18 h and then washed and conditioned in 70% ethanol. Subsequently, a gill arch in each fish was removed and subdivided into three samples for histological processing (n = 8 fish per treatment). The samples were then dehydrated in an increasing sequence of ethyl alcohol and xylol for 1 h, immersed in paraffin for 3 h, and then embedded in paraffin blocks at 56°C. The blocks were stored in a refrigerator (4 oC) for 12 h to cut posteriorly in a microtome (Zeiss Hyrax M15, Germany) with cross-sections (3 µm). After this, they were fixed on slides and submitted to staining using the hematoxylin-eosin technique (Luna 1968).
The slides were mounted in Entellan® (Merck, Darmstadt, Germany) and analyzed with a 40x objective light microscopy. Ten microphotographs per replicate were examined, totaling 80 micrographs per treatment. A digital camera (Axio Cam ICc3, Zeiss, Germain) coupled to the microscope (Axio Scope, Zeiss) and a computer was used; for analysis, we used the Axion Vision Le® 4.8.2 (Zeiss) software.
Analysis and quantification of the microphotographed samples were performed using the Cavalieri method (Mandarim-de-Lacerda 2003; Fiedler et al. 2020), which consists of an isotropic point counting system derived from a grid of points superimposed on the histological image to be analyzed. Then, the volume density (Vv) of the histological components was estimated using the following equation (adapted from Mandarim-de-Lacerda 2003):
VV (%) = PP/PT x 100, where PP is the part number of test points counted on a section in relation to the total points and PT is the total number of test points lying over the references space.
A 21.0 x 29.7-cm rectangular grid of stitches was used to count the points, with 1 cm between the points to estimate the VV of gill tissue, secondary lamella (SL), and filament epithelium. The following VV of gills lesions were also estimated using the same grid size, according to Monteiro et al. (2008): hyperplasia, SL fusion, epithelial displacement, ionocyte proliferation, mucous cell proliferation, edema, hemorrhage, aneurysm, fibrosis (fibrous connective tissue), and necrosis.
Photomicrographs were also used to quantify the histopathology of gills, which were classified into three stages: I (alterations that do not affect organ functions), II (mild to moderate alterations that do not affect organ functions), and III (severe or irreversible alterations). The histopathological alteration index (HAI) was calculated according to Poleksic and Mitrovic−Tutundzic (1994), using the following equation:
HAI = Ʃ ai+ 10 Ʃ bi+ 102Ʃ ci, where HAI is the degree of alterations in a single gill, a = first-stage alterations, b = second-stage alterations, and c = third-stage alterations.
The HAI values (%) were considered in five categories: < 0.10 = functionally normal gills; 0.11−0.20 = slightly to moderately damaged gills; 0.21−0.50 = moderately to heavily damaged gills; 0.51−1.00 = severe injuries on gills, and > 1.00 = irreparably damaged gills (adapted from Poleksic and Mitrovic−Tutundzic 1994).
Ventilatory rate (VR)
Juveniles II (n = 40, one individual per each 5-L tank), which were not used in the previous experiments, were used to assess the VR. The fish were exposed for 8 h to the same concentrations used in the transport procedure (0 (control), 10 and 15 mg L−1 of each essential oil). The evaluation times were: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h. Each fish was used only once. The VR was quantified by visually counting 20 successive opercular/mouth movements, measuring the elapsed time with a stopwatch (adapted from Hohlenwerger et al. 2017).
All data are expressed as the mean ± SEM (standard error of the mean). All data were subjected to Levene’s test and the Shapiro-Wilk test to verify the homogeneity of the variances and the normality of the data, respectively. Sedation and anesthetic activity were evaluated by power regression analysis (concentration × time). Two-way analysis of variance (ANOVA) was performed (time X treatment), followed by Tukey's post-hoc test for VR analysis. One-way ANOVA was performed followed by Tukey's post-hoc test to compare treatments after transport or Dunnett's test to compare these treatments with the treatment before transport. Significance was set at a critical level of 95% (p < 0.05).