Temperature-induced changes in the hematological and biochemical parameters of Nile tilapia anesthetized with Ocimum basilicum

Temperature-induced changes in the hematological and biochemical parameters of Nile tilapia anesthetized with Ocimum basilicum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Temperature-induced changes in the hematological and biochemical parameters of Nile tilapia anesthetized with Ocimum basilicum Paola Capistrano Santos, Emilly Monteiro Lopes, Arlene Sobrinho Ventura, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4713885/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The study aimed to evaluate the effect of temperatures of 23 and 27 ºC on the hematology of Nile tilapia juveniles ( Oreochromis niloticus ) subjected to anesthesia using the essential oil of basil ( Ocimum basilicum ) at a concentration of 250 mg L -1 . A total of 112 fish were divided into 4 treatments and distributed as follows: fish anesthetized with 250 mg L -1 at a temperature of 23ºC (n=42); fish anesthetized with 250 mg L -1 at a temperature of 27ºC (n=42); and for each temperature, a control group fish exposed to diluent solution (95% ethanol) (n=14, seven per temperature); a control group fish (water) (n=14, seven per temperature). They were exposed to 10 min for both temperatures. Blood collection was performed after the anesthetic induction at each temperature to determine the hematocrit, hemoglobin concentration (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), relative values of leukocytes and absolute values of leukocytes and thrombocytes, in addition to evaluating plasma chloride and total calcium ions. The analysis of chemical composition by gas chromatography coupled with mass spectrometry (GC-MS) demonstrated methyl chavicol (70.04%) and linalool (24.59%) as the major components of basil essential oil. Fish anesthetized at a temperature of 23 ºC showed lower neutrophil counts (p<0.05) compared to the other tested groups. For the temperature of 27 ºC, values of MCV, MCHC, and RBC values decreased (p<0.05) following exposure to basil essential oil. The calcium ion showed a statistical difference (p<0.05) between the treatments about the water control group. It is concluded that basil essential oil presented a positive anesthetic effect. The use of O. basilicum essential oil as an anesthetic in Nile tilapia was the best at 23ºC, causing less hematological and biochemical stress. Aquaculture Hematology Essential oil Anesthesia Stress Figures Figure 1 1 Introduction The Nile tilapia stands out as the main fish species worldwide. It has high versatility, adapting to semi-intensive and intensive farming, and demonstrates the ability to adapt to various production technologies, including recirculation systems and the biofloc system (Suáres-Puerto et al. 2023). However, indifferent production systems, and management activities such as transportation, vaccination, and biometrics promote physiological changes, including stress in the animals (Silva et al. 2015a; Medeiros-Junior and Brito 2021). Stress is described as alternations in the physiological state of fish due to environmental conditions that pose a threat to their survival (Ross and Ross 2008). On this view, anesthetics have been used in aquaculture since the 20th century to promote safe handling practices for the animals, being effective in reducing stress and pain (Priborsky and Velisek 2018; Bianchini et al. 2019) Roohi and Imanpoor (2015) emphasize that stressful situations during production procedures result in reduced immune response and growth, increasing the vulnerability of fish to the onset of diseases. The use of anesthetics in fish is influenced by biological and physiological variables, resulting in varied responses even with the same dose of anesthetic (Gressler et al. 2020). In this regard, temperature is a significant variable due to its influence on anesthetic concentration and induction (Medeiros-Junior and Brito 2021). According to Aydın et al. (2015), the time of induction and anesthetic recovery has a direct relationship with the metabolism of fish. The physiological processes of fish are associated with temperature, playing a significant role in the relationship between absorption and elimination of anesthetic compounds. Furthermore, temperature variation can increase the metabolism of fish, promoting increased blood flow in the gills (Zahl et al. 2011). Essential oils can be obtained from the distillation of plants and represent an efficient and safe alternative for conducting anesthetic procedures in fish (Barbas et al. 2017; Aziz et al. 2018; Souza et al. 2019; Gurkan and Hayaloglu, 2023). The genus Ocimum sp has more than 150 cataloged species (Kholiya et al. 2022). Ocimum basilicum , belonging to the Lamiaceae family, is commonly known as basil and has great economic importance in the pharmaceutical and food industries (Martin et al. 2010; Pravuschi et al. 2010; Gurkan and Hayaloglu 2023). In aquaculture, the essential oil of O. basilicum is used due to its antimicrobial, antioxidant, sedative, and anesthetic properties (Souza et al. 2019). The effects provided by anesthetic induction with the essential oil have been demonstrated in studies with tambaqui ( Colossoma macropomum ) (Ventura et al. 2021a), tambacu (male Piaractus masepotamicus x female Colossoma macropomum ) (Limma-Netto et al. 2016), and Nile Tilapia ( O. niloticus ) (Ventura et al. 2020). The use of essential oils for handling procedures in fish farming, in addition to reducing stress, provides reduced sensitivity to visual and mechanical stimuli and ensures the preservation of water quality variables (Aydin and Barbas 2020). Thus, the essential oil of O. basilicum is considered a promising anesthetic for use in fish farming. However, more information is needed on the effect of temperature during anesthesia on hematological parameters. Therefore, the essential oil of O. basilicum is considered a potential anesthetic for use in fish farming due to its anesthetic and sedative properties. However, this essential oil is not yet regulated for use due to the lack of knowledge about possible physiological alterations caused by it (Ventura et al. 2020). The immune stress response is characterized by a series of physiological changes, including the release of the neuroendocrine hormones catecholamines and cortisol into the circulatory system, which cause variations in the balance of plasma electrolytes and, in freshwater fish, modify gill permeability, resulting in the loss of blood electrolytes and osmoregulatory disturbances (Silva et al. 2012; Gressler et al. 2015). The present study aimed to evaluate the hematology of juvenile Nile tilapia subjected to anesthesia with the essential oil of O. basilicum at different temperatures. 2 Material and methods The experiment was conducted at the Laboratory of Aquatic Organism Health (AQUOS) at the Federal University of Santa Catarina (UFSC). All animal procedures were approved by the Ethics Committee on Animal Use (CEUA/UFSC nº 7363211122). 2.1 essential oil of Ocimum basilicum The essential oil was commercially acquired (Phytoterápica®, Nova Cantareira, Brazil). Chemical composition analyses of the essential oil were conducted using gas chromatography coupled with mass spectrometry (CG-MS) according to Ventura et al., (2023). 2.2 animals The Nile tilapia juveniles were from the Experimental Station of Epagri in Itajaí, EEI/EPAGRI-SC with an average weight ranging from 20 to 30 g. They were transported to AQUOS, where they remained for seven days in quarantine, distributed in a circular polyethylene tank with a total volume of 1 m 3 at a density of 4.38 kg/m 3 . Subsequently, the fish were distributed into 10 circular polyethylene tanks of 0.1 m 3 each for 15 days in the recirculation water system, with a settler, mechanical filter, and biological filter containing porous sponges, for acclimatization at a density of 0.04 kg/0.1 m 3 . The tanks were maintained under constant aeration, temperature control with heaters, and a water renewal rate of 10% per day. The monitoring of water quality variables including temperature (23.19 ± 0.09 ºC; 26.98 ± 0.31 ºC), dissolved oxygen (6.29 ± 0.53 mg L − 1 ; 6.49 ± 0.18 mg L − 1 ), and pH (7.28 ± 0.19; 7.27 ± 0.07) was conducted daily using a multiparameter meter (Hanna HI98190®). Total ammonia (1.04 ± 0.78 mg L − 1 ; 1.58 ± 1.16 mg L − 1 ), toxic ammonia (0.003 ± 0.002 mg L − 1 ; 0.004 ± 0.003 mg L − 1 ), and nitrite (0.54 ± 0.57 mg L − 1 ; 0.71 ± 0.58 mg L − 1 ) concentrations were determined using commercial colorimetric kits (Alfakit®). The animals were fed three times a day with Acqua Line® 1.7 mm (Crude protein 46%, Ether extract 8%, Crude fiber 3%, Ash 14%, Calcium 1.5–3%, Phosphorus 1%) until apparent satiety and subjected to a 24 h fasting period before the experiment. 2.2 Experimental design Juvenile Nile tilapia (64.99 ± 13.90 g and 15.63 ± 1.16 cm) (n = 112) were randomly distributed in 30 L aquariums and divided into 4 treatments: fish anesthetized with 250 mg L − 1 at a temperature of 23ºC (n = 42); fish anesthetized with 250 mg L − 1 at a temperature of 27ºC (n = 42); and for each temperature, a control group fish exposed to diluent solution (95% ethanol) (n = 14, seven per temperature); a control group fish (water) (n = 14, seven per temperature). After immersion bath with essential oil for 10 min, the fish exposed to EO were transferred to fourteen 30 L aquaria, with six fish per aquarium, along with the ethanol control group fish and water control group fish, with seven fish per aquarium free of anesthetic and diluent solution, at temperatures of 23 and 27ºC. The fish were not fed during the course of the experiment, and animal survival was recorded. The anesthetic concentration of the essential oil used in the present study was defined based on a previous study that determined the concentration capable of inducing anesthesia in less than 180 seconds, followed by a recovery time of less than 300 seconds according to the criteria of ideal anesthetic efficacy by Ross and Ross (2008). 2.3 blood collection Blood samples were collected after the anesthetic induction at each temperature. For the control groups, blood samples were collected from all fish at both temperatures (23 and 27ºC) without the use of anesthesia. The fish were individually captured and restrained with a damp cloth. Blood was collected by puncturing the caudal vein using syringes containing 10% EDTA anticoagulant solution (ethylenediaminetetraacetic acid). Blood smears were prepared in duplicate and stained with May Grunwald Giemsa-Wright (Ranzani-Paiva et al., 2013) for the determination of absolute values of leukocytes and thrombocytes, as well as relative values of leukocytes. For the determination of the total number of erythrocytes, a 5 µL aliquot of blood was diluted (1:200) in Dacies’s fluid (sodium citrate, 37–40% formaldehyde, toluidine blue) modified according to Blaxhall and Daisley (1973), then erythrocyte quantification was performed using a Neubauer chamber. A blood aliquot was used to determine the hematocrit percentage by the microhematocrit method; furthermore, another portion was used for the analysis of hemoglobin concentration with commercial reagents (LabTest Diagnóstica®). Hematimetric indices mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Ranzani-Paiva et al. (2013). 2.3.1 Biochemical analysis of chloride and total calcium ions Blood samples were centrifuged at 3000 rpm for five minutes to obtain plasma for the determination of plasma ions chloride (Cl − ) and total calcium (Ca 2+ ). The tests were performed using colorimetric methodology with commercial reagents (LabTest Diagnóstica®). In the testes, 10 µL of pooled plasma (six fish from each aquarium) were used, following the manufacturer’s kit instructions. 3 Statistical analysis For hematological and biochemical analyses, the data were subjected to Shapiro-Wilk and Levenes tests to assess normal distribution and variance homoscedasticity, respectively. Non-homogeneous data were log 10-transformed to achieve homogeneity. Subsequently, the data were subjected to One-way ANOVA analysis, and when appropriate, means were separated by Tukey’s test (p < 0,05). The analyses were performed using Statistica 10.0 software . The significance level was set at 5%. 4 Results 4.1 Essential oil composition The main components found in the essential oil were methyl chavicol (70.04%) and linalool (24.59%) as major constituents, while the minor constituents totaled 5.37% (Table 1 ). The detection and quantification limits for methyl chavicol were 0.007 µg kg − 1 and 0.020 µg kg − 1 , respectively. Table 1 Compounds present in basil essential oil O. basilicum essential oil Composition Total percentage (%) Methyl Chavicol 70.04 Linalool 24.59 δ-Carene 1.20 1,8-Cineol 1.14 Eugenol 1.25 γ-Elemene 0.88 Spatulenol 0.90 Total 100 4.2 survival analysis after anesthetic induction During the experiment, a mortality rate of 95.24% was recorded for fish subjected to anesthetic induction with O. basilicum essential oil at a water temperature of 23ºC. At a temperature of 27ºC, a survival rate of 76.6% was recorded for fish subjected to anesthetic induction. In the water control group, the survival rate was 85.72%. In the 95% ethanol control group, a survival rate of 100% was recorded for the fish at both temperatures. 4.3 Hematologial parameters The water temperature at 23ºC did not affect (p > 0.05) the hematimetric indices of Nile tilapia anesthetized with O. basilicum essential oil. Ethanol did not affect the erythrocyte parameters of the species. In the white series, neutrophil values were lower in fish from the 95% ethanol group (p < 0.05) (Table 2 ). After exposure to O. basilicum essential oil at a water temperature of 27ºC, a statistical difference (p < 0.05) was observed in MCV, MCH, and the number of RBC between the fish subjected to the anesthetic and 95% ethanol. The fish subjected to anesthetic induction with O. basilicum oil had higher MCV values than the fish in the water and 95% ethanol control groups (Table 3 ). Similarly, the same pattern was observed for MCH, where the values in the fish subjected to anesthetic induction were slightly higher than those in the water and ethanol control groups. The RBC values in fish exposed to anesthetic induction at a water temperature of 27ºC showed no difference between the 95% ethanol and essential oil treatments compared to the control group (p > 0.05). However, the 95% ethanol group had the highest average number of RBCs, while the group subjected to induction with the essential oil showed considerably lower averages (Table 3 ). The percentage of hematocrit, MCHC, as well as the total leukocyte and thrombocyte values, and the relative leukocyte values showed no significant difference (p > 0.05) (Table 3 ). Table 2 Hematological parameters (mean ± standard deviation) of Nile tilapia exposed to anesthetic induction with Ocimum basilicum essential oil at a water temperature of 23 ºC Parameters Temperature 23ºC Control 95% ethanol O. basilicum p value Hemoglobin (g dL − 1 ) 7.66 ± 0.61 7.81 ± 1.00 9.10 ± 2.83 0.2737 Hematocrit (%) 29.29 ± 2.75 30.29 ± 3.68 28.37 ± 3.83 0.4210 MCV (fL) 1.20 ± 0.24 1.31 ± 0.22 1.16 ± 0.22 0.2587 MCH ( g dL − 1 ) 0.33 ± 0.07 0.37 ± 0.10 0.37 ± 0.12 0.6292 MCHC (g dL − 1 ) 27.72 ± 3.82 26.24 ± 1.70 32.50 ± 9.08 0.9803 RBC (x 10 6 µL − 1 ) 2.52 ± 0.97 2.38 ± 0.56 2.48 ± 0.51 0.8632 WBC (x 10 3 µL − 1 ) 251.92 ± 43.15 237.85 ± 56.05 248.67 ± 51.55 0.7745 Thrombocytes (x 10 3 µL − 1 ) 35.23 ± 14.48 29.97 ± 14.51 31.08 ± 16.10 0.7885 Lymphocytes (x 10 3 µL − 1 ) 206.02 ± 40.81 196.95 ± 52.25 196.40 ± 40.73 0.9221 Monocytes (x 10 3 µL − 1 ) 20.17 ± 13.34 29.15 ± 18.60 25.24 ± 16.71 0.2898 Neutrophils (x 10 3 µL − 1 ) 20.10 ± 9.80 a 7.95 ± 1.34 b 19.76 ± 15.68 a 0.0000 Basophiles (x 10 3 µL − 1 ) 5.59 ± 3.12 2.27 ± 0.84 5.87 ± 6.68 0.3592 MCV – mean corpuscular volume, MCH – mean corpuscular hemoglobin, MCHC – mean corpuscular hemoglobin concentration, RBC – red blood cells, WBC – white blood cells. Different letters indicate significant differences by Tukey test (p < 0.05). Table 3 Hematological parameters (mean ± standard deviation) of Nile tilapia exposed to anesthetic induction with Ocimum basilicum essential oil at a water temperature of 27 ºC Parameters Temperature 27ºC Control 95% ethanol O. basilicum p value Hemoglobin (g dL − 1 ) 7.55 ± 1.33 7.46 ± 0.71 7.57 ± 1.29 0.9828 Hematocrit (%) 25.83 ± 1.21 26.79 ± 2.83 27.08 ± 4.60 0.7922 MCV (fL) 1.41 ± 0.47 ab 1.12 ± 0.15 b 1.68 ± 0.13 a 0.0493 MCH (g dL − 1 ) 0.39 ± 0.09 ab 0.32 ± 0.07 b 0.46 ± 0.13 a 0.0196 MCHC (g dL − 1 ) 29.34 ± 5.71 28.13 ± 4.21 27.89 ± 5.34 0.8259 RBC (x 10 6 µL − 1 ) 2.06 ± 0.80 ab 2.42 ± 0.35 a 1.76 ± 0.48 b 0.0198 WBC (x 10 3 µL − 1 ) 205.83 ± 80.43 242.41 ± 35.93 176.17 ± 49.76 0.9828 Thrombocytes (x 10 3 µL − 1 ) 32.76 ± 19.68 36.31 ± 25.76 22.39 ± 13.42 0.1299 Lymphocytes (x 10 3 µL − 1 ) 165.59 ± 60.46 202.66 ± 41.76 146.49 ± 38.64 0.2165 Monocytes (x 10 3 µL − 1 ) 18.60 ± 8.10 27.16 ± 11.08 19.25 ± 14.81 0.2469 Neutrophils (x 10 3 µL − 1 ) 13.97 ± 13.47 9.09 ± 5.57 7.12 ± 5.81 0.3198 Basophiles (x 10 3 µL − 1 ) 7.66 ± 9.55 3.29 ± 3.62 4.60 ± 4.85 0.6909 MCV – mean corpuscular volume, MCH – mean corpuscular hemoglobin, MCHC – mean corpuscular hemoglobin concentration, RBC – red blood cells, WBC – white blood cells. Different letters indicate significant differences by Tukey test (p 0.05) the plasma ions chloride and total calcium in juvenile Nile tilapia among the different treatments (Fig. 1 ). However, in the evaluation of the calcium ion, there was a statistical difference (p 0.05) between the tested treatments at both temperatures. 5 Discussion Essential oils are complex combinations composed of various organic chemical substances, whose constituent composition can be influenced by several factors (Abdoul-Latif et al. 2022). The compounds methyl chavicol and linalool were identified from chemical analysis as the main constituents of the essential oil of O. basilicum . In agreement with these findings, Oliveira et al. (2013), Ventura et al. (2020), and Yigit et al. (2022) observed the predominant presence of both compounds in the essential oil of O. basilicum cultivated in Brazil. Additionally, Costa et al. (2015) reported the predominant presence of these compounds, and other components, in 38 genotypes of basil cultivated in the United States. Fish undergo hematological variations due to the influence of factors such as temperature, pH, salinity, and dissolved oxygen. Additionally, using anesthetics can influence hematological parameters, resulting in stressful situations for the animals (Ranzani-Paiva et al. 2013). In this study, anesthetic induction in Nile tilapia showed distinct responses for MCV and MHC parameters among groups subjected to different temperatures. Anesthesia with the essential oil of O. basilicum promoted an increase in MCV and MCH parameters at a temperature of 27ºC, with the same parameters showing lower averages among groups tested at a temperature of 23ºC. Tambaquis kept at a temperature of 29,6ºC and anesthetized with clove oil and benzocaine (Pádua et al. 2013) showed the same trend for MCV observed in the present study. Similar results were observed in goldfish ( Carassius auratus ) anesthetized with propofol and clove oil at a temperature of 22ºC (Gholipourkanani and Ahadizadeh, 2013). Gholipourkanani and Ahadizadeh (2013) observed an increase in MCV value with the use of the synthetic anesthetic propofol and a reduction with the natural anesthetic clove oil, while for MCH, an opposite trend was observed. In rainbow trout ( Oncorhynchus mykiss ) anesthetized with citronellal at a temperature of 14.2ºC, an increase in the concentration of the essential oil also led to a reduction in these parameters (Hoseini et al. 2021). Erythrocytes are the most abundant cells in fish, and variations in their count can influence the metabolic rate and hematological parameters of fish (Witeska 2013). These cells play an essential role in the transport of oxygen and carbon dioxide (Ranzani-Paiva et al. 2013). The decrease in the number of RBCs in the present study was observed at a temperature of 27ºC, while at 23ºC there was no difference among the treatments tested. This may explain the mortalities observed during the anesthetic induction procedure performed at 27ºC. The combination of O. basilicum essential oil with an increase in temperature may have caused a considerable level of stress in the fish, leading to an imbalance in the immune system, and resulting in mortality and suppression of RBC count. Similar to the present study, Nile tilapia ( O. niloticus ) anesthetized with the essential oil of Lippia sidoides at a temperature of 27.67ºC also showed a reduction in the number of RBC (Hashimoto et al. 2016). Anesthetics tend to induce an increase in the number of RBC, as observed in Nile tilapia ( O. niloticus ) anesthetized with thymol (2-isopropyl-5-methylphenol) and eugenol (Yousefi et al. 2022), and with propofol and eugenol (Zahran et al. 2021), with differs from the present study. Neutrophils are phagocytic cells that play a role in the host’s defense system (Witeska et al. 2022). An increase in the number of these cells in Nile tilapia when exposed to anesthetic administration could be related to an imbalance in homeostasis as the animal attempts to restore normal conditions. In the present study, neutrophils were influenced by the treatment with water combined with 95% ethanol when compared to the control group with water and essential oil, indicating that the alcohol contained in the essential oil may influence the activity of these phagocytic cells. Ventura et al. (2021b) demonstrated that juvenile pacu ( Piaractus mesopotamicus ) anesthetized with O. basilicum essential oil at a temperature of 26.4ºC also did not show an increase in neutrophil values when subjected to anesthesia with O. basilicum essential oil. This trend was also observed with the essential oils of melaleuca and clove at a temperature of 28ºC (Santos et al. 2020), as well as in fish subjected to anesthesia with O. gratissimum and Z. officinale at a temperature of 26.3ºC (Silva et al. 2020). The main blood ions are directly influenced by stressors during the exchange process between the internal and external environments of fish (Silva et al. 2015b). In the present study, the use of O. basilicum essential oil resulted in lower calcium ion levels at a temperature of 27ºC. However, fish exposed to induction with essential oil did not differ from either of the control groups. Many physiological changes are related to exposure to anesthetic, so this result may be associated with the temperature increase during the procedure, as this trend was not observed at a temperature of 14.6ºC which did not show differences between the treatments (Taheri-Mirghaed et al. 2018). In Nile tilapia, it was observed that O. basilicum essential oil prevented calcium ion efflux during the experiment. Osmoregulatory disturbances tend to cause stress in fish, leading to a reduction in ion levels under stressful conditions. In response to these conditions, the animals tend to increase blood flow in the gills and cellular permeability, resulting in a reduction of plasma ions (Becker et al. 2012). 6 Conclusion The increase in water temperature combined with the use of O. basilicum essential oil tends to negatively influence the osmoregulatory and hematological systems of tilapia. The use of O. basilicum essential oil at a temperature equal to or similar to 23ºC could be the most recommended for use as an anesthetic in Nile tilapia, as it causes less stressful effects on the hematological and biochemical responses of the species. Declarations Acknowledgements The authors thank the National Council for Scientific and Technological Development (CNPq) for the research grant from the Institutional Program for Scientific Initiation Grants (PIBIC) to P.C. Santos, the research grant to M.L. Martins (CNPq 303822/2022-8, 409821/2021-7), C.A.L. Cardoso (312671/2021-0) and post-doctoral scholarships to A.S. Ventura (150256/2023-0), G.T. Jerônimo (314239/2020-0), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for master's scholarship to E.M. Lopes and financial support (CAPES finance code 001). 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Boletim do Instituto de Pesca 42(3):727-733. https://doi.org/10.20950/1678-2305.2016v42n3p727 Martins AGLA, Nascimento AR, Mouchrek-Filho JE, Mendes-Filho NE, Souza AG, Aragão NE, Silva DSV (2010) Atividade antibacteriana do óleo essencial do manjericão frente a sorogrupos de Escherichia coli enteropatogênica isolados de alfaces. Ciência rural 40(48):1791-1796. Medeiros-Junior EF, Brito OS (2021) Uso de anestésicos na aquicultura como prática de manejo e os fatores que interferem na anestesia. In: Cordeiro CAM, Dioniso SS, Holanda FCAF . Engenharia de Pesca: aspectos teóricos e práticos, 2nd ed., pp 216-228. Oliveira RA, Moreira IS, Oliveira FF (2013). Linalool and methyl chavicol present basil ( Ocimum sp.) cultivated in Brazil. Rev. Bras.Pl. Med. 15:309-311. Pádua SB, Dias-Neto J, Sakabe R, Claudiano GS, Chagas EC, Pilarski F (2013) Hematologic variables in tambaquis anesthetized with clove oil and benzocaine. Pesquisa Agropecuária Brasileira 48(8):1171-1174. https://doi.org/10.1590/S0100-204X2013000800056 Pravuschi PR, Marques PAA, Ringolin BHM, Santos ACP (2010) Efeito de diferentes lâminas de irrigação na produção de óleo essencial do manjericão ( Ocimum basilicum L.). Acta Scientiarum Agronomy 32(4):687-693. https://doi.org/10.4025/actasciagron.v32i4.3160 Priborsky J, Velisek J (2018) A Review of Three Commonly Used Fish Anesthetics. Reviews in Fisheries Science and Aquaculture 26(4):417-442. https://doi.org/10.1080/23308249.2018.1442812 Ranzani-Paiva MJT, Pádua SB, Tavares-Dias M, Egami MI (2013) Métodos para análise hematológica em peixes, Universidade Estadual de Maringá, Maringá, pp.135. Roohi Z, Imanpoor MR (2015) The efficacy of the oils of spearmint and methyl salicylate as new anesthetics and their effect on glucose levels in common carp ( Cyprinus carpio L., 1758) juveniles. Aquaculture 437:327-332. http://dx.doi.org/10.1016/j.aquaculture.2014.12.019 Ross LG, Ross B (2008) Anaesthetic and Sedative Techniques for Aquatic Animals , 3rd ed. Blackwell Publishing, Oxford, pp 179-190. Santos ELR, Rezende FP, Moron SE (2020) Stress-related physiological and histological responses of tambaqui ( Colossoma macropomum ) to transportation in water with tea tree and clove essential oil anesthetics. Aquaculture 523:735164. https://doi.org/10.1016/j.aquaculture.2020.735164 Silva LA, Martins MA, Santo FE, Oliveira FC, Chaves FCM, Chagas EC, Martins ML, Campos CM (2020) Essential oils of Ocimum gratissimum and Zingiber officinale as anesthetics for the South American catfish Pseudoplatystoma reticulatum . Aquaculture 528:735595. https://doi.org/10.1016/j.aquaculture.2020.735595 Silva LL, Garlet QI, Koakoski G, Abreu MS, Mallmann CA, Baldisserotto B, Barcellos LJG, Heinzmann BM (2015a) Anesthetic activity of the essential oil of Ocimum americanum in Rhamdia quelen (Quoy & Gaimard, 1824) and its effects on stress parameters. Neotropical Ichthyology 13(4):715-722. https://doi.org/10.1590/1982-0224-20150012 Silva LL, Garlet QI, Koakoski G, Oliveira TA, Barcellos LJG, Baldisserotto B, Pereira AMS, Heinzmann BM (2015b) Effects of anesthesia with the essential oil of Ocimum gratissimum L. in parameters of fish stress. Revista Brasileira de Plantas Medicinais 17(2):215-223. https://doi.org/10.1590/1983-084X/13_034 Silva RD, Rocha LO, Fortes BDA, Vieira D, Fioravantti MCS (2012) Hematological parameters of Nile tilapia ( Oreochromis niloticus L.) under air exposure stress. Pesquisa Veterinária Brasileira 32:99-107. Simões LN, Lombardi DC, Gomide ATM, Gomes LC (2011) Efficacy of clove oil as anesthetic in handling and transportation of Nile tilapia, Oreochromis niloticus (Actinopterygii: Cichlidae) juveniles. Zoologia 28(3):285-290. https://doi.org/10.1590/S1984-46702011000300001 Souza EM, Souza RC, Melo JFB, Costa MM, Souza AM, Copatti CE (2019) Evaluation of the effects of Ocimum basilicum essential oil in Nile tilapia diet: growth, biochemical, intestinal enzymes, haematology, lysozyme and antimicrobial challenges. Aquaculture 504:7-12. https://doi.org/10.1016/j.aquaculture.2019.01.052 Suárez-Puerto B, Araneda M, Gullian-Klanian M (2023) Bioeconomic analysis of the commercial production of Nile tilapia with biofloc and green water technologies. Aquaculture and Fisheries. https://doi.org/10.1016/j.aaf.2023.07.004 Taheri-Mirghaed A, Ghelichpour M, Zargari A, Yousefi M (2018) Anaesthetic efficacy and biochemical effects of 1, 8‐cineole in rainbow trout ( Oncorhynchus mykiss , Walbaum, 1792). Aquaculture Research 49(6):2156-2165. https://doi.org/10.1111/are.13671 Tavares-Dias M, Oliveira SR (2009) A review of the blood coagulation system of fish. Brazilian Journal of Biosciences 7(2):205-224. Ventura AS, Jerônimo GT, Oliveira SN, Gabriel AMA, Cardoso CAL, Teodoro GC, Corrêa-Filho RAC, Povh JA (2020) Natural anesthetics in the transport of Nile tilapia: Hematological and biochemical responses and residual concentration in the fillet. Aquaculture 526:735365. https://doi.org/10.1016/j.aquaculture.2020.735365 Ventura AS, Jerônimo GT, Corrêa-Filho RAC, Souza AI, Stringhetta GR, Cruz MG, Torres GS, Gonçalves LU, Povh JA (2021a) Ocimum basilicum essential oil as an anesthetic for tambaqui Colossoma macropomum : Hematological, biochemical, non-specific immune parameters and energy metabolism. Aquaculture 533:736124. https://doi.org/10.1016/j.aquaculture.2020.736124 Ventura AS, Gabriel AMA, Gandra FR, Noia IZ, Povh JA, Jerônimo GT (2021b) Thermal dynamics and physiological implications in pacu Piaractus mesopotamicus anaesthetised with Ocimum basilicum essential oil. International Aquatic Research 13(4):261-270. https://doi.org/10.22034/IAR.2021.1938212.1183 Witeska M (2013) Erythrocytes in teleost fishes: a review. Zoology and Ecology 23(4):275-281. http://dx.doi.org/10.1080/21658005.2013.846963 Witeska M, Kondera E, Ługowska K, Bojarski (2022) Hematological methods in fish–Not only for beginners. Aquaculture 547:737498. https://doi.org/10.1016/j.aquaculture.2021.737498 Yigit NO, Metin S, Sabuncu OF, Didinen BI, Didinen H, Ozmen O, Koskan O (2021) Efficiency of Ocimum basilicum and Eucalyptus globulus essential oils on anesthesia and histopathology of rainbow trout, Oncorhynchus mykiss . Journal of the World Aquaculture Society 53(5):1051-1061. https://doi.org/10.1111/jwas.12911 Yousefi M, Hoseini SM, Aydın B, Mirghaed AT, Kulikov EV, Drukovsky SG, Seleznev SB, Rudenko PA, Hoseinifar SH, Doan HV (2022) Anesthetic efficacy and hemato-biochemical effects of thymol on juvenile Nile tilapia, Oreochromis niloticus . Aquaculture 547:737540. https://doi.org/10.1016/j.aquaculture.2021.737540 Zahl IH, Kiessling A, Samuelsen OB, Hansen MK (2011) Anaesthesia of Atlantic halibut ( Hippoglossus hippoglossus ) Effect of pre‐anaesthetic sedation, and importance of body weight and water temperature. Aquaculture Research 42(9):1235-1245. https://doi.org/10.1111/j.1365-2109.2010.02711.x Zahran E, Risha E, Rizk A (2021) Comparison propofol and eugenol anesthetics efficacy and effects on general health in Nile Tilapia. Aquaculture 534:736251. https://doi.org/10.1016/j.aquaculture.2020.736251 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4713885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332672124,"identity":"c5dca711-fc13-43f5-bc5b-638d438fa383","order_by":0,"name":"Paola Capistrano Santos","email":"data:image/png;base64,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","orcid":"","institution":"Universidade Federal de Santa Catarina - 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UFSC","correspondingAuthor":false,"prefix":"","firstName":"Maurício","middleName":"Laterça","lastName":"Martins","suffix":""}],"badges":[],"createdAt":"2024-07-09 17:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4713885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4713885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61648339,"identity":"f7788bb5-c7e8-46e5-a101-a279bf5ca2fb","added_by":"auto","created_at":"2024-08-02 11:44:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94575,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical evaluations of chloride (\u003cstrong\u003eA\u003c/strong\u003e) and calcium ions (\u003cstrong\u003eB\u003c/strong\u003e) (mean ± standard deviation) in Nile tilapia exposed to anesthetic induction with \u003cem\u003eO. basilicum\u003c/em\u003eessential oil at water temperatures of 23 and 27ºC\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4713885/v1/f8b8930baecc20f859705b86.png"},{"id":61648340,"identity":"0861f39f-32d2-450e-af10-c9f3719fd973","added_by":"auto","created_at":"2024-08-02 11:44:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":731222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4713885/v1/14a8861e-47b5-4873-8d0d-da3dbd8b3c4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Temperature-induced changes in the hematological and biochemical parameters of Nile tilapia anesthetized with Ocimum basilicum","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe Nile tilapia stands out as the main fish species worldwide. It has high versatility, adapting to semi-intensive and intensive farming, and demonstrates the ability to adapt to various production technologies, including recirculation systems and the biofloc system (Su\u0026aacute;res-Puerto et al. 2023). However, indifferent production systems, and management activities such as transportation, vaccination, and biometrics promote physiological changes, including stress in the animals (Silva et al. 2015a; Medeiros-Junior and Brito 2021). Stress is described as alternations in the physiological state of fish due to environmental conditions that pose a threat to their survival (Ross and Ross 2008). On this view, anesthetics have been used in aquaculture since the 20th century to promote safe handling practices for the animals, being effective in reducing stress and pain (Priborsky and Velisek 2018; Bianchini et al. 2019) Roohi and Imanpoor (2015) emphasize that stressful situations during production procedures result in reduced immune response and growth, increasing the vulnerability of fish to the onset of diseases.\u003c/p\u003e \u003cp\u003eThe use of anesthetics in fish is influenced by biological and physiological variables, resulting in varied responses even with the same dose of anesthetic (Gressler et al. 2020). In this regard, temperature is a significant variable due to its influence on anesthetic concentration and induction (Medeiros-Junior and Brito 2021). According to Aydın et al. (2015), the time of induction and anesthetic recovery has a direct relationship with the metabolism of fish. The physiological processes of fish are associated with temperature, playing a significant role in the relationship between absorption and elimination of anesthetic compounds. Furthermore, temperature variation can increase the metabolism of fish, promoting increased blood flow in the gills (Zahl et al. 2011).\u003c/p\u003e \u003cp\u003eEssential oils can be obtained from the distillation of plants and represent an efficient and safe alternative for conducting anesthetic procedures in fish (Barbas et al. 2017; Aziz et al. 2018; Souza et al. 2019; Gurkan and Hayaloglu, 2023). The genus \u003cem\u003eOcimum\u003c/em\u003e sp has more than 150 cataloged species (Kholiya et al. 2022). \u003cem\u003eOcimum basilicum\u003c/em\u003e, belonging to the Lamiaceae family, is commonly known as basil and has great economic importance in the pharmaceutical and food industries (Martin et al. 2010; Pravuschi et al. 2010; Gurkan and Hayaloglu 2023).\u003c/p\u003e \u003cp\u003eIn aquaculture, the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e is used due to its antimicrobial, antioxidant, sedative, and anesthetic properties (Souza et al. 2019). The effects provided by anesthetic induction with the essential oil have been demonstrated in studies with tambaqui (\u003cem\u003eColossoma macropomum\u003c/em\u003e) (Ventura et al. 2021a), tambacu (male \u003cem\u003ePiaractus masepotamicus\u003c/em\u003e x female \u003cem\u003eColossoma macropomum\u003c/em\u003e) (Limma-Netto et al. 2016), and Nile Tilapia (\u003cem\u003eO. niloticus\u003c/em\u003e) (Ventura et al. 2020). The use of essential oils for handling procedures in fish farming, in addition to reducing stress, provides reduced sensitivity to visual and mechanical stimuli and ensures the preservation of water quality variables (Aydin and Barbas 2020). Thus, the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e is considered a promising anesthetic for use in fish farming. However, more information is needed on the effect of temperature during anesthesia on hematological parameters. Therefore, the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e is considered a potential anesthetic for use in fish farming due to its anesthetic and sedative properties. However, this essential oil is not yet regulated for use due to the lack of knowledge about possible physiological alterations caused by it (Ventura et al. 2020). The immune stress response is characterized by a series of physiological changes, including the release of the neuroendocrine hormones catecholamines and cortisol into the circulatory system, which cause variations in the balance of plasma electrolytes and, in freshwater fish, modify gill permeability, resulting in the loss of blood electrolytes and osmoregulatory disturbances (Silva et al. 2012; Gressler et al. 2015). The present study aimed to evaluate the hematology of juvenile Nile tilapia subjected to anesthesia with the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e at different temperatures.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cp\u003eThe experiment was conducted at the Laboratory of Aquatic Organism Health (AQUOS) at the Federal University of Santa Catarina (UFSC). All animal procedures were approved by the Ethics Committee on Animal Use (CEUA/UFSC n\u0026ordm; 7363211122).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 essential oil of \u003cem\u003eOcimum basilicum\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe essential oil was commercially acquired (Phytoter\u0026aacute;pica\u0026reg;, Nova Cantareira, Brazil). Chemical composition analyses of the essential oil were conducted using gas chromatography coupled with mass spectrometry (CG-MS) according to Ventura et al., (2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 animals\u003c/h2\u003e \u003cp\u003eThe Nile tilapia juveniles were from the Experimental Station of Epagri in Itaja\u0026iacute;, EEI/EPAGRI-SC with an average weight ranging from 20 to 30 g. They were transported to AQUOS, where they remained for seven days in quarantine, distributed in a circular polyethylene tank with a total volume of 1 m\u003csup\u003e3\u003c/sup\u003e at a density of 4.38 kg/m\u003csup\u003e3\u003c/sup\u003e. Subsequently, the fish were distributed into 10 circular polyethylene tanks of 0.1 m\u003csup\u003e3\u003c/sup\u003e each for 15 days in the recirculation water system, with a settler, mechanical filter, and biological filter containing porous sponges, for acclimatization at a density of 0.04 kg/0.1 m\u003csup\u003e3\u003c/sup\u003e. The tanks were maintained under constant aeration, temperature control with heaters, and a water renewal rate of 10% per day.\u003c/p\u003e \u003cp\u003eThe monitoring of water quality variables including temperature (23.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026ordm;C; 26.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 \u0026ordm;C), dissolved oxygen (6.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and pH (7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19; 7.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) was conducted daily using a multiparameter meter (Hanna HI98190\u0026reg;). Total ammonia (1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), toxic ammonia (0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 0.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and nitrite (0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) concentrations were determined using commercial colorimetric kits (Alfakit\u0026reg;). The animals were fed three times a day with Acqua Line\u0026reg; 1.7 mm (Crude protein 46%, Ether extract 8%, Crude fiber 3%, Ash 14%, Calcium 1.5\u0026ndash;3%, Phosphorus 1%) until apparent satiety and subjected to a 24 h fasting period before the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design\u003c/h2\u003e \u003cp\u003eJuvenile Nile tilapia (64.99\u0026thinsp;\u0026plusmn;\u0026thinsp;13.90 g and 15.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 cm) (n\u0026thinsp;=\u0026thinsp;112) were randomly distributed in 30 L aquariums and divided into 4 treatments: fish anesthetized with 250 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a temperature of 23\u0026ordm;C (n\u0026thinsp;=\u0026thinsp;42); fish anesthetized with 250 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at a temperature of 27\u0026ordm;C (n\u0026thinsp;=\u0026thinsp;42); and for each temperature, a control group fish exposed to diluent solution (95% ethanol) (n\u0026thinsp;=\u0026thinsp;14, seven per temperature); a control group fish (water) (n\u0026thinsp;=\u0026thinsp;14, seven per temperature). After immersion bath with essential oil for 10 min, the fish exposed to EO were transferred to fourteen 30 L aquaria, with six fish per aquarium, along with the ethanol control group fish and water control group fish, with seven fish per aquarium free of anesthetic and diluent solution, at temperatures of 23 and 27\u0026ordm;C. The fish were not fed during the course of the experiment, and animal survival was recorded. The anesthetic concentration of the essential oil used in the present study was defined based on a previous study that determined the concentration capable of inducing anesthesia in less than 180 seconds, followed by a recovery time of less than 300 seconds according to the criteria of ideal anesthetic efficacy by Ross and Ross (2008).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 blood collection\u003c/h2\u003e \u003cp\u003eBlood samples were collected after the anesthetic induction at each temperature. For the control groups, blood samples were collected from all fish at both temperatures (23 and 27\u0026ordm;C) without the use of anesthesia.\u003c/p\u003e \u003cp\u003eThe fish were individually captured and restrained with a damp cloth. Blood was collected by puncturing the caudal vein using syringes containing 10% EDTA anticoagulant solution (ethylenediaminetetraacetic acid). Blood smears were prepared in duplicate and stained with May Grunwald Giemsa-Wright (Ranzani-Paiva et al., 2013) for the determination of absolute values of leukocytes and thrombocytes, as well as relative values of leukocytes. For the determination of the total number of erythrocytes, a 5 \u0026micro;L aliquot of blood was diluted (1:200) in Dacies\u0026rsquo;s fluid (sodium citrate, 37\u0026ndash;40% formaldehyde, toluidine blue) modified according to Blaxhall and Daisley (1973), then erythrocyte quantification was performed using a Neubauer chamber. A blood aliquot was used to determine the hematocrit percentage by the microhematocrit method; furthermore, another portion was used for the analysis of hemoglobin concentration with commercial reagents (LabTest Diagn\u0026oacute;stica\u0026reg;). Hematimetric indices mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Ranzani-Paiva et al. (2013).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Biochemical analysis of chloride and total calcium ions\u003c/h2\u003e \u003cp\u003eBlood samples were centrifuged at 3000 rpm for five minutes to obtain plasma for the determination of plasma ions chloride (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) and total calcium (Ca\u003csup\u003e2+\u003c/sup\u003e). The tests were performed using colorimetric methodology with commercial reagents (LabTest Diagn\u0026oacute;stica\u0026reg;). In the testes, 10 \u0026micro;L of pooled plasma (six fish from each aquarium) were used, following the manufacturer\u0026rsquo;s kit instructions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Statistical analysis","content":"\u003cp\u003eFor hematological and biochemical analyses, the data were subjected to Shapiro-Wilk and Levenes tests to assess normal distribution and variance homoscedasticity, respectively. Non-homogeneous data were log 10-transformed to achieve homogeneity. Subsequently, the data were subjected to \u003cem\u003eOne-way\u003c/em\u003e ANOVA analysis, and when appropriate, means were separated by Tukey\u0026rsquo;s test (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05). The analyses were performed using \u003cem\u003eStatistica\u003c/em\u003e 10.0 \u003cem\u003esoftware\u003c/em\u003e. The significance level was set at 5%.\u003c/p\u003e"},{"header":"4 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Essential oil composition\u003c/h2\u003e \u003cp\u003eThe main components found in the essential oil were methyl chavicol (70.04%) and linalool (24.59%) as major constituents, while the minor constituents totaled 5.37% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The detection and quantification limits for methyl chavicol were 0.007 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.020 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCompounds present in basil essential oil\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eO. basilicum\u003c/em\u003e essential oil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eComposition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal percentage (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl Chavicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinalool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eδ-Carene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1,8-Cineol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEugenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eγ-Elemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpatulenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.2 survival analysis after anesthetic induction\u003c/h2\u003e \u003cp\u003eDuring the experiment, a mortality rate of 95.24% was recorded for fish subjected to anesthetic induction with \u003cem\u003eO. basilicum\u003c/em\u003e essential oil at a water temperature of 23\u0026ordm;C. At a temperature of 27\u0026ordm;C, a survival rate of 76.6% was recorded for fish subjected to anesthetic induction. In the water control group, the survival rate was 85.72%. In the 95% ethanol control group, a survival rate of 100% was recorded for the fish at both temperatures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Hematologial parameters\u003c/h2\u003e \u003cp\u003eThe water temperature at 23\u0026ordm;C did not affect (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) the hematimetric indices of Nile tilapia anesthetized with \u003cem\u003eO. basilicum\u003c/em\u003e essential oil. Ethanol did not affect the erythrocyte parameters of the species. In the white series, neutrophil values were lower in fish from the 95% ethanol group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter exposure to \u003cem\u003eO. basilicum\u003c/em\u003e essential oil at a water temperature of 27\u0026ordm;C, a statistical difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in MCV, MCH, and the number of RBC between the fish subjected to the anesthetic and 95% ethanol. The fish subjected to anesthetic induction with \u003cem\u003eO. basilicum\u003c/em\u003e oil had higher MCV values than the fish in the water and 95% ethanol control groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, the same pattern was observed for MCH, where the values in the fish subjected to anesthetic induction were slightly higher than those in the water and ethanol control groups.\u003c/p\u003e \u003cp\u003eThe RBC values in fish exposed to anesthetic induction at a water temperature of 27\u0026ordm;C showed no difference between the 95% ethanol and essential oil treatments compared to the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the 95% ethanol group had the highest average number of RBCs, while the group subjected to induction with the essential oil showed considerably lower averages (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe percentage of hematocrit, MCHC, as well as the total leukocyte and thrombocyte values, and the relative leukocyte values showed no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematological parameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) of Nile tilapia exposed to anesthetic induction with \u003cem\u003eOcimum basilicum\u003c/em\u003e essential oil at a water temperature of 23 \u0026ordm;C\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTemperature 23\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% ethanol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eO. basilicum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2737\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematocrit (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCV (fL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2587\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCH ( g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHC (g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC (x 10\u003csup\u003e6\u003c/sup\u003e\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.8632\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e251.92\u0026thinsp;\u0026plusmn;\u0026thinsp;43.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e237.85\u0026thinsp;\u0026plusmn;\u0026thinsp;56.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e248.67\u0026thinsp;\u0026plusmn;\u0026thinsp;51.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7745\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThrombocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.23\u0026thinsp;\u0026plusmn;\u0026thinsp;14.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.97\u0026thinsp;\u0026plusmn;\u0026thinsp;14.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.08\u0026thinsp;\u0026plusmn;\u0026thinsp;16.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7885\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e206.02\u0026thinsp;\u0026plusmn;\u0026thinsp;40.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e196.95\u0026thinsp;\u0026plusmn;\u0026thinsp;52.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e196.40\u0026thinsp;\u0026plusmn;\u0026thinsp;40.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9221\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.17\u0026thinsp;\u0026plusmn;\u0026thinsp;13.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.15\u0026thinsp;\u0026plusmn;\u0026thinsp;18.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.24\u0026thinsp;\u0026plusmn;\u0026thinsp;16.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2898\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.10\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.76\u0026thinsp;\u0026plusmn;\u0026thinsp;15.68\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasophiles (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.59 \u0026plusmn; 3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3592\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMCV \u0026ndash; mean corpuscular volume, MCH \u0026ndash; mean corpuscular hemoglobin, MCHC \u0026ndash; mean corpuscular hemoglobin concentration, RBC \u0026ndash; red blood cells, WBC \u0026ndash; white blood cells. Different letters indicate significant differences by Tukey test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematological parameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) of Nile tilapia exposed to anesthetic induction with \u003cem\u003eOcimum basilicum\u003c/em\u003e essential oil at a water temperature of 27 \u0026ordm;C\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eTemperature 27\u0026ordm;C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% ethanol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eO. basilicum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9828\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematocrit (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7922\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCV (fL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0493\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCH (g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHC (g dL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.34\u0026thinsp;\u0026plusmn;\u0026thinsp;5.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.89\u0026thinsp;\u0026plusmn;\u0026thinsp;5.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.8259\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC (x 10\u003csup\u003e6\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003e\u003cb\u003eab\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e205.83\u0026thinsp;\u0026plusmn;\u0026thinsp;80.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e242.41\u0026thinsp;\u0026plusmn;\u0026thinsp;35.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e176.17\u0026thinsp;\u0026plusmn;\u0026thinsp;49.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.9828\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThrombocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.76\u0026thinsp;\u0026plusmn;\u0026thinsp;19.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.31\u0026thinsp;\u0026plusmn;\u0026thinsp;25.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.39\u0026thinsp;\u0026plusmn;\u0026thinsp;13.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1299\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e165.59\u0026thinsp;\u0026plusmn;\u0026thinsp;60.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e202.66\u0026thinsp;\u0026plusmn;\u0026thinsp;41.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e146.49\u0026thinsp;\u0026plusmn;\u0026thinsp;38.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocytes (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.60\u0026thinsp;\u0026plusmn;\u0026thinsp;8.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.16\u0026thinsp;\u0026plusmn;\u0026thinsp;11.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.25\u0026thinsp;\u0026plusmn;\u0026thinsp;14.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2469\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.97\u0026thinsp;\u0026plusmn;\u0026thinsp;13.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasophiles (x 10\u003csup\u003e3\u003c/sup\u003e \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;9.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;4.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6909\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMCV \u0026ndash; mean corpuscular volume, MCH \u0026ndash; mean corpuscular hemoglobin, MCHC \u0026ndash; mean corpuscular hemoglobin concentration, RBC \u0026ndash; red blood cells, WBC \u0026ndash; white blood cells. Different letters indicate significant differences by Tukey test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe temperature of 23\u0026ordm;C did not affect (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) the plasma ions chloride and total calcium in juvenile Nile tilapia among the different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, in the evaluation of the calcium ion, there was a statistical difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at a temperature of 27\u0026ordm;C, with fish exposed to the 95% ethanol control showed higher average compared to the control group. The evaluation of the chloride ion showed no difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the tested treatments at both temperatures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5 Discussion","content":"\u003cp\u003eEssential oils are complex combinations composed of various organic chemical substances, whose constituent composition can be influenced by several factors (Abdoul-Latif et al. 2022). The compounds methyl chavicol and linalool were identified from chemical analysis as the main constituents of the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e. In agreement with these findings, Oliveira et al. (2013), Ventura et al. (2020), and Yigit et al. (2022) observed the predominant presence of both compounds in the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e cultivated in Brazil. Additionally, Costa et al. (2015) reported the predominant presence of these compounds, and other components, in 38 genotypes of basil cultivated in the United States.\u003c/p\u003e \u003cp\u003eFish undergo hematological variations due to the influence of factors such as temperature, pH, salinity, and dissolved oxygen. Additionally, using anesthetics can influence hematological parameters, resulting in stressful situations for the animals (Ranzani-Paiva et al. 2013). In this study, anesthetic induction in Nile tilapia showed distinct responses for MCV and MHC parameters among groups subjected to different temperatures. Anesthesia with the essential oil of \u003cem\u003eO. basilicum\u003c/em\u003e promoted an increase in MCV and MCH parameters at a temperature of 27\u0026ordm;C, with the same parameters showing lower averages among groups tested at a temperature of 23\u0026ordm;C. Tambaquis kept at a temperature of 29,6\u0026ordm;C and anesthetized with clove oil and benzocaine (P\u0026aacute;dua et al. 2013) showed the same trend for MCV observed in the present study. Similar results were observed in goldfish (\u003cem\u003eCarassius auratus\u003c/em\u003e) anesthetized with propofol and clove oil at a temperature of 22\u0026ordm;C (Gholipourkanani and Ahadizadeh, 2013). Gholipourkanani and Ahadizadeh (2013) observed an increase in MCV value with the use of the synthetic anesthetic propofol and a reduction with the natural anesthetic clove oil, while for MCH, an opposite trend was observed. In rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) anesthetized with citronellal at a temperature of 14.2\u0026ordm;C, an increase in the concentration of the essential oil also led to a reduction in these parameters (Hoseini et al. 2021).\u003c/p\u003e \u003cp\u003eErythrocytes are the most abundant cells in fish, and variations in their count can influence the metabolic rate and hematological parameters of fish (Witeska 2013). These cells play an essential role in the transport of oxygen and carbon dioxide (Ranzani-Paiva et al. 2013). The decrease in the number of RBCs in the present study was observed at a temperature of 27\u0026ordm;C, while at 23\u0026ordm;C there was no difference among the treatments tested. This may explain the mortalities observed during the anesthetic induction procedure performed at 27\u0026ordm;C. The combination of \u003cem\u003eO. basilicum\u003c/em\u003e essential oil with an increase in temperature may have caused a considerable level of stress in the fish, leading to an imbalance in the immune system, and resulting in mortality and suppression of RBC count. Similar to the present study, Nile tilapia (\u003cem\u003eO. niloticus\u003c/em\u003e) anesthetized with the essential oil of \u003cem\u003eLippia sidoides\u003c/em\u003e at a temperature of 27.67\u0026ordm;C also showed a reduction in the number of RBC (Hashimoto et al. 2016). Anesthetics tend to induce an increase in the number of RBC, as observed in Nile tilapia (\u003cem\u003eO. niloticus\u003c/em\u003e) anesthetized with thymol (2-isopropyl-5-methylphenol) and eugenol (Yousefi et al. 2022), and with propofol and eugenol (Zahran et al. 2021), with differs from the present study.\u003c/p\u003e \u003cp\u003eNeutrophils are phagocytic cells that play a role in the host\u0026rsquo;s defense system (Witeska et al. 2022). An increase in the number of these cells in Nile tilapia when exposed to anesthetic administration could be related to an imbalance in homeostasis as the animal attempts to restore normal conditions. In the present study, neutrophils were influenced by the treatment with water combined with 95% ethanol when compared to the control group with water and essential oil, indicating that the alcohol contained in the essential oil may influence the activity of these phagocytic cells. Ventura et al. (2021b) demonstrated that juvenile pacu (\u003cem\u003ePiaractus mesopotamicus\u003c/em\u003e) anesthetized with \u003cem\u003eO. basilicum\u003c/em\u003e essential oil at a temperature of 26.4\u0026ordm;C also did not show an increase in neutrophil values when subjected to anesthesia with \u003cem\u003eO. basilicum\u003c/em\u003e essential oil. This trend was also observed with the essential oils of melaleuca and clove at a temperature of 28\u0026ordm;C (Santos et al. 2020), as well as in fish subjected to anesthesia with \u003cem\u003eO. gratissimum\u003c/em\u003e and \u003cem\u003eZ. officinale\u003c/em\u003e at a temperature of 26.3\u0026ordm;C (Silva et al. 2020).\u003c/p\u003e \u003cp\u003eThe main blood ions are directly influenced by stressors during the exchange process between the internal and external environments of fish (Silva et al. 2015b). In the present study, the use of \u003cem\u003eO. basilicum\u003c/em\u003e essential oil resulted in lower calcium ion levels at a temperature of 27\u0026ordm;C. However, fish exposed to induction with essential oil did not differ from either of the control groups. Many physiological changes are related to exposure to anesthetic, so this result may be associated with the temperature increase during the procedure, as this trend was not observed at a temperature of 14.6\u0026ordm;C which did not show differences between the treatments (Taheri-Mirghaed et al. 2018). In Nile tilapia, it was observed that \u003cem\u003eO. basilicum\u003c/em\u003e essential oil prevented calcium ion efflux during the experiment. Osmoregulatory disturbances tend to cause stress in fish, leading to a reduction in ion levels under stressful conditions. In response to these conditions, the animals tend to increase blood flow in the gills and cellular permeability, resulting in a reduction of plasma ions (Becker et al. 2012).\u003c/p\u003e"},{"header":"6 Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe increase in water temperature combined with the use of \u003cem\u003eO. basilicum\u003c/em\u003e essential oil tends to negatively influence the osmoregulatory and hematological systems of tilapia. The use of \u003cem\u003eO. basilicum\u003c/em\u003e essential oil at a temperature equal to or similar to 23\u0026ordm;C could be the most recommended for use as an anesthetic in Nile tilapia, as it causes less stressful effects on the hematological and biochemical responses of the species.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe authors thank the National Council for Scientific and Technological Development (CNPq) for the research grant from the Institutional Program for Scientific Initiation Grants (PIBIC) to P.C. Santos, the research grant to M.L. Martins (CNPq 303822/2022-8, 409821/2021-7), C.A.L. Cardoso (312671/2021-0) and post-doctoral scholarships to A.S. Ventura (150256/2023-0), G.T. Jer\u0026ocirc;nimo (314239/2020-0), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for master\u0026apos;s scholarship to E.M. Lopes and financial support (CAPES finance code 001). This research was partially financed by Funda\u0026ccedil;\u0026atilde;o de Apoio ao Desenvolvimento do Ensino, Ci\u0026ecirc;ncia e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT 901/2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eAll procedures were approved by the Ethics Committee on Animal Use of Universidade Federal de Santa Catarina (UFSC), under protocol 7363211122.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdoul-Latif FM, Elmi A, Merito A, Nour M, Risler A, Ainane A, Bignon J, Ainane T (2022) Essential oils of \u003cem\u003eOcimum basilicum\u003c/em\u003e L. and \u003cem\u003eOcimum americanum\u003c/em\u003e L. from Djibouti: Chemical composition, antimicrobial and cytotoxicity evaluations. 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Blackwell Publishing, Oxford, pp 179-190.\u003c/li\u003e\n\u003cli\u003eSantos ELR, Rezende FP, Moron SE (2020) Stress-related physiological and histological responses of tambaqui (\u003cem\u003eColossoma macropomum\u003c/em\u003e) to transportation in water with tea tree and clove essential oil anesthetics. Aquaculture 523:735164. https://doi.org/10.1016/j.aquaculture.2020.735164\u003c/li\u003e\n\u003cli\u003eSilva LA, Martins MA, Santo FE, Oliveira FC, Chaves FCM, Chagas EC, Martins ML, Campos CM (2020) Essential oils of \u003cem\u003eOcimum gratissimum\u003c/em\u003e and \u003cem\u003eZingiber officinale\u003c/em\u003e as anesthetics for the South American catfish \u003cem\u003ePseudoplatystoma reticulatum\u003c/em\u003e. Aquaculture 528:735595. https://doi.org/10.1016/j.aquaculture.2020.735595\u003c/li\u003e\n\u003cli\u003eSilva LL, Garlet QI, Koakoski G, Abreu MS, Mallmann CA, Baldisserotto B, Barcellos LJG, Heinzmann BM (2015a) Anesthetic activity of the essential oil of \u003cem\u003eOcimum americanum\u003c/em\u003e in \u003cem\u003eRhamdia quelen\u003c/em\u003e (Quoy \u0026amp; Gaimard, 1824) and its effects on stress parameters. Neotropical Ichthyology 13(4):715-722. https://doi.org/10.1590/1982-0224-20150012\u003c/li\u003e\n\u003cli\u003eSilva LL, Garlet QI, Koakoski G, Oliveira TA, Barcellos LJG, Baldisserotto B, Pereira AMS, Heinzmann BM (2015b) Effects of anesthesia with the essential oil of \u003cem\u003eOcimum gratissimum\u003c/em\u003e L. in parameters of fish stress. Revista Brasileira de Plantas Medicinais 17(2):215-223. https://doi.org/10.1590/1983-084X/13_034\u003c/li\u003e\n\u003cli\u003eSilva RD, Rocha LO, Fortes BDA, Vieira D, Fioravantti MCS (2012) Hematological parameters of Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e L.) under air exposure stress. Pesquisa Veterin\u0026aacute;ria Brasileira 32:99-107.\u003c/li\u003e\n\u003cli\u003eSim\u0026otilde;es LN, Lombardi DC, Gomide ATM, Gomes LC (2011) Efficacy of clove oil as anesthetic in handling and transportation of Nile tilapia, \u003cem\u003eOreochromis niloticus\u003c/em\u003e (Actinopterygii: Cichlidae) juveniles. 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International Aquatic Research 13(4):261-270. https://doi.org/10.22034/IAR.2021.1938212.1183\u003c/li\u003e\n\u003cli\u003eWiteska M (2013) Erythrocytes in teleost fishes: a review. Zoology and Ecology 23(4):275-281. http://dx.doi.org/10.1080/21658005.2013.846963\u003c/li\u003e\n\u003cli\u003eWiteska M, Kondera E, Ługowska K, Bojarski (2022) Hematological methods in fish\u0026ndash;Not only for beginners. Aquaculture 547:737498. https://doi.org/10.1016/j.aquaculture.2021.737498\u003c/li\u003e\n\u003cli\u003eYigit NO, Metin S, Sabuncu OF, Didinen BI, Didinen H, Ozmen O, Koskan O (2021) Efficiency of \u003cem\u003eOcimum basilicum\u003c/em\u003e and \u003cem\u003eEucalyptus globulus\u003c/em\u003e essential oils on anesthesia and histopathology of rainbow trout, \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e. Journal of the World Aquaculture Society 53(5):1051-1061. https://doi.org/10.1111/jwas.12911\u003c/li\u003e\n\u003cli\u003eYousefi M, Hoseini SM, Aydın B, Mirghaed AT, Kulikov EV, Drukovsky SG, Seleznev SB, Rudenko PA, Hoseinifar SH, Doan HV (2022) Anesthetic efficacy and hemato-biochemical effects of thymol on juvenile Nile tilapia, \u003cem\u003eOreochromis niloticus\u003c/em\u003e. Aquaculture 547:737540. https://doi.org/10.1016/j.aquaculture.2021.737540\u003c/li\u003e\n\u003cli\u003eZahl IH, Kiessling A, Samuelsen OB, Hansen MK (2011) Anaesthesia of Atlantic halibut (\u003cem\u003eHippoglossus hippoglossus\u003c/em\u003e) Effect of pre‐anaesthetic sedation, and importance of body weight and water temperature. Aquaculture Research 42(9):1235-1245. https://doi.org/10.1111/j.1365-2109.2010.02711.x\u003c/li\u003e\n\u003cli\u003eZahran E, Risha E, Rizk A (2021) Comparison propofol and eugenol anesthetics efficacy and effects on general health in Nile Tilapia. Aquaculture 534:736251. https://doi.org/10.1016/j.aquaculture.2020.736251\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aquaculture, Hematology, Essential oil, Anesthesia, Stress","lastPublishedDoi":"10.21203/rs.3.rs-4713885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4713885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study aimed to evaluate the effect of temperatures of 23 and 27 ºC on the hematology of Nile tilapia juveniles (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) subjected to anesthesia using the essential oil of basil (\u003cem\u003eOcimum basilicum\u003c/em\u003e) at a concentration of 250 mg L\u003csup\u003e-1\u003c/sup\u003e. A total of 112 fish were divided into 4 treatments and distributed as follows: fish anesthetized with 250 mg L\u003csup\u003e-1\u003c/sup\u003e at a temperature of 23ºC (n=42); fish anesthetized with 250 mg L\u003csup\u003e-1\u003c/sup\u003e at a temperature of 27ºC (n=42); and for each temperature, a control group fish exposed to diluent solution (95% ethanol) (n=14, seven per temperature); a control group fish (water) (n=14, seven per temperature). They were exposed to 10 min for both temperatures. Blood collection was performed after the anesthetic induction at each temperature to determine the hematocrit, hemoglobin concentration (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), relative values of leukocytes and absolute values of leukocytes and thrombocytes, in addition to evaluating plasma chloride and total calcium ions. The analysis of chemical composition by gas chromatography coupled with mass spectrometry (GC-MS) demonstrated methyl chavicol (70.04%) and linalool (24.59%) as the major components of basil essential oil. Fish anesthetized at a temperature of 23 ºC showed lower neutrophil counts (p\u0026lt;0.05) compared to the other tested groups. For the temperature of 27 ºC, values of MCV, MCHC, and RBC values decreased (p\u0026lt;0.05) following exposure to basil essential oil. The calcium ion showed a statistical difference (p\u0026lt;0.05) between the treatments about the water control group. It is concluded that basil essential oil presented a positive anesthetic effect. The use of \u003cem\u003eO. basilicum\u003c/em\u003e essential oil as an anesthetic in Nile tilapia was the best at 23ºC, causing less hematological and biochemical stress.\u003c/p\u003e","manuscriptTitle":"Temperature-induced changes in the hematological and biochemical parameters of Nile tilapia anesthetized with Ocimum basilicum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-02 11:44:07","doi":"10.21203/rs.3.rs-4713885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-28T05:48:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T11:23:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T17:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169634700193179706838330980323350916847","date":"2024-07-12T09:36:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169686574598622586078637491087420977408","date":"2024-07-12T00:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320523954783127646426918487455317262315","date":"2024-07-12T00:01:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-11T15:30:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T15:29:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-10T10:10:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2024-07-09T17:57:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e829094e-b21a-4e57-910c-bf9b94b17670","owner":[],"postedDate":"August 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T12:21:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-02 11:44:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4713885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4713885","identity":"rs-4713885","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}