Use of the essential oil of Thymus vulgaris (thyme) and its nanoemulsion as an anesthetic during the cultivation of tambaqui (Colossoma macropomum): Anesthesia induction and recovery curve, physiology, performance and feed consumption

Use of the essential oil of Thymus vulgaris (thyme) and its nanoemulsion as an anesthetic during the cultivation of tambaqui (Colossoma macropomum): Anesthesia induction and recovery curve, physiology, performance and feed consumption | 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 Use of the essential oil of Thymus vulgaris (thyme) and its nanoemulsion as an anesthetic during the cultivation of tambaqui (Colossoma macropomum): Anesthesia induction and recovery curve, physiology, performance and feed consumption Túlio Pacheco Boaventura, Camila Gomes Oliveira, Fábio Aremil Costa Santos, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594736/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) and its nanoemulsion (NEOTV-T) were investigated as anesthetics in the management of Colossoma macropomum . The stability of nanoemulsions was analyzed by zeta potential and dynamic light scattering for an interval of 37 days prior to carrying out two anesthesia experiments. The first experiment determined anesthesia induction and recovery intervals for juveniles anesthetized with different concentrations of EOTV-T and NEOTV-T. Based on the obtained induction and recovery curve, the second experiment adopted concentrations of 75 mg/L of EOTV-T and 50 mg/L of NEOTV-T for use during biometric management every 10 days for 60 days of cultivation of juvenile C. macropomum . The results showed that NEOTV-T had an average diameter of less than 100 nm for up to 22 days and was more effective at inducing anesthesia than EOTV-T. The continuous use of anesthetics during biometrics did not affect juvenile performance in terms of growth, feed consumption and survival. Furthermore, no significant differences were observed in hematological and biochemical parameters between juveniles of the control treatment and those subjected to biometric management using 75 mg/L of EOTV-T and 50 mg/L of NEOTV-T. It can be concluded that the essential oil of T. vulgaris and its nanoemulsion can be used to facilitate the management of juvenile C. macropomum without compromising their performance or well-being. More studies need to be carried out to improve the stability of nanoemulsions of the essential oil of T. vulgaris and to evaluate their effects on acute stress. Biochemistry fish hematology thymol welfare Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Tambaqui, Colossoma macropomum (family Characidae), is a species of freshwater fish originating from the Amazon basin that is of economic importance in aquaculture (Brian et al. 2004 ). This scaled fish has dark spots on the ventral region (Boaventura et al. 2021 ), a robust body and tasty meat and can reach considerable sizes (Woynárovich and Van Anrooy. 2019). The majority of C. macropomum production is in the northern region of South America, and it is the second most produced fish species in Brazil (Peixe BR. 2023 ). Due to its performance in captivity, its production has expanded to several tropical Asian countries, including China, Indonesia, Malaysia, Myanmar and Vietnam (Woynárovich and Van Anrooy. 2019). Concern for animal welfare and the search for sustainable aquaculture practices has led to increased exploration of the use of essential oils to replace synthetic anesthetics during fish handling procedures (Aydın and Barbas. 2020; Purbosari et al. 2019 ). The use of these substances helps prevent physical damage during handling and even mitigates fish stress responses (Ross et al. 2009; Stoskopf and Posner. 2008). Thymus vulgaris , popularly known as thyme, is a plant species of the family Lamiacea (Azad et al. 2014 ). The essential oil extracted from this plant can present different types of major components, such as thymol, which is a promising substance for use as an anesthetic for several species of fish (Azad et al. 2014 ; Bagamboula et al. 2004 ; Brandão et al. 2021 ; Yousefi et al. 2018 ), even tambaqui (Boaventura et al. 2022a ). In addition to the use of traditional essential oils, several studies have evaluated the use of their nanoparticles or nanoemulsions as anesthetic agents (Ferreira et al. 2020 ; López-Cánovas et al. 2020 ; Rodrigues et al. 2021 ; Yostawonkul et al. 2019 ). The use of nanoparticles and nanoemulsions obtained from these essential oils increases their solubility in water, which improves their absorption and thus reduces the amount of substance needed to induce anesthesia, thereby mitigating possible side effects in fish (Ferreira et al. 2020 ; López-Cánovas et al. 2020 ). Despite these studies using essential oils and their nanoemulsions as anesthetics, no study has evaluated the influence of the constant use of these substances during biometric management and their possible effects on animal performance. Therefore, this study aimed to determine the ideal concentrations of the essential oil of T. vulgaris and its nanoemulsion to be used during biometric management of juvenile C. macropomum by evaluating the effects of their continuous use during cultivation on feed consumption, performance, physiology and metabolism. 2. Material and methods 2.1 Thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) The thymol chemotype of the essential oil of Thymus vulgaris used in the experiments was acquired from the company Laszlo ( https://www.emporiolaszlo.com.br ), which also provided its composition characterized by gas chromatography (Table 1 ). Table 1 Composition of the essential oil of Thymus vulgaris , Composition (%) α- thujeno 0.6 α-pineno 1.4 canphene 0.9 β-pineno 0.1 myrcene 1.6 p-cymene 31.2 limonene 0.2 1.8 cineole 1.2 y-terpinene 5.4 linalool 0.7 tymol 55.9 β -caryophyllene 0.3 The essential oil of Thymus vulgaris was acquired from the company Laszlo Aromoterapia LTDA, which also supplied their composition, obtained using a HP 5890 gas chro- matograph. Peaks less than 0.1% were disregarded. 2.2 Preparation of the nanoemulsion of the thymol chemotype of the essential oil of Thymus vulgaris (NEOTV-T). The nanoemulsion of the essential oil of Thymus vulgaris (NEOTV-T) was prepared following the method described by Candido et al. ( 2022 ), with adaptations. Initially, an emulsion was prepared containing 5%w/w of EOTV, 1%w/w of Tween 80 and 94%w/w of deionized water. The water and oils were first mixed using a mechanical mixer (Ultraturrax, IKA T10) at 14,500 rpm for 10 minutes at a temperature of 27°C. For nanoemulsion, the emulsion was then subjected to an ultrasonic tip sonicator (Ultronique, Desruptor, Indaiatuba-SP) operating at 70% amplitude, using a 4 mm diameter probe immersed halfway into the emulsion. Five cycles of 4 minutes (2 minutes on and 2 minutes off) were used during the process to avoid heating the emulsion. 2.3 Characterization of the nanoemulsion of the thymol chemotype of the essential oil of Thymus vulgaris. 2.3.1 Dynamic Light Scattering (DLS) and Zeta Potential Diameter measurements were made with DLS and zeta potential of T. vulgaris nanoparticle samples at a concentration of 0.4 mg/mL. Both DLS and zeta potential tests were performed with ZetaPALS equipment (Brookhaven Instruments). Analyses were performed on days 1, 2, 3, 6, 15, 22 and 37 after sample preparation. Means and standard deviations were obtained by taking 30 readings of DLS and Zeta Potential for each analysis day. 2.4. Animals and laboratory acclimatization Three hundred and ten juvenile C. macropomum (2.78 ± 0.88g and 5.43 ± 0.61 cm) were distributed and acclimatized in 30 L tanks filled with 28 L of water and maintained in recirculating aquaculture systems (RAS) for 7 days. During acclimatization, the juveniles were fed an extruded commercial diet (40% crude protein, WeaN Prime 1.8 to 2.3 mm – Bernaqua) twice a day (08:00 and 16:00 h) until apparent satiety. Water quality parameters were measured daily during this period, and were as follows: temperature (28.9 ± 0.2°C) and pH (7.05 ± 0.21) were measured using a COMBO portable pH meter (HANNA-HI 98107), dissolved oxygen (6.33 ± 0.27 mg/L) by a digital oximeter (HANNA-HI9146), and total ammonia (0.25 ± 0.00 mg/L) using Alcon colorimetric kits. 2.5. Experimental design Two experiments were carried out at the Laboratório de Aquicultura of the Universidade Federal de Minas Gerais (UFMG). The methodology was approved by the Comissão de Ética no Uso de Animais of UFMG, process CEUA: 37/2024. The anesthetic solutions were prepared by weighing the essential oil of T. vulgaris (EOTV-T) and diluting it 1:10 (w/w) in 99% ethanol. The nanoemulsion (NEOTV-T) was diluted directly in water. 2.5.1 Experiment 1. Anesthesia induction and recovery for Colossoma macropomum undergoing anesthesia with EOTV-T and NEOTV-T One-hundred and thirty juvenile C. macropomum were fasted for 24 hours. Anesthesia induction time was evaluated using 13 2-L aquariums, one being the control (containing water with the same amount of alcohol used to dilute the highest concentration of EOTV-T tested), six with different concentrations of EOTV-T ( 50, 75,100, 150, 200 and 300 mg/L) and another six with different concentrations of NEOTV-T (50, 75, 100, 150, 200 and 300 mg/L). Another 13 2-L aquariums containing only clean water were used to determine anesthesia recovery time. Ten juveniles were used for each concentration tested. Induction and recovery times were determined according to the criteria described by (Schoettger and Julin. 1967), whereby induction time was determined when a juvenile presented loss of balance and absence of external stimuli, while recovery time was determined through recovery of these responses. After recovery, the juveniles were transferred again to their respective RAS tanks for determination of survival rate 24 hours after the procedure. 2.5.2 Experiment 2. Performance and physiological responses of juvenile Colossoma macropomum subjected to constant biometric management with and without the use of EOTV-T and NEOTV-T Based on the results of Experiment 1, a new experiment was carried out with three treatments for biometrics with four repetitions each: control (0 mg/L), 75 mg/L of thyme essential oil (EOTV-T) and 50 mg/L of thyme nanoemulsion (NEOTV -T). These concentrations were chosen because they present induction and recovery intervals that do not differ significantly from those indicated in the literature (less than 3 minutes for induction and less than 5 minutes for recovery) (Keene et al. 1998 ; Marking and Meyer. 1985). One-hundred and eighty juveniles (2.78 ± 0.88g and 5.43 ± 0.61 cm) were used for this experiment. Before starting the experiment, the juveniles underwent biometric procedures with and without the use of anesthetics. The juveniles were then distributed in 12 150-L tanks (useful volume of 100 L) at 15 fish/tank kept in a RAS where they were cultivated for 60 days. Biometrics were performed every 10 days. The NEOTV-T used in the biometric procedures was used within 20 days of preparation. During the experiment, juveniles were fed until apparent satiety twice a day (08:00 and 16:00 h) with the same extruded commercial diet used during acclimatization. The food provided was previously weighed for each tank. Leftovers were collected 30 minutes after feeding, which were dried and weighed to estimate consumption and feed conversion. After 60 days, all juveniles were weighed and measured (without the use of anesthesia) for blood collection. Water quality parameters relating to temperature and pH were measured daily using a multi-parameter probe (HANNA brand, model HI98129). Dissolved oxygen (DO) and ammonia were measured every 3 days using a digital oximeter (HANNA brand, model HI9146-04) and colorimetric kits from Alcon, respectively. Biometric procedures were performed individually for each repetition every 10 days and at the end of 60 days. During biometrics, the juveniles were captured in the culture tanks with the aid of a pulsar and placed in a 20-L bucket (filled with 10 L of water from the culture tank itself) containing or not the addition of anesthetic (EOTV-T or NEOTV-T). The juveniles were collected individually in the bucket, contained in a damp cloth, weighed using a semi-analytical scale (Marte AD 2000 0.01 g) and measured for total length with a ruler to determine final weight (PF) and total length (TC). After the biometrics process, the juveniles were returned to the cultivation tank where recovery from anesthesia took place. Biometric data and feed consumption were used to calculate the following: Weight gain (WG) ((final weight - initial weight)/initial weight)*100); Feed consumption (FE.C (g) = (offered food (g) - dry leftover food (g))/ number of animals); Apparent feed conversion (FCR) (feed consumption/weight gain). Survival was also determined every 10 days and at the end of the experiment. The survival rate was determined by counting the number of initial and final animals in each phase and calculating the following: Survival (%) = (number of live fish x 100) / (total initial number of fish per tank) Fish physiology and stress were evaluated by collecting blood from 12 animals of each treatment, 3 from each repetition. Two collections were made from each animal: the first collected about 1 mL (0.7 mL using heparinized syringes, to which 10% heparin was subsequently added; the second collected about 0.3 mL in syringes without heparin). Part of the aliquots containing heparin were used to determine hemoglobin (Hb) (Quibasa-Bioclin, Belo Horizonte, MG, Brazil) and hematocrit (HTC) using the micro-hematocrit method with capillary tubes centrifuged in a microcentrifuge followed by reading in an appropriate table (Goldenfarb et al. 1971 ). Serum (without heparin) was used to determine total plasma protein (TPP) and albumin. The remainder of the collected blood was centrifuged at 4000 rpm for 5 minutes, after which the plasma was separated and used for biochemical analyses and determination of glucose (GLU), triglycerides (TRY), cholesterol (CHO) and the enzymes alanine aminotransferase enzymes (ALT) and aspartate aminotransferase (AST), using commercial colorimetric kits (Quibasa-Bioclin, Belo Horizonte, MG, Brazil) followed by reading on a spectrophotometer (Bioclin 100® semi-automatic analyzer). 2.6. Statistical analysis All data were subjected to the Shapiro-Wilk normality test and Levene's test for homoscedasticity. Anesthesia induction and recovery times were subjected to regression analysis. Parametric data were analyzed by One-way ANOVA followed by Tukey's test at 5% probability. Data regarding Zeta Potential, pH, hematocrit and albumin were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test at 5% probability. SigmaPlot and Infostat software were used for data analysis. 3. Results 3.1. Dynamic Light Scattering (DLS) and Zeta Potential The average diameter of the particles present in the nanoemulsion increased during storage, but did not exceed 15nm after 35 days of storage. (Fig. 1 A). The zeta potential of the particles was approximately − 19mV and reduced slightly to -10mV after 35 days of storage. A decrease in zeta potential contributes to increasing the average diameter of nanoparticles due to instability processes such as coalescence observed in nanoemulsions with zeta potential lower than 35mV in modulus. The zeta potential of the particles remained unchanged in the last days of storage, day 23 to day 37 (Fig. 1 B) (P > 0.05). Experiment 1. 3.2. Anesthesia induction and recovery times No mortality was observed during nor 24 hours after anesthesia. The induction (Fig. 2 A and 2 C) and recovery (Fig. 2 B and 2 D) curves for the juveniles and the average data analyzed by ANOVA (Table 2 ) show that the increase in EOTV-T concentration reduced induction time until the concentration of 150 mg/L. Table 2 Induction and recovery times for juvenile tambaqui ( Colossoma macropomum ) subjected to different concentrations of the essential oil of Thymus vulgaris and its nanoemulsion. Concentration mg/L Induction time (s) Recuperation time (s) EOTV NEOTV EOTV NEOTV 50 201.4 ± 23.44aA 116.11 ± 5.35bA 94.85 ± 11.53aC 109.86 ± 18.11aD 75 95.89 ± 12.44aB 76.5 ± 7.20bB 131.87 ± 26.40aCB 118.50 ± 19.98aD 100 51.5 ± 6.15aC 47.49 ± 6.30aC 135.25 ± 35.05aCB 123.28 ± 11.65aD 150 34.9 ± 6.20aD 34.00 ± 6.68aD 113.62 ± 17.45bC 170.14 ± 32.52aC 200 23.90 ± 5.08aD 26.44 ± 3.61aE 167.84 ± 31.00bB 260.83 ± 41.52aB 300 24.55 ± 3.35aD 23.9 ± 1.20aE 286.00 ± 32.68bA 382.16 ± 31.25aA Results expressed as Means ± Standard Deviation. Thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) and its nanoemulsion (NEOTV-T). Results followed by different lowercase letters indicate a significant difference between treatments at the same concentration. Results with different capital letters indicate a significant difference between concentrations of the same treatment by ANOVA followed by Tukey’s test (P < 0.05). Juveniles anesthetized with 50, 75, 100 and 150 mg/L of EOTV-T had similar anesthesia recovery times, while those anesthetized with 200 mg/L of EOTV-T had a longer recovery time than did those anesthetized with 50 mg/L. The longest recovery time using EOTV-T was for juveniles anesthetized with 300 mg/L (Table 2 ). Increasing NEOTV-T concentration reduced anesthesia induction time up to a concentration of 200 mg/L. The concentration of 300 mg/L of NEOTV-T provided an induction time similar to that of 200 mg/L (Table 2 ). Juveniles anesthetized with 50, 75 and 100 mg/L of NEOTV-T had similar anesthesia recovery times. From 150 mg/L onwards, increasing NEOTV-T concentration increased recovery time, reaching its greatest with 300 mg/L NEOTV-T. When comparing EOTV-T with NEOTV-T at the same concentration, 50 and 75 mg/L of NEOTV-T caused faster anesthesia induction than did the same concentrations of EOTV-T (Table 1 ). No differences in induction time were observed between EOTV-T and NEOT-V at concentrations of 100, 150, 200 and 300 mg/L. Table 2 shows the recovery times from anesthesia and the lack of differences between juveniles anesthetized with EOTV-T and NEOTV-T at concentrations 50, 75 and 100 mg/L. Juveniles anesthetized with 150, 200 and 300 mg/L of NEOTV-T had longer anesthesia recovery times than did those anesthetized with the same concentration of EOTV-T. Experiment 2 3.3 Water quality parameters There were no significant differences in water quality parameters (dissolved oxygen, temperature, ammonia and pH) among treatments during the 60 days of cultivation (P > 0.05) (Table 3 ). Table 3 Water quality parameters (mean ± standard deviation) during the cultivation of juvenile Colossoma macropomum subjected to biometric management with and without the use of the thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) and its nanoemulsion (NEOTV -T). Treatment Temperature (°C) OD (mg/L) Ammonia Total (mg/L) pH Control 27.35 ± 0.88A 6.02 ± 0.93A 0.46 ± 0.55A 7.11 ± 0.97A EOTV-T 27.30 ± 0.83A 6.18 ± 0.69A 0.46 ± 0.55A 6.98 ± 1.05A NEOTV-T 27.28 ± 0.80A 5.89 ± 0.82A 0.46 ± 0.55A 6.99 ± 1.07A Different capital letters indicate a significant difference (P < 0.05) between treatments. One-way ANOVA and Tukey’s test were used to determine statistical significance. Data relating to pH were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test. 3.4 Performance There were no significant differences for final weight, final length, weight gain, apparent feed conversion, feed consumption and survival among treatments throughout the 60 days of cultivation (P > 0.05) (Table 4 ). Table 4 Performance parameters (mean ± standard deviation) obtained during the cultivation of juvenile Colossoma macropomum subjected to biometric management with and without the use of the thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) and its nanoemulsion (NEOTV -T). Treatment FW (g) FL (cm) WG (%) FC (%) FE.C (g/fish) SUR (%) 1 at 10 days of cultivation Control 6.66 ± 0.80a 7.22 ± 0.24a 132.04 ± 12.34a 0.62 ± 0.05a 2.31 ± 0.18a 100.0 ± 0.0a EOTV-T 6.38 ± 1.35a 7.15 ± 0.47a 130.33 ± 21.28a 0.66 ± 0.09a 2.30 ± 0.43a 100.0 ± 0.0a NEOTV-T 6.61 ± 1.28a 7.08 ± 0.45a 139.17 ± 16.95a 0.59 ± 0.05a 2.24 ± 0.38a 100.0 ± 0.0a 11 at 20 days of cultivation Control 12.11 ± 1.47a 8.50 ± 0.35a 81.88 ± 20.5a 0.81 ± 0.05a 4.43 ± 0.70a 100.0 ± 0.0a EOTV-T 11.33 ± 2.64a 8.24 ± 0.60a 77.40 ± 12.61a 0.80 ± 0.08a 3.97 ± 1.15a 100.0 ± 0.0a NEOTV-T 12.11 ± 2.37a 8.28 ± 0.45a 82.88 ± 6.90a 0.74 ± 0.07a 4.03 ± 0.77a 100.0 ± 0.0a 21 at 30 days of cultivation Control 18.35 ± 2.23a 10.25 ± 0.35a 51.76 ± 10.01a 0.83 ± 0.10a 10.25 ± 0.35 100 ± 00a EOTV-T 16.03 ± 3.16a 9.35 ± 0.54a 40.34 ± 10.51a 0.89 ± 0.07a 9.35 ± 0.54 100 ± 00a NEOTV-T 16.50 ± 3.11a 9.72 ± 0.52a 34.80 ± 11.01a 1.07 ± 0.29a 9.72 ± 0.52 98.33 ± 2.89a 31 at 40 days of cultivation Control 26.84 ± 4.48a 11.06 ± 0.42a 45.54 ± 8.61a 0.83 ± 0.06a 7.00 ± 1.48 100.0 ± 0.0a EOTV-T 23.10 ± 4.93a 11.08 ± 0.75a 46.18 ± 3.79a 0.87 ± 0.05a 6.13 ± 1.79 100.0 ± 0.0a NEOTV-T 25.15 ± 5.40a 10.81 ± 0.75a 48.84 ± 5.52a 0.75 ± 0.07a 6.40 ± 1.48 100.0 ± 0.0a 41 at 50 days of cultivation Control 36.70 ± 7.03a 12.30 ± 0.67a 36.17 ± 4.52a 0.79 ± 0.09a 12.30 ± 0.67a 100.0 ± 0.0a EOTV-T 30.18 ± 6.45a 11.57 ± 0.70a 30.96 ± 6.64a 0.85 ± 0.09a 11.57 ± 0.70a 100.0 ± 0.0a NEOTV-T 34.35 ± 6.52a 11.92 ± 0.83a 40.38 ± 12.16a 0.79 ± 0.18a 11.92 ± 0.83a 100.0 ± 0.0a 51 at 60 days of cultivation Control 44.09 ± 10.37a 13.26 ± 1.05a 19.85 ± 2.06a 0.95 ± 0.11a 7.13 ± 2.86a 100.0 ± 0.0a EOTV-T 35.01 ± 10.01a 12.41 ± 1.01a 15.34 ± 5.22a 1.29 ± 0.35a 5.62 ± 1.84a 100.0 ± 0.0a NEOTV-T 41.17 ± 9.76a 13.03 ± 0.94a 19.44 ± 4.41a 1.05 ± 0.18a 6.94 ± 2.10a 100.0 ± 0.0a Total 1 at 60 days of cultivation Control 44.09 ± 10.37a 13.26 ± 1.05a 1431.3 ± 260.1a 0.81 ± 0.02 a 35.53 ± 7.58a 100 ± 00a EOTV-T 35.01 ± 10.01a 12.41 ± 1.01a 1165.0 ± 187.8a 0.88 ± 0.04 a 28.61 ± 7.62a 100 ± 00a NEOTV-T 41.17 ± 9.76a 13.03 ± 0.94a 1381.1 ± 291.7a 0.68 ± 0.22 a 26.54 ± 11.66a 98.33 ± 2.89a Results followed by different letters indicate a significant difference (P < 0.05) by ANOVA followed by Tukey's test at 5%. Final weight (g) (FW), final length (cm) (FL), weight gain (WG), feed conversion (FC), feed consumption (FE.C) and survival (%) (Sur). Thymol chemotype of the essential oil of Thymus vulgaris (EOTV-T) and its nanoemulsion (NEOTV-T) 3.5. Hematology and blood biochemistry There were no significant differences for hemoglobin (Fig. 3 A), hematocrit (Fig. 3 B), glucose (Fig. 4 A), cholesterol (Fig. 4 B), triglycerides (Fig. 4 C), total protein (Fig. 4 D), globulin (Fig. 4 E), albumin (Fig. 4 F), alanine aminotransferase (Fig. 4 G) and alanine aminotransferase (Fig. 4 H) among treatments during the 60 days of cultivation (P > 0.05). 4. Discussion The zeta potential and average diameter of the nanoparticles present in a nanoemulsion can vary depending on the type of essential oil and the proportions of oil and emulsifier used (Zhao et al. 2018 ). The nanoemulsion produced here had a negative charge and a diameter of less than 150 nm, yet slight variation was observed in average diameter and zeta potential during storage for 35 days. The stability and size of a nanoparticle in a nanoemulsion is related to the zeta potential of its surface, which leads to an electrostatic repulsion between the particles, preventing their coalescence (Guo et al.2024). No mortality was observed during nor 24 hours after management and anesthesia procedures. However, 50 mg/L of EOTV-T and 300 mg/L of NEOTV-T exceeded the induction and recovery times, respectively, recommended by the literature. Previous studies have safely anesthetized C. macropomum with EOTV-T (Boaventura et al. 2022a ), but there have been no studies of NEOTV-T, or any other thymol chemotype essential oil, as an anesthetic in fish. However, other authors have safely used nanoparticles (Ferreira et al. 2020 ) and nanoemulsions of other chemotypes of essential oils as anesthetics for fish (Kheawfu et al. 2022 ; Rodrigues et al. 2021 ; Yostawonkul et al. 2019 ). Increasing the concentration of EOTV-T or NEOTV-T reduced induction time and increased recovery time. NEOTV-T was able to induce anesthesia within the range recommended by the literature (less than 2 minutes) (Keene et al. 1998 ) at concentrations lower than EOTV-T could. Furthermore, higher concentrations of NEOTV-T increased anesthesia recovery time when compared to the same doses of EOTV-T. The increased efficiency of essential oils in nanoparticle form is related to their greater miscibility in water and better absorption by the body (Kheawfu et al. 2017 ; López-Cánovas et al. 2020 ; Rodrigues et al. 2021 ). Ferreira et al. ( 2020 ) compared the use of zein nanoparticles containing eugenol (positive charge) during anesthesia of Oreochromis niloticus and found that the anesthesia induction time for fish anesthetized with 80 mg/L of eugenol was similar to that for animals anesthetized with 40 mg/L of nanoparticles. Kheawfu et al. ( 2017 ) compared the efficiency of clove oil and its nanoemulsion (negative charge) as an anesthetic for O. niloticus and also observed a shorter anesthesia induction time for animals anesthetized with the nanoemulsion. The present study found no significant difference in induction and recovery intervals using 50 mg/L of NEOTV-T and 75 mg/L of EOTV-T, and so they were selected to carry out the second experiment. The constant use of anesthetics (50mg/L of NEOTV-T and 75 mg/L of EOTV-T) during biometrics in the cultivation of C. macropomum did not influence its performance, showing that these substances can be used to facilitate its management without causing economic loss to production. Several studies have been carried out in recent years to evaluate the influence of anesthetics on the physiological responses of animals (Boaventura et al. 2022a ; Souza et al. 2018 ; Souza et al. 2019 ; Iversen et al. 2009 ; Purbosari et al. 2019 ). The present study, however, was the first to evaluate responses related to production parameters (performance) with the continuous use of anesthetics during animal handling. Pirhonen and Schreck. (2003) studied feed consumption of Oncorhynchus mykiss after handling with and without the use of anesthesia (MS-222, clove oil and CO 2 ) and observed the lowest feed consumption for animals anesthetized with MS- 222 and oil. Boaventura et al. ( 2022a ) studied the influence of two chemotypes (thymol and linalool) of the essential oil of T. vulgaris on feed consumption of C. macropomum after transport and observed no difference in feed consumption between fish transported with and without the presence of the anesthetic. However, these studies only evaluated the influence of these anesthetics on feed consumption in a short period of time (5 days), without presenting data on zootechnical performance. No differences were observed in the analyzed hematological and biochemical parameters. The concentrations of EOTV-T and NEOTV-T used in biometric management did not cause stress or chronic metabolic or physiological changes in the juveniles. Despite being used to mitigate the effects of stress, the use of anesthetics can also induce acute stress and hematological and biochemical changes in animals (Aydın and Barbas. 2020; Souza et al. 2019 ; Purbosari et al. 2019 ). However, the influence of these anesthetics on acute responses, which are generally observed shortly after anesthesia, were not evaluated in the present study. Hematological parameters are indicative of animal health and stress status, and may indicate the ability of a fish to adapt to new environmental conditions (Ananias et al. 2024 ). As there was no change in hemoglobin and hematocrit levels during the experiment, we suggest that the use of the anesthetics did not influence the oxygen transport of the studied fish during production. Increased hematocrit and hemoglobin levels are generally observed in situations of stress, physical exertion, or hypoxia exposure (Witeska et al. 2022 ). When subjected to stress, an organism goes through a series of physiological changes to mobilize energy reserves to increase energy production (Boaventura et al. 2022b ; Iversen et al. 2009 ). Increased levels of cholesterol, triglycerides and glucose may be indicative of stress, as they can be attributed to the production of cortisol (Assis et al. 2024 ; Godavarthy et al. 2012 ) and mobilization of fatty acids in the body to increase production energy (Lepic et al. 2014 ; Santos et al. 2021 ). In addition to stress-related changes, cholesterol and triglyceride levels can also provide important information about the dietary and metabolic conditions of animals (Santos et al. 2021 ). As no differences were observed in these parameters in the present study, we suggest that the use of anesthetics did not affect the metabolism of the juveniles, nor did it cause them stress. The enzymes ALT and AST are used as indicators of tissue damage and are also involved in the mobilization of amino acids through gluconeogenesis for the production of glucose (Boaventura et al. 2020 b; Chatterjee et al. 2006 ). An increase in ALT and AST levels could suggest a response related to stress or tissue damage caused by the use of anesthetics. Therefore, the use of EOTV-T and NEOTV-T did not show signs of tissue damage that would alter ALT and AST levels in the body. Furthermore, EOTV-T and NEOTV-T also did not cause changes in the levels of total plasma protein, including albumin and globulins, which could be an indication of changes in osmotic pressure, transport substances (Metcalf et al. 2007 ) and immunological response (Khalil et al. 2023 ) or intoxication to contaminants (Javed and Usmani. 2015), which in this case could be the anesthetics used. 5. Conclusions We conclude that the nanoemulsion of the thymol chemotype of the essential oil of Thymus vulgaris has a diameter smaller than 100 nm for up to 22 days. The nanoemulsion induces anesthesia at concentrations lower than the essential oil dissolved in alcohol and water. The continuous use of EOTV-T and NEOTV-T during biometric management did not influence performance and stress and can be used to facilitate animal management without causing economic losses to production. Future evaluations are necessary to increase the stability of the nanoemulsion for a longer period of time, and to evaluate its influence on acute stress and its use in larger animals. Declarations Author Contribution Túlio Pacheco Boaventura: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – Original Draft, Writing- Reviewing and Editing, Visualization.Fabio Aremil Costa dos Santos,Camila Gomes de Oliveira,Ricardo de Oliveira Correia,Sidney dos Santos Silva,Andre de Sena Souza,Imaculada de Morais Carvalho Ananias and Nárcia Carolina Santos da Silva:Conceptualization, Methodology, Validation, Formal analysis, Investigation.Ronald Kennedy Luz and Juliano Elvis de Oliveira:Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Investigation, Writing – Original Draft, Writing- Reviewing and Editing, Visualization, Supervision, Project Administration, Funding Acquisition.All authors reviewed the manuscript Acknowledgement We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq-Brazil (CNPq-Brasil – 402952/2021-9 and 402840/2023-2), Fundação de Amparo a Pesquisa de Minas Gerais – FAPEMIG (FAPEMIG-Brasil – APQ-01531-21 and BPD-00406-22), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES-Brazil. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 Jul, 2024 Reviews received at journal 29 Jun, 2024 Reviewers agreed at journal 19 Jun, 2024 Reviewers agreed at journal 18 Jun, 2024 Reviewers agreed at journal 18 Jun, 2024 Reviewers invited by journal 18 Jun, 2024 Editor assigned by journal 18 Jun, 2024 Submission checks completed at journal 17 Jun, 2024 First submitted to journal 17 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4594736","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321720634,"identity":"857c3a43-5eed-404a-9501-23c0c70405d5","order_by":0,"name":"Túlio Pacheco Boaventura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFAC5oYDDAwHePglwDwJGYIaeBgYwVpkJGcAWUAtPERpAVIHbAxuQFiEtdhLJDYe+LjjDo/x7ebjj27UWPAwsB8+ugGvLRKJDQdnnnnGY3bnWGJzzjGgw3jS0m4Q0nKYt+0wj9mNHMPmHDagFgkgmygtxjNAWv6RosVAAqglt40YLWceQvwicSMtcXZunwQPGyG/sLcnH/4ADDF7/hnJBz7nfKuT42c/fAyvFjAARwgMsBFUjqFlFIyCUTAKRgE6AACcYUxs6cOk7wAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Lavras","correspondingAuthor":true,"prefix":"","firstName":"Túlio","middleName":"Pacheco","lastName":"Boaventura","suffix":""},{"id":321720635,"identity":"85b8f468-3680-4227-8f9d-ed3745b6b2cf","order_by":1,"name":"Camila Gomes Oliveira","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"Gomes","lastName":"Oliveira","suffix":""},{"id":321720639,"identity":"8d9aee1b-0707-4deb-9804-521d5bd0dd15","order_by":2,"name":"Fábio Aremil Costa Santos","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Fábio","middleName":"Aremil Costa","lastName":"Santos","suffix":""},{"id":321720642,"identity":"be19ab28-3b57-4a46-a019-c14659f93c7a","order_by":3,"name":"Nárcia Carolina Santos Silva","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Nárcia","middleName":"Carolina Santos","lastName":"Silva","suffix":""},{"id":321720643,"identity":"43dd704a-cffd-455f-ae23-4f4392c30987","order_by":4,"name":"Juliano Elvis Oliveira","email":"","orcid":"","institution":"Federal University of Lavras","correspondingAuthor":false,"prefix":"","firstName":"Juliano","middleName":"Elvis","lastName":"Oliveira","suffix":""},{"id":321720644,"identity":"d0c39460-9fbb-4a57-abef-790796eb4cbb","order_by":5,"name":"Ronald Kennedy Luz","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"Kennedy","lastName":"Luz","suffix":""},{"id":321720645,"identity":"d53e04cc-36c2-4f0d-9ea1-335ef53b1985","order_by":6,"name":"Ricardo Correia","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Correia","suffix":""},{"id":321720646,"identity":"acdaf917-4d10-4a8b-932d-dd111cb90f41","order_by":7,"name":"Sidney Silva","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Sidney","middleName":"","lastName":"Silva","suffix":""},{"id":321720647,"identity":"06174faa-db15-4f27-b054-2299d4fde39f","order_by":8,"name":"Ándre Souza","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Ándre","middleName":"","lastName":"Souza","suffix":""},{"id":321720648,"identity":"74208671-7853-4a40-88ff-e95a981cf33f","order_by":9,"name":"Imaculada Ananias","email":"","orcid":"","institution":"Federal University of Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Imaculada","middleName":"","lastName":"Ananias","suffix":""}],"badges":[],"createdAt":"2024-06-17 14:25:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4594736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4594736/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59585100,"identity":"df75c4dd-f9e6-46ee-b072-f497256f603f","added_by":"auto","created_at":"2024-07-03 13:32:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":684847,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size by DLS (A) and zeta potential (mV) (B) (mean ± standard deviation) of \u003cem\u003eThymus vulgaris\u003c/em\u003e nanoparticles analyzed on different days after nanoemulsification. Results followed by different letters differ significantly between days (P\u0026lt;0.05). One-way ANOVA and Tukey's test were used to determine statistical significance for particle size. Data regarding zeta potential were classified as non-parametric and so were analyzed using the Kruskal-Wallis test followed by Dunn's post-test.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4594736/v1/589756c4600ba1b31fd9b580.jpg"},{"id":59585097,"identity":"adace6c4-18b7-4c59-b34c-f88657b95acf","added_by":"auto","created_at":"2024-07-03 13:32:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125352,"visible":true,"origin":"","legend":"\u003cp\u003eAnesthesia induction and recovery curve. Induction (A, B) and recovery (C, D) times for juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e (n = 10 per treatment) subjected to different concentrations of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV-T).\u0026nbsp; The equations for EOTV-T were fitted to the data: Induction: Y=-33.5201+(10845.1284/x) R\u003csup\u003e2\u003c/sup\u003e=0.90 (A); Recovery Y=55.6857+0.6921*X, R\u003csup\u003e2\u003c/sup\u003e=0.86 (B). The equations for NEOTV-T were fitted to the data: Induction (Y= -1.6087 +(5738.3008/x), R\u003csup\u003e2\u003c/sup\u003e=0.95 (C); Recovery: (Y=28.8568+1.1323*X), R\u003csup\u003e2\u003c/sup\u003e=0.95 (D).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4594736/v1/161cdf2cabdc5a52238e1d38.jpg"},{"id":59585098,"identity":"5c9566f4-3249-40df-b518-7a89fbf3471b","added_by":"auto","created_at":"2024-07-03 13:32:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2139849,"visible":true,"origin":"","legend":"\u003cp\u003eHematological analyses (mean ± standard deviation) for juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e after 60 days of cultivation with subjection to biometric management with and without the use of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e of the (EOTV-T) and its nanoemulsion (NEOTV-T). Hemoglobin (A) and hematocrit (B). Results followed by different letters differ significantly between treatments (P\u0026lt;0.05). Data relating to hematocrit were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test. Data for hemoglobin were analyzed by One-way ANOVA and Tukey’s test.\u003c/p\u003e","description":"","filename":"FIgure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4594736/v1/4e0c62267145a3796810c1c8.jpg"},{"id":59585099,"identity":"65f5b385-5082-48b3-a75b-1d6d98194cea","added_by":"auto","created_at":"2024-07-03 13:32:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7339960,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical and enzymatic analyses (mean ± standard deviation) for juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e after 60 days of cultivation with subjection to biometric management with and without the use of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV-T). Glucose (A), cholesterol (B), triglycerides (C), total protein (D), globulin (E), albumin (F), alanine aminotransferase (G) and aspartate aminotransferase (H). Results followed by different letters differ significantly between treatments (P\u0026lt;0.05). Data relating to albumin were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test. The remaining results were analyzed by One-way ANOVA and Tukey’s test.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4594736/v1/34a2ca4c197bdc21e94ef1f3.jpg"},{"id":59585498,"identity":"f858a251-4c8f-49f5-9d2f-e5eeb5c69931","added_by":"auto","created_at":"2024-07-03 13:40:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11156085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594736/v1/15bd8c5b-e7aa-4cf7-8315-745f47b43df5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Use of the essential oil of Thymus vulgaris (thyme) and its nanoemulsion as an anesthetic during the cultivation of tambaqui (Colossoma macropomum): Anesthesia induction and recovery curve, physiology, performance and feed consumption","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTambaqui, \u003cem\u003eColossoma macropomum\u003c/em\u003e (family Characidae), is a species of freshwater fish originating from the Amazon basin that is of economic importance in aquaculture (Brian et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This scaled fish has dark spots on the ventral region (Boaventura et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), a robust body and tasty meat and can reach considerable sizes (Woyn\u0026aacute;rovich and Van Anrooy. 2019). The majority of \u003cem\u003eC. macropomum\u003c/em\u003e production is in the northern region of South America, and it is the second most produced fish species in Brazil (Peixe BR. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to its performance in captivity, its production has expanded to several tropical Asian countries, including China, Indonesia, Malaysia, Myanmar and Vietnam (Woyn\u0026aacute;rovich and Van Anrooy. 2019).\u003c/p\u003e \u003cp\u003eConcern for animal welfare and the search for sustainable aquaculture practices has led to increased exploration of the use of essential oils to replace synthetic anesthetics during fish handling procedures (Aydın and Barbas. 2020; Purbosari et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The use of these substances helps prevent physical damage during handling and even mitigates fish stress responses (Ross et al. 2009; Stoskopf and Posner. 2008). \u003cem\u003eThymus vulgaris\u003c/em\u003e, popularly known as thyme, is a plant species of the family Lamiacea (Azad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The essential oil extracted from this plant can present different types of major components, such as thymol, which is a promising substance for use as an anesthetic for several species of fish (Azad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bagamboula et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Brand\u0026atilde;o et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yousefi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), even tambaqui (Boaventura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to the use of traditional essential oils, several studies have evaluated the use of their nanoparticles or nanoemulsions as anesthetic agents (Ferreira et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; L\u0026oacute;pez-C\u0026aacute;novas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yostawonkul et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The use of nanoparticles and nanoemulsions obtained from these essential oils increases their solubility in water, which improves their absorption and thus reduces the amount of substance needed to induce anesthesia, thereby mitigating possible side effects in fish (Ferreira et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; L\u0026oacute;pez-C\u0026aacute;novas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite these studies using essential oils and their nanoemulsions as anesthetics, no study has evaluated the influence of the constant use of these substances during biometric management and their possible effects on animal performance.\u003c/p\u003e \u003cp\u003eTherefore, this study aimed to determine the ideal concentrations of the essential oil of \u003cem\u003eT. vulgaris\u003c/em\u003e and its nanoemulsion to be used during biometric management of juvenile \u003cem\u003eC. macropomum\u003c/em\u003e by evaluating the effects of their continuous use during cultivation on feed consumption, performance, physiology and metabolism.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1 Thymol chemotype of the essential oil of Thymus vulgaris\u003c/em\u003e (EOTV-T)\u003c/h2\u003e \u003cp\u003eThe thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e used in the experiments was acquired from the company Laszlo (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.emporiolaszlo.com.br\u003c/span\u003e\u003cspan address=\"https://www.emporiolaszlo.com.br\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which also provided its composition characterized by gas chromatography (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eComposition of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e,\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=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComposition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα- thujeno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-pineno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecanphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-pineno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emyrcene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-cymene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elimonene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.8 cineole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ey-terpinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.4\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=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etymol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ -caryophyllene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eThe essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e was acquired from the company Laszlo Aromoterapia LTDA, which also supplied their composition, obtained using a HP 5890 gas chro- matograph. Peaks less than 0.1% were disregarded.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e2.2 \u003cem\u003ePreparation of the nanoemulsion of the thymol chemotype of the essential oil of Thymus vulgaris (NEOTV-T).\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe nanoemulsion of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (NEOTV-T) was prepared following the method described by Candido et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with adaptations. Initially, an emulsion was prepared containing 5%w/w of EOTV, 1%w/w of Tween 80 and 94%w/w of deionized water. The water and oils were first mixed using a mechanical mixer (Ultraturrax, IKA T10) at 14,500 rpm for 10 minutes at a temperature of 27\u0026deg;C. For nanoemulsion, the emulsion was then subjected to an ultrasonic tip sonicator (Ultronique, Desruptor, Indaiatuba-SP) operating at 70% amplitude, using a 4 mm diameter probe immersed halfway into the emulsion. Five cycles of 4 minutes (2 minutes on and 2 minutes off) were used during the process to avoid heating the emulsion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of the nanoemulsion of the thymol chemotype of the essential oil of Thymus vulgaris.\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Dynamic Light Scattering (DLS) and Zeta Potential\u003c/h2\u003e \u003cp\u003eDiameter measurements were made with DLS and zeta potential of \u003cem\u003eT. vulgaris\u003c/em\u003e nanoparticle samples at a concentration of 0.4 mg/mL. Both DLS and zeta potential tests were performed with ZetaPALS equipment (Brookhaven Instruments). Analyses were performed on days 1, 2, 3, 6, 15, 22 and 37 after sample preparation. Means and standard deviations were obtained by taking 30 readings of DLS and Zeta Potential for each analysis day.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Animals and laboratory acclimatization\u003c/h2\u003e \u003cp\u003eThree hundred and ten juvenile \u003cem\u003eC. macropomum\u003c/em\u003e (2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88g and 5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 cm) were distributed and acclimatized in 30 L tanks filled with 28 L of water and maintained in recirculating aquaculture systems (RAS) for 7 days. During acclimatization, the juveniles were fed an extruded commercial diet (40% crude protein, WeaN Prime 1.8 to 2.3 mm \u0026ndash; Bernaqua) twice a day (08:00 and 16:00 h) until apparent satiety. Water quality parameters were measured daily during this period, and were as follows: temperature (28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C) and pH (7.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21) were measured using a COMBO portable pH meter (HANNA-HI 98107), dissolved oxygen (6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mg/L) by a digital oximeter (HANNA-HI9146), and total ammonia (0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 mg/L) using Alcon colorimetric kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experimental design\u003c/h2\u003e \u003cp\u003eTwo experiments were carried out at the Laborat\u0026oacute;rio de Aquicultura of the Universidade Federal de Minas Gerais (UFMG). The methodology was approved by the Comiss\u0026atilde;o de \u0026Eacute;tica no Uso de Animais of UFMG, process CEUA: 37/2024.\u003c/p\u003e \u003cp\u003eThe anesthetic solutions were prepared by weighing the essential oil of \u003cem\u003eT. vulgaris\u003c/em\u003e (EOTV-T) and diluting it 1:10 (w/w) in 99% ethanol. The nanoemulsion (NEOTV-T) was diluted directly in water.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Experiment 1. Anesthesia induction and recovery for \u003cem\u003eColossoma macropomum\u003c/em\u003e undergoing anesthesia with EOTV-T and NEOTV-T\u003c/h2\u003e \u003cp\u003eOne-hundred and thirty juvenile \u003cem\u003eC. macropomum\u003c/em\u003e were fasted for 24 hours. Anesthesia induction time was evaluated using 13 2-L aquariums, one being the control (containing water with the same amount of alcohol used to dilute the highest concentration of EOTV-T tested), six with different concentrations of EOTV-T ( 50, 75,100, 150, 200 and 300 mg/L) and another six with different concentrations of NEOTV-T (50, 75, 100, 150, 200 and 300 mg/L). Another 13 2-L aquariums containing only clean water were used to determine anesthesia recovery time.\u003c/p\u003e \u003cp\u003eTen juveniles were used for each concentration tested. Induction and recovery times were determined according to the criteria described by (Schoettger and Julin. 1967), whereby induction time was determined when a juvenile presented loss of balance and absence of external stimuli, while recovery time was determined through recovery of these responses. After recovery, the juveniles were transferred again to their respective RAS tanks for determination of survival rate 24 hours after the procedure.\u003c/p\u003e \u003cp\u003e2.5.2 Experiment 2. Performance and physiological responses of juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e subjected to constant biometric management with and without the use of EOTV-T and NEOTV-T\u003c/p\u003e \u003cp\u003eBased on the results of Experiment 1, a new experiment was carried out with three treatments for biometrics with four repetitions each: control (0 mg/L), 75 mg/L of thyme essential oil (EOTV-T) and 50 mg/L of thyme nanoemulsion (NEOTV -T). These concentrations were chosen because they present induction and recovery intervals that do not differ significantly from those indicated in the literature (less than 3 minutes for induction and less than 5 minutes for recovery) (Keene et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Marking and Meyer. 1985). One-hundred and eighty juveniles (2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88g and 5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 cm) were used for this experiment. Before starting the experiment, the juveniles underwent biometric procedures with and without the use of anesthetics. The juveniles were then distributed in 12 150-L tanks (useful volume of 100 L) at 15 fish/tank kept in a RAS where they were cultivated for 60 days. Biometrics were performed every 10 days. The NEOTV-T used in the biometric procedures was used within 20 days of preparation.\u003c/p\u003e \u003cp\u003eDuring the experiment, juveniles were fed until apparent satiety twice a day (08:00 and 16:00 h) with the same extruded commercial diet used during acclimatization. The food provided was previously weighed for each tank. Leftovers were collected 30 minutes after feeding, which were dried and weighed to estimate consumption and feed conversion.\u003c/p\u003e \u003cp\u003eAfter 60 days, all juveniles were weighed and measured (without the use of anesthesia) for blood collection.\u003c/p\u003e \u003cp\u003eWater quality parameters relating to temperature and pH were measured daily using a multi-parameter probe (HANNA brand, model HI98129). Dissolved oxygen (DO) and ammonia were measured every 3 days using a digital oximeter (HANNA brand, model HI9146-04) and colorimetric kits from Alcon, respectively.\u003c/p\u003e \u003cp\u003eBiometric procedures were performed individually for each repetition every 10 days and at the end of 60 days. During biometrics, the juveniles were captured in the culture tanks with the aid of a pulsar and placed in a 20-L bucket (filled with 10 L of water from the culture tank itself) containing or not the addition of anesthetic (EOTV-T or NEOTV-T). The juveniles were collected individually in the bucket, contained in a damp cloth, weighed using a semi-analytical scale (Marte AD 2000 0.01 g) and measured for total length with a ruler to determine final weight (PF) and total length (TC). After the biometrics process, the juveniles were returned to the cultivation tank where recovery from anesthesia took place. Biometric data and feed consumption were used to calculate the following:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eWeight gain (WG) ((final weight - initial weight)/initial weight)*100);\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFeed consumption (FE.C (g) = (offered food (g) - dry leftover food (g))/ number of animals);\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eApparent feed conversion (FCR) (feed consumption/weight gain).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eSurvival was also determined every 10 days and at the end of the experiment. The survival rate was determined by counting the number of initial and final animals in each phase and calculating the following:\u003c/p\u003e \u003cp\u003eSurvival (%) = (number of live fish x 100) / (total initial number of fish per tank)\u003c/p\u003e \u003cp\u003eFish physiology and stress were evaluated by collecting blood from 12 animals of each treatment, 3 from each repetition. Two collections were made from each animal: the first collected about 1 mL (0.7 mL using heparinized syringes, to which 10% heparin was subsequently added; the second collected about 0.3 mL in syringes without heparin). Part of the aliquots containing heparin were used to determine hemoglobin (Hb) (Quibasa-Bioclin, Belo Horizonte, MG, Brazil) and hematocrit (HTC) using the micro-hematocrit method with capillary tubes centrifuged in a microcentrifuge followed by reading in an appropriate table (Goldenfarb et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSerum (without heparin) was used to determine total plasma protein (TPP) and albumin. The remainder of the collected blood was centrifuged at 4000 rpm for 5 minutes, after which the plasma was separated and used for biochemical analyses and determination of glucose (GLU), triglycerides (TRY), cholesterol (CHO) and the enzymes alanine aminotransferase enzymes (ALT) and aspartate aminotransferase (AST), using commercial colorimetric kits (Quibasa-Bioclin, Belo Horizonte, MG, Brazil) followed by reading on a spectrophotometer (Bioclin 100\u0026reg; semi-automatic analyzer).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were subjected to the Shapiro-Wilk normality test and Levene's test for homoscedasticity. Anesthesia induction and recovery times were subjected to regression analysis. Parametric data were analyzed by One-way ANOVA followed by Tukey's test at 5% probability. Data regarding Zeta Potential, pH, hematocrit and albumin were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test at 5% probability. SigmaPlot and Infostat software were used for data analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Dynamic Light Scattering (DLS) and Zeta Potential\u003c/h2\u003e \u003cp\u003eThe average diameter of the particles present in the nanoemulsion increased during storage, but did not exceed 15nm after 35 days of storage. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The zeta potential of the particles was approximately \u0026minus;\u0026thinsp;19mV and reduced slightly to -10mV after 35 days of storage. A decrease in zeta potential contributes to increasing the average diameter of nanoparticles due to instability processes such as coalescence observed in nanoemulsions with zeta potential lower than 35mV in modulus. The zeta potential of the particles remained unchanged in the last days of storage, day 23 to day 37 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExperiment 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Anesthesia induction and recovery times\u003c/h2\u003e \u003cp\u003eNo mortality was observed during nor 24 hours after anesthesia. The induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) curves for the juveniles and the average data analyzed by ANOVA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) show that the increase in EOTV-T concentration reduced induction time until the concentration of 150 mg/L.\u003c/p\u003e \u003cp\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\u003eInduction and recovery times for juvenile tambaqui (\u003cem\u003eColossoma macropomum\u003c/em\u003e) subjected to different concentrations of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e and its nanoemulsion.\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=\"left\" 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\u003eConcentration\u003c/p\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInduction time (s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eRecuperation time (s)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEOTV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNEOTV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEOTV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNEOTV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e201.4\u0026thinsp;\u0026plusmn;\u0026thinsp;23.44aA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35bA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.85\u0026thinsp;\u0026plusmn;\u0026thinsp;11.53aC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e109.86\u0026thinsp;\u0026plusmn;\u0026thinsp;18.11aD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.89\u0026thinsp;\u0026plusmn;\u0026thinsp;12.44aB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.20bB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e131.87\u0026thinsp;\u0026plusmn;\u0026thinsp;26.40aCB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e118.50\u0026thinsp;\u0026plusmn;\u0026thinsp;19.98aD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15aC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.49\u0026thinsp;\u0026plusmn;\u0026thinsp;6.30aC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e135.25\u0026thinsp;\u0026plusmn;\u0026thinsp;35.05aCB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e123.28\u0026thinsp;\u0026plusmn;\u0026thinsp;11.65aD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.20aD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68aD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e113.62\u0026thinsp;\u0026plusmn;\u0026thinsp;17.45bC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e170.14\u0026thinsp;\u0026plusmn;\u0026thinsp;32.52aC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.90\u0026thinsp;\u0026plusmn;\u0026thinsp;5.08aD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61aE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e167.84\u0026thinsp;\u0026plusmn;\u0026thinsp;31.00bB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e260.83\u0026thinsp;\u0026plusmn;\u0026thinsp;41.52aB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35aD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20aE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e286.00\u0026thinsp;\u0026plusmn;\u0026thinsp;32.68bA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e382.16\u0026thinsp;\u0026plusmn;\u0026thinsp;31.25aA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eResults expressed as Means\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation. Thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV-T). Results followed by different lowercase letters indicate a significant difference between treatments at the same concentration. Results with different capital letters indicate a significant difference between concentrations of the same treatment by ANOVA followed by Tukey\u0026rsquo;s test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eJuveniles anesthetized with 50, 75, 100 and 150 mg/L of EOTV-T had similar anesthesia recovery times, while those anesthetized with 200 mg/L of EOTV-T had a longer recovery time than did those anesthetized with 50 mg/L. The longest recovery time using EOTV-T was for juveniles anesthetized with 300 mg/L (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreasing NEOTV-T concentration reduced anesthesia induction time up to a concentration of 200 mg/L. The concentration of 300 mg/L of NEOTV-T provided an induction time similar to that of 200 mg/L (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Juveniles anesthetized with 50, 75 and 100 mg/L of NEOTV-T had similar anesthesia recovery times. From 150 mg/L onwards, increasing NEOTV-T concentration increased recovery time, reaching its greatest with 300 mg/L NEOTV-T.\u003c/p\u003e \u003cp\u003eWhen comparing EOTV-T with NEOTV-T at the same concentration, 50 and 75 mg/L of NEOTV-T caused faster anesthesia induction than did the same concentrations of EOTV-T (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No differences in induction time were observed between EOTV-T and NEOT-V at concentrations of 100, 150, 200 and 300 mg/L. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the recovery times from anesthesia and the lack of differences between juveniles anesthetized with EOTV-T and NEOTV-T at concentrations 50, 75 and 100 mg/L. Juveniles anesthetized with 150, 200 and 300 mg/L of NEOTV-T had longer anesthesia recovery times than did those anesthetized with the same concentration of EOTV-T.\u003c/p\u003e \u003cp\u003eExperiment 2\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Water quality parameters\u003c/h2\u003e \u003cp\u003eThere were no significant differences in water quality parameters (dissolved oxygen, temperature, ammonia and pH) among treatments during the 60 days of cultivation (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=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWater quality parameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) during the cultivation of juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e subjected to biometric management with and without the use of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV -T).\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003cp\u003e(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOD\u003c/p\u003e \u003cp\u003e(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmmonia Total (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent capital letters indicate a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between treatments. One-way ANOVA and Tukey\u0026rsquo;s test were used to determine statistical significance. Data relating to pH were classified as non-parametric and were analyzed using the Kruskal-Wallis test followed by Dunn's post-test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Performance\u003c/h2\u003e \u003cp\u003eThere were no significant differences for final weight, final length, weight gain, apparent feed conversion, feed consumption and survival among treatments throughout the 60 days of cultivation (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePerformance parameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) obtained during the cultivation of juvenile \u003cem\u003eColossoma macropomum\u003c/em\u003e subjected to biometric management with and without the use of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV -T).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFW (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFL (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWG (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFE.C (g/fish)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSUR (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e1 at 10 days of cultivation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e132.04\u0026thinsp;\u0026plusmn;\u0026thinsp;12.34a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130.33\u0026thinsp;\u0026plusmn;\u0026thinsp;21.28a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e139.17\u0026thinsp;\u0026plusmn;\u0026thinsp;16.95a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e11 at 20 days of cultivation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.88\u0026thinsp;\u0026plusmn;\u0026thinsp;20.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.40\u0026thinsp;\u0026plusmn;\u0026thinsp;12.61a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.88\u0026thinsp;\u0026plusmn;\u0026thinsp;6.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e21 at 30 days of cultivation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.76\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.34\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.80\u0026thinsp;\u0026plusmn;\u0026thinsp;11.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e98.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31 at 40 days of cultivation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.84\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.54\u0026thinsp;\u0026plusmn;\u0026thinsp;8.61a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.40a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.84\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e41 at 50 days of cultivation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.70\u0026thinsp;\u0026plusmn;\u0026thinsp;7.03a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.18\u0026thinsp;\u0026plusmn;\u0026thinsp;6.45a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.96\u0026thinsp;\u0026plusmn;\u0026thinsp;6.64a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.35\u0026thinsp;\u0026plusmn;\u0026thinsp;6.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.38\u0026thinsp;\u0026plusmn;\u0026thinsp;12.16a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003e51 at 60 days of cultivation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.09\u0026thinsp;\u0026plusmn;\u0026thinsp;10.37a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.01\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.34\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.17\u0026thinsp;\u0026plusmn;\u0026thinsp;9.76a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.10a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal 1 at 60 days of cultivation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.09\u0026thinsp;\u0026plusmn;\u0026thinsp;10.37a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1431.3\u0026thinsp;\u0026plusmn;\u0026thinsp;260.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.01\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1165.0\u0026thinsp;\u0026plusmn;\u0026thinsp;187.8a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.61\u0026thinsp;\u0026plusmn;\u0026thinsp;7.62a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eNEOTV-T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.17\u0026thinsp;\u0026plusmn;\u0026thinsp;9.76a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1381.1\u0026thinsp;\u0026plusmn;\u0026thinsp;291.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.54\u0026thinsp;\u0026plusmn;\u0026thinsp;11.66a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e98.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eResults followed by different letters indicate a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by ANOVA followed by Tukey's test at 5%. Final weight (g) (FW), final length (cm) (FL), weight gain (WG), feed conversion (FC), feed consumption (FE.C) and survival (%) (Sur). Thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV-T)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Hematology and blood biochemistry\u003c/h2\u003e \u003cp\u003eThere were no significant differences for hemoglobin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), hematocrit (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), cholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), triglycerides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), total protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), globulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), albumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), alanine aminotransferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) and alanine aminotransferase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) among treatments during the 60 days of cultivation (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe zeta potential and average diameter of the nanoparticles present in a nanoemulsion can vary depending on the type of essential oil and the proportions of oil and emulsifier used (Zhao et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The nanoemulsion produced here had a negative charge and a diameter of less than 150 nm, yet slight variation was observed in average diameter and zeta potential during storage for 35 days. The stability and size of a nanoparticle in a nanoemulsion is related to the zeta potential of its surface, which leads to an electrostatic repulsion between the particles, preventing their coalescence (Guo et al.2024).\u003c/p\u003e \u003cp\u003eNo mortality was observed during nor 24 hours after management and anesthesia procedures. However, 50 mg/L of EOTV-T and 300 mg/L of NEOTV-T exceeded the induction and recovery times, respectively, recommended by the literature. Previous studies have safely anesthetized \u003cem\u003eC. macropomum\u003c/em\u003e with EOTV-T (Boaventura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), but there have been no studies of NEOTV-T, or any other thymol chemotype essential oil, as an anesthetic in fish. However, other authors have safely used nanoparticles (Ferreira et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and nanoemulsions of other chemotypes of essential oils as anesthetics for fish (Kheawfu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yostawonkul et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreasing the concentration of EOTV-T or NEOTV-T reduced induction time and increased recovery time. NEOTV-T was able to induce anesthesia within the range recommended by the literature (less than 2 minutes) (Keene et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) at concentrations lower than EOTV-T could. Furthermore, higher concentrations of NEOTV-T increased anesthesia recovery time when compared to the same doses of EOTV-T. The increased efficiency of essential oils in nanoparticle form is related to their greater miscibility in water and better absorption by the body (Kheawfu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; L\u0026oacute;pez-C\u0026aacute;novas et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ferreira et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) compared the use of zein nanoparticles containing eugenol (positive charge) during anesthesia of \u003cem\u003eOreochromis niloticus\u003c/em\u003e and found that the anesthesia induction time for fish anesthetized with 80 mg/L of eugenol was similar to that for animals anesthetized with 40 mg/L of nanoparticles. Kheawfu et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) compared the efficiency of clove oil and its nanoemulsion (negative charge) as an anesthetic for \u003cem\u003eO. niloticus\u003c/em\u003e and also observed a shorter anesthesia induction time for animals anesthetized with the nanoemulsion. The present study found no significant difference in induction and recovery intervals using 50 mg/L of NEOTV-T and 75 mg/L of EOTV-T, and so they were selected to carry out the second experiment.\u003c/p\u003e \u003cp\u003eThe constant use of anesthetics (50mg/L of NEOTV-T and 75 mg/L of EOTV-T) during biometrics in the cultivation of \u003cem\u003eC. macropomum\u003c/em\u003e did not influence its performance, showing that these substances can be used to facilitate its management without causing economic loss to production. Several studies have been carried out in recent years to evaluate the influence of anesthetics on the physiological responses of animals (Boaventura et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Iversen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Purbosari et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The present study, however, was the first to evaluate responses related to production parameters (performance) with the continuous use of anesthetics during animal handling. Pirhonen and Schreck. (2003) studied feed consumption of \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e after handling with and without the use of anesthesia (MS-222, clove oil and CO\u003csub\u003e2\u003c/sub\u003e) and observed the lowest feed consumption for animals anesthetized with MS- 222 and oil. Boaventura et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) studied the influence of two chemotypes (thymol and linalool) of the essential oil of \u003cem\u003eT. vulgaris\u003c/em\u003e on feed consumption of \u003cem\u003eC. macropomum\u003c/em\u003e after transport and observed no difference in feed consumption between fish transported with and without the presence of the anesthetic. However, these studies only evaluated the influence of these anesthetics on feed consumption in a short period of time (5 days), without presenting data on zootechnical performance.\u003c/p\u003e \u003cp\u003eNo differences were observed in the analyzed hematological and biochemical parameters. The concentrations of EOTV-T and NEOTV-T used in biometric management did not cause stress or chronic metabolic or physiological changes in the juveniles. Despite being used to mitigate the effects of stress, the use of anesthetics can also induce acute stress and hematological and biochemical changes in animals (Aydın and Barbas. 2020; Souza et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Purbosari et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the influence of these anesthetics on acute responses, which are generally observed shortly after anesthesia, were not evaluated in the present study.\u003c/p\u003e \u003cp\u003eHematological parameters are indicative of animal health and stress status, and may indicate the ability of a fish to adapt to new environmental conditions (Ananias et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As there was no change in hemoglobin and hematocrit levels during the experiment, we suggest that the use of the anesthetics did not influence the oxygen transport of the studied fish during production. Increased hematocrit and hemoglobin levels are generally observed in situations of stress, physical exertion, or hypoxia exposure (Witeska et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen subjected to stress, an organism goes through a series of physiological changes to mobilize energy reserves to increase energy production (Boaventura et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Iversen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Increased levels of cholesterol, triglycerides and glucose may be indicative of stress, as they can be attributed to the production of cortisol (Assis et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Godavarthy et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and mobilization of fatty acids in the body to increase production energy (Lepic et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to stress-related changes, cholesterol and triglyceride levels can also provide important information about the dietary and metabolic conditions of animals (Santos et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As no differences were observed in these parameters in the present study, we suggest that the use of anesthetics did not affect the metabolism of the juveniles, nor did it cause them stress. The enzymes ALT and AST are used as indicators of tissue damage and are also involved in the mobilization of amino acids through gluconeogenesis for the production of glucose (Boaventura et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003eb; Chatterjee et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). An increase in ALT and AST levels could suggest a response related to stress or tissue damage caused by the use of anesthetics. Therefore, the use of EOTV-T and NEOTV-T did not show signs of tissue damage that would alter ALT and AST levels in the body.\u003c/p\u003e \u003cp\u003eFurthermore, EOTV-T and NEOTV-T also did not cause changes in the levels of total plasma protein, including albumin and globulins, which could be an indication of changes in osmotic pressure, transport substances (Metcalf et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and immunological response (Khalil et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) or intoxication to contaminants (Javed and Usmani. 2015), which in this case could be the anesthetics used.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe conclude that the nanoemulsion of the thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e has a diameter smaller than 100 nm for up to 22 days. The nanoemulsion induces anesthesia at concentrations lower than the essential oil dissolved in alcohol and water. The continuous use of EOTV-T and NEOTV-T during biometric management did not influence performance and stress and can be used to facilitate animal management without causing economic losses to production. Future evaluations are necessary to increase the stability of the nanoemulsion for a longer period of time, and to evaluate its influence on acute stress and its use in larger animals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT\u0026uacute;lio Pacheco Boaventura: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing \u0026ndash; Original Draft, Writing- Reviewing and Editing, Visualization.Fabio Aremil Costa dos Santos,Camila Gomes de Oliveira,Ricardo de Oliveira Correia,Sidney dos Santos Silva,Andre de Sena Souza,Imaculada de Morais Carvalho Ananias and N\u0026aacute;rcia Carolina Santos da Silva:Conceptualization, Methodology, Validation, Formal analysis, Investigation.Ronald Kennedy Luz and Juliano Elvis de Oliveira:Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Investigation, Writing \u0026ndash; Original Draft, Writing- Reviewing and Editing, Visualization, Supervision, Project Administration, Funding Acquisition.All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico-CNPq-Brazil (CNPq-Brasil \u0026ndash; 402952/2021-9 and 402840/2023-2), Funda\u0026ccedil;\u0026atilde;o de Amparo a Pesquisa de Minas Gerais \u0026ndash; FAPEMIG (FAPEMIG-Brasil \u0026ndash; APQ-01531-21 and BPD-00406-22), and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; CAPES-Brazil.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnanias IMC, Silva SS, Santos FAC, Souza AS, Magalh\u0026atilde;es TB, Reis PAR, Favero GC, Luz RK (2024) Tambaqui Production at Different Stocking Densities in RAS: Growth and Physiology. 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Food Chem 269:577\u0026ndash;587. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2018.07.062\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2018.07.062\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Biochemistry, fish, hematology, thymol, welfare","lastPublishedDoi":"10.21203/rs.3.rs-4594736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe thymol chemotype of the essential oil of \u003cem\u003eThymus vulgaris\u003c/em\u003e (EOTV-T) and its nanoemulsion (NEOTV-T) were investigated as anesthetics in the management of \u003cem\u003eColossoma macropomum\u003c/em\u003e. The stability of nanoemulsions was analyzed by zeta potential and dynamic light scattering for an interval of 37 days prior to carrying out two anesthesia experiments. The first experiment determined anesthesia induction and recovery intervals for juveniles anesthetized with different concentrations of EOTV-T and NEOTV-T. Based on the obtained induction and recovery curve, the second experiment adopted concentrations of 75 mg/L of EOTV-T and 50 mg/L of NEOTV-T for use during biometric management every 10 days for 60 days of cultivation of juvenile \u003cem\u003eC. macropomum\u003c/em\u003e. The results showed that NEOTV-T had an average diameter of less than 100 nm for up to 22 days and was more effective at inducing anesthesia than EOTV-T. The continuous use of anesthetics during biometrics did not affect juvenile performance in terms of growth, feed consumption and survival. Furthermore, no significant differences were observed in hematological and biochemical parameters between juveniles of the control treatment and those subjected to biometric management using 75 mg/L of EOTV-T and 50 mg/L of NEOTV-T. It can be concluded that the essential oil of \u003cem\u003eT. vulgaris\u003c/em\u003e and its nanoemulsion can be used to facilitate the management of juvenile \u003cem\u003eC. macropomum\u003c/em\u003e without compromising their performance or well-being. More studies need to be carried out to improve the stability of nanoemulsions of the essential oil of \u003cem\u003eT. vulgaris\u003c/em\u003e and to evaluate their effects on acute stress.\u003c/p\u003e","manuscriptTitle":"Use of the essential oil of Thymus vulgaris (thyme) and its nanoemulsion as an anesthetic during the cultivation of tambaqui (Colossoma macropomum): Anesthesia induction and recovery curve, physiology, performance and feed consumption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 13:32:00","doi":"10.21203/rs.3.rs-4594736/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-07-02T12:38:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-29T09:39:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185274833216488002523194044735870582665","date":"2024-06-19T04:42:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78949881509189795078431845710173191846","date":"2024-06-18T08:10:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191820666255407868527707725564122172311","date":"2024-06-18T07:52:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-18T05:32:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-18T05:27:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-17T22:15:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2024-06-17T14:23:43+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":"084b7d44-fb86-4aa3-9ef0-6390fff5b013","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-11T15:36:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 13:32:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4594736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594736","identity":"rs-4594736","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}