Anaesthetic efficacy of clove oil,benzocaine, 2‐phenoxyethanol and tricaine methanesulfonate in juvenile marbled spinefoot (Siganus rivulatus)

Anaesthetics are used in aquaculture and fisheries to facilitate routine procedures, such as capture, handling, transportation, tagging, grading and measurements that can often cause injury or induce physiological stress. Two experiments were performed to assess the efficacies of four anaesthetic agents, clove oil, benzocaine, 2‐phenoxyethanol and MS‐222 on juvenile marbled spinefoot rabbitfish (Siganus rivulatus). In the first experiment we tested the lowest effective doses that produced induction and recovery times in 3 min or less and 5 min or less respectively. Dosages were 70 mg L^?1 for clove oil, 60–70 mg L^?1 for benzocaine, 400 μL L^?1 for 2‐phenoxyethanol and 100–125 mg L^?1 for MS‐222. In the second experiment, we determined optimal concentrations of the four anaesthetics if they were to be used to transport rabbitfish fry. Anaesthetic concentrations suitable for handling and transport were: 10–15 mg L^?1 of MS‐222, 5–10 mg L^?1 of benzocaine, 5 mg L^?1 of clove oil and 50–100 μL L^?1 of 2‐phenoxyethanol. All anaesthetic agents are acceptable for use on S. rivulatus, however, 2‐phenoxyethanol, MS‐222 and clove oil appear to be more suitable than benzocaine. Further studies need to be conducted on effects of high and low doses of anaesthetic agents on physiology of marbled spinefoot.     

The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss Walbaum)

The effects of 60‐mg L^?1 clove oil and 60‐mg L^?1 tricaine methanesulphonate (MS‐222) on the blood chemistry of rainbow trout were compared after exposure to handling stress via caudal puncture blood sampling. Fish sampled by caudal puncture and subsequently exposed to anaesthetics showed a typical handling stress response over a 48‐h period. There were no significant differences between the responses of fish exposed to equal concentrations of clove oil and MS‐222, with the following exceptions: the blood glucose at full anaesthesia, and lactate at full recovery increased significantly in the clove oil‐exposed fish. In a subsequent experiment, the stress response observed in fish sampled by caudal puncture and exposed to clove oil and MS‐222 was compared with a non‐anaesthetized control group. The increases in plasma cortisol levels were significantly lower at recovery in fish treated with either anaesthetic compared with the control fish. Fish exposed to MS‐222 had significantly higher cortisol levels at 1 h. These findings show that few differences exist between the anaesthetic effects of clove oil and MS‐222 on the physiological response of fish to stress. However, clove oil is more effective at reducing the short‐term stress response induced by handling and blood sampling, and is recommended as an alternative fish anaesthetic.     

Anaesthetic efficacy and physiological responses to clove oil-anaesthetized kelp grouper Epinephelus bruneus

The efficacy of clove oil as an anaesthetic and at producing a physiological response (plasma cortisol and glucose) was evaluated in the kelp grouper, Epinephelus bruneus . To acquire complete anaesthesia in less than 3 min and recovery in <10 min, three doses of clove oil were tested at 18, 22 and 26 °C. Although higher anaesthetic doses resulted in shorter induction times and longer recovery times, and a lower temperature resulted in longer anaesthesia induction and slower recovery, we found the optimal dose and administering temperature of clove oil to be 250–300 mg L⁻¹ at water temperature of 18 °C, 150–200 mg L⁻¹ at water temperature of 22 °C and 50–100 mg L⁻¹ at water temperature of 26 °C respectively. Following the administration of 150 mg L⁻¹ of clove oil at 22 °C, the plasma cortisol level was highest (4.24 ± 1.571 μg dL⁻¹) after 12 h and the plasma glucose was highest (92.7 ± 9.61 mg dL⁻¹) after 2 h. These results should be useful to the aquaculture industry, where anaesthesia is necessary for a range of activities.     

Clove oil as an anaesthetic for common octopus (Octopus minor, Sasaki)

We evaluated the anaesthetic effect of clove oil [2‐methoxy‐4‐2‐(2‐propenyl)‐phenol] on the common octopus (Octopus minor), in terms of the time required to become anaesthetized (‘anaesthetic time’) and recovery time. We used a factorial experimental design and administered clove oil at different temperatures (15, 20 and 25°C) and concentrations (50, 100, 150, 200, 250 and 300 mg L⁻¹). We observed a significant (P<0.05) relationship between concentration and temperature, and each variable was effective (P<0.05). Anaesthetic time linearly decreased as the concentration and temperature increased. However, recovery time increased as the concentration increased and temperature decreased. There was no mortality. A concentration of 200 mg L⁻¹ clove oil showed rapid anaesthetic and recovery times in the common octopus, indicating its suitability for this species.     

Effect of anaesthesia with clove oil in fish (review)

Clove oil is an effective, local and natural anaesthetic. Many hatcheries and research studies use clove oil to immobilize fish for handling, sorting, tagging, artificial reproduction procedures and surgery and to suppress sensory systems during invasive procedures. Clove oil may be more appropriate for use in commercial aquaculture situations. Improper clove oil use can decrease fish viability, distort physiological data or result in mortalities. Because animals may be anaesthetized by unskilled labourers and released in natural water bodies, training in the proper use of clove oil may decrease variability in recovery and experimental results and increase fish survival. Here, we briefly describe many aspects of clove oil, including the legal uses of it, anaesthesia mechanism and what is currently known about the preparation and behavioural and pathologic effects of the anaesthetic. We outline methods and precautions for administration and changes in fish behaviour during progressively deeper anaesthesia and discuss the physiological effects of clove oil, its potential for compromising fish health and effectiveness of water quality parameters.     

Comparison of tricaine methanesulphonate (MS222) and clove oil anaesthesia effects on the physiology of juvenile chinook salmon Oncorhynchus tshawytscha (Walbaum)

This study investigated the feasibility of using clove oil as an alternative to tricaine methanesulphonate as a fish anaesthetic, particularly in fish stress research. The physiological stress responses of juvenile chinook salmon Oncorhynchus tshawytscha (Walbaum) anaesthetized with either tricaine (50 mg L^?1) or clove oil (20 p.p.m.) were compared using unanaesthetized fish as controls. Haematocrit, serum cortisol and serum glucose concentrations, serum lysozyme activity and differential leucocyte counts were measured from blood samples collected before, during and upon recovery from anaesthesia and at specified intervals up to 72 h after recovery. Differences between the two anaesthetic groups were not significant for most of the physiological traits measured. Serum lysozyme activity of control fish, however, was significantly suppressed relative to the treated fish for 72 h after stress. Clove oil may be a safe and cost‐effective alternative to tricaine without significantly affecting study results. Furthermore, clove oil may be more practical for field‐based research, because a withdrawal period is unnecessary, and clove oil does not pose an environmental hazard.     

Effects of water temperature and body weight on anaesthetic efficiency in marbled rabbitfish (Siganus rivulatus)

The effects of four anaesthetic agents, tricaine methanesulphonate (MS‐222) (112.5 mg L^?1), 2‐phenoxyethanol (400 μL L^?1), clove oil (70 mg L^?1) and benzocaine (65 mg L^?1) on juvenile marbled spinefoot (Siganus rivulatus) of three mean body weights (7.3 g, 19.1 g, 55.5 g) and at three temperatures (20, 25, 30°C) were evaluated. In addition, the relationship between body lipid content and efficacy of the four anaesthetic agents was evaluated in juvenile S. rivulatus. Times necessary for induction and recovery were recorded. Significant effects of temperature on induction and recovery times were observed. Induction and recovery times decreased with increasing water temperature. No uniform relationship between body weight of juvenile marbled spinefoot and anaesthetic efficacy was observed. Body fat content was positively correlated with induction time only when MS‐222 was used but did not affect induction times of fish exposed to 2‐phenoxyethanol, clove oil or benzocaine. Recovery times were generally longer for all fish containing more body fat. Results of the study show that anaesthetic efficiency increases with increasing water temperature but is not strongly affected by body weight for juvenile marbled spinefoot. In addition, body fat in fish affected the efficacy of the various anaesthetic agents tested in this study, generally slowing down recovery.     

Olfactory nerve response of masu salmon (Oncorhynchus masou Brevoort) and rainbow trout (O. mykiss Walbaum) to clove oil and MS-222

Two anaesthetics, clove oil and methane sulphonate (MS-222), were examined for their effects on the olfactory nerve response of masu salmon ( Oncorhynchus masou Brevoort) and rainbow trout ( O. mykiss Walbaum). Exposing both species to clove oil for 3 min at concentrations of 50 and 100 mg L⁻¹, or for 10 min at 50 mg L⁻¹, did not significantly reduce their olfactory response. Directly applying clove oil anaesthesia to the olfactory epithelium significantly reduced olfactory response though after 20 min, olfactory response recovered to 70% and 52% of pre-treatment levels in masu salmon and rainbow trout respectively. Compared with the post-anaesthetic recovery of responses after clove oil (50 mg L⁻¹), buffered MS-222 (100 mg L⁻¹) with NaHCO₃ (100 mg L⁻¹), and unbuffered MS-222 (100 mg L⁻¹) treatment for 3 min, the response after MS-222 treatment declined gradually and significantly, but not after clove oil and MS-222+NaHCO₃ treatments. Clove oil appears to be an effective and relatively safe anaesthetic for salmonids with little long-term impact on their olfactory response, which plays a crucial role in their life history.     

Effects of clove oil and MS-222 on blood hormone profiles in rainbow trout Oncorhynchus mykiss, Walbaum

Clove oil has been demonstrated to be an effective, inexpensive anaesthetic and euthanizing agent for a number of fish species, including rainbow trout, used in aquaculture and fisheries research. However, the potential for clove oil to cause perturbations in important plasma hormone concentrations has not been investigated. The effect of anaesthesia and euthanasia in trout with eugenol (the active ingredient in clove oil) on plasma cortisol, glucose, growth hormone (GH) and two thyroid hormones [tri‐iodothyronine (T₃) and thyroxine (T₄)] was compared with tricaine methanesulfonate (MS‐222) anaesthesia, and stunning by cranial concussion in two experiments. Effects on blood chemistry were different when comparing the particular anaesthetic method being used. Stunning fish significantly increased plasma cortisol and glucose levels (both P<0.05), while euthanizing fish using either clove oil or MS‐222 had no effect on these hormone levels. In contrast, the levels of GH, T₃ and T₄ hormones were unaffected regardless of whether fish were euthanized by stunning, MS‐222 or clove oil. Variation in effects between hormones were observed using clove oil eugenol. In fish sampled 10 min after anaesthetizing with 150 mg L^?1 of eugenol, cortisol levels were significantly decreased (P<0.03), while there were no differences in either glucose or GH levels. Tri‐iodothyronine and T₄ also showed significantly elevated levels (P<0.05) after 10‐min exposure to eugenol. These results highlight the importance of investigating the potential effects of any new anaesthetic or euthanizing compounds on blood plasma parameters, prior to using them in a research setting, or when comparing results to other studies which have utilized alternative anaesthetic compounds.     

Comparison of the effects of four anaesthetics on biochemical blood profiles of perch

This study investigated the feasibility of using clove oil, 2-phenoxyethanol, or Propiscin as an alternative to tricaine methane sulphonate (MS 222) as a fish anaesthetic, particularly in regard to reducing fish stress. The biochemical blood profiles of perch Perca fluviatilis L. anaesthetized with either MS 222 (100 mg L⁻¹), clove oil (33 mg L⁻¹), 2-phenoxyethanol (0.40 mL L⁻¹) or Propiscin (1.0 mL L⁻¹), and a non-anaesthetized control group were compared. Biochemical profiles were determined from blood samples collected before treatment in controls. For each anaesthetic tested, fish were divided into two groups, one sampled immediately after 10-min anaesthesia and a second, sampled 24 h after 10-min anaesthesia. The values determined in the present study suggested that internal organs and tissues of perch were slightly altered by MS 222, clove oil and 2-phenoxyethanol anaesthesia, but not by Propiscin anaesthesia.     

Clove oil attenuates stress responses in lambari,Astyanax altiparanae

The effects of clove oil anaesthetic on mitigating the physiological responses to air exposure, a stressful and routine situation in fish farming, laboratory conditions and sport fishing (catch and release), were evaluated in lambari (A. altiparanae). Adult females (n = 80) were randomly sorted to receive one of four treatments: control, anaesthesia (clove oil 50 mg/L), stress (5 min air exposure) and pre‐anaesthesia associated to stress. Their cortisol, glucose, lactate and haematocrit levels, the hepatosomatic index (HSI), liver and muscle glycogen, lipid peroxidation and the enzymatic activity of lactate dehydrogenase (LDH), catalase (CAT) and glutathione reductase (GR), were recorded. Glucose levels increased (53.9%) after anaesthesia and/or stress. The stress situation increased plasma cortisol (146.6%), lactate (294.6%) and lipid peroxidation in white muscle (45%) and decreased glycogen in white muscle (40.1%). The haematocrit increased after stress or anaesthesia (7.9%) while the liver glycogen and HSI did not change. Anaesthesia or stress did not affect the LDH but reduced the activity of CAT (46.1%) and GR (30.3%). We concluded that the anaesthetic clove oil in the concentration 50 mg/L modulates the physiological responses to air exposure stress improving the welfare; air exposure and clove oil affect the antioxidant defences of lambari; the activity of CAT and GR and the concentration of MDA can be used as biomarkers of stress in A. altiparanae.     

The use of clove oil, metomidate, tricaine methanesulphonate and 2-phenoxyethanol for inducing anaesthesia and their effect on the cortisol stress response in black sea bass (Centropristis striata L.)

Juvenile and adult black sea bass (Centropristis striata L.) were exposed to various concentrations of four anaesthetics to determine practical dosages for handling as well as for procedures such as bleeding, ovarian biopsy or tag implantation. In experiment 1, juveniles exposed to either 2.0 mg L^?1 metomidate, 15 mg L^?1 clove oil, 70 mg L^?1 tricaine methanesulphonate (TMS) or 200 mg L^?1 2‐phenoxyethanol (2‐PE) reached stage II of anaesthesia in 3–5 min and could be handled for weighing and measuring. All fish had completed recovery to stage III within 6 min. In experiment 2, the established concentrations of each anaesthetic were tested on juveniles to determine their ability to prevent a reflex to a subcutaneous needle puncture. All of the fish exposed to clove oil (20 mg L^?1) and 40% of the TMS‐treated (70 mg L^?1) fish reacted while none of the fish anaesthetized in metomidate (2.0 mg L^?1) or 2‐PE (200 mg L^?1) responded to the needle puncture. In experiment 3, metomidate (5.0 mg L^?1), clove oil (30 mg L^?1) TMS (125 mg L^?1) or 2‐PE (300 mg L^?1) were all effective for performing an ovarian biopsy or tag implantation on adults. In experiment 4, TMS (125 mg L^?1) exacerbated the cortisol response to a short handling stressor during a 30 min exposure. Fish anaesthetized in 2‐PE (300 mg L^?1), metomidate (5.0 mg L^?1) or clove oil (40 mg L^?1) had increased cortisol levels associated with the handling stressor but there were no further increases during the remainder of the experimental period. The results demonstrate that these anaesthetics are effective for sedation and anaesthesia of black sea bass and that the best choice is dependant upon the procedures to be performed.     

The anaesthetic effect of camphor (Cinnamomum camphora), clove (Syzygium aromaticum) and mint (Mentha arvensis) essential oils on clown anemonefish,Amphiprion ocellaris (Cuvier 1830)

The aim of this study was to evaluate the use of clove (Syzygium aromaticum), camphor (Cinnamomum camphora) and mint (Mentha arvensis) essential oils as anaesthetics during the management of clown anemonefish (Amphiprion ocellaris). For 15 min, the animals were subjected to concentrations of 5, 10, 20, 27 and 35 μL L^?1 of clove oil, 17, 35, 50, 70 and 100 μL L^?1 of mint oil, and 200, 400, 500, 550 and 600 μL L^?1 of camphor oil (tested in 10 animals per concentration). A control group (without anaesthetic) and a complementary group, which was exposed to ethanol, were also evaluated. After exposure to the anaesthetic, the fish were transferred to clean water to assess recovery. The mortality and feeding behaviour of the fish were then observed for 48 h after exposure to the oils. All of the essential oils produced an anaesthetic effect on A. ocellaris. The 27, 70 and 500 μL L^?1 concentrations of clove, mint, and camphor oils promoted surgical anaesthesia after 310.5, 312.0, and 535.0 s (medians) respectively. The recovery times of fish exposed to these same concentrations were 396, 329.5 and 229 s respectively. The decision of which oil to use is dependent on the management situation and the consideration of the induction and recovery times of each essential oil.     

Cortisol and haematological response in sea bream and trout subjected to the anaesthetics clove oil and 2-phenoxyethanol

Clove oil has been tested for anaesthesia induction and recovery time as well as for haematology and stress indicators in the gilthead sea bream and rainbow trout. The former parameters were compared with those generated by 2‐phenoxyethanol. The results showed only slight differences between both anaesthetics in terms of anaesthetic efficiency and physiological effects. In addition, clove oil does not block the cortisol response to stress, as happens with other anaesthetics.     

Effects of anaesthesia with MS-222, clove oil and CO2 on feed intake and plasma cortisol in steelhead trout (Oncorhynchus mykiss)

We examined the effects tricaine methanesulfonate (MS-222), clove oil and CO₂ on feed intake and cortisol response in steelhead trout, Oncorhynchus mykiss. Even though a body of literature exists about the effects of different anaesthetics on fish, no comparative information seems to be available about their effects on feed intake after anaesthesia, which would be important to know especially in aquaculture research. We anaesthetised juvenile steelhead trout with these three anaesthetics, and then sampled them 4, 24 and 48 h later. Fish in all groups ate relatively well already 4 h after anaesthesia. However, feed intake in fish treated with clove oil or MS-222 was lower than in the controls. There were no differences in feed intake among anaesthetised groups. Plasma cortisol concentrations were elevated 48 h after anaesthetisation, but the treatment means were equal throughout the experiment. Our results support previous findings that clove oil is a reasonable alternative to MS-222.     

Daily rhythms of cortisol and glucose and the influence of the light/dark cycle on anaesthesia in Nile tilapia (Oreochromis niloticus): Does the timing of anaesthetic administration affect the stress response?

Although daily variations in drug pharmacokinetics have been reported for a variety of teleost species, the influence of this daily variation on the cortisol response following anaesthesia remains poorly understood. To address this, two experiments were performed. The first experiment described the daily patterns of cortisol and glucose secretion in tilapia (Oreochromis niloticus). The second experiment investigated how the timing of anaesthetic administration (specifically at mid‐light [ML] or at mid‐dark [MD]) affects the induction and recovery times and plasma cortisol and glucose levels of juvenile Nile tilapia exposed to benzocaine, clove oil or tricaine methanesulphonate (MS‐222). The results revealed that the effect on the stress response associated with the moment when anaesthesia took place (ML or MD) varied according to the treatment (p < 0.05). Cortisol levels were significantly higher at ML for MS‐222 (ML = 116.23 ± 25.55; MD = 48.25 ± 22.33 ng/dl) (p < 0.05) and clove oil (ML 59.73 ± 14.27; MD 38.26 ± 12.07 ng/dl) (p < 0.05), whereas no significant differences were found between ML and MD cortisol levels for the control treatment (ML = 72.91 ± 18.42; MD = 64.80 ± 10.68 ng/dl) (p > 0.05) or in the benzocaine‐treated group (ML = 38.7 ± 4.90; MD = 38.60 ± 3.69 ng/dl) (p > 0.05). The highest plasma cortisol level in ML was found in the MS‐222‐treated group. All the tested anaesthetics had similar cortisol levels at MD (p > 0.05).     

Efficacy of tricaine methanesulphonate and clove oil as anaesthetics for juvenile cobia Rachycentron canadum

Six experiments were designed to determine the optimal anaesthetic dosage of tricaine methanesulphonate (TMS) and clove oil that could be used safely on juvenile cobia Rachycentron canadum of two sizes [G1=4.9±0.8 g; G2=13.9±3.1 g]. We documented the stage of anaesthesia and the acute toxicity as 96 h LC₅₀ (lethal concentration 50% population) at various exposure times of the two anaesthetics. At 10 min induction time, the TMS 96 h LC₅₀ was 93.9 mg L⁻¹ in G1 and 97.0 mg L⁻¹ in G2. Compared with clove oil, the 96 h LC₅₀ was 60.0 mg L⁻¹ in G1 and 69.8 mg L⁻¹ in G2. The difference between the two groups (G1, G2) did not influence anaesthesia safety ( P >0.05). Rachycentron canadum achieved stage 3 anaesthesia more rapidly at a lower clove oil concentration level (40 mg L⁻¹, 10 min) than TMS (60 mg L⁻¹, 10 min), but the recovery period of clove oil, was significantly longer. Clove oil was the most effective in reducing the short-term stress induced by routine biometry (20 mg L⁻¹, 10 min) and also by transporting (1 mg L⁻¹, 8 h). Whereas, for long-term exposure, 40 mg L⁻¹ TMS was found to be safe.     

Efficacy and effects of clove oil and MS‐222 on the immune‐biochemical responses of juvenile rohu Labeo rohita

The present study determined the effective concentrations of clove oil and MS‐222 in juvenile rohu Labeo rohita for quick induction and recovery. The immune‐biochemical responses due to 0, 1 and 24 hr exposure to those anaesthetics were also evaluated. Of four concentrations of the anaesthetics examined, the lowest effective concentration of clove oil and MS‐222 were 50 µl/L and 125 mg/L respectively. Clove oil and MS‐222 significantly increased the myeloperoxidase, total protein and alkaline phosphatase activity at some of the holding durations. However, superoxide anion production (after 0 and 1 hr) and antiprotease activity (after 24 hr) were significantly reduced in fish exposed to clove oil. Serum glucose content was significantly elevated in the MS‐222‐treated group. Furthermore, the clove oil‐treated group showed significantly higher levels of serum Na⁺ and K⁺, while the aspartate and alanine aminotransferase activities were significantly enhanced in the MS‐222 group. The use of both clove oil and MS‐222 is advised as an anaesthetic agent for rohu with a bias towards clove oil, considering its economic and operational feasibility.     

A meta-analytic review of monoterpene for fish anaesthesia

The use of anaesthetic agents has been increasing to address the welfare needs of different fish species in scientific and aquaculture practices. MS-222 is the most used synthetic anaesthetic but some limitations and side effects have been reported. Natural substances have been used as potential substitutes with clove oil playing an important role due to its eugenol content. Yet, other monoterpenes have shown anaesthetic properties in different fish species. As such, a quantitative assessment has been conducted to explicitly relate and summarise the use of monoterpenes with described anaesthetic properties in fish. A combined PubMed, Web of Science, Scopus, PubMed Central and Europe PMC electronic database search was performed, from January 2002 to August 2022, following the PRISMA guidelines. Out of 1555 articles retrieved from the literature search, 30 English reports met the criteria for inclusion in the meta-analysis through a generic inverse-variance method (random-effects model) and according to the fish age. The findings show a total of 10 different monoterpenes with high homogeneity and effectiveness in inducing anaesthesia and recovery according to the fish anaesthetic criteria. Further subgroup analysis showed these effects were independent of fish age. Meta-regression of the included studies revealed an inverse linear association between anaesthesia induction and recovery time and water bath temperature while no further correlation was found with other confounding factors (body weight, water pH and dissolved oxygen levels). Although further studies will be required, this meta-analysis provides robust evidence that different monoterpenes serve as an attractive and effective alternative for fish anaesthesia although water bath temperature also played an important role in anaesthesia outcome.     

Simultaneous effect of sex and dose on efficacy of clove oil,tricaine methanesulfonate, 2‐phenoxyethanol and propofol as anaesthetics in guppies,Poecilia reticulata (Peters)

We studied the simultaneous effect of sex and dose on anaesthesia efficacy to estimate, if possible, the lowest effective dose (LED) for clove oil, tricaine methanesulphonate (MS‐222), 2‐phenoxyethanol (2‐PE) and propofol in mature guppies. LED is the lowest dose needed to reach A5 stage in a mean time of 3 min, with mean recovery (R5) time of 5 min. We used four doses/anaesthetic: 25, 50, 75 and 100 mg/L for clove oil; 120,140,160 and 180 mg/L for MS‐222; 800, 1,000, 1,200 and 1,400 mg/L for 2‐PE, and 7.50, 8.25, 10.00 and 11.25 mg/L for propofol. Each dose was tested on 10 females and 10 males. Morbidity, mortality and behavioural changes were checked on two control groups (10 males and 10 females/group). Sedation (A3), A5 and R5 times were recorded. Significant interactive effect dose*sex on A5 time was found for each anaesthetic agent (p_dose*sex < .05). Except for MS‐222 (p_dose*sex = .284), significant interactive effect dose * sex on R5 time was found (p_dose*sex < .05). A5 time in females tended to be greater than in males, but, in general, R5 times were longer in males. Body size differences between males and females could explain these differences in MS‐222 on A5 time and for clove oil, 2‐PE and propofol on R5 time. No dose simultaneous meet LED′s conditions relating to both A5 and R5 times; therefore the lowest doses inducing A5 in a mean time of 3 min could be a safe guideline for anaesthetic procedure in both male and females.