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.     

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.     

Effects of repeated handling, with or without anaesthesia, on feed intake and growth in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum)

The effects of weekly anaesthetization with clove oil and tricaine methanesulphonate (MS‐222) on feed intake and growth were examined in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), held individually. Repeated handling without anaesthetics significantly reduced feed intake and weight gain compared with an unhandled control group during an 8‐week experiment. When anaesthetics were used during handling the feed consumption and weight gain were significantly (MS‐222) or not significantly (clove oil) higher than in fish handled without anaesthesia. When compared with the unhandled control group, neither of these two anaesthetics had significant effects on feed intake but, in contrast to MS‐222, repeated anaesthesia with clove oil had a significant negative effect on growth. However, the effects of MS‐222 and clove oil on the growth were not significantly different from each other. Feed conversion ratio (feed/gain) of MS‐222‐anaesthetized fish was significantly higher compared with unhandled control and handled control fish but was not significantly different from fish anaesthetized with clove oil. These results suggest that both MS‐222 and clove oil alleviate handling stress in juvenile rainbow trout, and that these two anaesthetics are rather similar with respect to their effects during repeated exposures.     

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.     

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.     

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.     

Does the time of feeding affect the diurnal rhythms of plasma hormone and glucose concentration and hepatic glycogen content of rainbow trout?

The diurnal rhythms of plasma glucose, cortisol, growth hormone (GH) and thyroid hormone (T₄, T₃) concentrations and hepatic glycogen content were measured in rainbow trout that had been entrained to a specific time of daily feeding (post-dawn, midday, pre-dusk); the purpose of the study was to investigate the significance of feeding time on hormones and metabolite patterns. Plasma GH, cortisol and T₄ concentrations all showed evidence of a diurnal rhythm in some treatment groups. There was a significant interaction between the time of feeding and plasma GH and cortisol concentration rhythms; for GH, this appeared to be related to the phase-shifting of the post-prandial increases in plasma GH concentrations, and for cortisol, the rhythms were only evident in fish fed in the post-dawn period [diurnal rhythms were not evident in treatment groups fed in at midday or pre-dusk]. Peak plasma T₄ concentrations were evident during the photophase in all three treatment groups; however, the time of feeding had a negligible effect on the timing of those peaks. There were no apparent diurnal rhythms of plasma T₃ and glucose concentrations, hepatic glycogen content or hepatosomatic index in any of the three treatment groups. To whom correspondence should be addressed     

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).     

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.     

The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum)

The anaesthetic effects of clove-oil-derived eugenol were studied in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Acute lethality and the effects of multiple exposures to eugenol were measured. The estimated 8-96 h LC₅₀ for eugenol was found to be approximately 9 p.p.m. Times to induction and recovery from anaesthesia were measured and compared with MS-222 under similar conditions. Eugenol generally induced anaesthesia faster and at lower concentrations than MS-222. The recovery times for fish exposed to eugenol were six to 10 times longer than in those exposed to similar concentrations of MS-222. Clove oil eugenol was determined to be an acceptable anaesthetic with potential for use in aquaculture and aquatic research. Doses of 40-60 p.p.m. eugenol were found to induce rapid anaesthesia with a relatively short time for recovery in juvenile trout.