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.     

Menthol as anaesthetic for lambari Astyanax altiparanae (Garutti & Britski 2000): attenuation of stress responses

The anaesthetic potential of menthol was evaluated in lambari Astyanax altiparanae by exposing fingerlings to concentrations 50, 100, 150, 200, 250 and 300 mg L^?1 and measuring the induction and recovery times to deep anaesthesia, the mortality rates during and 96 h after procedure and after 6 min of continuous exposure. The effect of menthol on stress responses were evaluated by comparing glucose and cortisol levels of juveniles subjected to anaesthesia (50 mg L^?1), stress (air exposure) or pre‐anaesthesia associated to stress. All concentrations induced deep anaesthesia within 0.5 to 1 min, with recovery between 1.83 and 4.16 min, without mortality during the induction or up to 96 h after exposure. Induction time decreased and recovery time increased linearly as the menthol concentration increased. Continuous exposure to 50, 100 and 150 mg L^?1 concentrations resulted in mortality rates of 0%, 20% and 80% respectively. Anaesthesia or air exposure increase blood glucose but prior anaesthesia with menthol suppressed the elevation of cortisol caused by stress. Menthol has an anaesthetic effect and attenuates the stress response in lambari and 50 mg L^?1 is the most effective concentration for inducing deep anaesthesia in 1.0 min, safe for up to 6 min exposure.     

Assessment of induction and recovery times of anaesthesia in Astyanax bimaculatus using 2‐phenoxyethanol and the essential oils of Melaleuca alternifolia and Ocimum gratissimum

This study aimed to determine the lowest effective concentration (LEC) of 2‐phenoxyethanol (2‐Phe) and essential oils (EOs) of Melaleuca alternifolia and Ocimum gratissimum able to induce anaesthesia in Astyanax bimaculatus. LEC was assumed to be the required concentration to carry out anaesthesia in less than 3 min with recovery in <5 min and sufficient to carry out routine aquaculture procedures. The following concentrations were tested: 2‐Phe (100, 200, 300, 400, 500 and 600 µl/L); M. alternifolia (200, 300, 400, 500 and 600 µl/L); and O. gratissimum (10, 20, 30, 40, 50, 60, 70, 100, 150, 250 and 300 µl/L). Results show that all three agents evaluated have effective anaesthetic properties for use in Astyanax bimaculatus with LEC for 2‐Phe, M. alternifolia and O. gratissimum of 200, 300 and 60 µl/L respectively.     

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.     

Optimal dietary methionine + cystine requirement for finishing lambari,Astyanax altiparanae (Garutti and Britski, 2000)

A 95‐day feeding trial was conducted to quantify the methionine + cystine requirement for finishing lambari, Astyanax altiparanae (6.10 ± 0.11 g). Six extruded isoproteic (310.14 g/kg crude protein) and isoenergetic (19.76 MJ/kg gross energy) diets were prepared to contain 6.71, 8.31, 11.31, 13.12, 15.59 and 19.74 g/kg dry diet of methionine + cystine. Quadruplicate groups of female lambari were randomly assigned to 24 aquaria (70 L each) and fed to apparent satiety six times daily. The methionine + cystine requirement was determined by quadratic regression analysis of growth performance, whole body composition, muscle development, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity against dietary methionine + cystine concentrations, at 5% significance. Fish fed 6.71–11.31 g/kg dry diet of methionine + cystine showed increased weight gain, per cent weight gain, specific growth rate and protein efficiency ratio. There were no significant differences in whole body composition, muscle growth and activity of AST and ALT in fish fed the dietary treatments. In conclusion, according to the second‐order polynomial analysis of weight gain, the optimum dietary methionine + cystine requirements for finishing lambari were estimated to be 13.66 g/kg dry diet (4.40% dietary protein).     

Impact of inducing general anaesthesia with MS‐222 on haematological and biochemical parameters of pikeperch (Sander lucioperca)

The aim of this study was to determine the impact of MS‐222 on the haematological and biochemical parameters of juvenile pikeperch. The experiment was conducted on fish with body weight of 112.50 ± 13.01 g and body length of 22.5 ± 1.8 cm. The fish were exposed to MS‐222 via immersion at two concentrations (100, 150 mg/L) and for two exposure periods (3, 10 min). Significantly higher values of white blood cells, red blood cells, haemoglobin and haematocrit were noted in all groups of fish from which blood samples were drawn immediately after exposure. After 24 hr, only in group V (100/3/24) did the values of these parameters return to initial values. The analysis of biochemical parameters signalled significant differences in three parameters: glucose, total protein and lactates. Twenty‐four hours after exposure, the values of these three parameters returned to initial levels. Significant differences were also noted in four ion concentrations: potassium, phosphorus, sodium and iron. Twenty‐four hours later, potassium and phosphorus concentrations had returned to initial values, while that of sodium still exhibited significant differences. Twenty‐four hours later, differences were also noted in iron ion concentrations in the groups subjected to the longest exposure or the highest concentration. All of the MS‐222 immersion parameters were effective. However, the smallest changes in the analysed parameters of pikeperch blood were noted in individuals from group I (100/3/0). The current study indicated, however, that longer pikeperch immersion in the anaesthetic solution (>3 min) or applying concentrations exceeding 100 mg/L are not recommended.     

The fatty acid compositions of total,neutral and polar lipids in wild and farmed lambari (Astyanax altiparanae) (Garutti & Britski, 2000) broodstock

One way to develop broodstock fish diets is to determine the compositions of wild broodstock tissues and attempt to replicate these compositions in the eggs of farmed fish via dietary manipulation. We collected 30 wild and 30 farmed lambari females that were in the reproductive stage. The extraction and separation of polar and neutral fractions and the saponification and methylation of lipids were performed and analysed in the muscles, livers and ovaries to determine the compositions of the fatty acids via gas chromatography. Regardless of habitat, lambaris mobilize large amounts of fat to the ovaries during the reproductive period, in addition to highly unsaturated fatty acids, such as arachidonic, eicosapentaenoic and docosahexaenoic acids. The wild lambaris were observed to contain higher levels of various fatty acids, including linoleic acid, which is an essential fatty acid. The most abundant fatty acid that was observed in the commercial diet was linoleic acid, which was supplied in all fish farmed tissues. The commercial diet has low AA, EPA and DHA contents, and, higher levels of these fatty acids were recorded in the tissues of farmed lambari, which suggests that this species are able to elongate and desaturate precursors, linoleic and linolenic acids, into highly unsaturated fatty acids.     

Anaesthetic efficacy of MS‐222 and AQUI‐S® in advanced size fry of rohu,Labeo rohita, (Hamilton‐Buchanan)

Anaesthetic agents are very useful for reducing the stress caused by handling, sorting, transportation, artificial reproduction, tagging, administration of vaccines and surgical procedures in fish. The efficacy of two anaesthetics: MS‐222 and AQUI‐S^® were tested on rohu, Labeo rohita advanced size fry. The lowest effective doses that produced induction in 3 min or less and recovery times 5 min or less and meet the most criteria of good anaesthetic characteristics were 125 mg L^?1 of MS‐222, and 30 mg L^?1 of AQUI‐S^® in rohu, Labeo rohita advanced size fry. Induction times were significantly decreased with increased in the concentrations of any of the two tested anaesthetic agents. The lowest doses suitable for transportation of rohu advanced size fry observed were: 10–15 mg L^?1 of MS‐222 and 2.5 mg L^?1 AQUI‐S^®. Both anaesthetics showed promising to be used as anaesthetics for handling and transportation in rohu (Labeo rohita) advanced fry.     

Determining the tranquilization dose and residue of tricaine methanesulfonate (MS-222) in sea bass <Emphasis Type="Italic">Lates calcarifer</Emphasis> tissue

Although tricaine methanesulfonate (MS-222) is often used to tranquilize fish, the guidelines for its use in sea bass, a brackish species, have not been established. The aim of the study reported here was to establish the tranquilizing concentration of MS-222, based on the time required for MS-222 residue elimination and withdrawal. Thirty-six fish (6/group) were immersed in different concentrations of MS-222 (0, 30, 50, 60, 70 and 90 mg/l) to evaluate the fish physiological behavior. After 200 fish were anesthetized at 90 mg/l, the fish achieved a healthy recovery within 72 h after the administration of saline. The 10 fish in the control group were subject to the same treatment without anesthesia, 3 out of 10 died. After 108 fish (54/group) were immersed in 30 or 60 mg/l of MS-222, the sedated fish were healthy during and after the 8 h of transport. However, all the 10 fish in the control group died within 3 days. By high-performance liquid chromatography, the residue of MS-222 was assessed. In the skinned muscle and liver, the elimination half-life was 5.54 and 5.27 h (30 mg/l) and 8.72 and 7.15 (60 mg/l), respectively, and the withdrawal time was at least 4.5 days at 30 mg/l and 7.5 days at 60 mg/l.     

Comparative efficacy of MS-222 and benzocaine as anaesthetics under simulated transport conditions of a tropical ornamental fish Puntius filamentosus (Valenciennes)

There is a growing commercial interest in the fish, Puntius filamentosus , in the ornamental fish trade in India and elsewhere. The trade is, however, hampered by severe mortalities during transport of the fish owing to insufficient data available on the use of anaesthetics. To resolve this problem, we evaluated the efficacy of two anaesthetics, MS-222 and benzocaine, in sedating P. filamentosus in simulated transportation experiments and used stress response parameters such as cortisol and blood glucose levels to perform assessments. We observed that MS-222 at 40 mg L⁻¹ and benzocaine at 20 mg L⁻¹ were sufficient to induce sedation for 48 h. Above these concentrations, both the anaesthetics adversely affected the fish and resulted in mortalities. Both anaesthetics significantly lowered the blood cortisol and glucose levels compared with the unsedated controls. Importantly, the anaesthetics treatment significantly lowered the post-transport mortality in the fish. The results of the study show that MS-222 and benzocaine could be used as sedatives to alleviate transport-related stress in P. filamentosus to improve their post-transport survival and hence reduce economic loss.     

Impact of fish size,water temperature,and MS‐222 concentration on inducing general anesthesia in pikeperch (Sander lucioperca)

The aim of the study was to determine the optimum concentration of MS‐222 for given size groups of pikeperch and water temperatures. The study considered three size groups of pikeperch (body weight [BW] 8.56, 15.72, 52.91 g), an MS‐222 water solution (50, 100, and 150 mg/L), and two temperatures (20 or 23°C). It was revealed that the optimum MS‐222 concentration depended largely on the size of the pikeperch and on water temperature. For fish with a BW <10 g the recommended concentration is 100 mg/L but only at 23°C. For fish with a BW of 10–40 g at an immersion temperature of 20°C the safe MS‐222 concentration ranges from 100 to 150 mg/L. However, at a temperature of 23°C the recommended concentration of the anesthetic is 100 mg/L. Similarly for larger fish, that is, fish with a BW >40 g, the optimum MS‐222 concentration at a water temperature of 20°C is in the range of 100–150 mg/L, but at a temperature of 23°C is it approximately 100 mg/L. Additionally, when exposure to the anesthetic is shorter (several min), a concentration of 150 mg/L is also safe for juvenile pikeperch of this size.     

Menthol and 1,8‐cineole as new anaesthetics in common carp,Cyprinus carpio (Linnaeus, 1758)

This study describes anaesthetic efficacy of menthol and 1,8‐cineole in common carp, Cyprinus carpio, in comparison with eugenol. Common carp fingerlings were exposed to eugenol: 5, 10, 15, 25, 35, 50, 75, 100, 150 and 200 ppm; menthol: 5, 10, 15, 25, 50, 100, 200, 300, 400, 500 and 600 ppm; 1,8‐cineole: 100, 150, 200, 300, 400, 500, 600, 700 and 800 ppm. Induction time and recovery time were recorded. Results showed that menthol and 1,8‐cineole anesthetized the fish at higher concentrations compared to eugenol. The fish exposed to menthol showed common fish behavioural responses to anaesthesia, similar to eugenol. But, 1,8‐cineole‐exposed fish showed tail‐up swimming, which was not observed before. Also, 1,8‐cineole failed to completely cease muscle tone. Exposure to 200 ppm eugenol and 600 ppm menthol resulted in 40% and 20% mortality, respectively. Induction time was exponentially dependent on anesthetic concentrations. Recovery time was linearly correlated to eugenol and menthol, but not 1,8‐cineole concentrations. Recovery time was exponentially dependent on induction time in the fish anesthetized with eugenol and menthol, but not 1,8‐cineole. Menthol and 1,8‐cineole are recommended for carp anaesthesia. Menthol is capable to anesthetize common carp within 1–3 min at 118–512 ppm. Common carp anesthetized with 108–133 ppm menthol recovers within 5 min. 1,8‐cineole failed to anesthetize common carp within less than 150 s at 300–800 ppm concentrations. However, it anesthetizes carp within 3 min at 595 ppm concentration. Also, 1,8‐cineole is not recommended for fish surgery. Both menthol and 1,8‐cineole were less efficacious than eugenol.     

Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status

Responses to anaesthesia with essential oil (EO) of Aloysia triphylla (135 and 180 mg L^?1) and tricaine methanesulfonate (MS222) (150 and 300 mg L^?1) were assessed in silver catfish. Exposure to the anaesthetics elicited a stress response in the species. In the case of MS222, it was displayed as a release of cortisol into bloodstream, elevation in hematocrit and plasma ion loss. The EO presented cortisol‐blocking properties, but increased haematocrit and disturbances of hydromineral balance were observed. Liver antioxidant/oxidant status of EO and MS222‐anaesthetized silver catfish was also estimated. The synthetic anaesthetic induced lipoperoxidation, notwithstanding increased catalase contents, whereas the naturally occurring product was capable of preventing the formation of lipid peroxides, possibly due to combined actions of catalase and glutathione‐S‐transferase. Anaesthetic efficacy was also tested via induction and recovery times. Overall, the promising results obtained for the physiological parameters of the EO‐treated fish counterbalanced the slight prolonged induction time observed for 180 mg L^?1. As for 135 mg L^?1, both induction and recovery times were lengthy; despite that, the EO was able to promote oxidative protection and mitigate stress. None of the MS222 concentrations prompted such responses concomitantly.     

Conservation status of the San Christóbal pupfish Profundulus hildebrandi Miller (Teleostei: Profundulidae) in the face of urban growth in Chiapas,Mexico

1. Profundulus hildebrandi, the San Cristóbal pupfish, is a small cyprinodontiform fish endemic to the closed basin of San Cristóbal de Las Casas, a fast‐growing city in highland Chiapas, Mexico. This study constitutes the first population study of this endangered taxon. 2. In order to judge its conservation status, its distribution and habitat were described, and its population size was estimated through a capture–recapture technique. To the extent possible, present conditions of its habitat were compared with historic information, mainly old city maps and photographs. 3. Introduction of largemouth bass (Micropterus salmoides) coincided with local extirpation of P. hildebrandi. Other risk factors related to urban growth include pollution of streams, as well as habitat destruction, fragmentation, and modification; about 60% of the original pupfish habitat is heavily polluted by sewage. 4. Although its population size is apparently large (probably more than 8000), its very restricted distribution, its shrinking habitat and the increasing risk factors provide justification to consider P. hildebrandi as an endangered fish, according to the Mexican Official Norm and the Red Book of the IUCN. Copyright © 2004 John Wiley & Sons, Ltd.     

Effects of insecticide Thiodan® on the morphology and quantification of ovarian follicles in lambaris Astyanax bimaculatus (Linnaeus, 1758) in different treatments

Thiodan^® is an insecticide which can cause morphological changes in fish tissues, dependent on the concentration. To investigate possible morphological changes, this study evaluated the number and diameter of ovarian follicles in lambaris Astyanax bimaculatus exposed to Thiodan^® for 96 h in different treatments at three sub‐lethal concentrations: 1.15, 2.3 and 5.6 μg L^?1 and a control group without Thiodan^®. Females exposed to Thiodan^® were divided into four treatments: (i) without acclimation and without food, (ii) without acclimation and with food, (iii) with acclimation for 10 days and without food; (iv) with acclimation for 10 days and with food. In this study, it was noted that the action of Thiodan^® at sub‐lethal concentrations in a static system did not affect the morphology of follicular development. However, the treatments with acclimation showed lower numbers of primary and secondary follicles when compared to fish from treatments without acclimation. For the groups exposed to Thiodan^®, a greater number of atretic follicles were observed compared to the control groups. The follicular diameter of secondary follicles in fish exposed to Thiodan^® in the treatment with acclimation and with food was highest in the control group (P < 0.05). Furthermore, the mean gonadosomatic (GSI) and hepatosomatic (HSI) indices of treatments with acclimation were lower than those for treatments without acclimation. These data suggest that the fish may be being affected by the pesticide to which the fish were submitted, and may cause impairment to the follicular development.     

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.     

Diet of the Eurasian otter (Lutra lutra) on the River Gradac,Serbia: Predation in a brown trout‐dominated stream

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Protein‐sparing effect by dietary lipid increase in juveniles of the hybrid fish tambatinga (♀Colossoma macropomum × ♂Piaractus brachypomus)

This research assessed four levels of crude protein—200, 250, 300 and 350 g/kg—associated with two crude lipid concentrations—110 and 140 g/kg—in diets for juveniles of tambatinga (♀Colossoma macropomum × ♂Piaractus brachypomus) over 63 days. The fish (15.2 ± 0.2 g) were distributed in 24–160 L tanks (11 fish/tank) in a 4 × 2 completely randomized factorial design (n = 3). Increasing the lipid content in the diets reduced (p < 0.05) the protein requirements for weight gain from 326 g/kg (diets with 110 g/kg LIP) to 255 g/kg (diets with 140 g/kg). Protein retention in the fish increased (p < 0.05) when they were fed up to 293 g/kg CP and 140 g/kg LIP; in the diets with 110 g/kg LIP, protein retention decreased linearly (p < 0.05) as the protein content in the diets increased. Feed efficiency increased (p < 0.05) when the fish received up to 308 g/kg CP regardless of lipid concentration (p > 0.05). In conclusion, tambatinga juveniles at 15–120 g have a minimum requirement of 255 g/kg CP and 140 g/kg CL and a protein:energy ratio of 17.4 g CP kJ/digestible energy (DE).     

Using Bacillus subtilis E20‐fermented soybean meal as replacement for fish meal in the diet of orange‐spotted grouper (Epinephelus coioides,Hamilton)

The potential of Bacillus subtilis E20‐fermented soybean meal (FSBM) as a partial alternative component of fish meal (FM) in fed diets of orange‐spotted grouper (Epinephelus coioides) was evaluated in this study. An FM‐based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal (SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively, based on broken‐line analyses. Histopathological changes in the liver and distal intestine, and significantly lower activity levels of digestive enzymes, including pepsin in the stomach and trypsin, chymotrypsin, amylase and lipase in the distal intestine, were found in fish fed a diet containing 30% of FM replaced by SBM. However, these parameters were improved by the substitution of FSBM. It is therefore believed that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM.