Background

There are many occasions in fisheries and aquaculture requiring some form of sedation or calming of fish in order to facilitate their handling without harming or stressing them markedly (Summerfelt and Smith, 1990; Ross and Ross, 1999). Stress caused by handling and transport of fish can result in immune suppression, physical injuries or even death. Anesthesia is required for several applications such as measuring or weighing fish, sorting and tagging, administration of vaccines, live transport, sampling for blood or gonadal biopsies and collection of gametes (Mylonas et al., 2005). Many chemicals are used as anesthetics to immobilize fishes to avoid this stress through adequate manipulation (Marking and Meyer, 1985; Gilderhus and Marking, 1987; Summerfelt and Smith, 1990). An ideal anesthetic should induce anesthesia rapidly with minimum hyperactivity or stress. It should be easy to administrate and should maintain the animal in the chosen state (Shawn et al., 2004).

A number of different anesthetics have been used for aquaculture applications. Tricaine methanesulfonate (MS-222), Benzocaine, Quinaldine, 2-Phenoxyethanol, Metomidate, Eugenol and Etomidate are widely used drugs for inhalation anaesthesia (Weyl et al., 1996; Ross and Ross, 1999; Jasna et al., 2005; Mylonas et al., 2005). Clove oil has been reported and recommended strongly as an anesthetic (Anderson et al., 1997; Keene et al., 1998; Waterstrat, 1999). Eugenol is the main component in clove oil (70-90%) which is obtained by distillation of the flowers, stems and leaves of the clove tree (Eugenia aromatica or caryophyllata) (Filiciotto et al., 2012). In addition to its worldwide use as a food flavoring, it has also been employed for centuries as a topical analgesic in dentistry (Curtis, 1990; Soto and Burhanuddin, 1995) and its major advantage is that it is inexpensive in comparison to others. However using clove oil in high concentrations may lead to the decrease of neurosensory function, respiratory failure, medullary collapse and eventually death (Schreck and Moyle, 1990). Clove oil concentrations above 0.05 mL per 500 mL of water increase considerably the mortality rates and are only suitable for studies in which fish are needed to be sampled and sacrificed for later laboratory analyses (e.g., dietary or reproductive analysis) (Fernandes et al., 2017). The MS-222 chemical name is tricaine methanesulfonate. It is sold as Tricaine-S and Finquel. It comes as a white, crystalline powder that can be dissolved in water at up to an 11% solution and characterized by rapid sedation induction (Shawn et al., 2004).

2-Phenoxyethanol (2-PE) (1-hydroxy-2-phenoxyethane) is a moderately water-soluble, colourless, oily, aromatic liquid often used as a topical anesthetic and commonly used in closed fish transport systems. It is suitable for aquaculture operations because of its easy preparation, low cost, rapid induction and rapid uneventful recovery, and has bactericidal and fungicidal properties (Tsantilas et al., 2006). Nevertheless, there are some indications that 2-Phenoxyethanol may be a probable hazard to the handler causing fatigue and drowsiness (Velisek et al., 2007).

Mugil cephalus and Sparus aurata are of the foremost preferable fish species by the local Egyptian consumers. Marine fish farming in Egypt began in 1976 with the culture of gilthead sea bream. The main source of cultured sea bream in Egypt is the extensive and semi-intensive commercial earthen ponds, which are estimated at 8,000 ha (Sadek, 2000). Mullets are also an important component of Egyptian fisheries and are considered as one of the most important cash crops from artisanal fisheries in the numerous lagoons throughout the country. Mullet are cultured in a large number of countries worldwide, usually in extensive and semi-intensive pond systems. Egypt has a long history of mullet aquaculture, which was traditionally practiced in the “hosha” system in the Nile Delta region for centuries (Eisawy and El-Bolok, 1975). Currently, Egypt is a leading country in mullet aquaculture with a record production of 156,400 tonnes in 2005 (Saleh, 2008).

The objective of the current study was to examine the efficacy of different doses of clove oil, Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol as a calming agent in order to determine the optimal dose for short time fish handling of juveniles and adults Mugil cephalus and Sparus aurata as an example of a common highly economic species in the Egyptian market.

1 Materials and Methods

The current study was carried out starting from October 2013 to February 2014 in El Max research station located in Alexandria Governorate affiliated to the National Institute of Oceanography and Fisheries, Egypt. Juveniles and adults of both M. cephalus and S. aurata were chosen as test animals based on their high economic value in the Egyptian market.

1.1 Maintenance of test animals

Fish were maintained in circular fiberglass tanks (1000 L³). Water quality parameters were as follows: pH 7.55, Do 6.25 mg/L, ammonia 0.22 mg/L, nitrite 0.07 mg/L, alkalinity 600 mg/L, water temperature 22±1°C and total hardness 780 mg/L. Mugil cephalus fish average weight (mean ± SD) was 39 ± 15 g for juveniles, while average weight was 329 ± 31 g for adults. Sparus aurata fish average weight (mean ± SD) was 52 ± 12 g for juveniles, while average weight 358 ± 21 g for adults.

1.2 Experimental design and procedure

Fish had been starved for 24 hrs prior to the experiment. Three types of anesthetics clove oil, Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol were used. Fish were exposed to (5, 10, 15 mL^-1 for juvenile) and (15, 20, 25 mL^-1 for adults) concentration of clove oil, (50, 75, 100 mL^-1 for juvenile) and (100, 125, 150 mL^-1 for adults) concentrations of MS-222 (200, 250, 300 mL^-1 for juvenile) and (300, 350, 400 mL^-1 for adults) concentrations of 2-Phenoxyethanol. The stock solution of clove oil was prepared according to (Ghanawi et al., 2013) as clove oil is poorly soluble in water so it was mixed with Ethanol 96% (1 : 9) then concentrations were prepared. MS-222 is readily soluble in water so different concentrations were easily prepared; however, 2-Phenoxyethanol was added directly into the anesthetic tank (Shaik, 1999).

The fish were exposed to anesthetics till stage 3 for full induction time and full recovery time. The induction and recovery times were recorded using a stopwatch for each fish species as (mean ± SD). Each fish was let until it reached S3 and kept in the same aquarium until it starts the recovery stage (Table 1). The time of induction was recorded for each fish when fish lost total equilibrium, its opercular movement rate was reduced and fish did not respond to pressure on its body (S). Also, the time of recovery was recorded when fish started swimming in a normal manner (R) (Table 1). Following recovery, fish were placed into maintenance aquarium and were observed for 48 h for any adverse effects. Experiments were fulfilled using 15 fish for each species and stage in triplicate (n=45).

1.3 Statistical analysis

The data was collected and entered into the personal computer. Statistical analysis was completed using Statistical Package for Social Sciences (SPSS/version 17) software. The statistical test used as follow: mean and standard deviation (mean ± SD) for sedation and recovery duration according to different anesthetic treatment in both species, Mann Whitney test was applied for comparison between unpaired signed ranks test in respect to the response of each anesthetic on both species at different stages separately, then applied to compare different doses of anesthetic on separate species in different stages.

2 Results

2.1 The response of fish stage in both species to different anesthetics and recovery time variation

Variation in fish species showed significant differences in time duration for sedation using clove oil as an anesthetic for adults and juveniles. Mugil cephalus recorded higher sedation and lower recovery times than that recorded for Sparus aurata at all concentrations and stages (p≤0.001). Also, the minimal sedation time and maximum recovery time were recorded at the highest concentration (25 mL^-1) for both species. All durations were showing a significant difference in both species using clove oil in sedation for juveniles and adult stages except for the recovery time at (20 mL^-1) for adults as time durations were showing insignificant differences (Figure 1).

Both species were responding, in the same manner, using MS-222 in respect to sedation and induction duration. The maximum sedation time was recorded conducting the least (100 mL^-1) concentration of MS-222 in M. cephalus with a minimum recovery duration, while all sedation durations were lower in S. aurata and recovery durations were higher than that recorded for M. cephalus for the entire concentrations (Figure 2). Sedation and recovery duration at all concentrations were significantly different in both species at adult stage (p≤0.001). The response of both species at juvenile stage showed insignificant difference using (50 mL^-1) concentration at sedation time, while, recovery duration was significantly different in both juvenile species at the same concentration. Higher concentrations (75, 100 mL^-1) of MS-222 showed significantly different sedation and induction durations when conducted in both species at juvenile stage (Figure 2).

Both species at all stages were responding the same manner on conducting different concentrations of 2-Phenoxyethanol as M. cephalus recorded higher sedation and lower recovery times than that recorded for S. aurata at all concentrations and stages. The response of adults of both species to induction was significantly different on the conduction of (300, 400 mL^-1) 2-Phenoxyethanol and was not significant using (350 mL^-1) concentration in both species. The sedation and recovery times recorded by juveniles of both species showed significantly different durations on conducting (200, 250 mL^-1) 2-Phenoxyethanol (p≤0.001), while, insignificant sedation time was recorded when using (300 mL^-1) of the same anesthetic at the same stage but showing significant recovery time for both species (Figure 3).

2.2 Dose-response in both species to anesthetics in respect to different stages

The response of adult M. cephalus fish to different concentrations of clove oil reveals that sedation time was significantly different at all concentrations displaying inverse relation to the concentration (p≤0.001) (Figure 4). The maximum sedation time was recorded at (15 mL^-1) concentration and the minimum sedation time was recorded at (25 mL^-1). The recovery duration on using the same concentrations reveals that the minimum recovery duration was recorded after 1.8 ± 0.232 min. Using (15 mL^-1) concentration and was significantly different from durations recorded on using the other two concentrations (Table 2). On the other hand using clove oil at (20, 25 mL^-1) concentrations reveals that no significant difference was recorded concerning the recovery duration, however, they display high significant difference form the first concentration (Table 2).

The duration recorded till complete sedation for adults S. aurata using the same concentrations of clove oil reveals that none of the three concentrations affects the sedation time as no significant differences were recorded (p≤0.001) (Table 2). Although sedation time did not show any significant difference using different concentrations of clove oil, recovery time represented high significant difference on using (15 mL^-1) concentration than using (20, 25 mL^-1) concentrations (p≤0.001).

The conduction of the three selected doses of MS-222 to induce complete sedation in adults M. cephalus not only resulted in insignificant sedation durations, but also the recovery durations (p≤0.001). In contrast, the response of adults S. aurata to MS-222 (100 mL^-1) concentration resulted in significantly different sedation and recovery durations using (125, 150 mL^-1) concentrations. However, the latter concentrations showed no significant difference in sedation and recovery durations for S. aurata adults (Table 3).

Juveniles M. cephalus and S. aurata response to the entire selected concentrations (50, 75 and 100 mL^-1) to induce complete sedation were insignificantly different (p≤0.001). Whereas, juveniles S. aurata showed significantly different durations reaching recovery in response to the entire concentrations (p≤0.001). The response of M. cephalus to reach recovery using (50 and 75 mL^-1) were in significantly different, while using higher concentrations (75 and 100 mL^-1) of MS-222 displayed significantly different recovery duration than recorded using (50 mL^-1).

The induction durations till complete sedation in adults M. cephalus were significantly different at all concentration (300, 350 and 400 mL^-1) of 2-Phenoxyethanol (Table 4). However recovery durations were significantly lower using (300 mL^-1) than that recorded after using (350 and 400 mL^-1), but the latter concentrations did not show any significant differences in recovery durations in adults M. cephalus. The induction response of adults S. aurata was slightly different than that recorded in the other species as (300 and 350 mL^-1) did not differ significantly to induce sedation, while, the highest concentration of 2-Phenoxyethanol (400 mL^-1) accelerated the induction time as it was reached after 0.613 ± 0.023 min. On the contrary, the recovery time recorded after conduction of (300 and 350 mL^-1) were not significantly different in adults S. aurata, nevertheless, (400 mL^-1) concentration delayed recovery time significantly reaching 0.955 ± 0.130 min. The response of juveniles in both species did not show any significant variation in conducting (200, 250 and 300 mL^-1) of 2-Phenoxyethanol either to induce complete sedation or to completely recover from this anesthetic. Significant differences of time duration (efficiency) according to the conduction of different concentrations of anesthetics in relation to the two fish species were recoreded.

3 Discussion

Although there are many instances to use anesthetics in aquaculture and fisheries activities, inappropriate doses may induce undesirable stress (Ghanawi et al., 2013). Therefore it is very much important to evaluate the proper doses of anesthetics in respect to species and fish stage response. The current study investigated the efficacies of clove oil, Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol which can be used as anesthetics for M. cephalus and S. aurata at different stages. Mugil cephalus recorded higher sedation and lower recovery times than that recorded for Sparus aurata at all concentrations and stages (p≤0.001). The entire types of anesthetic demonstrated a dose-response concerning sedation and recovery duration regardless of fish species or stage. Induction durations decreased significantly with increase in concentration of any of the three examined anesthetic agents. Similar induction trends were reported for other fish species including Senegalese sole (Solea senegalensis) (Weber et al., 2009), and European seabass (Dicentrarchus labrax). The current results revealed that the recovery durations were more different than sedation duration in response to different doses of anesthetics. Clove oil led to longer calming duration for both species inducing complete sedation in about a minute for adults and juveniles and recovery duration was longer than the recorded using the two other anesthetics. In contrast, higher anesthetic doses resulted in similar or shorter recovery times in gilthead sea bream (Sparus aurata) and seabass (Dicentrarchus labrax) (Mylonas et al., 2005). Although large difference in recovery times between MS-222 and clove oil was reported and probably caused by the stimulatory effect of MS-222 on the respiratory system and heart of fish resulting in increased respiratory and heart rate which in turn removes excess anesthetic, clove oil has an inhibitory effect on the respiratory rate and a lesser ability to remove excess anesthetic from the fish’s system results in longer recovery times (Keene et al., 1998). Clove oil consistently yields similar levels of physiological disturbance and minimizes responses to external stressors to that observed with MS-222 (Cho and Heath, 2000; Sladky et al., 2001; Wagner et al., 2003). More studies proposed that clove oil is an effective alternative for the sedation of fish and may have several benefits over other methods including its low cost (Cooke et al., 2004).

Phenoxyethanol can induce sedation in both species, however, more concentrations should be used to reach complete sedation and lower recovery durations were recorded.

The quick induction, long recovery time and high recovery rate were obtained at concentrations between 0.25~0.50 mL^-1 of clove oil. The hematological indices in goldfish while clove oil was used up to concentration of 75 ppm, were not changed however using clove oil at 150 ppm concentration could increased RBC value after 24 h (Abdolaziz et al., 2011). This oil is equally effective against the catfish M. vittatus. So, clove oil is a user-safe, eco-friendly and alternative to the chemical fish anaesthetic and can be used in the aquaculture practice (Alam et al., 2012).

In conclusion, clove oil was the most effective anesthetic for juveniles and adults M. cephalus and S. aurata handling in addition to its low price. Accordingly, authors strongly recommend and encourage people working in aquaculture and fisheries sector to routinely use it in fish handling. Anesthetics are important to assist in reducing stress during fish handling, however, more appropriate protocols are still necessary.

Authors’ contributions

Both authors designed the experiment, collected data, and references then carried out the statistical analysis, while the corresponding author was responsible of manuscript writing and editing. Both authors read and approved the final manuscript.

Acknowledgements

Authors would like to thank colleagues in El Max research station for their collaboration to bring this research paper out.

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