African catfish (Clarias gariepinus) is the main aquaculture specie in Nigeria and many African countries [1]. This specie contributes more than 70% of the inland aquaculture production in Nigeria and is  considered as the major provider of fish protein through aquaculture to the populace [2]. It is extensively cultured in different parts the world due to its fast growth, omnivorous feeding nature and tolerance to wide water quality and temperature ranges [3]. During aquaculture operations, fishes are faced with several potential stressors such as transportation, capture and handling procedures [4]. Conversely,  a highly crowded and confined farming environment, possible air exposure and variation in water quality are all factors that may increase the stress level of organisms [5] and have significant effects on fish physiology and survival (Green, 2008). Gabriel and Akinrotimi [4] opined that stress can cause significant losses of resources and productivity in fish in reared different culture systems. 

Anesthesia is a term that described the biological reversible state of an organism, induced by an external agent, which results in the partial or complete loss of sensation or loss of voluntary neuromotor control, through chemical or non-chemical means [6-7]. Anesthesia is frequently applied in aquaculture being a valuable tool that helps to minimize fish stress and to prevent physical injuries to fish while handling them during a routine practices in aquaculture [8], Anesthesia is required for measuring or weighing fish, sorting and tagging,  live transport, sampling for blood or gonadal biopsies and collecting of gametes, surgical procedures [9]. Anaesthetics are normally utilized to reduce the incidence of stress associated with aquaculture operations [10]. Hence, anaesthetizing fish prior to transport and breeding can reduce metabolic rate, oxygen demand, reduce general activity and mitigate the incidence of stress response [11]. Knowledge about the ideal and optimum concentration of anesthetics for various fish species is necessary because inappropriate concentrations may lead to adverse effects such as stress; therefore, access to safe and effective fish sedatives is a critical need of fisheries researchers, managers, and culturists [12-13].

When choosing anesthetics, a number of considerations are important, such as efficacy, cost, availability and ease of use, as well as toxicity to fish, humans and the environment and the choice may also depend on the nature of the experiment and species of fish [14]. When the fish is removed from the anesthetic, the recovery should be rapid, the anesthetic should be effective at low doses [15]. Different anesthetic agents such as MS222, quinaldine, benzocaine, 2-phenoxyethanol, methomidate, Isoeugenol and propofol are used to anesthetize fishes in aquaculture, most researchers have tried to find the best and most efficient anesthetic agent with the least side effects [16]. Overdose of an anesthetics or retaining the fish in an anesthetics bath for a long period leads to the fading of ventilation, hypoxia, and finally respiratory and cardiac collapse, the fading of ventilation is an important warning sign suggesting that the exposure should be terminated [17].

Organic farming of agricultural and horticultural crops is being used as a popular venture in the direction of sustained and eco-friendly food production activity in different parts of the world. Organic farming looks for alternatives to those chemicals that are currently being used in aquaculture, and the anesthetics are one such important input, as a result, different natural anesthetics were investigated to compare their effectiveness with chemical products [18]. Conversely, Plant extracts represent a potential source of new and effective anaesthetics for use in fish handling and transportation in intensive aquaculture. With the recent awareness on safe aquaculture practices, to develop “green” anaesthetics with low environmental and health risks, coupled with the prohibitive cost and scarcity of conventional anaesthetics [19], there is the need therefore to develop a viable alternative anaesthetics of plant origin which could be used in fish transportation. In this study the main substance that will be used is an aqueous extract of leaves of Indian almond tree (Terminalia catappas). This is considered an appropriate anaesthetic for fish because of its low cost, availability and safety to fish and humans [20]. 

A number of studies have tested leaves of Indian almond tree as anesthetics in several aquatic animals both marine and fresh water species with interesting results [19]. Leaves of Indian almond tree is also considered less expensive compare to the other fish anesthetic such as tricaine methanesulfonate. To the best of my knowledge there are no reports on the anaesthetic efficacy of different maturity stages of leaves of Indian almond tree in C. gariepinus Hence, the need for the study. The aim of the research is to investigate the efficacy of Indian almond tree leaf extracts as anaesthetic for C.gariepinus.

Sources of Experimental Fish 

A total of 180 specimens of Clarias gariepinus fingerlings were procured from African Regional Aquaculture Centre, (ARAC), Aluu, Rivers State of Nigeria. 

Acclimation of Experimental Fish 

The fish were transferred in 50L jerry cans to Fish Disease Laboratory at the Center. The fish were acclimated for a period of seven days. During this period they were fed with commercial feed (55.0% CP) at 5% body weight, twice a day. The water in acclimation tanks were renewed every two days. 

Indian almond tree leaf

Various maturity stages such as newly bud; matured (green) and dead (brown) leaves of Indian almond tree (Terminalia catappas) were collected from the back of Fisheries Department, in University of Port Harcourt, Port Harcourt.  The leaves were identified using The Tree Identification Book by [21].  The leaves were rinsed twice in clean water and were also air dried in a shade for 24hours. The leaves were later crushed using kitchen knife.  The leaves were later milled into paste using an electric grinder (6.5 HP Grinder, by Honda Electronics, Japan).  2kg of milled leaf was later be weighed using weighing scale and dissolved in 2L of water for a period of 12 hours.  The solution was later filtered through a 0.2µ nylon mesh to produce the crude extracts as described by.

Experimental Design

The experiment was a 3x3x5x1 factorial design, having three factors (three different maturity stages of Indian almond tree leaves) and three replicates with five concentrations for one size of C. gariepinus.

Preparation of Test Solution

A stock solution of the anaesthetics was prepared by adding 1ml of the anaesthetic concentrate to 1 litre of water.  Exposure concentration of anaesthetics will be 0.00 (control); 10.00, 20.00, 30.00, 40.00 and 50.00 mg/L,  Fifteen 30L plastic containers was  labeled and each was filled with water from the borehole to the 20L mark, and another 15 plastic containers was filled with fresh water without anaesthetics,   were placed side by side.  The different concentrations were prepared by serial dilution by measuring 10.00, 20.00, 30.00, 40.00 and 50.000, of the stock solutions (x20) with the borehole water to give the desired concentrations. The same thing was repeated for each of Indian almond leaf maturity stage.

Experimental Procedure

The anaesthetic solution was then stirred with a glass rod (50cm in length) for homogeneous mixture.  Within 10 minutes the tanks were stocked randomly with 10 fingerlings per tank, using a scoop net. Three tanks each were used for each concentration and the control in each.  The tanks were not aerated during the experimental period.  Duration of fish exposure to various anaesthetics at different concentrations depend on the induction and recovery time.  The same procedure was for each of Indian almond leaf maturity stage.

Determination of Induction and Recovery Time

The time for onset of anaesthesia for the exposed fish was measured in all the three leaf maturity stages, using a digital stopwatch.  Fish behaviour was monitored individually through the induction and recovery stages in each life stage and concentrations for all the three anaesthetics.  In the induction stage, four different behaviours was observed such as : slow swimming, slight increase in opercula beat frequency, loss of equilibrium, reflexes and movement, and lastly deep anaesthesia, where the fish was found laid on one side bottom of the tanks.After the anaesthsia, fish was removed individually using a scoop net and was transferred into a clean water tank.  Recovery time which followed the following stages; reappearance of opercula movements, partial recovery of equilibrium, irregular balance, total recovery of equilibrium and lastly, normal swimming was observed and recorded.

Evaluation of Water Quality Parameters 

During the study, in the experimental tanks, different water quality parameters were evaluated.   Water temperature measurement was taken by using mercury in glass thermometer (°C). Hydrogen ion concentration (pH) was determined by the use of a pH meter (Model HI 9812, Hannah Products, Portugal).  Dissolved oxygen levels in the experimental tanks were evaluated using the Winkler method (APHA, 1998). The value of nitrite and ammonia were measure using a fresh water test kit with colorimetric chart (Model, AQ - 2 - Code 363303) by Lamotte Company, Chester Town, Maryland, USA.

Data Analysis 

Data from this experiment were subjected to one way analysis of variance (ANOVA) test at 0.05 probability. Difference among means where they exist was determined by Tukey Honest significant difference (HSD) using statistical package for the Social Sciences (SPSS) version 22. [22]. 

The water quality in experimental tanks of C.gariepinus exposed to newly bud indian almond leaf extracts are presented in Table 1.  The results indicated a significant reduction (p<0.05) in the values of dissolved oxygen which reduced with increasing concentration of the anaesthetics. Also, the values of nitrite, sulphide and ammonia significantly increased with increasing concentrations of the plant extracts. While other water quality parameters such as temperature, and pH were within the same range were within the same range with no significant different in relation to the concentration of the anaesthetics  (p>0.05). The same trend was observed water quality in experimental tanks of C.gariepinus exposed to matured indian and Dead almond leaf extracts (Table 2 and 3). The induction time (s) in fingerlings of C.gariepinus exposed to newly bud, Matured and Dead   Indian almond tree leaf  extracts are presented in Table 4,5 and 6. The different maturity stages of Indian almond tree extracts as anaesthetics resulted in different induction times depending on the dosage and maturity stage of the leaf. The various stages of induction which include:  decrease in caudal fin strokes; decrease in swimming ability; loss of equilibrium; cessation in Operculum beat frequency and immobilization, decreased with the increase in the concentrations of the leaf extracts (Table 4, 5 and 6). The results of recovery time (s) in fingerlings of C.gariepinus exposed to newly bud, matured and Dead Indian almond tree leaf extracts are presented in Tables 7, 8 and 9. The result indicated a significant (p<0.05) increased in the recovery time, as the concentrations of the leaf extracts increased, The various stages of recovery in the exposed fish differs significantly at various concentrations. 

The comparative induction time (time taken for the fish to be anaesthetized) in C.gariepinus exposed to newly bud, matured and Dead Indian almond tree leaf extracts were shown in Figure 1. The highest induction time was recorded in fish exposed to newly bud extracts. While the lowest was recorded in fingerlings exposed to mature leaf. (Figure 1).  For the recovery time, (Figure 2) the longest recovery time was observed in the fish exposed to newly bud leaf extracts. The recovery time for all the life stages generally increased as the concentrations of the anaesthetics increased. However, a shortest recovery time was observed in fingerlings exposed to matured leaf extracts in all concentrations of exposure (Figure 2).

Table 1: Water Quality in Experimental Tanks of C.gariepinus Exposed to Newly Bud Indian Almond Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05)

Table 2: Water Quality in Experimental Tanks of C.gariepinus Exposed to Matured Indian almond Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05)

Table 3: Water Quality in Experimental Tanks of C.gariepinus Exposed to Dead Indian almond Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05)

Table 4: Induction time (s) in Fingerlings of C.gariepinus Exposed to Newly Bud Indian almond Tree Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Decrease in caudal fin strokes; II- Decrease in swimming ability; III- Loss of equilibrium; IV- Immobilization

Table 5: Induction time (s) in Fingerlings of C.gariepinus Exposed to Matured Indian almond Tree Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Decrease in caudal fin strokes; II- Decrease in swimming ability; III- Loss of equilibrium;  IV- Immobilization

Table 6: Induction time (s) in Fingerlings of C.gariepinus Exposed to Dead Indian almond Tree Leaf Extracts (Mean±SD)

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Decrease in caudal fin strokes; II- Decrease in swimming ability; III- Loss of equilibrium; IV- Immobilization

Table 7: Recovery Time (s) in Fingerlings of C.gariepinus Exposed to Newly Bud Indian almond Tree Leaf Extracts (Mean±SD) 

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Reappearance of Opercula movement; II- Fin movement resumes; III- Partial Swimming Resumes; IV- Fish regains full and active swimming

Table 8: Recovery Time (s) in Fingerlings of C.gariepinus Exposed to Matured Indian almond Tree Leaf Extracts (Mean±SD) 

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Reappearance of Opercula movement; II- Fin movement resumes; III- Partial Swimming Resumes IV- Fish regains full and active swimming 

Table 9: Recovery Time (s) in Fingerlings of C.gariepinus Exposed to Dead Indian almond Tree Leaf Extracts (Mean±SD) 

Means within the same roll with different superscripts are significantly different (p<0.05) KEY:   I- Reappearance of Opercula movement; II- Fin movement resumes; III- Partial Swimming Resumes;  IV- Fish regains full and active swimming

Figure 1: Comparitive Values of Induction Time in C.gariepinus Exposed to Different Types of Indian Almond Leaf Extracts

Figure 2: Comparitive Values of Recovery Time in C.gariepinus Exposed to Different Types of Indian Almond Leaf Extracts

The results of this study which showed that T. cattapa extracts used as anaesthetic agent on fingerlings of C.gariepinus  at various concentrations, exhibited various induction and recovery time of anaesthetized fish followed the typical patterns of fish exposed to different doses of anaesthetics (Marking and Meyer 1985; Agokei and Adebisi, 2010; Akinrotimi et al., 2014b). This study agreed with some studies which reported that fish exposed to anaesthetics usually exhibit decrease in opercula movement, swimming movements, loss of reflection and hyperactivities (Agbon et al., 2002; Akinbulumo, 2005; Olufayo and Fagbenro, 2007; Olufayo and David, 2013). Moreover, Omoniyi et al. (2002) reported that decreased in opercula movement was caused by decreased efficiency oxygen uptake or oxygen transport and the behavior within the induction time. According to Ross and Ross, (2008), anaesthesia in fish should be quickly induced, and the appropriate stage should be achieved in less than 3-5min to avoid stress and hyperactive behaviour. The recovery time should also to be no longer than 5-10 min subsequent to the transfer to clean water. Considering the concentrations that provoked stage IV in fingerlings of C. gariepinus exposed to T. cattappa at different maturity stages. Fish exposed to matured leaf extracts reached the anaesthetics stage faster, this was followed by the fish exposed to dead leaves extracts. However, newly bud leaf extracts took a longer time to induce anaesthesia in the exposed fish. This discrepancy may be explained by the differences in maturity of the leaf that produces the extracts. Ghanawi et al. (2013) reported that maturity of plant affects its efficacy.. The ideal extracts of T. cattappa concentration established in this study is relatively close to that the one reported by Akinrotimi et al. (2014a), using the same plant in the same species, but at different concentrations. 

The time taken for the fish to enter the desired stage of anaesthesia (induction time) decreased with increasing concentration of the plant extracts as reported in other studies [23-25]. This observation also agrees with the submission of Akinrotimi et al.   [10] that the degree of anaesthesia is influenced by the concentration of the anaesthetic in the central nervous system (CNS) of the exposed organism. Therefore, in the present study the increase in time taken to sedate  the experimental fish with increased concentration of the T. cattappa leaf extract may be attributed to the accumulation of the active ingredients, in this case alkaloids , tannins and sapponins , in the body of the fish which impairs the activity of the CNS at a much faster rate. Conversely, the failure of anaesthetized fish to enter deep anaesthesia (anaesthetic stage 4) at different time as obtained in this study could be due to the immaturity of the leaf used. Akinrotimi [19] observed that immature leaf generally contains lesser amount of active ingredients, and usually require a greater concentration of the anaesthetic than the mature leaves. The result further indicated that the effective concentration of the aqueous leaf extracts of T. catappa needed to induce anaesthetics was higher in extracts from newly bud leave and very low in the fish exposed to matured leaves extracts.  The induction time obtained in the fish exposed to matured leaves is similar to the report of Durville and Collet, [26] in  Valamugil cunnesius and Monodactylus argenteus  exposed to clove oil. This result is in tandem with rapid induction time (3–5 minutes) required of an ideal anaesthetic [24,27-28].

Moreover, Chemical agents have been used in the handling and transportation of fish to reduce mortality which occurs as a result of excitement and hyperactivity [24]. The long induction and recovery time of newly bud leaf extracts obtained in this study could be an added advantage in activities such as morphological evaluation, biopsy and stripping which require long handling periods outside water. It is has also been suggested that light sedation is desirable during transportation of fish. This is because fish anaesthetized at deep sedation (anaesthetic stage 4) levels lose equilibrium and may sink to the bottom, pile up and finally suffocate to death [29]. Since transportation often involve long distances, the long induction time of dead and newly bud leaves of  T. catappa leaf extracts could be considered for use as a tranquilizer in the delivery of fish over long distances and other handling procedures in aquaculture operations.  

The three stages of maturity in the leaves of T. catappa used in this study induced anaesthesia and recovery at different times at the same concentration. When comparing the different leaves tested in this study, the rapidity in which the matured leaves promoted deep anaesthesia is an advantage related to the dead and newly bud leaves, especially for use in fast handling cases, such as biometrics procedures. While the dead and newly bud leaves could be used for longer aquaculture procedures. From the results obtained in this study,  extracts of matured leaves should be used for quick and deep sedation while dead and newly bud leaves could used for light and long sedation activities such as stripping band transportation of fish in aquaculture.

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