Fish transportation, which entails moving small or large quantities of fish over various distances to the seas where they are to be stocked, is a crucial component of aquaculture operations [1, 2]. Fish are frequently moved for a variety of purposes, including the gathering and transportation of brood stock, fingerlings, juveniles, and adult fish to stocking sites or markets, as well as the introduction of fish species into a new culture setting [3.4.5]. According to several writers, moving fish from one place to another can cause stress, which has a detrimental impact on the fish's performance and reduces its survival in the culture medium [6.7.8]. Thus, acute stimuli including handling and transport have been linked to significant losses and high mortality in newly stocked fish farms [9.10]. Transport times might vary greatly based on the distance traveled as part of the handling procedures in intensive fish farming. In general, juveniles and fingerlings are moved from the hatchery to the culture site. To achieve the requirements set forth by the farmer, the fish must enter the farm in acceptable physiological condition [11].

To reduce the anxiety caused by aquaculture treatments, anesthetics are employed. Fish that have been anesthetized before being transported have lower metabolic rates, lower oxygen demands, less general activity, less impact from handling, and less likelihood of stress reactions. The primary materials employed in this investigation are an aqueous extract of the clove plant's seed (Syzygium aromaticum) and nutmeg. Eugenol (70–90%) is the primary and active component. Due to their accessibility, affordability, and safety for both humans and fish, clove extracts are regarded as an acceptable anesthetic for fish [12, 13]. Clove seed is being utilized as a food additive in Nigeria. An important cultured species in Nigeria is Oreochromis niloticus, and fish farmers typically transfer these fish to the location where they will be cultivated. They typically arrive to a particular location in appalling physiological circumstances, which has a detrimental effect on their chances of surviving and doing well in the cultural system. Therefore, this study will be conducted to evaluate the effectiveness of certain plant extracts from clove calyxes as anesthetics in Oreochromis niloticus transportation.

For usage in handling and transporting fish in intensive aquaculture, plant extracts have the potential to yield new and strong anesthetics. It is therefore necessary to develop a workable alternative anesthetics of plant origin which could be used in fish transportation. With the recent awareness on safe aquaculture practices, to develop "green" anesthetics with low environmental and health risks, coupled with the prohibitive cost and scarcity of conventional anesthetics [14]. Many farmers reported fatalities when moving various mullet, tilapia, and clariid species. In addition to raising awareness of the effectiveness of these plant extracts that could be used as an alternative to pharmaceutical anesthetics, this effort will provide information on the use of local anesthetics such as clove seed and nut Meg seed extracts. The results of this study will aid in managing the strain associated with fish farming and increase production in numerous fish farms in Nigeria, namely in the Niger Delta, as well as in other locations where such negative effects of transportation have been noted. The purpose of the study is to determine whether clove seed and nutmeg extracts are effective as anesthetics when transporting Oreochromis niloticus.

Fish Experimental Sources

A total of 450 O. niloticus fingerling specimens were obtained from the production ponds at the African Regional Aquaculture Centre (ARAC), Aluu, Rivers State, Nigeria (mean length 87cm; standard deviation, 1.54; and mean weight 10.23g; standard deviation, 123).

Fish Used In Experiments Are Acclimated 

They were brought in 50L jerry cans to be acclimated for seven days in the Fish Disease Laboratory at the Center. They received ARAC feed (35.0% CP) at 3% body weight  during this time. Every two days, the water in the acclimation tanks was replaced [15].  Preparation of Plant Extracts Nutmeg (Myristica fragrans), and dried buds of clove plant, (Syzigium aromaticum) was purchased from Choba Market in Obio Akpor Local Government Area of Rivers State. Plants authentication was done using the keys of Agbaje, [16]. These seeds were taken to the laboratory and ground into power using a kitchen blender (Model H2, Ken Wood, Japan). The milled seeds were sieved using 0.1 micro nylon mesh to obtain the fine powder.

Experimental Design

The design of the experiment was Completely Randomized Design (CRD) having five treatments levels each with three replicates for each of the life stages. A total of 75 plastic basins of dimension (52 x 44 x 34 cm3) each were used for the experiments. The 75 basins were labeled based on life stage of the fish, treatment levels and replicates. Each basin was stocked with five (5) fish per tank. A total of 225 (two hundred and twenty five) fish were stocked.

Experimental Procedure

The powder was weighed into different concentrations (10.0, 20.0, 30.0, 40.0 and 50mg/l) using a sensitive weighing balance. It was applied directly in three replicates into the water (10L) level in 30L experimental plastic aquaria. The mixtures were stirred vigorously to ensure homogenous mixture. The fish was weighed with 20 kg round top weighing scale (Model 1123HK, Digital Scales, Ltd, Beijing, China). While the length was measured with transparent meter rule. They were then be introduced into prepared experimental aquaria, containing five concentrations of each of the powdered plant seeds (10.00; 20.00; 30.00; 40.00 and 50.00 mg/l) at the rate of five fish per tank in triplicates. The Fish was then transported from the ARAC farm in Aluu to Rivers State University fish farm in department of Fisheries and Aquatic Environment, over a distance of 60km. Before and after transportation, blood samples (l ml) will be taken for glucose test from the Vena caudalis of the fish using a 5ml syringe fitted with 21G needle. The glucose levels were evaluated with glucose meter (Accuchek model RS 910, China). The survival of the experimental fish was observed and recorded in each of the concentration.

Determination of Induction and Recovery Time

The time for onset of anaesthesia for the exposed fish was measured in all the plant extracts using a digital stopwatch.  Fish behaviour was monitored individually through the induction and recovery stages in each life stage and concentrations [Table 3.1].  In the induction stage, five different behaviours were observed [17]. After the anaesthsia, fish was removed individually using a scoop net and 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

The water pH was determined in situ in each of the aquarium with a pH meter (Hanna Products, Portugal).  This was achieved by dipping the end of the electrode into the test solution and the mode button was selected and reading was taken. The temperature of the water was measured by placing the mercury in glass thermometer in the water and taking a reading after five minutes at 15cm depth. While the values of Ammonia, dissolved oxygen and sulphide were evaluated using LaMotte fresh water test kit (Model AQ4, Chestown, Maryland, USA).

Statistical Analysis

The data obtained from the study was collated and analyzed using statistics software 8.0 for windows. Data was first tested for normality (Kolmogorov - Smirnov test) and homosesdasticity of variance (Bartetts test). When these conditions were satisfied, a two way analysis of variance (ANOVA) was employed to reveal significant differences in measured variables among control and experimental groups. When a difference are detected (P<0.05), Tuckey’s multiple comparison test was applied to identify which treatment are significantly different.

The water quality parameters in experimental tanks of O. niloticus exposed and transported with clove seed extracts are presented in [Table 1].  The results indicated a significant reduction (P<0.05) in the values of dissolved oxygen, in the water without anaesthetics (0.00mg/l). Whereas, higher values of ammonia and sulphide were also recorded in the control. While other water quality parameters were within the same range with no significant different in relation to the concentration of the anaesthetics  (P>0.05). Mean values for the water quality variables such as temperature, pH, dissolved oxygen (DO), ammonia and sulphide obtained during exposure of O.niloticus to nut meg seed extracts are presented in [Table 2]. Lower values of dissolved oxygen were observed in the control, when compared to other concentrations. Whereas, higher values of ammonia and  sulphide were also recorded in the control.    While temperature and pH values were within the same range with no significant different (P>0.05) 

The survival in the fingerlings of O.niloticus exposed to nut Meg seed extracts is presented in Table 3. The survival of the fish decreased significantly (P<0.05) as the transportation time increased.  The lowest survival rate (20.00%) was recorded in the  fish  with no anaesthetics (control), in 80 minutes,   while the highest  values of survival  were observed in  the fish transported with  40.00mg/l  concentration of  nut meg. The same trend was observed in the juveniles of O. niloticus transported with nut Meg [Table 4]. After 80 minutes, the lowest survival rate (10.0%) was recorded in the control. While the highest survival (95.0) was recorded at 40.0 mg/l of the anaesthetics. 

The percentage survival in the fingerlings of O.niloticus transported with clove seed extracts are presented in Table 4.5. Generally, the survival of the fish decreased significantly (P<0.05) as the transportation time increased, except at 40.0 mg/l concentration of the anaesthetics.  The lowest survival rate (30.00%) was recorded in the fish with no anaesthetics (control), in 80 minutes,   while the highest values of survival (100.0 %) were observed in the fish transported with 40.00mg/l concentration of clove seed extracts. The same trend was however observed in the juveniles of O. niloticus transported with clove seed extracts (Table 4.6). At the end of   80 minutes, the lowest survival rate (10.0%) was recorded in the control. While the highest survival (100.0) was recorded at 40.0 mg/l of the extracts. 

The comparative glucose levels in O.niloticus juveniles transported with nut Meg and clove seed extracts are presented in Table 4.7. Before transportation the glucose levels were within the same range in all concentrations of exposure. The glucose levels in the fish exposed to both nut Meg and clove decreased significantly (P<0.05), with increasing concentrations of the anaesthetics. Comparative survival of in the fingerlings and juveniles of O.niloticus transported with nut Meg and clove seed extracts are shown in Figure 4.1 and 4.2. The results indicated that higher survival rate was recorded in fish transported with clove seed extracts when compared to nut Meg extracts in both sizes. 

Table 1: Water Quality Parameters in Experimental Tanks of O.niloticus Fingerlings Exposed to Clove Bud Extracts (Mean ± SD).

Table 2: Water Quality Parameters in Experimental Tanks of O.niloticus Fingerlings Exposed to Nut Meg Extracts (Mean ± SD).

Table 3: Survival (%) in O.niloticus Fingerlings Transported with Nut Meg Extracts.

Table 4: Survival (%) in O.niloticus Juveniles Transported with Nut Meg Extracts.

Table 5: Survival (%) in O.niloticus Fingerlings Transported with Clove Bud Extracts.

Table 6: Survival (%) in O.niloticus Juveniles Transported with Clove Bud Extracts.

Table 7: Comparative Glucose Levels in O.niloticus Juveniles Transported with Nut Meg and Clove Seed Extracts (Mean ± SD).

Figure 1: Comparative Survival in  Fingerlings of O.niloticus exposed  to Nut meg and Clove Seed Extracts.

            Figure 2: Comparative Survival in Juveniles of O. niloticus  exposed to  Nut meg and Clove Seed Extracts.

Figure 3: Comparative Glucose Levels   in O.niloticus Juveniles Transported with Nut Meg and Clove Seed Extracts.

Long recognized is the necessity for a method of immobilizing aquatic species without harming the victim. According to documents, fish immobilization has been the topic of basic and applied research that has been especially relevant to aquatic animals employed in aquaculture. Nowadays, immobilizing fish using anesthesia is a standard practice. Anesthesia usage may increase transit survivability [18]. In this study, fish exposed to various doses of clove and nutmeg seed extracts had good survival rates. This finding supported that made by Adamek et al. [19] about the movement of Indian shrimp (Fenneropenaeus indicus). This, according to Akinrotimi et al. [20], is a result of the fish being able to breathe adequately, maintaining homeostasis during transit, and increasing its survival due to the light anesthesia caused by clove application for the alleviation of transportation-related stress. Along with decreased activity, aquatic animals are transported with their metabolic rates reduced using mild doses of anesthetics. This could lessen physiological stress, oxygen consumption, and the formation of CO2 and ammonia [21, 22], which would reduce fatalities during and after transportation.

The water quality parameters in experimental tanks results indicated a significant reduction in the values of dissolved oxygen, in the water without anaesthetics (0.00mg/l). Whereas, higher values of ammonia and sulphide were also recorded in the control. While other water quality parameters were within the same range with no significant different in relation to the concentration of the anaesthetics  Similar result was observed in the transportation waters of Clarias gariepinus using  clove [23] This is because use of anaesthetics in fish transport minimizes its activity, and the excretion of ammonia through the gills. Hence clove and nut Meg  seed extracts maintain a relatively good water quality during fish transportation [24].  However, Winton [25] reported that typical oxygen consumption rates of spring Chinook salmon, Oncorhynchus tshawytscha smolts are 210 mg kg/1h/1 in untreated transportation tank water and 190 mg kg/1h/1 when 10 mg/L MS-222 is added. Kolarova et al.,[26] have also observed that 2-phenoxyethanol did not affect the oxygen consumption or ammonia production of goldfish, Carassius auratus during transport; on the other hand Lays et al., [27] have showed that it suppressed oxygen consumption rates of guppy, Poecilia reticulata, in simulated transportation experiment. In simulated air transport of platy fish, Xiphophorus maculates, 2-phenoxyethanol and quinaldine sulphate were efficient in decreasing the excretion of CO2 and ammonia, MS-222 reduced ammonia but not CO2 production and metomidate had no effect on excretion of metabolic wastes [28]. Iversen et al. [29] observed that the use of anesthetic (ethynelglycol-monophenylether) during simulated transportation of red porgy, Pagrus pagrus fry had no significant effect on CO2, NH3 and NH4 concentrations of exposure water. The results showed by Leji et al., [30] experiment indicate wide variation in oxygen consumption between six species of fish in response to low concentrations of clove oil during extended exposure. These examples indicate that different anesthetics may differ in their efficiency and it may also indicate the presence of inter fish-species variability in metabolic rates during exposure to low sedation concentration of anesthetics.

In aquaculture practices the understanding of fish stress response is essential to avoid stress-related problems, and to improve fish quality in rearing conditions so as to optimize production. As in other vertebrates, fish experiencing stress show a number of physiological changes that are expressed through a number of particular indicators [31]. Stress has been described as an energy drain of energy that might be utilized in growth diverted to catabolic utilization [32]. However, mobilization of readily available energy in the form of glucose is suggested to enhance the survival of fish.  It is perhaps, not surprising, therefore, that elevation of plasma glucose has been recognized as a part of generalized stress response in fish. The reduction in glucose levels of O. niloticus  juveniles  with increased concentrations of clove and nut Meg extracts in this work is similar to the findings of Akinrotimi et al.  et al. [33] in Sarotherodon melanotheron transported with clove oil. However, Lambooij et al. [34] observed an increase in glucose levels of common carp (Cyprinuscarpio) transported with anaesthetic metomidate, this contradictory trend according to Akinrotimi et al. [35] lies in the ability of clove to block cortisol secretion which stimulate glucose production. As cortisol is considered to be a major mediators of the increase in plasma glucose levels seen in stressful fish (Barton, 2002).

In conclusion, results indicated that    glucose levels in O.niloticus juveniles transported with nut Meg and clove seed extracts decreased significantly (P<0.05), with increasing concentrations of the anaesthetics. Comparative survival of in the fingerlings and juveniles of O.niloticus transported with nut Meg and clove seed extracts indicated that higher survival rate was recorded in fish transported with clove seed extracts when compared to nut Meg extracts in both sizes.  The water quality parameters in experimental tanks results indicated a significant reduction (P<0.05) in the values of dissolved oxygen, in the water without anaesthetics (0.00mg/l). Whereas, higher values of ammonia and sulphide were also recorded in the control. While other water quality parameters were within the same range with no significant different in relation to the concentration of the anaesthetics.From the results obtained in this study,  a concentration of 40.0 mg/l of nut Meg extracts is ideal for transportation of both fingerlings and juveniles of O. niloticus . While a lower concentration of 30.0 mg/l of clove seed extracts is recommended for transportation of O.niloticus in aquaculture with little or no mortality.

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