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Research Article
 

Effect of Cymbopogon citratus Essential Oil (EO) on Handling Stress in Giant Freshwater Prawn (Macrobrachium rosenbergii)



Wan Noazira Wan Adnan, Nurul Ulfah Karim, Nor Asma Husna Yusoff, Mohd Ihwan Zakariah and Marina Hassan
 
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ABSTRACT

Background and Objective: Effects of Cymbopogon citratus essential oil (EO) was tested on minimizing handling stress in Macrobrachium rosenbergii through the evaluation of their metabolite responses [glucose, lactate, glycogen, protein, Lactate Dehydrogenase (LDH), Malate Dehydrogenase (MDH), Acetylcholinesterase (AChE) and Alanine Aminotransferase (ALT)]. This study aimed to investigate the efficacy of C. citratus extract in the anaesthetization of M. rosenbergii. Materials and Methods: Three treatments including control, prawn exposed to stress alone (T1) and prawn exposed to stress in the presence of C. citratus EO (T2) were tested. A C. citratus EO at 500 μL L1 had been determined in a previous study and was selected as the critical dose to be applied as an anesthetic agent. Handling stress was induced into prawns by netting, at 2 min interval for 30 min and their hemolymph were collected to determine the metabolite responses. Results: The increase of glucose, lactate and LDH of M. rosenbergii when exposed to handling stress alone (T1) in comparison to T2 (stress with anesthetic C. citratus EO) were identified. Further, a low glycogen level in parallel with low AChE activity was observed which indicates the involvement of secondary metabolites to cope with the energy demand in T1 over T2. Conclusion: This study indicates the efficiency of C. citratus EO to reduce stress during handling in M. rosenbergii.

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Wan Noazira Wan Adnan, Nurul Ulfah Karim, Nor Asma Husna Yusoff, Mohd Ihwan Zakariah and Marina Hassan, 2021. Effect of Cymbopogon citratus Essential Oil (EO) on Handling Stress in Giant Freshwater Prawn (Macrobrachium rosenbergii). Pakistan Journal of Biological Sciences, 24: 13-18.

DOI: 10.3923/pjbs.2021.13.18

URL: https://scialert.net/abstract/?doi=pjbs.2021.13.18
 
Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Anesthetic is the calmness-causing agents which are commonly applied during capture, handling, sorting and transporting of fishes and shellfishes to minimize stress1. In crustaceans, the increase of Crustacean Hyperglycemic Hormone (CHH) due to stress environment resulting in changes in metabolic enzymes and physiological performances of the animals2,3. Several chemicals have been applied to mitigate the anesthetic effects in prawns including tricaine methanesulfonate (MS222), however, it was not effective in crustaceans and produce side effects which make the prawns unsafe for human consumption4. Salin5 has reported the efficient use of cold-anaesthetization using chilled sawdust to minimize stress in M. rosenbergii but only for a short-transportation period.

Cymbopogon citratus is usually called lemongrass and comes from a family of Graminaceae. It can be found in tropical and subtropical countries including Malaysia, Indo-China, Sri Lanka, Africa and South Africa6. Citral and geraniol are commonly reported as major compounds in C. citratus compound and possess several biological activities7-9. Interestingly, C. citratus has been found can act as a relaxant and antidepressant in forced swimming rats10. Besides, Blanco11 had reported the use of C. citratus leaves in treating nervous excitement. Based on those mentioned above, it was then hypothesized that C. citratus extract may exhibit anesthetic effects on stressed M. rosenbergii. Thus, a study was conducted to investigate the efficacy of C. citratus extract in anaesthetization of M. rosenbergii.

MATERIALS AND METHODS

Preparation of the experimental animals: This study was carried out in the Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, Malaysia from 2014-2018. Adult M. rosenbergii with an average weight of 18-23 g were used in this study without regard to gender. Prawns procured from a local farm in Jelebu, Negeri Sembilan were brought in an aerated fiberglass tank (1000 L) at ambient water temperature. During this period, prawns were fed with commercial pellets by Cargill Sdn. Bhd., twice per day for 15 days before use in experiments.

Plant collection and extraction: A Cymbopogon citratus leaves were collected from a local garden in Kuala Terengganu and dried in an oven (25°C) for 3-5 days. Dried leaves were powdered using a heavy-duty blender (Waring, USA) and extracted by a hydro-distillation method as according to Kulkarni12, with slight modifications. The crude essential oil of C. citratus was concentrated in a rotary evaporator (Buchi, Flawil, Switzerland) into 1 mL and stored in an amber bottle at 4°C until further use.

Preparation of C. citratus essential oil anesthetic: A Cymbopogon citratus essential oil was diluted in ethanol in the ratio of 1:10. A stock solution was made up by diluting 20 mL of essential oil in 200 mL ethanol. An appropriate volume of stock solution was added into a freshwater aquarium to obtain the concentrations needed. In this study, 500 μL L1 C. citratus essential oil was determined previously as a minimum concentration to cause induction in M. rosenbrgii. Hence, 500 μL L1 C. citratus essential oil was prepared and used in this stress analysis to determine the changes in hemolymph content after stress. Sole ethanol was used as a control in this study.

Metabolic responses of M. rosenbergii to handling in presence of C. citratus essential oil as anesthetic: Three different groups of prawns were performed in separate 15 L aquarium contained 10 L of freshwater. Six prawns were used in each treatment with five replicates each. The stress test was designed according to Saydmohammed and Pal13, with slight modifications. In the T1 group, test prawns were exposed to handling stress without anesthetic. In the T2 group, prawns were exposed to handling stress with the presence of anesthetic at 500 μL L1, whilst in the control group, prawns were maintained without handling stress and no presence of anesthetic in the aquarium. Handling stress in T1 and T2 groups were created by disturbing the prawns by netting for 2 min interval in 30 min. Prawns were sampled after 30 min and their hemolymph were collected separately for analysis.

Hemolymph preparation: Hemolymph was collected from each prawn through the ventral sinus, placed in a centrifuge tube containing anticoagulant solution (0.114 M trisodium citrate and 1 M sodium chloride at pH 7.45) and centrifuged at 5000 g for 15 min. After separation, the supernatant was stored at -80°C until further biochemical analysis. All procedures were performed in the ice-cold condition (4°C).

Hemolymph biochemical analysis: The concentration of glucose, lactate and glycogen contents were determined using standard kits (Abnova, Taiwan), according to the manufacturer’s protocols. The activities of LDH, ALT and AChE were also determined using commercial assay kits from a similar manufacturer (Abnova, Taiwan). The MDH activities were determined using another commercial kit from a different manufacturer (Sigma Aldrich, USA). The protein content was determined using a method derived by Bradford14. A 100 μL hemolymph was diluted with 900 μL distilled water in a clean test tube. Then, 1 mL of Bradford reagent was added and color development was observed in 10 min. Another clear test tube was filled with bovine serum albumin (BSA) (DifcoTM, USA) standard at a similar amount of Bradford reagent (DifcoTM, USA). The absorbance of each mixture was measured using a UV/Vis Spectrophotometer (Thermo Fisher Scientific, USA) at 595 nm wavelength and the amount of total protein in the hemolymph sample was calculated based on the plotted calibration curve graph.

Statistical analysis: The hemolymph chemistry parameters were homogenous (Levene’s test) and normally distributed (Kolmogorov-Smirnov test). Therefore, a one-way ANOVA test was performed and p<0.05 was considered as significant. All tests were performed using MINITAB 16.0 software.

RESULTS AND DISCUSSION

In this present study, data of glucose, lactate and glycogen and protein concentration in M. rosenbergii hemolymph after 30 min handling stress with and without C. citratus EO was presented in Fig. 1-4. A significantly higher glucose concentration with a simultaneously lower glycogen level was observed in the stressed group without C. citratus EO (T1) as compared to T2 and control. Similarly, an increase in lactate concentration, whilst stagnant in protein level was also found in the T1 group as compared to T2 and control group.

Meanwhile, data on different enzyme activities of M. rosenbergii after handling stress for 30 min with and without C. citratus EO was tabulated in Table 1. A significantly lower LDH activity was found in the stressed-treated group (T2) as compared to the non-treated stress group (T1). In contrast, MDH activity was found to increase in the stressed-treated group (T2) as compared to the control and T1 group, however, its increment was not significant. An increase of ALT activity was found in the stress-treated group (T2) as compared to T1 and control. Further, AChE activity was decreased in the stressed group (T1) and found to increase in the stressed-treated group (T2), with no significant difference.

Exposure to types of stress in captive animals will affect their physiological performance and immune-competence, which in turn reduced their current market value15.

Fig. 1:
Glucose content of M. rosenbergii hemolymph in response to handling stress
 
Different small letters (a, b, c) indicate significant differences amongst different treatments, T1: Stressed group, T2: Stressed group in the presence of C. citratus EO, p<0.05 was considered significant

Fig. 2:
Lactate content of M. rosenbergii hemolymph in response to handling stress
 
Different small letters (a, b) indicate significant differences amongst different treatments, T1: Stressed group, T2: Stressed group in the presence of C. citratus EO, p<0.05 was considered significant

Table 1:Enzymes of M. rosenbergii hemolymph in response to handling stress
LDH: Lactate dehydrogenase, MDH: Malate dehydrogenase, AChE: Acetylcholin-esterase, ALT: Alanine aminotransferase, LDH: μm mg1, MDH: Protein min1, AChE: mg protein min1 at 37°C, ALT: Nanomoles of pyruvate formed mg1 protein min1 at 37°C, different superscripts in the same row (a, b) indicate significant differences amongst different treatments, p<0.05 was considered significant

In crustaceans, stressful conditions will induce the regulation of Crustacean Hyperglycemic Hormones (CHH), elevate the glucose level thus results in gross changes in animal physiological performance2,16. Despite the vastness of techniques applied to mitigate stress in crustaceans5,17, one in demand currently is the use of natural anesthetic comes from plant herbs13,18,19.

This current study reported the potential use of C. citratus EO as stress minimizing agent in M. rosenbergii. A 500 μL L1 of C. citratus EO was used to anesthetize M. rosenbergii in this study, which had been previously determined able to promote anesthetic effect to M. rosenbergii, with a high survival rate (92%).

Fig. 3:
Glycogen content of M. rosenbergii hemolymph in response to handling stress
 
Different small letters (a, b) indicate significant differences amongst different treatments, T1: Stressed group, T2: Stressed group in the presence of C. citratus EO, p<0.05 was considered significant

Fig. 4:
Protein content of M. rosenbergii hemolymph in response to handling stress
 
Similar small letters (a) indicate no significant differences amongst different treatments, T1: Stressed group, T2: Stressed group in the presence of C. citratus EO, p<0.05 was considered significant

A significant increase of glucose level with a concomitant decrease of glycogen level (p<0.05) in stressed prawn alone (T1), over stress-treated prawn with anesthetic C. citratus EO (T2), respectively has been shown in Fig. 1-3. The high level of glucose in the T1 group was likely maintained by utilizing the stored glycogen in the hemolymph due to stress handling. The finding was similar to Stentiford et al.20, where they observed the utilization of glucose by the glycogenolysis process in Norway lobsters when exposed to parasites. Glycogenolysis is a process of breaking down the glycogen molecule into glucose21. Interestingly, glucose and glycogen levels showed an inverse trend in the T2 group over T1 which indicates the efficiency of C. citratus EO as a stress-relieving agent by providing calmness to the stressed prawns. As been reported by Vale et al.22, C. citratus EO is commonly high in citral compound which is capable to produce anxiolytic, sedative and motor relaxant effects in mice. Nevertheless, the glucose level in T2 was depicted higher than the control group, 2.34±0.71 and 0.27±0.25 nmol μL1, respectively. This situation might be due to the addition of C. citratus EO itself which exhibits stressor effects to the prawn, thus induce the stress response mechanisms23. Readman et al.24 had stated the aversive effects of plant essential oils in aquatic fish even applied at low concentrations.

Further, the lactate level in the T2 group was decreased significantly over T1, 1.13±0.39 and 0.71±0.24 nmol μL1, respectively. The reduction might be due to the less stress experienced by the prawn in the T2 group with the presence of C. citratus EO as compared to the stress-alone group (T1). The formation of lactate is when the glycolysis process is performed under the anaerobic condition to maintain adequate energy during stress condition25. The decrease of the lactate level was parallel to the decreasing of LDH activity in T2. This LDH enzyme is important in converting pyruvate into a lactate molecule particularly when rapid energy is needed during several stressors such as handling stress and environmental stress26. Ribas et al.27, had reported the ability of 1000 mg L1 clove oil to anesthetize seawater Senegal sole (Solea senegalensis) which resulted in a decrease of glucose and lactate hemolymph.

MDH is involved in aerobic metabolisms and plays an important role in metabolite exchange26. However, results show no significant difference in MDH activity in all treatment groups (Table 1). Findings were contradicted with Saydmohammed and Pal13 which reported the increase of MDH activity in M. rosenbergii due to stress handling. However, results might vary as the sample used are also different. Readman et al.24 also clarify that the results of the essential oil itself may acts differently in different procedures and animals used. Hemolymph protein in the present study was more or less similar among all treatment groups (Fig. 4), which revealed the protein was not affected by handling stress or anesthesia effects from C. citratus EO. The result was supported by Nagahama et al.29, where the plasma protein in silver catfish (Rhamdia quelen) anesthetized with both Hesperozygis ringens and Lippia alba EO were not change. Typically the unaffected protein in hemolymph resulted in no changes in ALT activities represents no protein metabolism involved during treatment.

Acetylcholine is considered as an inhibitory transmitter in crustaceans which are stored and released from synaptic vesicles30. In the present study, AChE activity was found to be inhibited in the T1 group, over control and T2 (Table 1), which results in an increased level of acetylcholine at postsynaptic ends31. This enzyme inhibition in synaptic buds during handling was reported previously to cause erratic behavior in rats32. The addition of C. citratus EO in stressed prawn increased AChE activity in the T2 group as compared to T1 which indicates the ability of C. citratus EO to ameliorate the chlorogenic effect due to stress handling in M. rosenbergii.

It seems that C. citratus essential oil used in this study can provide a relaxation effect to stressed prawns as depicted in their metabolites hemolymph. However, the hematological analysis also would be an advantage if could be determined, which important to support the finding, become our limitation in this study. Furthermore, the determination of their mode of actions also is crucial to provide a thorough understanding of the anesthetic mechanisms in crustaceans.

CONCLUSION

In conclusion, results indicate the potentiality of C. citratus essential oil as an anesthetic agent by minimizing handling stress in M. rosenbergii. However, the dosage during application may vary among species and the size of animals used needs to be further investigated.

SIGNIFICANCE STATEMENT

This study discovers the metabolite’s responses in prawn hemolymph after been exposed to stress handling with the presence of C. citratus essential oil as an anesthetic agent. Findings would be beneficial for other researchers and also people in aquaculture industries to uncover the potentiality of C. citratus essential oil for further development as the demand for natural anesthetic is at high stake currently. Thus, a new natural anesthetic from other plant sources also can be developed and possibly with the rise of any other plant anesthetics or combination of them, may be arrived at.

ACKNOWLEDGMENT

I would like to thank the Institute of Tropical Aquaculture and Fisheries (AKUATROP), Universiti Malaysia Terengganu (UMT) for the support of this project. Appreciation extended to MyBrain15 (MyPhD) for providing a scholarship to Wan Noazira Wan Adnan. A special thanks to the late Associate Prof. Dr. Safiah Jasmani for her guidance and supports.

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