Lemongrass essential oil (Cymbopogon flexuosus) as a dietary additive in Nile tilapia (Oreochromis niloticus)ABSTRACT
The objective of the work was to evaluate the effects of adding lemongrass essential oil on growth and oxidative stress in the rearing phase of Oreochromis niloticus. Two hundred Nile tilapia fingerlings (0.974±0.007g) were used, at a density of 10 fish per aquarium (70 L). The treatments consisted of five levels of lemongrass essential oil (EO) (0.00; 0.89; 1.78; 2.67; and 3.56mg kg-1) added to the feed, with four replicates. The animals were fed ad libitum, four times a day, for 37 days, at low temperatures. At the end of the experiment, the fish were anesthetized, weighed, euthanized, and dissected to obtain vicerosomatic and hepatosomatic index parameters. Liver samples from five animals from each experimental unit were collected for biochemical analysis of lipoperoxidation, catalase, glutathione S-transferase, superoxide dismutase, and protein carbonylation. The feeding with lemongrass essential oil influenced fish survival, and the other productive performance parameters did not show significant differences. At redox equilibrium, a decrease in glutathione S-transferase and increased superoxide dismutase activity were observed. It is concluded that the addition of up to 3.56mg kg-1 of lemongrass EO does not interfere with the productive performance improves survival and influences the redox balance of the animals.
keywords:
nutritional additives; fish health; fish farming; redox equilibrium
RESUMO
O objetivo do trabalho foi avaliar os efeitos da adição de óleo essencial de capim-limão sobre o crescimento e o estresse oxidativo na fase de recria de Oreochromis niloticus. Foram utilizados 200 alevinos de tilápia-do-nilo (0,974±0,007g), em uma densidade de 10 peixes por aquário com volume de 70 litros. Os tratamentos consistiram em cinco níveis de óleo essencial (OE) de capim-limão (0,00; 0,89; 1,78; 2,67; e 3,56mg kg-1) adicionados à ração, com quatro repetições. Os animais foram alimentados ad libitum, quatro vezes ao dia, durante 37 dias, em baixas temperaturas. Ao final do experimento, os peixes foram anestesiados, pesados, eutanasiados e dissecados para obtenção dos parâmetros do índice vicessomático e hepatossomático. Amostras de fígado de cinco animais de cada unidade experimental foram coletadas para análises bioquímicas de lipoperoxidação, catalase, glutationa S-transferase, superóxido dismutase e carbonilação de proteínas. A alimentação com óleo essencial de capim-limão influenciou a sobrevivência dos peixes, e os demais parâmetros de desempenho produtivo não apresentaram diferenças significativas. No equilíbrio redox, foi observada diminuição da glutationa S-transferase e aumento da atividade da superóxido dismutase. Conclui-se que a adição de até 3,56mg kg-1 de OE de capim-limão não interfere no desempenho produtivo, melhora a sobrevivência e influencia o balanço redox dos animais.
Palavras-chave:
aditivos nutricionais; saúde dos peixes; piscicultura; equilíbrio redox
INTRODUCTION
Aquaculture has great potential to meet the demand for healthier food due to the stagnation of fish stocks, which is the agricultural activity that grows the most in the world (A Situação…, 2022). Freshwater fish are the largest group of aquatic organisms produced, with a production of 54.4 million tonnes in 2020, a growth of 2.7% between 2019 and 2020 (A Situação…, 2022). In the face of continued growth, fish farming will provide more than half of the world's fish for human consumption in the coming years (Barroso et al., 2019). Nile tilapia (Oreochromis niloticus) is the world's fourth most-produced freshwater species (A Situação…, 2022). This species is of great importance due to its high growth rate, rusticity, mastery of reproductive technology, and good organoleptic characteristics of its meat (Barroso et al., 2018).
Due to the intensification of production and the emergence of diseases, some chemical products are used in aquaculture, representing a high risk to the aquatic environment and the health of consumers (Rico and Van Den Brink, 2014). Given this, the production chain and researchers have been looking for alternatives, mainly in food additives used in natural medicine. Additives are substances added to diets that can intensify, preserve, or modify their properties without impairing their nutritional value (Rodrigues et al., 2015).
Herbal products help combat microbial and parasitic diseases, due to essential oil (EO) compounds that cause deformities and dysfunctions in bacterial and parasitic cells (Bandeira Junior et al., 2022). Therefore, these products have been the subject of research in aquaculture and can be used in baths or even added to fish diets (Bandeira Junior et al., 2022), as additives with an important role in the growth, health and resistance of animals (Zheng et al., 2009). Different types of medicinal herbs have demonstrated effects on fish diseases (Hoseinifar et al., 2020).
The reduction in pollution of aquatic environments, less toxicity for animals, and less deposition in tissues are characteristics that make EOs even more interesting among nutritional additives (Coimbra et al., 2006). Thus, extracts derived from plants, such as EOs, can replace or reduce the use of antibiotics and chemotherapy (Vaseharan and Thaya, 2014). In addition, they have anti-inflammatory properties and stimulate the health of animals (Tariq et al., 2019). Furthermore, several EOs have antioxidant activity, preventing oxidative stress in fish, which is directly linked to life strategies and the type of cultivation (Birnie-Gauvin et al., 2017).
Lemongrass (Cymbopogon flexuosus) belongs to the Poaceae family and is distributed in several tropical countries. Lemongrass essential oil can be easily extracted from the fresh plant through steam extraction (Malee et al., 2000). The antibacterial activity of lemongrass mainly resides in the α-citral and β-citral components present in the EO (Onawunmi et al., 1984; Adukwu et al., 2016). This EO is mainly composed of citral isomers, which represent approximately 61% of the oil's composition (Melo et al., 2020).
Citral is known to possess anti-inflammatory, immunomodulatory, antiseptic, antimicrobial, fungistatic, and antioxidant properties (Bachiega and Sforcin, 2011). Thus, this study aimed to evaluate the effects of adding lemongrass essential oil on the growth and oxidative stress of O. niloticus fingerlings raised in low temperatures.
ETHICAL ASPECTS
The research was submitted to the Ethics Committee on Animal experimentation - CEEAAP of the State University of West Paraná and approved under the number 06/10.
MATERIAL AND METHODS
The experiment was carried out after approval by the Animal Ethics Committee for the use of experimental animals at the State University of Western Paraná (14/2020). Two hundred O. niloticus fingerlings (0.974±0.007g) were used, at a density of 10 fish per aquarium. The animals were distributed in 20 aquariums of 70L, installed in static system with aeration, during the winter. A partial change of 30% of the water volume in each aquarium was performed daily. The experiment lasted 37 days and consisted of five treatments, which were lemongrass essential oil levels (0.00; 0.89; 1.78; 2.67; and 3.56mg kg-1), with four experimental units per treatment. The experimental unit was considered a 30L aquarium with 10 animals.
The experimental diets were formulated considering the chemical composition of each feed, with protein and digestible energy meeting the requirements of the species (Table 1). The EO from C. flexuosus was donated by GRASP (Curitiba, Paraná, Brazil). The main components of lemongrass essential oil are: α-citral, β-citral, Z-verbenol, citronellol, Z-geraniol and caryophyllene (Rampelotto et al., 2018). The software used in the formulation of the rations was SuperCrac®. The ingredients used in the manufacture of feeds were ground in a hammer mill with a 0.5mm sieve and mixed for 20 minutes. The feed was produced in pelletized form with a diameter of 1 mm. The animals were fed the experimental diets to apparent satiation, four times a day (8:00, 11:00, 14:00, and 17:00; hh:mm).
The water temperature was measured daily (max.: 22.02±0.72°C, min.: 20.75±0.95°C, Incoterm®). Dissolved oxygen (6.98±0.45mg L-1; Oximeter YSI® 550A), total ammonia (0.51±0.06mg L-1), nitrite (0.17±0.05mg L-1), nitrate (0.15±0.09mg L-1), turbidity (23.12±3.21 NTU), total phosphorus (5.12±0.48mg L-1), alkalinity (44.03±3.25mg of CaCO3 L-1) (AlfaKit AT 100 PB II® Multiparameter Photocolorimeter), and water pH (7.03±0.15; digital pHmeter Tecnal® Tec 5) were measured weekly at 6 am, being within the appropriate parameters for the species (Boyd e Trucker, 1998).
At the end of the experiment, the animals were anesthetized with 50mg L-1 of eugenol (Cunha et al., 2010). The zootechnical parameters of final weight, length, and survival were measured. Also, the parameters of weight gain, daily weight gain, feed conversion (considering only the feed consumed), and condition factor were calculated. The animals were euthanized with a lethal dose of 500mg L-1 of eugenol (Cunha et al., 2010), followed by spinal section and dissection. The liver and viscera were weighed to assess viscerosomatic and hepatosomatic indices.
After the animals were euthanized, liver samples were collected and stored at -80ºC (Ultra Freezer SS Scientific®). To perform the biochemical analyses, the liver samples were homogenized with saline phosphate buffer solution (pH 7.2). After breaking the cells in a homogenizer (T10 basic, IKA®), the samples were centrifuged at 12800g at 4ºC for 10 minutes. The supernatant was collected, divided into microtubes, and stored at -80ºC. A fraction of the prepared samples was used to carry out the protein quantification, by the method of Bradford (1976). After protein quantification, the samples were normalized to 1mg of protein mL-1, to carry out the assays. In the liver samples, the parameters of glutathione S-transferase (GST) (Habig et al., 1976), lipid peroxidation (LPO) (Jiang et al., 1992), catalase (CAT) (Aebi, 1984), carbonylation of proteins (PCO) (Levine et al., 1990) and superoxide dismutase (SOD) (Crouch et al., 1981) were measured. A microplate reader (Fisherscientific Accuskan Go®) was used to carry out the analyses.
The data were tested regarding the assumptions of normality and homogeneity, using the Shapiro-Wilk and Levene tests, respectively. Subsequently, the data was submitted to analysis of variance (ANOVA), when significant differences were observed, Tukey's multiple interval test was applied (p<0.05). All analysis was performed using the Statistica 7.1® software.
RESULTS AND DISCUSSION
Studies using EO in fish diets have been gaining ground, due to the numerous benefits already shown by other authors (Al-Sagheer et al., 2017; Valladão et al., 2017; Amer et al., 2018) and most of them do not have contraindications.
The growth (Table 2) of Nile tilapia fingerlings fed diets containing increasing levels of lemongrass essential oil was not influenced (p>0.05). The addition of 3.56mg kg-1 lemongrass EO (C. flexuosus) to the diets of juvenile Nile tilapia provided better survival (80.00±9.13%) (p<0.05). Liver redox balance indicators (Table 3) were influenced in animals fed diets containing increasing levels of lemongrass EO. Glutathione S-transferase was reduced (p<0.05) in the liver of animals fed with 3.56mg kg-1 of lemongrass EO. Superoxide dismutase activity increased (p<0.05) in the liver of animals fed diets containing 1.78mg kg-1 of lemongrass EO.
Antioxidants are important molecular protection mechanisms and stress biomarkers (Hellou et al., 2012). Cellular homeostasis can be maintained through the balance and degradation of reactive oxygen species (ROS), also called oxidative stress (Covarrubias et al., 2008). ROS are molecules formed as byproducts of aerobic respiration and other catabolic and anabolic processes (Wu et al., 2011). SOD can be considered a signaling enzyme, which can appear due to tissue inflammation and the presence of high concentrations of ROS (Zou et al., 2017). Enzymes that act against ROS are divided into extracellular or intracellular, where SOD acts as the first line of defense against ROS on the surface of a cell, and GST acts as an internal cell protection mechanism (Hellou et al., 2012). Decreased levels of GST with increasing concentrations of EOs show an antioxidant capacity in liver cells. GST is an enzyme that is directly related to the decrease of ROS and is also responsible for the detoxification of animals (Best et al., 2002). Studies on the effects of natural compounds in fish diets should be encouraged, due to their benefits and low impact on animals and the farming system.
Lemongrass EO is rich in α-citral and β-citral components (Onawunmi et al., 1984), having antibacterial and antimicrobial action, which causes deformities and breaks in the cell walls of parasites (Bandeira Junior et al., 2022), where hyperpolarization occurs, reducing intracellular pH and ATP concentration (Copatti et al., 2022), reducing its pathogenic action on animals. In addition, lemongrass EO has compounds such as 1,8-cineol, ÿ-pinene, and limonene (Oussalah et al., 2007), which are characterized by their hydrophobicity, facilitating their action on the lipid membranes of bacterial cells (Burt, 2004).
Trials using lemongrass essential oil in fish feed often fail to identify statistically significant differences in weight gain or feed conversion rate, as cited by Rampelotto et al., (2018). However, this does not preclude the presence of positive effects on other physiological fronts, which add value to its use in aquatic production. The low survival rate throughout the experiment may be directly related to the period in which the animals were cultivated at low temperatures. Furthermore, the intensification of cultivation systems and the use of high-density diets increase the occurrence of overload in the animals' organs, increasing their metabolic effort (Drew et al., 2007). The use of additives or supplements with antioxidant activity helps to reduce the damage caused to animals (Bombardelli et al., 2022). The better survival and health of the animals feeding with the higth levels may have occurred due to the immunomodulatory capacity and antioxidant activity of lemongrass EO (Bachiega and Sforcin, 2011). Furthermore, essential oils can modulate the innate and adaptive immune response, which is particularly important at low temperatures, when the efficiency of the fish's immune system typically declines. Studies in tilapia (Oreochromis niloticus) showed that diets supplemented with lemongrass and geranium essential oil resulted in higher serum IgM levels, antioxidant activity (SOD, CAT, GSH), and a reduction in enteric pathogens such as Aeromonas hydrophila (Al-Sagheer et al., 2017). These effects suggest that, even under heat stress, the additive can maintain immunocompetence and reduce mortality. The antioxidant activity of EO may be related to the citral content, known for its immunomodulatory and antioxidant activities (Djenane, 2015; Bachiega ans Sforcin, 2011). Therefore, in the present work, it can be observed that the addition of lemongrass EO influences the survival of the fish and influences the redox balance of the animals, altering the activity of antioxidant enzymes. Although the low survival results in the 1.78mg EO kg-1 treatment have no biological explanation, the analyses performed in this experiment did not allow elucidation of the mechanisms that led to these results. Thus, it is important to further studies on the effect of EOs as additives in fish diets.
ACKNOWLEDGEMENTS
We would like to thank the “Instituto Neotropial de Pesquisas Ambientais” (INEO).
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Data-available-upon-request - the research data are available upon request.
