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Effect of Lippia grata essential oil as a feed additive on the performance of tambatinga juveniles

ABSTRACT

Lippia grata (formerly known as Lippia gracillis) is an aromatic plant native to Brazil, with leaves rich in essential oils that possess significant biological activities. We evaluated the effect of essential oil of L. grata (EOLG) as a dietary additive on the growth, somatic indices, and biochemical parameters of juveniles (5.25 ± 0.26 g) of tambatinga, a hybrid fish obtained by crossing tambaqui (Colossoma macropomum) with pirapitinga (Piaractus brachypomum) of great economic importance in north and northeastern Brazil. We evaluated four dietary treatments, consisting of EOLG supplemented at 0.0, 0.5, 1.0, and 2.0 mL kg-1, over 60 days. Carcass yield was significantly higher in fish fed all EOLG diets compared to those fed the control diet (0.0 mL kg-1). Animals that received the 0.5 mL kg-1 treatment gained significantly more weight and showed a higher specific growth rate than those treated with 1.0 and 2.0 ml kg-1 EOLG, although none differed significantly from the control. The feed conversion rate was significantly lower in the 0.5 than in the 1.0 mL kg-1 treatment. Compared with higher concentrations, the diet containing 0.5 mL kg-1 EOLG increased the use of muscle glycogen, glucose, and lactate to meet energy demands, avoiding the use of muscle protein. Our results suggest that dietary supplementation with EOLG significantly improves carcass yield in tambatinga juveniles but that concentrations above 0.5 mL kg-1 may compromise growth rates and carbohydrate metabolism in this fish.

KEYWORDS:
diets; growth; Lippia gracillis; Colossoma macropomum; Piaractus brachypomum

RESUMO

Lippia grata (previamente conhecida como Lippia gracillis) é uma planta aromática nativa do Brasil, com folhas ricas em óleos essenciais que possuem atividades biológicas significativas. Avaliamos o efeito do óleo essencial de L. grata (OELG) como aditivo alimentar sobre o crescimento, índices somáticos e parâmetros bioquímicos de juvenis (5,25 ± 0,26 g) de tambatinga, um hibrido obtido do cruzamento de tambaqui (Colossoma macropomum) com pirapitinga (Piaractus brachypomum) com grande importância econômica no norte e nordeste do Brazil. Foram avaliados quatro tratamentos dietéticos consistindo na suplementação com OELG em 0,0; 0,5; 1,0 e 2,0 mL kg-1 durante 60 dias. O rendimento de carcaça foi significativamente maior nos peixes alimentados com todas as dietas contendo OELG em comparação à dieta controle. Os animais do tratamento 0,5 mL kg-1 ganharam significativamente mais peso e apresentaram maior taxa de crescimento específico do que aqueles tratados com 1,0 e 2,0 mL kg-1 OELG, embora nenhum tenha diferido significativamente do controle. A taxa de conversão alimentar foi significativamente menor no tratamento 0,5 mL kg-1 do que no tratamento 1,0 mL kg-1. Comparada com as concentrações mais altas, a dieta contendo 0,5 mL kg-1 OELG aumentou o uso de glicogênio muscular, glicose e lactato para suprir as demandas energéticas, evitando o uso de proteína muscular. Nossos resultados sugerem que a suplementação dietética com OELG melhora significativamente o rendimento de carcaça de juvenis de tambatinga, mas concentrações acima de 0,5 mL kg-1 podem comprometer as taxas de crescimento e metabolismo de carboidratos desses peixes.

PALAVRAS-CHAVE:
dietas; crescimento; Lippia gracillis; Colossoma macropomum; Piaractus brachypomum

INTRODUCTION

Plant-derived feed additives present beneficial effects on fish health, growth, and feeding efficiency in cultured fish due to the high quantity and variety of secondary bioactive metabolites (phytochemicals) they contain. The active properties of phytochemicals are due to their synthesis by secondary metabolism in plants (Sutili et al. 2018Sutili, F.J.; Gatlin III, D.M.; Heinzmann, B.M.; Baldisserotto, B. 2018. Plant essential oils as fish diet additives: benefits on fish health and stability in feed. Reviews in Aquaculture, 10: 716-726.; Souza et al. 2019Souza, C.F.; Baldissera, M.D.; Baldisserotto, B.; Heinzmann, B.M.; Martos-Sitcha, J.A.; Mancera, J.M. 2019. Essential oils as stress-reducing agents for fish aquaculture: A review. Frontiers in Physiology, 10: 785. ; Heluy et al. 2020Heluy, G.M.; Ramos, L.R.V.; Pedrosa, V.F.; Sarturi, C.; Figueiredo, P.G.P.; Vidal, L.G.P.; França, I.F.; Pereira, M.M. 2020. Oregano (Origanum vulgare) essential oil as an additive in diets for Nile tilapia (Oreochromis niloticus) fingerlings reared in salinized water. Aquaculture Research, 51: 3237-3243.; Kuralkar and Kuralkar 2021Kuralkar, P.; Kuralkar, S.V. 2021. Role of herbal products in animal production - An updated review. Journal of Ethnopharmacology, 278: 114246. ; Faehnrich et al. 2021Faehnrich, B.; Franz, C.; Nemaz, P.; Kau, H.P. 2021. Medicinal plants and their secondary metabolites −State of the art and trends in breeding, analytics and use in feed supplementation - with special focus on German chamomile. Journal of Applied Botany and Food Quality, 94: 61-74.). Phytochemicals act differently depending on the concentration of the active or major ingredients, the amount used in the diet, and the application form (Sutili et al. 2018; Souza et al. 2019; Chung et al. 2020Chung, S.; Lemos, C.H.D.P.; Teixeira, D.V.; Fortes-Silva, R.; Copatti, C.E. 2020. Essential oil from Ocimum basilicum improves growth performance and does not alter biochemical variables related to stress in pirarucu (Arapaima gigas). Anais da Academia Brasileira de Ciências, 92: e20181374.). Dietary addition of essential oils from plants such as Citrus sinensis (L.) Osbeck (Acar et al. 2015Acar, Ü.; Kesbiç, O.S.; Yilmaz, S.; Gültepe, N.; Türker, A. 2015. Evaluation of the effects of essential oil extracted from sweet orange peel (Citrus sinensis) on growth rate of tilapia (Oreochromis mossambicus) and possible disease resistance against Streptococcus iniae. Aquaculture, 437: 282-286.), Citrus limon (L.) Burm (Ngugi et al. 2016Ngugi, C.C.; Oyoo-okoth, E.; Muchiri, M. 2016. Effects of dietary levels of essential oil (EO) extract from bitter lemon (Citrus limon) fruit peels on growth, biochemical, haemato-immunological parameters and disease resistance in juvenile Labeo victorianus fingerlings challenged with Aeromonas hydrophila. Aquaculture Research, 48: 2253-2265.), Aloysia citriodora Palau (formerly A. triphylla) (Zeppenfeld et al. 2016Zeppenfeld, C.C.; Hernández, D.R.; Heinzmann, B.M.; Cunha, M.A.; Schmidt, D.; Baldisserotto, B. 2016. Essential oil of Aloysia triphylla as feed additive promotes growth of silver catfish (Rhamdia quelen). Aquaculture Nutrition, 22: 933-940.), Citrus x auranticus L. (Lopes et al. 2019Lopes, J.M.; Souza, C.F.; Saccol, E.M.H.; Pavanato, M.A.; Antoniazzi, A.; Rovani, M.T.; Heinzmann, B.M.; Baldisserotto, B. 2019. Citrus x auranticus oil as feed additive improved growth performance, survival, metabolic, and oxidative parameters of silver catfish (Rhamdia quelen). Aquaculture Nutrition, 25: 310-318. ), and Citrus x latifolia (Yu. Tanaka) Tanaka (Lopes et al. 2020) increase growth rates in fish and are viable alternatives to increase the productivity of intensive fish farming. Yet, essential oils of Lippia alba (Mill.) N. E. Brown, Lippia sinoides Cham, Ocimum gratissimum L., or Zingiber officinale Roscoe do not influence fish growth; rather, they can reduce lipid peroxidation and increase the tissue antioxidant response (Saccol et al. 2013Saccol, E.M.H.; Uczay, J.; Pes, T.S.; Finamor, I.A.; Ourique, G.M.; Riffel, A.P.K.; et al. 2013. Addition of Lippia alba (Mill) N. E. Brown essential oil to the diet of the silver catfish: An analysis of growth, metabolic and blood parameters and the antioxidant response. Aquaculture, 416: 244-254.) or improve the nonspecific immune response and survival (Monteiro et al. 2021Monteiro, P.C.; Brandão, F.R.; Farias, C.F.S.; Alexandre Sebatião, F.; Majolo, C.; Dairiki, J.K.; et al. 2021. Dietary supplementation with essential oils of Lippia sinoides, Ocimum gratissimum and Zingiber officinale on the growth and hemato-immunological parameters of Colossoma macropomum challenged with Aeromonas hydrophila. Aquaculture Reports, 19: 100561. ).

Several mechanisms have been proposed to explain the beneficial effects of essential oils on fish performance, such as changes in the gastrointestinal tract and its microbiota that improve digestibility, nutrient absorption, and the immune response (Talpur 2014Talpur, A.D. 2014. Mentha piperita (Peppermint) as feed additive enhanced growth performance, survival, immune response and disease resistance of Asian seabass, Lates calcarifer (Bloch) against Vibrio harveyi infection. Aquaculture, 420-421: 71-78.; Acar et al. 2015Acar, Ü.; Kesbiç, O.S.; Yilmaz, S.; Gültepe, N.; Türker, A. 2015. Evaluation of the effects of essential oil extracted from sweet orange peel (Citrus sinensis) on growth rate of tilapia (Oreochromis mossambicus) and possible disease resistance against Streptococcus iniae. Aquaculture, 437: 282-286.); increased antioxidant activity (Saccol et al. 2013Saccol, E.M.H.; Uczay, J.; Pes, T.S.; Finamor, I.A.; Ourique, G.M.; Riffel, A.P.K.; et al. 2013. Addition of Lippia alba (Mill) N. E. Brown essential oil to the diet of the silver catfish: An analysis of growth, metabolic and blood parameters and the antioxidant response. Aquaculture, 416: 244-254.; Lopes et al. 2019Lopes, J.M.; Souza, C.F.; Saccol, E.M.H.; Pavanato, M.A.; Antoniazzi, A.; Rovani, M.T.; Heinzmann, B.M.; Baldisserotto, B. 2019. Citrus x auranticus oil as feed additive improved growth performance, survival, metabolic, and oxidative parameters of silver catfish (Rhamdia quelen). Aquaculture Nutrition, 25: 310-318. ) and resistance to stress (Souza et al. 2019Souza, C.F.; Baldissera, M.D.; Baldisserotto, B.; Heinzmann, B.M.; Martos-Sitcha, J.A.; Mancera, J.M. 2019. Essential oils as stress-reducing agents for fish aquaculture: A review. Frontiers in Physiology, 10: 785. ) and direct effects on the development of pathogenic organisms that reduce their ability to colonize the digestive tract, thus preventing disorders that affect digestion and nutrient absorption (Harikrishnan et al. 2011Harikrishnan, R.; Balasundaram, C.; Heo, M.S. 2011. Impact of plant products on innate and adaptive immune system of cultured finfish and shellfish. Aquaculture, 317: 1-15.; Campagnolo et al. 2013Campagnolo, R.; Freccia, A.; Bergmann, R.R.; Meurer, F.; Bombardelli, R.A. 2013. Óleos essenciais na alimentação de alevinos de tilápia do Nilo. Revista Brasileira de Saúde e Produção Animal, 14: 565-573.). The interaction among the intestinal microflora, the morphology of the gastrointestinal mucosa, blood biochemical levels, the immune system, and the absorption of nutrients has a direct influence on the health and productive performance of fish (Ahmadifar et al. 2011Ahmadifar, E.; Falahatkar, B.; Akrami, R. 2011. Effect of dietary thymol-cravacrol on growth performance hematological parameters and tissue composition of juvenile rainbow trout Oncorhynchus mykkis. Journal of Applied Ichthyology, 1: 1057-1060.; Campagnolo et al. 2013; Lopes et al. 2020).

Lippia grata Schauer (Verbenaceae), formerly designated as Lippia gracillis Schauer, is an aromatic shrub up to 1.5 m in height, growing wild in areas of northern and northeastern Brazil (Albuquerque at al. 2007Albuquerque, U.P.; Medeiros, P.M.; Almeida, A.L.S.; Monteiro, J.M.; Lins Neto, E.M.F.; Melo, J.G.; Santos, J.P. 2007. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach. Journal of Ethnopharmacology, 114: 325-354.; Franco et al. 2014Franco, C.S.; Ribeiro, A.F.; Carvalho, N.C.C.; Monteiro, O.S.; Da Silva, J.K.R.; Andrade, E.H.A.; Maia, J.G.S. 2014. Composition and antioxidant and antifungal activities of the essential oil from Lippia gracilis Schauer. African Journal of Biotechnology, 13: 3107-3113.). Its aerial parts have antibacterial, antioxidant, and anti-inflammatory properties (Takeuchi and Cafe 2016Takeuchi, M.G.; Cafe, M.B. 2016. Aditivos Fitogênicos na Alimentação de Aves de Produção. 1st ed. online. Navegando Publicações, Uberlândia, 59p.; Mendes et al. 2020Mendes, S.S.; Bomfim, R.R.; Jesus, H.C.R.; Alves, P.B.; Blank, A.F.; Estevam, C.S.; Antoniolli, A.R.; Thomazzia, S.M. 2020. Evaluation of the analgesic and anti-inflammatory effects of the essential oil of Lippia gracilis leaves. Journal of Ethonopharmacology, 129: 391-397.) and are used to treat gastrointestinal, respiratory, and cutaneous infections (Albuquerque et al. 2006Albuquerque, C.C.; Camara, T.R.; Mariano, R. L.R.; Willadino L.; Júnior, C.M.; Ulisses, C. 2006. Antimicrobial action of the essential oil of Lippia gracillis Schauer. Brazilian Archives of Biology and Technology, 49: 527-535.). The main active components of the essential oil of L. gracillis are thymol, carvacrol, p-cymene, and γ-terpinene, which are molecules with antimicrobial properties (Gomes et al. 2011Gomes, S.V.F.; Nogueira, P.C.L.; Moraes, V.R.S. 2011. Aspectos químicos e biológicos do gênero Lippia enfatizando Lippia gracilis Shauer. Eclética Química, 36: 64-77.). Although it is used routinely in popular human medicine to treat infections, there are few studies on the action of this essential oil in animal production and as a performance enhancer (Cardoso Junior 2017Cardoso-Júnior, G.S. 2017. Óleo essencial de alecrim da chapada (Lippia gracilis schauer) em dietas de codornas japonesas em crescimento. Master´s dissertation, Universidade Federal de Sergipe, Brazil, 48p. (https://ri.ufs.br/jspui/handle/riufs/7040).
https://ri.ufs.br/jspui/handle/riufs/704...
; Rocha et al. 2020Rocha, G.F.; Del Vesco, A.P.; Santana, T.P.; Santos, T.S.; Cerqueira, A.S.; Zancanela, V.T.; Fernandes, R.P.M.; Oliveira Júnior, G.M. 2020. Lippia gracilis Schauer essential oil as a growth promoter for Japanese quail. Animal, 14: 2023-2031.). Considering other species of the genus Lippia, there are studies evaluating its anesthetic or growth effects in fish (Silva et al. 2013Silva, L.L; Da Silva, D.T.; Garlet, Q.I.; Cunha, A.L.; Mallmann, C.A.; Baldisserotto, B.; Longhi, S.J.; Pereira, A.M.S.; Heinzmann, B.M. 2013. Anesthetic activity of Brazilian native plants in silver catfish (Rhamdia quelen). Neotropical Ichthyology, 11: 443-451.; Toni et al. 2014Toni, C.; Becker, A.G.; Simões, L.N.; Pinheiro, C.G.; Silva, L.L.; Heinzmann, B.M.; Caron, B.O.; Baldisserotto, B. 2014. Fish anesthesia: effects of the essential oil of Hesperozygis ringens and Lippia alba on the biochemistry and physiology of silver catfish (Rhamdia quelen). Fish Physiology and Biochemistry, 40: 701‐714.; Ventura et al. 2019Ventura, A.S.; de Castro Silva, T.S.; Zanon, R.B.; Inoue, L.A.K.A.; Cardoso, C.A.L.; 2019. Physiological and pharmacokinetic responses in neotropical Piaractus mesopotamicus to the essential oil from Lippia sidoides (Verbenaceae) as an anesthetic. International Aquatic Research, 11: 1-12.; Monteiro et al. 2021Monteiro, P.C.; Brandão, F.R.; Farias, C.F.S.; Alexandre Sebatião, F.; Majolo, C.; Dairiki, J.K.; et al. 2021. Dietary supplementation with essential oils of Lippia sinoides, Ocimum gratissimum and Zingiber officinale on the growth and hemato-immunological parameters of Colossoma macropomum challenged with Aeromonas hydrophila. Aquaculture Reports, 19: 100561. ). However, to date, there are no published studies on the effect of L. grata essential oil on fish.

Tambatinga is a hybrid fish produced from the female of the tambaqui (Colossoma macropomum Cuvier) and the male of the pirapitinga (Piaractus brachypomus Cuvier) that achieves higher weight gain than its parent species (Hashimoto et al. 2012Hashimoto, D.T.; Senhorini, J.A.; Foresti, F.; Porto Foresti, F. 2012. Interspecific fish hybrids in Brazil: management of genetic resources for sustainable use. Reviews in Aquaculture, 4: 108-118.). It is also omnivorous and very resilient to rearing in intensive systems (Alencar Araripe et al. 2011Alencar Araripe, M.N.B.; Araripe, H.G.A.; Lopes, J.B.; Braga, T.E.A.; Andrade, L.S.; Monteiro, A. 2011. Relação treonina:lisina para alevinos de tambatinga (Colossoma macropomum x Piaractus brachipomum). Boletim do Instituto de Pesca, 37: 393-400.). Its culture is of great economic importance in northern and northeastern Brazil (Ribeiro et al. 2019Ribeiro, P.F.; Leite, L.A.; Quaresma, F.S.; Farias, W.R.L.; Sampaio, A.H. 2019. Dietary supplementation with Arthrospira platensis in tambatinga (Colossoma macropomum × Piaractus brachypomus). Revista Ciência Agronômica, 50: 600-608.); therefore, the aim of this study was to evaluate the potential effect of dietary supplementation with essential oil of L. grata on performance and health indicators in tambatinga juveniles.

MATERIAL AND METHODS

Fish and culture conditions

The experiment was conducted at Universidade Federal do Maranhão (UFMA), Maranhão state, Brazil. We used 160 juveniles of tambatinga with an initial weight of 5.25 ± 0.26 g and a length of 7.13 ± 0.15 cm. The fish were purchased from a local fish farm and remained in an outdoor tank for 10 days, fed with commercial feed (45% crude protein) three times a day. After this period, the fish were placed at random in twenty 150-L tanks (eight fish per tank) in a water recirculation system, fed with the control diet (Table 1), and acclimated to laboratory conditions for 10 days. The closed water recirculation system was also equipped with an air blower (pressure 1300 mm H2O), a peripheral water pump (maximum flow (Q) L h 2400), a decantation box (250 L), and a biofilter (250 L) to perform biological control of nitrogenous waste. The study was authorized by the Ethics Committee on Animal Experimentation of UFMA (process # 23115.004974/2016-46 CEUA/UFMA).

Source material, extraction, and characterization of the essential oil

The essential oil of L. grata (EOLG) was extracted from leaves collected in Chapada das Mesas National Park (07°07′47.1′′S, 4°25′36.8′′W), municipality of Carolina, Maranhão state, Brazil, in February 2016. The plant was identified, and a voucher specimen was deposited at the herbarium of Museu Emilio Goeldi, Belém, Pará state, Brazil (MG 230155). Collection was authorized under Brazilian legislation for the protection of biodiversity (SISGEN license # AD7DF67).

The leaves were dried for five days at room temperature, ground, and then submitted to hydrodistillation (3 h) using a Clevenger-type apparatus (Figueiredo et al. 2019Figueiredo, P.L.B.; Pinto, L.C.; Da Costa, J.S.; Da Silva, A.R.C.; Mourão, R.H.V.; Montenegro, R.C.; Da Silva, J.K.R.; Maia, J.G.S. 2019. Composition, antioxidant capacity and cytotoxic activity of Eugenia uniflora L. chemotype-oils from the Amazon. Journal of Ethnopharmacology, 232: 30-38.). The essential oil was dried over anhydrous sodium sulfate (Merck-Millipore, São Paulo, Brazil), and the yield per dry weight of plant material was calculated. The moisture content was calculated in duplicate using an Infrared Moisture Balance (Genaka, São Paulo, Brazil) for water loss measurement.

The EOLG was analyzed on a gas chromatograph coupled to a mass spectrometer (GCMS)QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan), equipped with the GCMS-Solution software containing the NIST 11, FFNSC 2, and Adams libraries, as described in detail by Fernandes et al. (2021Fernandes, Y.M.L.; Matos, J.V.S.; Lima, C.A.; Tardini, A.M.; Viera, F.A.P.; Maia, J.G.S.; Monteiro, O.S.; Longato, G.B.; Rocha, C.Q. 2021. Essential oils obtained from aerial Eugenia punicifolia parts: Chemical composition and antiproliferative potential evidenced through cell cycle arrest. Journal of the Brazilian Chemical Society, 32: 1381-1390.).

Experimental design and treatments

The experimental design was randomized with four treatments (test diets) and five replicates per treatment. The diet formulations were based on Zeppenfeld et al. (2016Zeppenfeld, C.C.; Hernández, D.R.; Heinzmann, B.M.; Cunha, M.A.; Schmidt, D.; Baldisserotto, B. 2016. Essential oil of Aloysia triphylla as feed additive promotes growth of silver catfish (Rhamdia quelen). Aquaculture Nutrition, 22: 933-940.), with a control diet and three diets with increasing concentrations of EOLG (0.5, 1.0, and 2.0 mL kg-1) (Table 1). The ingredients of the base diet were ground, weighed, and mixed to complete homogenization. The EOLG was added with canola oil for adequate homogenization. Water was added to aid mixing with the dried ingredients, and the mixtures were pelletized. The pellets were dried for 24 h at 40 ºC in a forced-air circulation oven. The diets were stored at -18 ºC until use. The analysis of diet composition followed AOAC (1995AOAC. 1995. AOAC Official Methods of Analysis. 16th ed. Association of Official Analytical Chemists, Washington, DC, 771p.) for crude protein, dry matter, and ash; Bligh and Dyer (1959Bligh, E.G.; Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37: 911-917.) for fat; and Van Soest et al. (1991Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74: 3583-3597.) for neutral detergent fiber.

Table 1
Composition and proximate analysis of the experimental diets with of the essential oil of Lippia grata (EOLG).

Feed management and water quality

Fish were fed with the experimental diets to apparent satiation three times a day (8:00, 12:00, and 17:00) for 60 days. Thirty minutes after the first meal, fecal residues were removed from the tanks by siphoning, and the water level was replaced.

The temperature (28.0 ± 1.5 °C) and dissolved oxygen levels (6.94 ± 0.92 mg L-1) were monitored daily with an oximeter (HANNA, T160). Weekly measures of pH (6.9 ± 0.2 units) were taken with a digital (DMPH-2) pH meter, and those of total ammonia (0.25 ± 0.03 mg L-1) and non-ionized ammonia (0.08 ± 0.02 mg L-1) using a commercial ammonia kit (LabconTest toxic ammonia). All water quality parameters were within the limits considered suitable for the species (Moro et al. 2013Moro, G.V.; Torati, L.S.; Luiz, D.B.; Matos, F.T. 2013. Monitoramento e manejo da qualidade da água em pisciculturas. In: Rodrigues, A.P.O.; Lima, A.F.; Alves, A.L.; Rosa, D.K.; Torati, L.S.; Santos, V.R.V (Ed.). Piscicultura de Água Doce: Multiplicando Conhecimentos. 1st ed. Embrapa Pesca e Aquicultura, Palmas, p.141-169. ; Ribeiro et al. 2019Ribeiro, P.F.; Leite, L.A.; Quaresma, F.S.; Farias, W.R.L.; Sampaio, A.H. 2019. Dietary supplementation with Arthrospira platensis in tambatinga (Colossoma macropomum × Piaractus brachypomus). Revista Ciência Agronômica, 50: 600-608.).

Performance parameters

At the end of the feeding trial, all fish were fasted for 24 h, anesthetized with eugenol (40 µL L-1) (Moraes et al. 2017Moraes, T.C.H.; Ferreira, C.M.; Gama, K.F.S.; Hoshiba, M.A.; Povh, J.A.; Abreu, J.S. 2017. Routine exposure to biometric procedures in fish farming reveals differences in stress response in tambaqui and hybrid tambatinga. Boletim do Instituto de Pesca, 44: 1-10. ), and then weighed and measured to determine growth variables. Two fish per replicate (tank) were randomly selected and euthanized by spinal cord sectioning behind the operculum, and their livers were removed, weighed, quickly placed on ice, and frozen at -20 ºC for biochemical analysis. After complete dissection, each carcass was weighed, the fillets were removed and weighed, and a sample of white muscle was taken and stored at -20 ºC for biochemical analysis.

We determined the following performance parameters: final weight (FW, g); final total length (FL, cm); condition factor (CF) = 100 x (body weight; g)/(body length; cm)3; feed conversion ratio (FCR) = feed intake/weight gain; specific growth rate (SGR, %/day) = (ln (final weight) - ln (initial weight)/period x 100; weight gain (WG, g) = final weight - initial weight; survival (S, %) = live animals at day 60/initial number of animals in the tank (Ribeiro et al. 2019Ribeiro, P.F.; Leite, L.A.; Quaresma, F.S.; Farias, W.R.L.; Sampaio, A.H. 2019. Dietary supplementation with Arthrospira platensis in tambatinga (Colossoma macropomum × Piaractus brachypomus). Revista Ciência Agronômica, 50: 600-608.; Lopes et al. 2020Lopes, J.M.; Marques, N.C.; Dos Santos, M.D.D.C.; Souza, C.F.; Baldissera, M.C.; Carvalho, R.C.; Santos, L.L.; Pantoja, B.T.S.; Heinzmann, B.M.; Baldisserotto, B. 2020. Dietary limon Citrus × latifolia fruit peel essential oil improves antioxidant capacity of tambaqui (Colossoma macropomum) juveniles. Aquaculture Research, 51: 4852-4862. ); hepatic somatic index (HSI, %) = (liver weight /whole fish weight) x 100; carcass yield (CY, %) = (eviscerated fish weight/whole fish weight) x 100 (Rampelotto et al. 2018Rampelotto, C.; De Lima, J.S.; Pinheiro, C.G.; Salbego, J.; Da Silva, L.P.; Emanuelli, T. 2018. Supplementation with microencapsulated lemongrass essential oil improves protein deposition and carcass yield in silver catfish (Rhamdia quelen). Acta Scientiarum Animal Sciences, 40: e36517.); fillet yield (FY, %) = (fillet weight/whole fish weight) x 100 (Geraldo et al. 2015Geraldo, A.M.R.; Da Cunha, L.; Hoshiba, M.A.; Cardoso, M.S.; Da Silva, V.C.; Tamajusuku, A.S.K. 2015. Fillet and carcass yield and fillet chemical composition of piava from fish farming and from the wild. Boletim do Instituto de Pesca, 41: 743-749.).

Biochemical parameters

Glycogen levels in the liver and muscle were determined following Bidinotto et al. (1997Bidinotto, P.M.; Moraes, G.; Souza, R.H.S. 1997. Hepatic glycogen and glucose in eight tropical freshwater teleost fish: A procedure for field determinations of micro samples. Boletim Técnico do CEPTA, 10: 53-60.). Subsamples of 50 mg were added to 1 mL potassium hydroxide (KOH) and 3 mL ethanol for hydrolysis and precipitation of glycogen. Tissue subsamples of 50 mg were heated at 100 ºC with KOH, and the supernatant was used to determine the total protein level following Lowry et al. (1951Lowry, O.H.; Rosebrough, N.J.; Farra, L.; Randall, R.J. 1951. Protein measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 193: 265-275.), with bovine serum albumin as the standard. Other tissue subsamples of 50 mg were homogenized with 10% trichloroacetic acid (1:20 dilution) using a motor-driven Teflon pestle and centrifuged at 1,000 g for 10 min to flocculate the proteins. The supernatant was used for glucose and lactate determination following Dubois et al. (1956Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28: 350-356. ) and Harrower and Brown (1972Harrower, J.R.; Brown, C.H. 1972. Blood lactic acid-a micromethod adapted to field collection of microliter samples. Journal of Applied Physiology, 32: 709-711.), respectively.

Statistical analysis

All variables showed homogeneity of variances (Levene test) and a normal distribution (Shapiro-Wilk test). The variables were compared among treatments using one-way analysis of variance and Duncan’s test (P < 0.05), with the statistical program SPSS 21.0.

RESULTS

Characterization of the essential oil

The major constituents of the EOLG were α-pinene (24.47%), 1,8-cineole (16.18%), β-pinene (11.89%), and limonene (9.64%) (Table 2).

Table 2
Chemical composition of the essential oil of Lippia grata leaves collected in Chapada das Mesas National Park (Maranhão, Brazil). IRCa = calculated retention index (on Rxi-5ms column); IRCb = retention index from literature (Adams, 2001Adams, R.P. 2001. Identification of essential oil components by gas chromatography/quadrupole mass spectroscopy. Allured Publishing Corporation, Illinois, 302p.).

Performance parameters

Overall, performance parameters in the EOLG treatments did not differ significantly from those of the control, except for carcass yield, which was significantly higher in all EOLG treatments (Table 3). The final weight, final total length, specific growth rate, and weight gain were significantly higher in the 0.5 mL kg-1 EOLG treatment than in the 1.0 or 2.0 mL kg-1 treatments, but no EOLG treatment differed significantly from the control. The condition factor was significantly lower in the 1.0 mL kg-1 treatment than in the control. The feed conversion rate was significantly lower in the 0.5 mL kg-1 EOLG treatment than in the 1.0 mL kg-1 treatment but did not differ significantly from the control. The EOLG treatments tended to produce a lower HSI than the control. Fillet yield was not affected significantly by dietary EOLG, and no mortality was observed in any treatments during the experimental period.

Table 3
Growth parameters and somatic indices of tambatinga juveniles fed diets supplemented with different concentrations of the essential oil of Lippia grata (EOLG). IW: initial weight; IL: initial length; FW: final weight; FL: final length; CF: condition factor; FCR: feed conversion rate; SGR: specific growth rate; WG: weight gain; HSI: hepatic somatic index; CY: carcass yield; FY: fillet yield.

Biochemical parameters

Biochemical parameters tended to vary more in the EOLG treatments than in the control (Table 4). Lactate in muscle was significantly higher in all EOLG treatments than in the control, while protein levels were significantly lower in the 1.0 and 2.0 mL kg-1 EOLG treatments. Glycogen levels in muscle were significantly higher in the 1.0 and 2.0 mL kg-1 than in the 0.5 mL kg-1 EOLG treatment but did not differ from the control. Muscle glucose levels were significantly lower in the 0.5 and 2.0 mL kg-1 EOLG treatments than in the other two treatments.

Table 4
Hepatic and muscular metabolic parameters of tambatinga juveniles fed diets supplemented with different concentrations of the essential oil of Lippia grata. Glucose and glycogen= µmol glucose g tissue-1; Lactate= µmol lactate g tissue-1; Protein= mg g tissue-1.

Hepatic lactate was significantly lower in the 0.5 mL kg-1 EOLG treatment than in the control. Hepatic glycogen was not affected by dietary supplementation with EOLG, but glucose was significantly lower in all EOLG treatments than in the control. A significantly lower hepatic protein level was observed only in the 2.0 mL kg-1 EOLG treatment.

DISCUSSION

Lippia grata varies in the composition of its volatile constituents throughout northeastern Brazil (Franco et al. 2014Franco, C.S.; Ribeiro, A.F.; Carvalho, N.C.C.; Monteiro, O.S.; Da Silva, J.K.R.; Andrade, E.H.A.; Maia, J.G.S. 2014. Composition and antioxidant and antifungal activities of the essential oil from Lippia gracilis Schauer. African Journal of Biotechnology, 13: 3107-3113.). The major constituents of EOLG from Chapada das Mesas National Park (α-pinene, 1,8-cineole, β-pinene, and limonene) in this study corroborated those described by Monteiro et al. (2021Monteiro, P.C.; Brandão, F.R.; Farias, C.F.S.; Alexandre Sebatião, F.; Majolo, C.; Dairiki, J.K.; et al. 2021. Dietary supplementation with essential oils of Lippia sinoides, Ocimum gratissimum and Zingiber officinale on the growth and hemato-immunological parameters of Colossoma macropomum challenged with Aeromonas hydrophila. Aquaculture Reports, 19: 100561. ) for EOLG from the same region of the state Maranhão but differed markedly from those from other localities. The essential oil of L. grata [L. gracilis] leaves from other northeastern Brazilian localities has components in common with our sample, even regarding low level constituents. EO of L. grata [L. gracilis] leaves collected in Crato, Ceará state, contained a minor percentage of α-pinene (Bitu et al. 2012Bitu, V.; Botelho, M.A.; Costa, J.G.M.; Rodrigues, F.F.G.; Veras, H.N.H.; Martins, K.T.; et al. 2012. Phythochemical screening and antimicrobial activity of essential oil from Lippia gracillis. Brazilian Journal of Pharmacognosy, 22: 69-75.), however, the oxygenated monoterpenes thymol and carvacrol were the primary constituents, as in other EOs from the Brazilian northeast (Teles et al. 2010Teles, T.V.; Bonfim, R.R.; Alves, P.B.; Blank, A.F.; Jesus, H.C.R.; Quintans-Jr. L.J. ; Serafini, M.R.; Bonjardim, L.R., Araújo, A.D.S. 2010. Composition and evaluation of the lethality of Lippia gracilis essential oil to adults of Biomphalaria glabrata and larvae of Artemia salina. African Journal Biotechnology, 9: 8800-8804.; Souza 2013Souza, F.H.O. 2013. Efeitos abióticos na composição do óleo essencial de Lippia gracilis: Influência na mortalidade e repelência de Sitophilus zeamais. Master´s dissertation, Universidade Federal de Sergipe, Brazil. 39p. (https://ri.ufs.br/jspui/handle/riufs/6560).
https://ri.ufs.br/jspui/handle/riufs/656...
; Santos et al. 2014Santos, M.M.; Peixoto, A.R.; Pessoa, E.S.; Nepa, H.B.S.; Paz, C.D.; Souza, A.V.V. 2014. Estudos dos constituintes químicos e atividade antibacteriana do óleo essencial de Lippia gracilis a Xanthomonas campestris pv. viticola “in vitro”. Summa Phytopathologica, 40: 277-280.). The chemical composition of our sample suggests the presence of a new L. grata chemotype from Maranhão state, with a predominance of α-pinene, β-pinene, 1,8-cineole, and limonene, instead of thymol and carvacrol. In a detailed review, Pascual et al. (2001Pascual, M.E.; Slowing, K.; Carretero, E.; Mata, D.S.; Villar, A. 2001. Lippia: traditional uses, chemistry and pharmacology: a review. Journal of Ethnopharmacology, 76: 201-214.) reported that the compounds most frequently found in essential oils of Lippia species are limonene, β-caryophyllene, ρ-cymene, camphor, linalool, α-pinene, and thymol. In fact, essential oil content and composition can differ greatly, even within the same genus, as well as amongs different ripening stage or in different organs (Tirado et al.1995Tirado, C.B.; Stashenko, E.E.; Combariza, M.Y.; Martinez, J.R. 1995. Comparative study of Colombian citrus oils by high-resolution gas chromatography and gas chromatography mass-spectrometry. Journal of Chromatography A, 697: 501-513.; Stashenko et al. 1996Stashenko, E.E.; Martínez, R.; Pinzdn, M.H.; Ramfrez, J. 1996. Changes in chemical composition of catalytically hydrogenated orange oil (Citrus sinensis). Journal of Chromatography A, 752: 217-222.). The variability in the constitution of Lippia essential oils owes to the large number of species in the genus and their wide geographic distribution (Monteiro et al. 2021Monteiro, I.N; Ferreira, L.O.G.; De Oliveira, A.K.M.; Favero, S.; Figueiredo, P.L.B.; Maia, J.G.S.; Monteiro, O.S.; Matias, R. 2021. Toxicity of the Lippia gracilis essential oil chemotype,pinene-cineole-limonene, on Spodoptera frugiperda (Lepidoptera: Noctuidae). International Journal of Tropical Insect Science, 4: 181-187.). Gomes et al. (2011Gomes, S.V.F.; Nogueira, P.C.L.; Moraes, V.R.S. 2011. Aspectos químicos e biológicos do gênero Lippia enfatizando Lippia gracilis Shauer. Eclética Química, 36: 64-77.) reported that the chemical composition of L. gracilis oil presents quantitative fluctuations of the major components which are probably owed to genetic variability, depending on where and in which conditions the plant was cultivated.

The results for FW, FL, WG, and SGR indicated that the use of EOLG concentrations greater than 0.5 mL kg-1 does not favor the growth of juvenile tambatinga. Similarly, Brum et al. (2017Brum, A.; Pereira, S.A.; Owatari, M.S.; Chagas, E.C.; Chaves, F.C.L.; Mourino, J.L.P.; Martins, M.L. 2017. Effect of dietary essential oil of clove basil and ginger on Nile tilapia (Oreochromis niloticus) following challenge with Streptococcus agalactiae. Aquaculture, 468: 253-243.) evaluated the effect of the essential oil of O. gratissimum and Z. officinale (40 and 16% 1,8-cineole, respectively) in the diet of Oreochromis niloticusLinnaeus, 1758, at concentrations of 0.5, 1.0 and 1.5%. The essential oil of O. gratissimum at 0.5% significantly improved food conversion in comparison with the control, while no improvements in the growth parameters were observed for Z. officinale. Likewise, diets supplemented with essential oil of O. gratissimum, Z. officinale (28.2% and 15.8% 1,8-cineole, respectively), and Lippia sidoides Cham. (0.7% 1.8-cineole) did not significantly affect growth parameters of tambaqui (Monteiro et al. 2021Monteiro, P.C.; Brandão, F.R.; Farias, C.F.S.; Alexandre Sebatião, F.; Majolo, C.; Dairiki, J.K.; et al. 2021. Dietary supplementation with essential oils of Lippia sinoides, Ocimum gratissimum and Zingiber officinale on the growth and hemato-immunological parameters of Colossoma macropomum challenged with Aeromonas hydrophila. Aquaculture Reports, 19: 100561. ).

The concentration and synergy of compounds in an essential oil and the level of dietary inclusion, as well as factors such as the form of administration, target species, and age of the individuals, explain variations in the responses observed to its use (Campagnolo et al. 2013Campagnolo, R.; Freccia, A.; Bergmann, R.R.; Meurer, F.; Bombardelli, R.A. 2013. Óleos essenciais na alimentação de alevinos de tilápia do Nilo. Revista Brasileira de Saúde e Produção Animal, 14: 565-573.). Essential oil components differ in their mechanisms of action, which may result in improved or antagonistic specific activity (Efferth and Koch 2011Efferth, T.; Koch, E. 2011. Complex interactions between phytochemicals. The multitarget therapeutic concept of phytotherapy. Current Drug Targets, 12: 122-132. ). The biological effect of essential oil is a result of interactions among its constituent compounds (Sonboli et al. 2006Sonboli, A.; Babakhani, B.; Mehrabian, A.R. 2006. Antimicrobial activity of six constituents of essential oil from Salvia. Zeitschrift für Naturforschung, 61: 160-164. ).

The evaluation of fish carcasses is of great economic and production importance (Fernandes et al. 2010Fernandes, T.R.C.; Doria, C.R.C.; Menezes, J.T.B. 2010. Características de carcaça e parâmetros de desempenho do Tambaqui (Colossoma macropomum, cuvier, 1818) em diferentes tempos de Cultivo e alimentado com rações comerciais. Boletim do Instituto da Pesca, 36: 45-52.). In this sense, supplementation with EOLG increased the carcass yield in tambatinga juveniles compared to the control. Fish carcass is an important indicator in nutrition studies, as it is mainly related to visceral fat or muscle deposition. In Salvelinus fontinalis Mitchill, Salmo trutta Linnaeus, 1758, and hybrid trout, differences in carcass yield were approximated by fat content (Şahin et al. 2011Şahin, S.A.; Başçınar, N.; Kocabaş, M.; Tufan, B.; Köse, S.; Okumuş, I. 2011. Evaluation of meat yield, proximate composition and fatty acid profile of cultured brook trout (Salvelinus fontinalis Mitchill, 1814) and black sea trout (Salmo trutta labrax Pallas, 1811) in comparison with their hybrid. Turkish Journal of Fisheries and Aquatic Sciences, 11: 261-271. ). The same method was used for Astronotus ocellatus Agassiz, Pellona castelnaeana Valenciennes, and Leporinus friderici Bloch (Barai et al. 2022Barai, A.A.; Souza, A.F.L.; Viana, A.P.; Inhamuns, A.J. 2022. Seasonal influence on centesimal composition and yield of Amazonian fish. Food Science and Technology, 42: e55320. ). We did not analyze fat content, but the EOLG may have influenced the fish body composition and, consequently, the carcass yield.

The levels of metabolites in different tissues such as plasma, liver, and muscle can be indicative of dietary nutrient utilization (Saccol et al. 2013Saccol, E.M.H.; Uczay, J.; Pes, T.S.; Finamor, I.A.; Ourique, G.M.; Riffel, A.P.K.; et al. 2013. Addition of Lippia alba (Mill) N. E. Brown essential oil to the diet of the silver catfish: An analysis of growth, metabolic and blood parameters and the antioxidant response. Aquaculture, 416: 244-254.). In our study, the response of biochemical parameters suggests that metabolic pathways varied with the EOLG concentration. Fish fed with 0.5 mL EOLG kg-1 apparently used muscle glycogen and glucose stores and liver glucose and lactate to meet energy demands and showed lower preference for the use of body protein. This strategy did not impair the growth of juveniles. However, fish in the 1 and 2 mL EOLG kg-1 treatments may have prioritized the use of body protein (reduced muscle protein concentrations) for conversion to energy and thus meet the need to maintain body tissues, blood glucose levels, and growth rates. In this case, the glycogen stores in the muscle and liver were maintained, but the possible use of amino acids through the gluconeogenic pathway and their conversion into glucose may be reflected in the lower growth rates presented by tambatinga juveniles in these treatments. According to Lehninger et al. (2006Lehninger, A.L.; Nelson, D.L.; Cox, M.M. 2006. Princípios de Bioquímica. 4th ed. Sarvier, São Paulo, 1202p.), the metabolic routes that maintain blood sugar levels in fish are gluconeogenesis, using substrates such as amino acids, lactate, and pyruvate, and the breakdown of glycogen reserves in the muscle and liver, which can occur aerobically or anaerobically. Still, the lactate produced in the muscle and released into the circulation and that available in the fish liver are metabolically converted into glucose (Rito et al. 2018Rito, J.; Viegas, I.; Pardal, M.A.; Metón, I.; Baanante, I.V.; Jones, J.G. 2018. Disposition of a glucose load into hepatic glycogen by direct and indirect pathways in juvenile seabass and seabream.Scientific Reports, 8: 464. ).

Different responses to essential oil concentrations were also observed in silver catfish (Rhamdia quelen Silfvergrip) fry treated with dietary supplementation of Citrus x aurantium essential oil (93.8% limonene, 2.6% linalool, and 1.7% β-pinene): fish treated with 2 mL kg-1 oil showed changes in carbohydrate and protein metabolism in the liver and muscle that resulted in a higher growth rate, while lower concentrations were associated with metabolic rearrangements to maintain blood sugar and tissue glycogen and lactate levels, impairing zootechnical performance (Lopes et al. 2019Lopes, J.M.; Souza, C.F.; Saccol, E.M.H.; Pavanato, M.A.; Antoniazzi, A.; Rovani, M.T.; Heinzmann, B.M.; Baldisserotto, B. 2019. Citrus x auranticus oil as feed additive improved growth performance, survival, metabolic, and oxidative parameters of silver catfish (Rhamdia quelen). Aquaculture Nutrition, 25: 310-318. ).

CONCLUSIONS

Our results suggest that dietary supplementation with EOLG significantly improves carcass yield in tambatinga juveniles but that concentrations above 0.5 mL kg-1 may compromise growth rates and carbohydrate metabolism in this fish.

ACKNOWLEDGMENTS

The present work was supported in part by Instituto Nacional de Ciência e Tecnologia - Centro de Estudos de Adaptações Aquáticas da Amazônia II (INCT-Adapta II) and a fellowship from Fundação de Amparo à Pesquisa do Estado do Maranhão (FAPEMA, Brazil) for Thaisa Sales Costa.

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  • CITE AS:

    Costa, T.S.; Silva, R.C.; Pretto, A.; Monteiro, O.S.; Siqueira, J.C.; Baldisserotto, B.; Lopes, J.M. 2022. Effect of Lippia grata essential oil as a feed additive on the performance of tambatinga juveniles. Acta Amazonica 52: 122-130.

Edited by

ASSOCIATE EDITOR:

Rodrigo do Valle

Publication Dates

  • Publication in this collection
    04 July 2022
  • Date of issue
    Apr-Jun 2022

History

  • Received
    19 Aug 2021
  • Accepted
    23 May 2022
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