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

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.


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. 2018;Souza et al. 2019;Heluy et al. 2020;Kuralkar and Kuralkar 2021;Faehnrich et al. 2021).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. 2020).Dietary addition of essential oils from plants such as Citrus sinensis (L.) Osbeck (Acar et al. 2015), Citrus limon (L.) Burm (Ngugi et al. 2016), Aloysia citriodora Palau (formerly A. triphylla) (Zeppenfeld et al. 2016), Citrus x auranticus L. (Lopes et al. 2019), 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. 2013) or improve the nonspecific immune response and survival (Monteiro et al. 2021).
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 2014;Acar et al. 2015); increased antioxidant activity (Saccol et al. 2013;Lopes et al. 2019) and resistance to stress (Souza et al. 2019) 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. 2011;Campagnolo et al. 2013).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. 2011;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. 2007;Franco et al. 2014).Its aerial parts have antibacterial, antioxidant, and anti-inflammatory properties (Takeuchi and Cafe 2016;Mendes et al. 2020) and are used to treat gastrointestinal, respiratory, and cutaneous infections (Albuquerque et al. 2006).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. 2011).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 2017; Rocha et al. 2020).Considering other species of the genus Lippia, there are studies evaluating its anesthetic or growth effects in fish (Silva et al. 2013;Toni et al. 2014;Ventura et al. 2019;Monteiro et al. 2021).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 (Hasimoto et al. 2012).It is also omnivorous and very resilient to rearing in intensive systems (Alencar Araripe et al. 2011).Its culture is of great economic importance in northern and northeastern Brazil (Ribeiro et al. 2019); 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.

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 H 2 O), 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 # 23115.004974/2016-46CEUA/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).

ACTA AMAZONICA
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. 2019).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. (2021).

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. (2016), 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 (1995) for crude protein, dry matter, and ash; Bligh and Dyer (1959) for fat; and Van Soest et al. (1991) for neutral detergent fiber.

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.

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. 2017), 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.

Biochemical parameters
Glycogen levels in the liver and muscle were determined following Bidinotto et al. (1997).Subsamples of 50 mg were added to 1 mL potassium hydroxide (KOH) and 3 mL ethanol for hydrolysis and precipitation of glycogen.Tissue Ingredients (g kg -1 ) EOLG concentration in diet (mL kg -1 ) ACTA AMAZONICA 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. (1951), 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. (1956) and Harrower and Brown (1972), 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.

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,  ACTA AMAZONICA 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.

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.

Parameter
EOLG concentration in diet (mL kg -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. 2014).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. (2021) 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. 2012), however, the oxygenated monoterpenes thymol and carvacrol were the primary constituents, as in other EOs from the Brazilian northeast (Teles et al. 2010;Souza 2013;Santos et al. 2014).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. (2001) 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.1995;Stashenko et al. 1996).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. 2021).Gomes et al. (2011) 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.Glucose and glycogen= µmol glucose g tissue -1 ; Lactate= µmol lactate g tissue -1 ; Protein= mg g tissue -1 .
Values presented as mean ± SD (n=8).Different letters in the rows indicate significant difference by Duncan test (P < 0.05).

ACTA AMAZONICA
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. (2017) 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 niloticus Linnaeus, 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. 2021).
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. 2013).Essential oil components differ in their mechanisms of action, which may result in improved or antagonistic specific activity (Efferth and Koch 2011).The biological effect of essential oil is a result of interactions among its constituent compounds (Sonboli et al. 2006).
The evaluation of fish carcasses is of great economic and production importance (Fernandes et al. 2010).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. 2011).The same method was used for Astronotus ocellatus Agassiz, Pellona castelnaeana Valenciennes, and Leporinus friderici Bloch (Barai et al. 2022).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. 2013).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. (2006), 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. 2018).
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. 2019).

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.

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

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.Values are the mean ± SD (n = 5).For all variables, except survival, data were analyzed as average per tank (replicate).Different letters in the rows indicate significant difference by the Duncan test (P < 0.05).

Table 4 .
Hepatic and muscular metabolic parameters of tambatinga juveniles fed diets supplemented with different concentrations of the essential oil of Lippia grata.