Essential oil from Ocimum basilicum improves growth performance and does not alter biochemical variables related to stress in pirarucu (Arapaima gigas)

Abstract

Diet supplementation with essential oil from sweet basil Ocimum basilicum (EOOB) can increase fish growth. So, this study aimed to evaluate the effect of EOOB in the diet on growth performance and plasmatic variables of pirarucu juveniles (Arapaima gigas) submitted to stressful condition (stocking density of 7.56 kg m^-3per tank and limited space). Four diets (in triplicates) were evaluated with increasing levels of EOOB (0.0 control; 0.5; 1.0; and 2.0 mL kg diet^-1) over 48 days. Linalool was the major constituent of EOOB (54.19%). The addition of 2.0 mL EOOB kg diet^-1 improved final weight, weight gain, specific growth rate, condition factor and feed conversion ratio; it also decreased plasma urea levels and increased plasma albumin and total proteins levels. Plasma glucose, cortisol, and acid uric levels were not influenced by the addition of EOOB to the fish diet. In conclusion, the addition of 2.0 mL EOOBkg diet^-1 is recommended for pirarucu juveniles, due to improved growth performance, and this supplementation did not compromise the homeostasis of fish rearing in a high stocking density.

Key words
fish diet; glucose; growth promoter; high stocking density; linalool; metabolic response

INTRODUCTION

Pirarucu (Arapaima gigas) is one of the largest freshwater fish species in the Amazon basin, an it is a new and promising species for cultivation in fish farms. This is due to the high marketability of its fillet, as well as its high rate of growth, rusticity and adjustment to artificial feeding (de Andrade et al. 2007, Drumond et al. 2010). In addition, pirarucu is an obligate air-breather (Baldisserotto et al. 2008), which can facilitate its existence in environments with low oxygen availability.

Stress can be caused by common farming practices, such as feeding, handling and stocking (Barcellos et al. 2003, 2004, Sena et al. 2016). Fish farmers tend to increase stocking density as a strategy to increase fish production in intensive fish farming (Lemos et al. 2018). Stocking density is one important biological factor in aquaculture because it influences survival, growth, health and production costs (Copatti et al. 2008). In addition, high stocking density is commonly associated with an increase in the concentration of nitrogen compounds in the water, which can impair water quality and fish development and causes stress (Lemos et al. 2018). In previous study, the economic analysis performed by De Oliveira et al. (2012) indicated that the aquaculture of pirarucu in cages at moderated stocking densities (0.26 kg m^-3) and without space limitation showed better zootechnical indices. Similarly, Cavero et al. (2003) verified that stocking densities of 0.15, 0.20 or 0.25 kg m^-3 can be used for pirarucu reared in confined environments. A high stocking density (1.00 kg m^-3) induced stress responses in pirarucu, and the magnitude of stress of the densification was even greater than that caused by transport (Brandão et al. 2006). However, these authors conducted their research with pirarucu juveniles around 10 or 100 g without space limitations.

In the present study, pirarucu juveniles were raised at a very high stocking density (7.56 kg m^-3 per tank) and limited space (0.5 m^-3), which can be considered stressful for fish (Barcellos et al. 2004). In previous study, Souza et al. (2015) evaluated the effect of the essential oil of Lippia alba as a feed additive in silver catfish (Rhamdia quelen), submitted to a very high stocking density of 10.6 kg m^-3 and limited space, and they indicated the use of 0.25 mL of essential oil of L. alba kg food^-1 for this species was effective. In addition, a proper diet may improve the response of aquatic animals to stressful farming conditions (Zheng et al. 2009). With the increasing demand for pirarucu outgrowing current operations, the determination of an appropriate diet that contributes to its growth and health is required.

In addition, diet supplementation with essential oils can improve growth performance of fish (Ngugi et al. 2017, Baba et al. 2018, De Souza et al. 2019a). In a recent study, De Souza et al. (2019b) found that essential oil from sweet basil Ocimum basilicum (EOOB) (2 mL kg diet^-1) is a growth promoter for Nile tilapia. This can be due the presence of linalool as the main chemical compound (53.35%) studied by these authors, because linalool is known to have antioxidant, antimicrobial, anti-inflammatory, and digestive-stimulant mechanisms of action (Moghaddam et al. 2011, Heldwein et al. 2014, De Souza et al. 2017). On the other hand, a diet with essential oils of L. Alba (55.25% of linalool) and Ocimum americanum (33.54% of linalool) did not alter the growth of silver catfish and red drum (Sciaenops ocellatus), respectively (Sacco et al. 2013, Sutili et al. 2016).

Sweet basil (O. basilicum Lamiaceae) is one of the most frequently grown aromatic herbs in the world. It is rich in natural antioxidants such as flavonoids (quercetin, kaempferol, rutin), phenolic acids (p-coumaric acid, caffeic acid, caftaric acid), steroids and vitamins (A, C, E, K) (Mustafa & Usman 2011); these substances could contribute to the growth and health of fish. The EOOB has been shown to be effective in aquaculture as an antioxidant (Lee et al. 2005), antimicrobial (Moghaddam et al. 2011, De Souza et al. 2018), and anesthetic agent (Limma-Neto et al. 2016, 2017). However, there is no previous study verifying the efficacy of adding EOOB to the pirarucu diet.

So, in light of its potential, EOOB may be an alternative for improving growth of pirarucu raised at very high stocking densities and limited space in artificial environments. Therefore, the objective of this study was to evaluate the effect including EOOB into a commercial diet on growth performance and plasmatic variables related to health in pirarucu juveniles reared in artificial environments.

MATERIALS AND METHODS

Essential oil from Ocimum basilicum

Leaves from O. basilicum cultivated in Três Passos, Rio Grande do Sul, Brazil, were collected in August 2016. EOOB was extracted from fresh leaves from the plants by hydrodistillation using a Clevenger-type apparatus for 2 hours according to guidelines set out by the European Pharmacopoeia (2007). Determination of the chemical compounds in the essential oils was carried out by gas chromatography/mass spectrometry according to De Souza et al. (2017). The yield was calculated as w/w (%); the EOOB constituents (Table I) were identified by comparing the mass spectra with a mass spectral library (NIST 2008). The GC-MS analysis was performed using an Agilent- 6890 gas chromatograph coupled to an Agilent 5973 mass selective detector and helium was used as the gas carrier. The capillary column choose was HP5-MS (Hewlett Packard, 5% fenilmetilsiloxane, 30 m x 0.25 mm, film thickness: 0.25 μm) and 70 eV of ionization energy. The experimental protocol was approved by the Ethical Committee of the School of Veterinary Medicine and Animal Science of the Universidade Federal da Bahia, Salvador, Bahia, Brazil (number 71/2017).

Table I
Chemical composition of essential oil of Ocimum basilicum. IKc: retention índex calculated. IKt: retention index reference (NIST 2008). %: relative percentage.

Chemical compound	IKc	IKt	%
β-Myrcene	990	985	0.59
Eucalyptol	1031	1031	4.91
β-trans-ocimene	1048	1048	0.80
Linalool	1099	1101	54.19
Camphor	1142	1146	0.86
(-)-Bornyl acetate	1285	1284	1.45
p-Eugenol	1356	1356	4.96
β-Elemen	1386	1394	0.54
α-bergamotene	1437	1436	5.24
Germacrene D	1455	1453	1.07
α-amorphene	1483	1490	2.45
δ-Guaiene (α-bulnesene)	1507	1505	2.08
γ-Cadinene	1515	1514	2.60
T-Cadinol	1640	1640	3.24
Identified Compounds			85.02

Animals

Pirarucu juveniles were purchased from Agua Vale Fish Farming (Ituberá, Bahia, Brazil) and the experiments were performed at the Fish Feeding Behavior and Nutrition Laboratory of the Universidade Federal do Recôncavo da Bahia, Cruz das Almas, Bahia, Brazil. Pirarucu juveniles (945.40 ± 18.06 g; 54.60 ± 0.30 cm, n = 4 per tank; stocking density of 7.56 kg m^-3 per tank) were housed in 12 continuously aerated, 500 L (0.5 m³) tanks with a recirculation aquaculture systems and chemical and biological filters and were fed with commercial extruded fish food (400 g kg^-1 crude protein, 9% ethereal extract, 13.40 MJ kg^-1 digestible energy and 14-16 mm-grain-size pellets; Pratigi Alimentos Company©, Castro Alves, Bahia, Brazil). Prior to the experiment, the animals underwent a fast for 24 h.

Experimental procedure

The experimental design was completely randomized, with four treatments and three replicates. Different concentrations of EOOB (0.0 – control, 0.5, 1.0, or 2.0 mL EOOB kg diet^-1) were added to the commercial extruded fish food (Pratigi Alimentos Company©). The EOOB was diluted with cereal alcohol 1:7.5 (EOOB: alcohol). These solutions were sprayed manually with the aid of a hand sprayer. The food was then dried at room temperature for 24 h before its use in feeding the fish, and it was kept under refrigeration (–20^oC) until use, following the methodology suggested by Dairiki et al. (2013). The groups (0.0, 0.5, 1.0, and 2.0), with the same initial stocking density described above (7.56 kg m^-3),were fed until apparent satiety into three meals (9:00 a.m., 12:30 p.m. and 5:00 p.m.) for 48 days. This stoking density was based on previously study (Pedrosa et al. 2018), which considered6.0 kg m−³ as high density. Feed consumption was measured every three days, and biometry was performed every 15 days by weighing all fish in each tank.

The physicochemical variables of the water, including pH (6.31 ± 0.41) (pH meter Hanna - HI 98130), temperature (26.92 ± 1.03^oC) and dissolved oxygen (7.24 ± 0.79 mgL^-1 O₂) (oximeter Politerm-POL 60) were monitored daily and total ammonia (0.91 ± 0.72 mg L^-1 N-NH₃), nitrite (0.66 ± 0.21 mg L^-1 N-NO₂) and alkalinity (24.89 ± 12.75 mg L^-1 CaCO₃) (kit Alfatecnoquímica, Florianópolis, SC) were monitored twice a week. In order to remove excess feces and other dirt, the tanks were cleaned by siphoning daily.

Growth performance

After 48 days, the following animal performance variables were analyzed at the end of the experiment: final weight (g), final length (cm), weight gain (WG) (g) = final body weight – initial body weight; specific growth rate (SGR) (% per day) = 100 * (ln final weight – ln initial weight)/days of experiment; feed conversion ratio (FCR) = consumed feed/weight gain; condition factor (CF) (g cm^-3 *100) = 100 * (final weight/final length³); and survival (SR) (%) = (final fish number/initial fish number)*100.

Sample collection and analysis

At the end of the experiment, the animals underwent a fast of 24 h and then blood was collected from all fish. Blood (2.0 mL per fish) was drawn from the caudal vasculature with the aid of pre-heparinized syringes (heparin sodium 5,000 I.U. mL^-1). The blood was centrifuged at 10000 x g for 5 min (4°C), and the plasma was carefully pipetted out. The samples were stored under refrigeration at –20°C.

The cortisol S kit (Bio Mérieux, France) was used for the determination of plasma cortisol levels (ng mL^-1) in the mini-VIDAS® equipment from the enzyme-linked fluorescent assay. The volume of plasma used was 200 µL. Both analyzers were cleaned, calibrated, and operated in accordance with the manufacturer’s instructions. The measurement values of the Vidas cortisol S kit range from 2 to 650 ng mL^-1. The repeatability, inter-run reproducibility and the inter-lot reproducibility were all calculated according to Tholen et al. (2004). The observed values of total precision, dependent from serum concentration, ranged from 7.42 to 12.98 for the coefficient of variance (%). The coefficient of variation for the fish ranged from 8.68 to 13.59%, and the detection limit of the assay was 2 ng mL^-1.

Plasma glucose levels (mg dL^-1) were determined enzymatically using glucose oxidase/glucose peroxidase according to the protocol by Sena et al. (2016). Determinations of the plasma urea (mg dL^-1), albumin (g L^-1), total proteins (g L^-1), and uric acid (mg dL^-1) were performed using commercial Kits (Labtest ® kits; Vista Alegre, MG, Brazil) in a semi-automatic biochemical analyzer (Doles®, model D-250).

Statistical analysis

The results are expressed as means ± standard error of the mean (S.E.M.), with tanks as the statistical units. Levene’s test was performed to evaluate the homogeneity of data variances. The data showing homogeneous variances were compared using one-way analysis of variance (ANOVA) (p< 0.05). The effects of EOOB on the growth performance and biochemical variables were evaluated based on linear regression. In addition, significant differences among treatments were determined by post-hoc Tukey tests (p< 0.05).

RESULTS

A linear effect (p< 0.05) was observed between the EOOB and thefinal weight (y = 1385.304 + 43.504x, R² = 0.75), WG (y = 437.990 + 43.883x, R² = 0.89), SGR (y = 0.784 + 0.069x, R² = 0.98), CF (y = 0.184 + 0.027x, R²=0.94) and FCR (y = 1.940 – 0.223x, R² = 0.90).Therefore, according to the linear effect, pirarucus fed with 2.0 mL EOOB kg diet^-1 increased their Final W, WG, SGR, and CF, and they decreased their FCR (Table II). In addition, the inclusion of 2.0 mL EOOB kg diet^-1 significantly increased the WG and SGR and decreased the FCR in comparison with the control group (0.0 mL EOOB kg diet^-1; p< 0.05) (Table II). Experimental conditions did not influence survival since there was no mortality during the experimental period for either the control or treatment group.

Table II
Growth performance (mean ± SEM) of pirarucu fed with diets containing different concentrations of the essential oil of Ocimum basilicum (EOOB).

Variables	EOOB (mL kg diet-1)				p
	0.0	0.5	1.0	2.0
Initial W	955.00±14.42	944.10±22.49	936.10±7.72	951.00±11.17	NS
Final W	1401.25±26.35	1406.00±59.28	1398.50±19.90	1515.00±5.07	NS
WG	446.25±23.86b	461.90±38.16ab	462.40±12.75ab	564.00±12.94a	0.037
Final L	61.00±1.13	61.80±0.88	60.70±0.67	61.60±0.68	NS
SGR	0.79±0.04b	0.82±0.08ab	0.84±0.02ab	0.97±0.03a	0.025
CF	0.19±0.01	0.19±0.02	0.21±0.01	0.24±0.01	NS
FCR	1.93±0.04a	1.78±0.12ab	1.81±0.08ab	1.46±0.11b	0.029

• Initial W (initial weight), Final W (final weight) and WG (weight gain) are expressed in g. Final L (final length) is expressed in cm, SGR (specific growth rate) is expressed as % per day and, and CF (condition factor) is expressed as g cm^-3*100. FCR = feed conversion ratio. Different letters indicate statistical difference between treatments (Tukey’s test, p< 0.05; n = 3 tanks per treatment). NS = No significant.

Another linear effect (p< 0.05) was observed between the EOOB and the plasma albumin (y = 0.630 + 0.049x, R² = 0.82), total proteins (y = 3.058 + 0.179x, R²=0.74) and urea levels (y = 7.744 – 0.713x, R² = 0.86). So, according to the linear effect, an increase in EOOB concentration in the diet proportionally decreased plasma urea levels and increased plasma albumin and total proteins levels in juveniles (Table III). Additionally, juveniles fed with the 2.0 mL EOOB kg diet^-1 presented significantly higher plasma albumin than those feed with 0.0 (control group) or 1.0 mL EOOB kg diet^-1 and total proteins than those receiving other concentrations of EOOB in the diet (p< 0.05). Plasma glucose, cortisol, and uric acid levels were not influenced by the addition of EOOB to the pirarucu diet (Table III).

Table III
Plasma biochemical variables (mean ± SEM) of pirarucu fed with diets containing different concentrations of the essential oil of Ocimum basilicum (EOOB).

Variables	EOOB (mL kg diet-1)				p
	0.0	0.5	1.0	2.0
Cortisol	42.70±3.73	53.03±4.04	46.69±3.75	45.82±3.92	NS
Glucose	46.64±1.68	52.11±2.42	46.56±3.02	52.73±2.99	NS
Albumin	0.64±0.02b	0.66±0.04ab	0.65±0.03b	0.74±0.02a	0.045
Total proteins	3.12±0.03b	3.13±0.05b	3.11±0.11b	3.48±0.06a	0.009
Urea	8.01±0.94	7.12±0.81	6.90±0.64	6.45±0.02	NS
Uric acid	0.15±0.01	0.17±0.02	0.17±0.02	0.16±0.02	NS

• Cortisol is expressed in ng mL^-1, glucose, urea and uric acid are expressed in mg dL^-1 and albumin and total proteins are expressed in g L^-1. Different letters indicate statistical difference between treatments (Tukey’s test, p< 0.05; n = 3 tanks per treatment). NS = No significant.

DISCUSSION

The present study showed that dietary supplementation of EOOB (2.0 mL kg diet^-1) exerted a positive effect on growth performance (final weight, WG, SGR, CF, and FCR) in pirarucus. In our study, pirarucus’ weight gain occurred within the range expected for juveniles, with initial weight above 500 g (Gandra et al. 2007, Pedrosa et al. 2018). The growth promotion effect of essential oils could be attributed to their distinctive aromatic flavor, which makes them strong appetite stimulants, that causes increased voluntary feed intake and results in improved weight gain (Abdel-Latif & Khalil 2014). According to El-Dakar et al. (2015), the olfactory feed ingredients of O. basilicum could enhance fish growth through their ability to act as feeding enhancers.

In addition, the mechanism attributed to the essential oil’s effect has been postulated to be a digestibility enhancer which balances the intestinal microbiota (Zheng et al. 2009, Reverter et al. 2014). Its consequence could be increase food consumption and better nutrient utilization and absorption (Radhakrishnan et al. 2015), which could improve growth performance. Another model that could explain the essential oil coumponds’ mode of action to promote growth could be the strong antibacterial effect. In this sense, De Souza et al. (2018) showed that EOOB showed an inhibitory effect on Aeromonas spp.

Previous studies reported similar results in which a diet with O. basilicum extract was a growth promoter for common carp (Cyprinus carpio) (400 mg kg^-1) (Amirkhani & Frouzbakhsh 2015) and gilthead sea bream (Sparus aurata) (20 g kg^-1) (El-Dakar et al. 2015). Supplementation with O. basilicum dried leaves improved the growth rate in hybrid tilapia (20 g kg^-1) (Oreochromis niloticus X Oreochromis aureus) (El-Dakar et al. 2008), and EOOB was effective at a concentration of 2.0 mL kg diet−¹ for improving growth performance in Nile tilapia (De Souza et al. 2019b).

In the current study, linalool was the major constituent of EOOB (54.19%), and linalool (or an interaction of the different components of EOOB with linalool) may have acted as a growth promoter (De Souza et al. 2019a). Linalool is known to have antioxidant, antimicrobial, anti-inflammatory, and digestion-stimulating mechanisms of action (Moghaddam et al. 2011, Heldwein et al. 2014, De Souza et al. 2017). Similarly, a higher growth performance was found in Nile tilapias fed with an added of 2.0 mL essential oil of L. alba kg diet^-1 or 2.0 mL EOOB kg diet^-1; where linalool was the major compound (81.64 and 53.35%, respectively) was found in these components (De Souza et al. 2019a, b). In addition, the specific growth rate of O. mossambicus was also greater when fed with lipid extract from ginger (Zingiber officinale) (Immanuel et al. 2009). Ginger is a rich source of volatile oil, and linalool is an important constituent of its oil (Shakya 2015), as it is in EOOB. However, dietary addition of other essential oils containing linalool as the main compound did not affect silver catfish or red drum growth (Saccol et al. 2013, Sutili et al. 2016).

Accordingly, it was found that dietary addition of EOOB possibly did not change metabolism in the pirarucu, since plasma glucose and cortisol levels were not affected (Lemos et al. 2018). Plasma cortisol is a modulator of various physiological processes that rise in response to stress (Lemos et al. 2018). An elevation in plasma cortisol levels induces secondary physiological responses, such as increased plasma glucose levels (Pankhurst 2011). In addition, the values found in the present study for plasma cortisol and glucose levels are close to or slightly above baseline values found in previous studies with non-stressed pirarucus (Brandão et al. 2006, Gomes 2007). Therefore, these results allow us to state that the addition of EOOB to the pirarucu did not impair the homeostasis of the fish during 48 days of rearing in a very high stocking density.

Similar results were record in pirarucu juveniles subjected to different feeding strategies (Pedrosa et al. 2018). Linalool has anti-inflammatory and antioxidant properties (Heldwein et al. 2014), but the exact mechanism of the linalool and/or EOOB effect on plasma cortisol has not been established. Accordingly, Souza et al. (2015) found that silver catfish that were fed diets supplemented with essential oil of L. alba (55.25% of linalool) also did not alter plasma cortisol and glucose levels. Similarly, Nile tilapia fed a 1.0 or 2.0 mL kg diet^-1 of essential oil of L. alba (81.64% of linalool) did not present with metabolic changes (De Souza et al. 2019a). However, Nile tilapia which received diets supplemented with 1.0 mL EOOB kg diet^-1 (53.35% of linalool) had reduced plasma glucose levels and experienced benefits in growth, intestinal enzymes, lysozyme, and hematological variables (De Souza et al. 2019b).

On the other hand, the plasma total proteins and albumin levels were high in fish that were fed a diet with 2.0 mL of EOOBkg diet^-1. Plasma total proteins levels area result of synthesis and degradation of whole-body nitrogen. In some cases, higher plasma protein levels could indicate a better nutritional status in these fish (Higuchi et al. 2011) which promotes protein deposition. For example, high plasma total proteins concentrations were related with growth performance and protein utilization in rainbow trout (Rumsey et al. 1994). Similarly, previous studies have found an increase in total proteins levels with the use of essential oils or plant extracts in fish diets (Dugenci et al. 2003, Gulec et al. 2013, El-Dakar et al. 2015, De Souza et al. 2019b). In addition, the metabolic importance of plasma albuminis limited, and the increase of plasma albumin levels can be considered a nonspecific immune response in fish (Amirkhani & Firouzbakhsh 2015, Reverter et al. 2014). Furthermore, the values found in the present study for plasma albumin and total proteins levels are close to the baseline values found in previous studies (Tavares-Dias et al. 2007, Hoshino et al. 2017) in pirarucu.

Finally, stress-induced protein mobilization may cause an increase in the amount of ammonia excreted by fish and consequently elevation in plasma uric acid and urea levels (Barcellos et al. 2003). However, this was not verified in the present study. Plasma urea levels decreased with an increase in the concentration of EOOB in the diet of pirarucu juveniles, and their levels remained within the values described as normal for pirarucu by Tavares-Dias et al. (2007). In addition, urea production does not reflect direct oxidative deamination of amino acids and, consequently, does not reflect increased metabolic cost (Wood et al. 2017).

CONCLUSIONS

The supplementation of 2.0 mL EOOB kg diet^–1 is the best indicated concentration for pirarucu juveniles reared in a very stocking density of 7.56 kg m^-3, since it improved growth performance, guaranteed health, and did not evoke changes related to stress in the metabolic profile.

ACKNOWLEGMENTS

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES) through a research grant awarded to the first author.

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Publication Dates

• Publication in this collection
  03 July 2020
• Date of issue
  2020

History

• Received
  7 Jan 2019
• Accepted
  7 May 2019