Efeitos do R-(+)-limoneno na dieta sobre o crescimento, metabolismo, estresse e respostas antioxidantes de peixes infectados ou não com Aeromonas hydrophila

Abstract

Intensive fish farming can lead to stressful situations that affect their growth. Aeromonas hydrophila is a challenging pathogen for the productive success of fish. In addition to causing outbreaks with mass death of fish and presenting multidrug resistance, it could form biofilm in aquaculture facilities. For this reason, the use of natural substances has gained special attention. Plants, through their secondary metabolism, originate essential oils, made up of various compounds with biological activities. The addition of essential oils to fish diets has been shown to improve production performance and preserve animal health. R-(+)-limonene is a monoterpene from plants of the Citrus genus and the main and most active constituent of the terpenoid fractions of lemon and orange oils. Several studies report that EOs containing limonene exhibit growth-promoting, antimicrobial, anti-inflammatory, anesthetic, antioxidant and immunostimulating activities. Thus, the objective of this study was to investigate the in vitro activities of R-(+)-limonene and its in vivo dietary effects on growth, metabolism, stress and antioxidant responses in Rhamdia quelen infected or not with Aeromonas hydrophila. In the in vitro test, the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), the antibacterial activity in the combined therapy test and the effect of biofilm formation on plates and in nylon cubes with the two limonene isoforms were verified. In the tests carried out, A. hydrophila (MF 372510) was the most sensitive of the strains tested. In the MIC and MBC results, limonene isoforms showed high antibacterial activity. In optical microscopy and atomic force microscopy, S-(-)-limonene and R-(+)-limonene showed strong antibiofilm activity. The two limonene isoforms associated with florfenicol showed activity against A. hydrophila. In the in vivo test, fish were fed for 67 days with different doses of R- (+)-limonene in the diet (control 0.0, L0.5, L1.0 and L2.0 mL/kg of diet). On the sixtieth day, the animals were challenged or not with A. hydrophila and after seven days they were euthanized. R-(+)-limonene supplementation in the diet increased the productive performance of fish, with an increase in weight, specific growth rate, feed conversion rate and igf1 mRNA levels in the liver. Metabolic responses showed that the addition of R-(+)-limonene to the diet was not harmful to the animals' health and increased resistance to challenge with A. hydrophila. R-(+)-limonene in the diet stimulated antioxidant defenses and revealed better performance against the pathogen. Histological findings showed that dietary R-(+)-limonene is non-toxic and has a hepatoprotective effect against A. hydrophila. The highest dose of R-(+)-limonene in the diet negatively regulated the expression levels of crh and hspa12a mRNA in the brain of fish infected with A. hydrophila. Limonene isoforms have anti-biofilm action on A. hydrophila. The association of R-(+) and S-(-)-limonene with florfenicol may be an alternative to reduce the dose of antimicrobials, in addition to inhibiting the activity of A. hydrophila. R-(+)- limonene in the fish diet increased productive performance and was beneficial to their health, in addition, it reduced stress and made them more resistant to A. hydrophila.