The improvement in the zootechnical performance of fish fed with diets containing essential oils has been attributed to the antioxidant, anti-inflammatory properties and the positive modulation exerted on the microbiota and intestinal morphology that allow the increase in the nutrient absorption capacity (Elgendy et al. 2024)). In addition, the use of these essential oils can reduce the impact caused by infectious agents on animal survival rates due to improved immunity and reduced stress levels (Yousefi et al. 2021). In this study, the inclusion of the commercial phytoadditive (Digestarom® PEP MGE 150) in the diet of the tambaqui had effects on growth and caused a decrease in total cholesterol and ionoregulatory enzymes, without the occurrence of mortality in the specimens. Our findings indicate that inclusion of this phytoadditive in dietary levels starting at 400 mg kg− 1 is able to promote better zootechnical performance, which results in an increase in final mass, weight gain, and a better condition factor and specific growth rate. One positive effect regarding the use of this phytoadditive was observed in giant gourami (Osphronemus goramy), although in higher concentrations (5 g kg− 1 up to 15 g kg− 1) than those tested in the present study (Karimifar et al. 2022).
In our study, the better feed conversion without alterations to the hepatosomatic index of the animals can be attributed to the increased protein and lipid retention already observed for the phytoadditive, even in diets with a low protein content (Gonçalves et al. 2019), since no variations in food consumption were noted. Similarly, Rodrigues et al. (2018) did not observe changes in the performance of gilthead seabream (Sparus aurata) when testing 200 mg kg− 1 in the diet compared to the control diet. This is similar to what was observed in the present study, although nutrient retention levels increase and nitrogen loss through feces decreases with the addition of the phytoadditive in the concentrations recommended by the manufacturer. We believe that the main consequence of this process was the increase in the loin eye area observed in fish fed the levels of D400 and D500, indicating better muscle development and performance in the tambaqui (See Fig. 1C).
These results are further reinforced by the study conducted by Carneiro et al. (2021), in which the use of higher levels of Origanum vulgare essential oil resulted in a significant increase in body protein retention and muscle hyperplasia (increased muscle fibers) in the neotropical catfish Lophiosilurus alexandri. These results are consistent with the literature, which suggests that compounds such as carvacrol and thymol, present in O. vulgare, can play an important role in gene regulation, especially in increasing the expression of factors such as growth hormone and insulin-like growth factors (IGF-1), as mentioned by Sah et al. (2016). These factors are closely related to the control of muscle growth, since they affect the processes of hyperplasia and hypertrophy in fish, as reported by Fuentes et al. (2013). In addition, Aanyu et al. (2018) observed that limonene also plays a role in the activation of growth-related genes, including IGF-1, and the regulation of nutrient uptake and transport in Oreochromis niloticus. Another study, conducted by Rafieepour et al. (2019) on rainbow trout (Oncorhynchus mykiss), demonstrated that the inclusion of dietary oregano extracts resulted in improvements in growth and increased levels of the T3 hormone.
Thus, our findings indicate that the inclusion of this phytoadditive contributes to the improvement in loin gain in tambaqui, and this cut is of interest in consumer markets and one of the basic characteristics for the development of genetic improvement programs (Perazza et al. 2019; Amanajás and Val 2023). In addition, Prado et al. (2004), used ultrasound and demonstrated a confidence level of 80% compared to other carcass evaluation techniques. In the study by Perazza et al. (2015), when measuring the loin eye area (LEA) of tambaqui, the methodology used was validated for the characterization of this region, since no significant differences were observed between the values obtained by ultrasound and those obtained by measurement using tracing paper. Although studies with the use of ultrasound in fish are on the rise, especially in terms of genetic improvements, the use of these techniques to quantify meat production in tropical species is still under-explored (Perazza et al. 2019). This practice of analyzing the eye area of the loin is more common in pigs, and an improvement in this index is found in animals fed with EOs and some natural herbs, also improving the quality of fat content in the muscle (Czech et al. 2009).
In addition to these factors related to growth performance, EOs also act as potential stress reducers (Souza et al. 2019). In this study, no changes were observed in the hematological responses of the tambaqui fed with the phytoadditive. Our results are similar to those reported for sturgeon (Huso huso) fed with Digestarom® PEP MGE at concentrations of up to 40 g kg− 1 in the diet for 70 days (Defaee et al. 2022). However, our results partially corroborate the findings of Chung et al. (2021) for tambaqui fed with Zingiber officinale EO (1.5 mL kg− 1 of feed), for which the authors observed an increase in hemoglobin levels and attributed the responses to the thermogenic effect caused by the consumption of the oil from this plant. The difference in the chemical composition of the plant may explain the observed variation because, in a study conducted with tambaqui using Lippia sidoides, Ocimum gratissimum and Zingiber officinale, that used a similar period and higher doses, no differences were observed in the hematological profile of the species (Monteiro et al. 2021). Thus, the data obtained in this study reinforce that the phytoadditive based on aniseed, oregano, citrus and thyme, even in small doses compared to other studies, does not cause hematological alterations that indicate respiratory changes for tambaqui in the evaluated period.
Furthermore, the present study shows a reduction in cholesterol levels of the tambaqui that received doses of 400 mg kg− 1 and 500 mg kg− 1 of the phytoadditive compared to those that were fed the control diet, which suggests a hypolipidemic effect of the essential oils used. Studies have shown that oregano and citrus essential oils reduce the levels of circulating lipids in the blood in different species due to the presence of carvacrol, thymol and limonene, which act on the lipid synthesis pathway, and act especially in the reaction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A, limiting the synthesis of cholesterol, thus explaining the present result (Kesbiç et al. 2020; and reviewed by Bahr et al. 2021). The same effect was not reported for the European sea bass (Dicentrarchus labrax) when up to 5 g kg− 1 of aniseed was included in the diet of the species (Ashry et al. 2022). Our data suggest that tambaqui fed with a mixture of essential oils containing citrus and oregano or even the active ingredient carvacrol show a decrease in lipid levels in the plasma (Silva et al. 2021).
Other physiological indicators, such as glucose, in the tambaqui showed no variations when fed with this commercial phytogenic. This molecule is often used to evaluate stress and energy metabolism in fish subjected to experimental diets. The study conducted by Silva et al. (2021) showed that carvacrol, one of the active ingredients present in Digestarom®, did not cause alterations in plasma glucose levels even at concentrations that were higher than those applied in the present study. In the Characidae Astyanax altiparanae, fed with oregano EO, no changes in plasma glucose levels were also observed (Ferreira et al. 2014). The same occurred with fish of the families Cichlidae (Oreochromis niloticus) and Cyprinidae (Cyprinus carpio) (Ahmadifar et al. 2011; Santo et al. 2020). These results show that this commercial phytoadditive in the concentrations studied does not alter the glucose levels of this species during the period observed in this study. However, contrasting results were observed for Oncorhynchus mykiss and Oreochromis mossambicus since they showed variations in blood glucose when fed with herbs composed of 80.6% carvacrol and with citrus EOs, respectively (Baba et al. 2016; Saei et al. 2016). In general, the biological effects of EOs on fish energy metabolism are conflicting, and there is a need for studies that evaluate the mechanisms of action of the isolated compounds and the mix of EOs (Dawood et al. 2022).
The dosage of total plasma protein is often used as an indicator of osmotic pressure, immune system response and the transport of several metabolites that are important for fish health. In addition, it provides information on the nutritional status of the animal (Frota et al. 2022). In this study, no changes in total plasma protein levels were observed. These results are also similar to those obtained when tambaqui were fed with Lippia sodoides (thymol being the main compound; 76.6% composition) since, at concentrations similar to those of this study, no changes in plasma levels were observed. Therefore, other plants with phytotherapeutic effects at higher concentrations also did not alter the serum protein values of tambaqui (Copatti et al. 2022). However, the results in the literature indicate divergences because a lower concentration of lemon EO resulted in increased plasma protein in tambaqui (Baba et al. 2016). Moreover, Cyprinus carpio fed 500 mg kg− 1 of oregano, a level similar to that of this study, showed an increase in plasma protein values (Abdel-Latif et al. 2020). This variation in results may be related to the synergism of compounds present in essential oils, the level of inclusion in the diet, the form of administration and the way these essential oils are added to the feed (microencapsulated or au naturel), the storage of the experimental feeds and the target species. These are some explanations for the various responses observed. For the present study, we believe that the commercial phytogenic in the concentrations studied did not alter the physiological state of plasma protein levels in the tambaqui (Chakraborty et al. 2014; ; Kesbiç et al. 2020).
The enzymes NKA and H+ATPase play an important role in ion regulation, blood acid-base balance and ammonia excretion in fish (reviewed by Souza et al. 2019; Tresgueres et al. 2023). The secretion of H+ into the external environment and catalyzed by H+ATPase enables the uptake of Na+ into the intracellular environment and is still linked to the mechanisms of ammonia excretion and Na+ channels (Nawata et al. 2007). On the other hand, the activity of the NKA enzyme in the basolateral portion creates a gradient for the uptake of sodium by the plasma and the injection of potassium into the branchial intracellular environment (Hwang et al. 2011).
In this study, the decrease in plasma sodium levels at concentrations of 400 mg kg− 1 and 500 mg kg− 1 and the increase in branchial activity of NKA when compared to the control group was observed (see Table 5 and Fig. 2). These data indicate that feeding tambaqui with a commercial phytoadditive composed of EOs can cause a reduction in plasma Na+ levels, which may be occurring by diffusion or non-renal reuptake, which would lead to osmoregulatory imbalance. However, the simultaneous increase in the activity of the enzyme H+ATPase in the gills (Table 5 and Fig. 2) indicates that there is a counterbalancing mechanism acting to prevent the continued loss of these ions. The effects caused by the use of EOs in the diet or management of fish in terms of osmoregulatory responses have been little explored and the mechanisms have not been elucidated. Toni et al. (2014) observed the increase in the activities of both enzymes in jundiá (Rhamdia quelen) when using anesthetic baths containing Hesperozygis ringens, as well as the reduction in sodium levels and elevation of plasma potassium, after four hours of recovery. Similar to the findings of these authors, the potassium levels observed in the present study were also elevated in the plasma of animals fed with the D400 and D500 diets. Based on some of the studies, we hypothesized that the increase in plasma potassium in the present study may be related to the vasodilator effect induced by carvacrol and thymol, which triggers the opening of voltage-gated potassium channels. This physiological response promotes the outflow of ions into the extracellular environment, resulting, therefore, in an increase in the plasma concentration of this K+ ion when it is incorporated into the bloodstream. This observation was confirmed in mammalian aortic preparations in vitro (Testai et al. 2016).
It is known that vasodilation can lead to a considerable increase in water perfusion, which in turn can result in ionic losses (Wood and Eom 2021). However, the literature supports the idea that EOs play a protective role by helping to mitigate losses in the net flow of ions in fish during stressful situations (Souza et al. 2019). This study is a pioneer in observing this physiological effect of an EO-based phytogenic compound on fish diets and its influence on ion regulation, which highlights the need for further research to elucidate the mechanisms of action of EOs in the diets of commercial fish species.