Introduction: Common carp (Cyprinus carpio) is one of the most important cultured species in aquaculture in the world and Iran, and has high economic value. Although this species is known as a fish resistant to environmental fluctuations, sudden temperature changes are considered one of the most important environmental stressors in culture systems (Engelsma et al., 2003; Takahara et al., 2011; Long et al., 2020). Temperature stress, especially cold stress, activates a set of physiological responses in fish, including increased cortisol secretion, increased blood glucose, and changes in lipid and protein metabolism (Takahara et al., 2011). At the cellular level, heat stress increases the production of reactive oxygen species (ROS) and induces oxidative stress, which is associated with an increase in MDA and changes in antioxidant enzyme activity (Ma et al., 2021). Suppression of the immune system is another consequence of heat stress, which increases the risk of infectious diseases (Engelsma et al., 2003). In recent years, the use of plant additives in aquatic diets has been considered as a safe and cost-effective solution to combat stress (Herrera et al., 2019). Oregano (Mentha pulegium) essential oil, with its active ingredient pulegone, has antioxidant, anti-inflammatory, and immunomodulatory properties (Boukhebti et al., 2011). Studies have shown that adding this essential oil to the diet of common carp increased growth performance, reduced stress indices, and improved immune responses (Yousefi et al., 2023). Also, in tilapia, menthol essential oil has been shown to improve growth, immunity, and stress resistance (Magouz et al., 2021; Dawood et al., 2020). Temperature stress in common carp mainly occurs in dense systems, transportation, and water exchange with temperature differences of more than 2-3 °C. Therefore, the present study was conducted to investigate the effects of different levels of mentha pulegium essential oil on growth, stress, and immunity indices of common carp under temperature stress conditions.
Materials and Methods: 180 healthy fish with an average weight of 31 ± 2.5 g were randomly distributed into 12 300-liter polyethylene tanks (15 fish per tank) and fed the experimental diets for 60 days. Water quality were monitored daily, and the water system was designed as a semi-flow system with 20% water volume change per day. Mentha pulegium essential oil at dietary levels of 100, 250 and 500 mg/kg (Yousefi et al. 2025) were prepared. Each level of essential oil was dissolved in 25 ml of soybean oil and sprayed on the basal diet, and the control diet received only soybean oil. The experiment was conducted in a completely randomized design with 4 treatments (control, MEO-100, MEO-250 and MEO-500) and 3 replications, and the fish were fed two meals a day for 60 days. Growth indices including weight gain percentage, specific growth rate, feed conversion ratio, survival percentage and protein efficiency ratio were calculated at the beginning, every 14 days and at the end of the period. After 60 days, the fish were subjected to acute cold stress by being transferred to 60-liter aquariums at a temperature of 17 ± 0.5°C for 24 hours and survival rates were recorded. Blood was collected from 3 fish per tank (9 fish per treatment) from the caudal vein using a heparinized syringe and the fish were anesthetized with 40 mg/L clove oil. Blood samples were centrifuged and plasma was stored at -20°C. Immune components, antioxidant enzyme activities, malondialdehyde, cortisol and glucose were measured using commercial kits.
Result and Discussion: The results showed that the MEO-250 and MEO-500 treatments had significant improvements in growth indices including weight gain, specific growth rate and protein efficiency ratio compared to the control group (P<0.01) and the feed conversion ratio in these treatments was significantly reduced. The survival rate in these two treatments reached 100%, which was significantly higher than the control (P<0.01). The activities of antioxidant enzymes (SOD, CAT and GPx) in the MEO-250 and MEO-500 treatments were significantly higher than the control and MEO-100 (P<0.01), while the MDA level was significantly reduced (P<0.01). The activity of lysozyme and the level of total immunoglobulin in the MEO-250 and MEO-500 treatments were significantly higher than the control (P<0.01). Plasma cortisol and glucose concentrations in these treatments were significantly lower than the control (P<0.01). After cold stress, survival rates in MEO-250 and MEO-500 treatments were significantly higher than the control (P<0.01). However, no significant difference was observed between the two treatments MEO-250 and MEO-500 in any of the studied indices (P>0.01). The improvement in growth was attributed to the presence of bioactive compounds such as polgon and menthol, which increase feeding efficiency by increasing diet attractiveness, stimulating appetite, and improving digestive enzyme activity. The antioxidant effects of the essential oil are attributed to phenolic and flavonoid compounds such as rosmarinic acid, which reduce oxidative stress by inhibiting free radicals and stimulating antioxidant enzymes. The immune system is enhanced through the activation of macrophages and neutrophils, increased lysozyme activity, and regulation of cytokine gene expression through the NF-κB and MAPK pathways. The reduction in cortisol and glucose indicates modulation of the hypothalamic-pituitary-adrenal axis by essential oil compounds, which reduces the energy expenditure associated with the stress response and allocates more energy for growth. The similar performance of 250 and 500 mg treatments indicates reaching a threshold level of effect or saturation of the biological system (Schulz, 1987), such that the bioactive compounds of the essential oil have their maximum inductive capacity at a dose of 250 mg/kg and further increase in dose does not have an additive effect. The doses used are within the safe range and are consistent with previous studies (Yousefi et al., 2023; Gardner and McGuffin, 2013).
Conclusion: The present study showed that the addition of oregano essential oil to the diet of common carp, especially at the level of 250 mg/kg feed, through mechanisms of strengthening the antioxidant system and innate immunity, reducing stress indices, improves growth, general health and resistance to the environmental stressor, namely temperature stress. Given the lack of significant differences between the levels of 250 and 500 mg/kg in most parameters, the level of 250 mg/kg is recommended as the optimal and cost-effective dose. |
