In the present study, the inclusion of nano-curcumin into the Nile tilapia diets produced significantly a higher growth performance compared to the diets containing free curcumin or the control diet. This study remarkably reveals the positive role of nano-curcumin that was superior to its free-form in improving the growth performance of Nile tilapia. In the current study, the best growth parameters (WG, SGR and ADG) and feed utilization (FCR and PER) of Nile tilapia was achieved at 100 mg kg-1 of nano-curcumin followed by 200 mg kg-1 of nano-curcumin. This is in agreement with Mahmoud et al. (2017) who found that feeding Nile tilapia (Oreochromis niloticus) with 50 or 100 mg kg-1 diet of curcumin enhanced growth performance and feed utilization. They linked this improvement in growth and feed utilization to improved activities of the digestive enzymes (amylase, protease, trypsin and lipase) and thus improved growth performance (Midhun et al., 2016, Jiang et al., 2016). In addition, curcumin can enhance other enzymes (Na+/K+-ATPase, intestinal alkaline phosphatase, gamma-glutamyl transpeptidase, and creatine kinase) which are responsible for nutrients degradation and assimilation (Jiang, et al., 2016). Moreover, curcumin might work as a prebiotic that promoted gut flora, and improved the intestinal digestion and absorption, resulting in enhancing the overall health and growth of fish (Midhun et al. 2016). Positive effects of dietary supplementation of curcumin have been revealed in different fishes such as grass carps (Ctenopharyngodon idells) (Hu et al., 2003), crucian carp (Carassius auratus) (Jiang et al., 2016), large yellow croaker (Pseudosciaene crocea) (Wang et al., 2007), rainbow trout (Yonar et al., 2019) and Asian see bass (Lates calcarifer) (Abdelwahab et al., 2012).
In fact, there is superiority in the present study for curcumin nanoparticles over curcumin in enhancing the growth rate of Nile tilapia. A lower dose of nano-curcumin can enhance the growth performance of Nile tilapia and give the same effects as a higher dose of curcumin. For instance, 50 mg kg-1 of nano-curcumin gave the same effect of 100 mg kg-1 of native curcumin. In support, nano-curcumin showed a 60-fold increase in biological half-life compared to the free curcumin in rat models (Ma et al., 2007). This may be because nano-curcumin has longer retention time, circulation time and mean residence time inside the body than free curcumin (Mythri et al., 2007). In addition, it has been proved that nano-curcumin shows higher systemic bioavailability in plasma and tissues compared to free curcumin (Zou et al., 2013). This may be due to the curcumin low solubility in water, therefore, it forms aggregates and is susceptible to opsonization, while nano-curcumin dissolves completely in water without aggregations due to its zeta potential (Muller and Keck, 2004). Following the same pattern, nano-curcumin showed superiority in all parameters measured in the current study, including heat stress resistance, liver health assessment (ALT and AST), hematological count, stress indicators and immunity.
On the other, the inclusion of free-curcumin, not nano-curcumin, at the highest level (200 mg kg-1 diet) resulted in a lower growth performance and feed utilization compared to CON in the present study. In fact, curcumin is a polyphenol. It has been reported that low doses of polyphenols can bio-accumulate in body tissues where they are not completely absorbed in the intestine (Mojzer et al. 2016). Thus, high levels of polyphenols may lead to growth regression (Omnes et al. 2017). This effect was lower in nano-curcumin treatments, indicating that the nano-curcumin might be safer than its free form. Recent animal studies revealed the bio-safety of nano-curcumin utilization (Lee et al., 2011).
In the present study, curcumin and nano-curcumin increased the protein and lipid body contents. Similarly, dietary supplementation with curcumin at the levels of 50 to 200 mg kg-1 diet significantly improved the crude lipid and protein deposition in Nile tilapia muscles (Mahmoud et al. 2017). This could be related to the regulation of the intestinal microbiota that enhanced the efficient nutrient utilization (Mahmoud et al. 2017). Also, the positive role of curcumin on enhancing the activity of fish digestive enzymes, such as trypsin, lipase and amylase might be another explanation (Jiang, et al., 2016). In addition, compared to the positively charged particles, negatively charged particles such as nano-curcumin reduce the adsorption rate of serum proteins, leading to longer circulation half-lives (Alexis et al., 2008). This may justify that nano-curcumin gave better protein content than free curcumin.
Any increase in liver functions enzymes activity such as ALT and AST is a biological marker of liver damage (Jahanbin et al., 2012, Li et al., 2011). In the current study, either before or after the heat stress, ALT and AST levels were reduced in the nano-curcumin groups and to some extent in the free curcumin treatments, confirming the superiority of nano-curcumin (this was justified earlier in this discussion). Moghaddam et al. (2015) demonstrated the protective effect of curcumin in rat through its ability to regulate the antioxidant enzymes imbalance and decrease lipid peroxidation levels under the influence of fluoride-induced hepatotoxicity. In similar, curcumin can prevent hepatotoxicity in rats induced by hydrogen peroxide and reduce levels of ALT and AST enzymes (Al-Rubaei et al. 2014). Moreover, experimental results in mice, with fibrosis caused by carbon tetrachloride (CCl4), showed that nano-curcumin significantly reduced ALT and AST levels (Son et al., 2013). Furthermore, histopathological analysis of mice liver showed that hepatic fibrosis treated with nano-curcumin was healed after 4 weeks (Son et al., 2013). Thus, the curcumin ability to reduce some markers of liver injury in the blood (such as ALT and AST) is an indication of its ability as an anti-inflammatory agent during the liver injury (Lin et al., 2012).
Despite the protective effects of curcumin against liver damage in this study at a moderate concentration (100 mg kg-1), adverse effects appeared at a higher concentration (200 mg kg-1). Following the same pattern, curcumin also showed a concentration-dependent effect in the gian carp (Cyprinus carpio), which suffered from CCl-induced liver damage (Cao et al., 2015). Likewise, it was found that curcumin has dual effects on alcoholic liver injury in male rats depending on the concentration as its protective effect was only obtained at the low concentration, but acceleration of liver injury was noticed using a higher concentration (Zhao et al., 2012). Female Swiss mice and Wistar rats were fed dietary turmeric (0, 1 and 5%) and ethanolic turmeric extract (0, 0.05 and 0.25%) for 14 or 90 days. The high dose of turmeric (5%) for a longer duration resulted in a significant body weight reduction, affecting the relative weights of the liver and causing liver toxicity in both mice and rats (Deshpande et al., 1998). A study in rats indicated that a high concentration with long-term intake of curcumin can decrease body weight, accelerate oxidative stress and inflammation and stimulate liver injury while increasing AST and γ-GGT levels (Qiu et al., 2016).
This dual-effect of curcumin may be linked to its ability to stimulate heme oxygenase-1 (HO-1) in non-toxic and toxic concentrations, and HO-1 induction has been found to be associated with the production of reactive oxygen species (ROS), suggesting a causal relationship (McNally et al., 2007). Similarly, Cao et al. (2006) found that at lower concentrations, curcumin have beneficial effects by inducing antioxidant activities, however, higher concentrations increase cellular ROS levels, with oxidative DNA damage in human hepatoma G2 cells.
Heat stress elevates cortisol and glucose levels. In the current study, the inclusion of curcumin (excluding the high dose 200 mg kg-1) or nano-curcumin in tilapia diets reduced both glucose and cortisol either before or after heat stress. The best cortisol levels were detected at CN50 and CN100. This is probably justified by the nature of curcumin as a polyphenol. Polyphenols have been shown to improve stress indicators, cortisol and glucose (Husni, et al. 2019, Guo et al. 2020). These effects can be also attributed to the anti-oxidation properties of curcumin which has been found to eliminate lipid radicals in the cell membrane and become a phenoxyl radical (Sharma, 1976, Ak and Gülçin 2008). Moreover, curcumin lowers hepatic glucose level by increasing the glucose uptake through upregulating GLUT2, GLUT3 and GLUT4 gene expressions (Ghorbani et al. 2014). In support, Wei et al. (2010) revealed that dietary curcumin significantly reduced stress-induced cortisol by one-third in pigs.
However, in this study, a higher concentration of curcumin (200 mg kg-1) gave a higher level of cortisol compared to the other treatments including the control group. In parallel, C200 produced a higher level of glucose similar to that obtained by the control group. The same effect was reported in laying hens where higher doses of curcumin did not reduce cortisol compared to lower concentrations that decreased cortisol levels before or after a high temperature exposure (Nawab et al., 2020). The authors linked this effect to the dual-action potential of curcumin, as it can act as an antioxidant and / or oxidant, depending on its dose (Evans and Halliwell, 2001). The high concentration of peroxides increases the release of ROS (reactive oxygen species) that damage cells and tissues (Evans and Halliwell, 2001).
In this study, the experimental diets had no effect on the hematological counts, except for C200 which gave higher leukocytes and lower neutrophils compared to the other experimental diets. An elevated count of leukocytes at high level of curcumin (200 mg kg-1) may emphasize the stressful status of fish at this concentration. Leukocytosis is directly proportional to the severity of the stress as well as the damage from stress that subsequently triggers the immune defense system (Wedemeyer et al. 1990, Javed and Usmani 2012).
Temperature affects hematological parameters in fish (Fazio et al. 2013). In this regard, after the heat stress, platelets, MCV, MCH, leukocytes and neutrophils counts were raised while the number of lymphocytes decreased and there was no effect on other blood parameters in the current study. In support, Grzelak et al. (2017) reported significant lymphopenia and neutrophilia in acutely stressed zebra fish (Danio rerio) compared to unstressed or control fish. Moreover, thermal stress results in hypoxia or anoxia (Carvalho and Fernandes 2006, Hedayati and Tarkhani 2014), and this effect was reported in the present study. In turn, it was found that hypoxia increases the number of leukocytes, platelets, and neutrophils and reduces the number of lymphocytes in red tilapia (Salem et al., 2021). This may be linked to the elevated level of cortisol (Salem et al., 2021). Neutrophilia and lymphopenia were apparent after treatment with cortisol in carp (C. carpio L.) (Wojtaszek et al. 2002). Crucian carp (Carassius carassius) produced a high ratio of neutrophils to lymphocytes in the blood after being subjected to stress, which is closely related to elevated glucocorticoid levels (Sula and Aliko 2017).
Immunoglobulin M and complements (C3 and C4) are major components of the innate immune system of fish and they are the first line of immune system defense and playing a crucial role in protecting fish health (Reddy and Corley, 1999, Tang et al., 2008, Ichiki et al., 2012, Ming et al., 2019). IgM and complements play an important role in protecting the animal body from infection during stress as well as promoting the engulfment of apoptotic and injured cells (Bouchama et al., 1996, Patz. et al., 2005, Roberts et al., 2008, Ehrenstein and Notley, 2010). In fact, before and after heat stress in the present study, levels of IgM, C3, and C4 increased with the inclusion of curcumin. Nano curcumin gave better improvement in these indicators rather than free curcumin. The results presented herein of IgM, C3 and C4 highlight two facts: low dose of curcumin is better and nano formulation is more effective (which were justified earlier in this discussion). In support, curcumin induced the innate immune response of Nile tilapia at a dose of 50 mg kg-1 but at higher levels this inhibited growth (Mahmoud et al. 2017). In this study, the levels of IgM, C3, and C4 showed an increase with increasing temperature indicating increased of induction anti-inflammatory factors to relieve the tissue damage which is proven by a significant increase in serum liver enzymes (ALT and AST) levels. It is known that heat stress leads to tissue damage and oxidative stress in fish, thus resulting in apoptosis and cell death (Robertson et al., 1997, Moulin and Arrigo, 2006, Wang et al., 2019). Previous studies revealed that the short-term acute heat stress leads to up-regulation of complements 3 and 4 (Sesay et al., 2017, Cheng et al., 2018). Moreover, deficiency in complement system has increased the vascular damage, vascular permeability and angioedema (Sarma and Ward, 2011). A gradual increase in plasma IgM levels was observed with increasing temperature (18, 23, 28, 33) in Nile tilapia except for the highest temperature (Dominguez et al. 2004). Similarly, rearing sea bass at high temperature (23℃) increased their IgM levels compared to fish reared at 17 ℃ (Varsamos et al., 2006). On the contrary, other studies revealed that HS decreases IgM, C3 and C4 levels in fish (Sunyer et al., 1995, Rotllant et al., 1997, Rebl et al., 2020). This discrepancy might be justified by the duration of heat stress where IgM, C3 and C4 levels will elevate under acute stress, but chronic stress will decrease these parameters ( Dominguez et al., 2004, Dang et al., 2012, Zhang et al., 2014). In support, Blunt snout bream (Megalobrama ambl ycephala Yih) which showed an increase in alternative complement activities and IgM levels after heat stress exposure for 6 h, then after 12 h these parameters decreased (Zhang et al., 2014).