1. Introduction
Recently, aquaculture production has witnessed a boom to fill the needs of the fish shop. Fish is the main food in most developing countries, which leads to increased demand [
1]. Worldwide production of tilapia (
Oreochromis spp.) has been increasingly growing at a rate of 10% since 2001, making it one of the main and rapidly developing aquaculture species [
2]. Nile tilapia (
Oreochromis niloticus) is one of the most widely cultured species in many tropical countries. The fish’s response to stress conditions depends on the stress factor (crowding, hypoxia, temperature, presence of heavy metals, etc.) and the fish features such as fish species, age, or gender. Such stressful conditions can increase the spread of pathogenic bacteria and cause severe disease outbreaks [
3,
4].
Staphylococcus aureus is a dangerous pathogen isolated from society and healthcare centers worldwide. The importance of
S. aureus is owing to the rapid occurrence of antibiotic resistance among most of its isolates and the secretion of virulence factors that donate to their invasiveness and ability to disease [
5]. Its pathogenicity is mostly related to genetic characteristics that mediate virulence, immune evasion, invasive capacity, and antibiotic resistance [
6]. It is the third most common reason for foodborne disease worldwide and is the most common factor in food poisoning outbreaks [
7]. The prohibition on antibiotics as feed additives has hastened research and resulted in extensive studies on alternative feed additives in aquaculture diets. These alternatives, such as phytogenic feed additives, include herbs, resins, or spices that enrich the diet with many volatile substances and aromatic compounds. These supplements enhance health status, growth performance, and fish immunity [
8,
9,
10].
Boswellia serrata resin, olibanum or frankincense, is obtained from the Burseraceae family [
11,
12], which grows in dry and arid regions Yemen and Oman, India, northeast Africa [
13]. This old medicine is thought to have antiseptic, anti-inflammatory, antimicrobial, anxiolytic, and anti-cancer effects [
14]. These medicinal effects are related to various aromatic compounds, such as the main active principle boswellic acid [
15,
16]. Moreover,
boswellia serrata resin contains volatile oils which composed of sesquiterpenes and monoterpenes [
17,
18], diterpenes such as cembrenol (serratol), incensole, and incensole acetate [
18,
19], lipophilic pentacyclic triterpene acids of the oleanane (α-boswellic acids), ursane-(β-boswellic acids) and lupane-type (lupeolic acids), and an ether-insoluble fraction containing polysaccharides (arabinose, galactose, xylose) [
20]. It is approved that
Boswellia serrata resin is safe, and its use as a feed additive is allowed by the US Food and Drug Administration (USFDA) [
21]. The medicinal abilities of
Boswellia serrata have been established by several investigators and have been well acknowledged in the works [
22,
23,
24,
25]. There are no data about their aptness for use in aquaculture and their effects on fish growth, immunity, and health status. So, this study, for the first time, evaluated the potential impacts of using
Boswellia serrata resin extract on the growth performance, immune response, disease resistance, and antioxidant status of
Oreochromis niloticus.
4. Discussion
The data available for the effects of
Boswellia serrata resin extract in fish diets are almost entirely scarce. Consequently, the current study was performed to illustrate the influence of BSRE as a feed additive on growth performance, general health, and immune response of
O. niloticus. As an initial experiment, feeding
O. niloticus on BSRE supplemented diets triggered no mortalities between all treated groups, which helped us eliminate the ID50. The present study reported improved FBW, TBWG, and total FI of fish fed on the BSRE5 diet. The PPV was increased in BSRE supplemented diets. However, no effect on the FCR by the addition of BSRE. The increased BW and BWG in the BSRE5 group in the current study may be due to the improved feed intake compared to the control group and other BSRE-complemented groups. As well, the improved growth performance in the BSRE5 diet may be attributed to the enhanced intestinal histology reported in this study that was indicated by increased villus height, villus width, and mucosal thickness, consequently improving the intestinal absorptive surface for nutrients. Amer et al. [
8] reported that using phytogenic feed additives, particularly medicinal plants, leads to improved fish performance efficiency as it can augment gut function [
9,
10,
48,
49,
50]. This coordinates with the intestinal morphometric measurements of the villus heights and the count of goblet cells, which secrete mucus to coat and keep the intestinal mucosa from damage, dehydration, and pathogens [
51,
52]. Caspary [
53] determined that the intestinal villus length influences the absorption, which increases the feed utilization. Additionally, the higher intestinal villus heights and goblet cells count indicating an improvement in nutrient absorption, resistance against intestinal pathogens due to enhancing capacity of absorptive surface area, and so considered as a growth promoter. Gabriel et al. [
54] recorded a marked improvement in the weight gain and specific growth rate in GIFT (genetically improved farmed tilapia) tilapia fed on aloe vera. Mukherjee et al. [
55] stated enhanced growth and innate immunity of Nile tilapia by the dietary addition of
Withania somnifera root extracts. However, higher levels of BSRE (10 and 15 g kg
−1) did not affect the fish growth, which may be due to the hypolipidemic effect of BSRE bioactive compounds (boswellic acid and oleanolic acid), which observed in the fish body composition in the present study. Wang et al. [
56] presented that oleanolic acid administration to mice (20 mg/kg/day) caused reduced body, fat, and liver weights.
Although the feed cost in the BSRE5 diet was increased, the feed cost/kg gain was not significantly different between all experimental diets. The increased feed cost was due to the increased feed intake in the BSRE5 group compared to other groups. However, due to the increased TBWG in the BSRE5 group compared to other groups, the feed cost/kg gain became not significantly different. Regarding the fish body composition results, dietary supplementation of BSRE increased the crude protein content and reduced the fat content in a level-dependent manner while the ash content was increased in the BSRE15 group. The obtained results may be attributed to the BSRE content from boswellic acid reported stimulating pancreatic enzyme secretion that improves protein and energy digestibility and decreases endogenous losses of nitrogen, ammonia production [
57], in addition to having an antihyperlipidemic activity [
58]. It was reported that oleanolic acid and boswellic significantly decreased visceral fat, plasma lipids, ghrelin, and increased leptin in obese Swiss mice [
59].
Serum biochemical and innate immune parameters are crucial health pointers [
60,
61]. The blood glucose level is a significant physiological indicator estimating fish health conditions. It is mobilized to provide metabolic energy in fish [
62] and used by fish to deal with physiological stress, acting as a useful stress tolerance indicator [
62]. The BSRE acts as a hypoglycemic agent regarding serum glucose levels, which is a good sign of lowered stress [
63]. The hypoglycemic effect of BSRE is due to its content from boswellic acid and oleanolic acid. The mechanism by which the boswellic acid induces the hypoglycemic effect is through increased peripheral glucose utilization and inhibition of intestinal glucose transporter activity, as reported in [
58]. Whereas the hypoglycemic activity of oleanolic acid is carried out by improving the insulin response, it maintains the function and survival of β-cells and prevents complications of diabetes. Oleanolic acid may control the enzymes involved in insulin biosynthesis, signaling, and secretion [
64]. Wang et al. [
56] demonstrated that oleanolic acid’s hypoglycemic activity occurs through glucose improvement, insulin tolerance, enhancement of insulin signaling, and inhibition of gluconeogenesis.
Moreover, our results indicated a hypolipidemic effect of BSRE in a level-dependent manner, which could also be because of its content from boswellic acid and oleanolic acid. Jadhav and Puchchakayala [
58] investigated the antihyperlipidemic activity of boswellic acid. Wang et al. [
56] stated that oleanolic acid administration to mice (20 mg/kg/day) reduced the serum total cholesterol, triglyceride, LDL, and free fatty acids and decreased the accretion of hepatic lipid through downregulation of lipogenic genes expression (stearoyl-CoA desaturase 2 [SCD2], acetyl-CoA carboxylase [ACC], acyl-CoA cholesterol acyltransferase [ACAT], and glycerol-3-phosphate acyltransferase [Gpam]) [
59]. Oleanolic acid and correlated triterpenes have important therapeutic properties such as antidiabetic, antioxidant, anti-inflammatory, microbicide, and hypolipidemic actions [
65,
66].
The current study showed a reduced ALT level in the BSRE5 group while its level was increased in the highest level of BSRE (BSRE15). Additionally, the creatinine level was reduced in the BSRE15 group. ALT is a standard indicator of liver disease, and increased serum levels indicate liver damage [
67]. The reduced liver function also led to reduced creatine production and decreased creatinine levels in the blood due to reduced storage of creatine and reduced conversion of creatine to creatinine [
68]. This indicates that increasing the level of BSRE badly affects liver function, which may be explained by liver overload by increasing the level of the bioactive compounds presents in BSRE that responsible for the stimulated immune system, hypoglycemic, and antihyperlipidemic effects where the liver is the site of production of serum total protein and its fractions and the organ of glucose and fat metabolism. The same was observed in the kidney and gill histoarchitectures, which showed moderate changes in BSRE10 and BSRE10 treatments.
Serum total protein, albumin, and globulin are effective indicators of humoral immunity and fish well-being as the essential components of blood serum, especially globulin, which is a protein fraction involved in the immune response [
63,
69], and its increase implies an enhancement in the fish immune system [
10]. In the current study, the increased total serum protein, albumin, total globulin, and immunological globulin suggesting the immune-modulatory effect of BSRE in a level-dependent manner. Similarly, Syrovets et al. (2000) attributed the potent induction of the non-specific immune response by BSRE to their bioactive compounds (polysaccharides, incensol acetate, and boswillic acids), which act as an immune stimulant [
12,
70]. This supports our results of the increased intraepithelial lymphocytic infiltration and the splenic cords (Billroth’s cords or red pulp cords) outstanding, especially in BSRE10. These splenic cords are reservoirs of monocyte clusters that total more than the total number of monocytes in the circulation. They can be rapidly organized to leave the spleen and help treat persistent infections [
71], indicating the improved ability to clear microbial antigens from the gut in BSRE10 and BSRE15, as the intestine is the primary infection route for pathogens in fish [
72,
73]. The melano-macrophage centers (MMC) were mild to moderately activated in splenic tissue of fish fed BSRE. El-Asely et al. [
74] recorded increased MMC in Nile tilapia fed a diet supplemented by
Echinacea purpurea. Additionally, Ledic-Neto et al. [
75] stated that feeding Nile tilapia on a diet complemented with propolis (2%) for 15 days displayed increased melano-macrophage centers, despite the difference between treatments disappeared seven days later, suggesting physiological adaptation to the supplements.
Conversely, Brum et al. [
76] found that at 55 days of feeding fish on a diet supplemented with 0.5% basil exhibited a reduction in the count of melano-macrophages centers. Despite this, there was no difference in the area of the spleen they engaged, and it could be said that the centers of macrophages and melanocytes in these animals were less distributed. Considering that their results were obtained after 55 days of feeding on a supplemented diet, it may be explained as a physiological adaptation to the supplemented diet. The current study results coordinate with Mukherjee et al. [
55] who detected an elevation in the plasma levels of total protein and total immunoglobulin of Nile tilapia by dietary addition of
W. somnifera root extracts.
The fish’s innate immunity is the main line of protection against invading pathogens. The lysozyme activity and nitric oxide (NO) are important components of the innate immune system that play a vital role in destroying pathogens [
77]. Our results illustrated that BSRE increased the lysozyme activity in a level-dependent manner, which coordinates with the result of total protein and maybe explained by the potent non-specific stimulation of the innate immune system by BSRE bioactive compounds; polysaccharides, incensol acetate, and boswillic acids, which considered as an immune stimulant as mentioned by [
12,
70]. Engstad et al. [
78] stated that increased lysozyme in the blood of stimulated fish is conducted either by the proliferated phagocytes or the increased productivity of lysosomes. Pratheepa and Sukumaran [
79] demonstrated that the increased lysozyme activity could be because of increasing the blood neutrophils and monocytes of fish fed diet fortified with plant extract compared to that found in the control diet. The results of the present study are coordinated with Mukherjee et al. [
55] who reported an increase in the lysozyme activity in Nile tilapia fed on a diet fortified with
W. somnifera root extract. Furthermore, myeloperoxidase (MPO) is a neutrophil released enzyme that is an essential constituent of protection against pathogens [
80] and has a vital function for the innate immune response [
81]. The current study results showed increased MPO production in BSRE supplemented groups in a level-dependent manner that also indicates the immune-modulating effect of BSRE. These results harmonize with that obtained by Kurian et al. [
81] who stated the activation of fish peroxidase when fed on herbal immune stimulants.
Staphylococcus aureus has been recently reported in Nile tilapia (
O. niloticus) triggering high mortality with various pathological alterations [
82]. It also poses health risks to fish handlers and consumers [
83]. In addition, in aquaculture, methicillin-resistant
Staphylococcus aureus (MRSA) has been isolated from tilapia in Malaysia [
84]. It has also been related to mortality and morbidity in the culture of Nile tilapia in northern Egypt [
85].
In the current study, BSRE supplementation increased the survivability of Nile tilapia challenged with
S. aureus in a dose-dependent manner. The increased survivability may be due to the stimulation of innate and non-specific immune responses by BSRE supplementation and its bioactive compounds, which have antibacterial, immune-modulatory, anti-inflammatory properties [
65,
66,
86]. Similarly, Kurian et al. [
81] indicated that Nile tilapia fed Leucas Aspera showed markedly improved resistance against Streptococcus agalactiae infection compared to the control. The dietary addition of BSRE decreased the serum level of NO in a level-dependent manner. The reduced NO production can be attributed to the inhibition of the NO production and on the generation of NO synthase caused by the bioactive components of BSRE as sesquiterpenes [
87], diterpenes [
88,
89], and triterpenes [
90]. Furthermore, Yoshikawa et al. [
91] indicated inhibition of nitric oxide production from lipopolysaccharide-activated macrophages caused by the mono and triterpenes constituents of
Boswellia carterii gum-resin.
The antioxidant enzymes protect the fish body from oxidative alterations that resulted from increased ROS production [
92,
93]. The superoxide dismutase activity, glutathione peroxidase level, and catalase activity are significant indicators for estimating the antioxidant activity in aquatic organisms [
94,
95]. Our study reported increased levels of antioxidant enzymes (CAT, SOD, and GSH) in BSRE supplemented groups in a level-dependent manner that can be attributed to the bioactive triterpenes (boswellic acids and oleanolic acid) that possess antioxidant activity [
96,
97,
98] through increasing the expression of the antioxidant enzymes [
64]. Similarly, Sharma et al. [
99] reported the antioxidant activity of aqueous extract of
B. serrate in a level-dependent manner. Afsar et al. [
100] demonstrated the invitro antioxidant activity of methanolic extract of
Boswellia serrate due to its content of flavonoids, terpenoids, tannins, saponins, anthraquinones.