1. Introduction
Intensive aquaculture is accompanied by numerous stressors that cause stressful and undesirable conditions for fish and may cause infectious diseases and significant financial losses [
1,
2]. The administration of antibiotics to prevent and treat aquatic animal disease has been restricted or forbidden because of its adverse consequences on target and non-target animals and natural habitats [
3,
4,
5]. Recently, there has been growing attention paid toward safer alternatives to antibiotics, such as natural active compounds, to control and treat diseases in aquaculture [
6,
7,
8].
Among marine organisms, photosynthetic microalgae and some macroalgae (seaweeds) can be considered the foundation of the aquatic food web [
9,
10]. Macroalgae can generate a broad spectrum of primary and secondary metabolites [
11,
12] and thus is known as one of the most valuable sources of natural antioxidants and antimicrobial agents [
13]. Seaweeds have recently received attention as dietary supplements in aquaculture because of their biologically active substances and chemical composition [
14,
15]. The dietary administration of suitable macroalgae and its extracts have been proven to enhance the growth performance and health status of different aquatic species with no adverse effects [
16,
17,
18,
19,
20]. For example, Vazirzadeh et al. [
18] stated that adding red macroalgae to rainbow trout feeds improved immunity, antioxidant status, and immune-related genes in a time- and dose-dependent manner. Moreover, Hoseinifar et al. [
19] reported that supplementing the diet with 0.5%
Halopithys incurva increased immunity and antioxidant defense in zebrafish (
Danio rerio). In another recent study involving red macroalgae, it has been reported that
Laurencia caspica extract enhances resistance to bacterial diseases and promotes immune system improvement in Nile tilapia (
Oreochromis niloticus) [
20]. Accordingly, red algae (Rhodophyta), as one category of macroalgae, are an excellent source of bioactive compounds, generally essential fatty acids (polyunsaturated fatty acids and sterols), terpenes, mycosporine-like amino acids, proteins, pigments, phenolic compounds, polysaccharides, vitamins, and minerals [
11]. Red seaweeds have been well recognized for their antioxidant, antitumor, antimicrobial, anti-inflammatory, anticancer, antidiabetic, and anti-amyloidogenic properties [
11,
19,
21].
Most Rhodophyta belong to the
Florideophyceae, which are chiefly multicellular [
22].
Galaxaura oblongata is a prevalent benthic and macroscopic marine algae belonging to phylum Rhodophyta, class
Florideophyceae, with expansive distribution in shallow tropical and subtropical marine environments [
23]. However, there is still a gap in understanding its structure, chemical composition, and bioactive compounds.
It has been proposed that
G. oblongata has been proposed as a natural pharmaceutical compound with possible therapeutic functions due to its potent anti-inflammatory, anti-edema, antitumor, anticancer, and antioxidant properties [
24,
25,
26]. With such promising attributes,
G. oblongata may promote fish health and welfare. The nutritional and anti-nutritional contents of macroalgae commonly differ between genera, according to the region where the algae are collected and the season. Therefore, not all macroalgae have the same beneficial effects on all aquatic species [
10]. In this sense, most of the research evaluating macroalgae’s effects in aquafeeds has focused on some genera such as
Ulva,
Sargassum,
Gracilaria, and
Porphyra species [
12]. To the best of our knowledge, no data is available regarding the effects of
G. oblongata on fish. Rainbow trout,
Oncorhynchus mykiss, is a leading fish cultivated globally. Its production reached 959,000 tons in 2020, accounting for 1.67% of the total finfish production (FAO, 2022). Hence, the current study aimed to investigate the possible impact of
G. oblongata on growth performance, serum, mucosal antioxidant status, and antioxidant-related gene expression of
O. mykiss.
4. Discussion
Previous works with red and brown seaweeds reported an excellent nutritional composition, which may be a functional and cost-effective replacement for proteins, minerals, and vitamins [
13,
38]. In this regard, several publications have addressed the potency of different red and brown seaweed species to ameliorate the growth performance and feed revenue of several aquatic animal models [
39,
40,
41,
42,
43,
44,
45,
46]. In addition, based on evidence, red algae contain higher PUFAs (n−3 (eicosapentaenoic acid) and n−6 (arachidonic acid)) than green algae and have a higher nutritional value than brown algae [
13], which in turn may have growth promotion effects on aquatic animals. Nevertheless, in this study, the fish that received dietary
G. oblongata exhibited no significant improvement in growth parameters (WG, SGR, and FCR). These findings are consistent with those obtained by Vazirzadeh et al. [
10], who claimed that the long-term (83-day) administration of 5% and 10% red algae (
Gracilariopsis persica and
Hypnea flagelliformis) and brown algae (
Sargassum boveanum) had no remarkable effect on the growth performance and FCR of rainbow trout. Similarly, the growth performance in zebrafish supplemented with
Gracilaria gracilis (0.25, 0.5, and 1%) [
47] and Persian sturgeon (
Acipenser persicus) supplemented with
G. persica (2.5, 5, and 10 g kg
−1 diet) [
48] was not significantly different from fish fed non-supplemented feeds. These discrepancies in the results reveal the theory that dietary macroalgae may exert species-specific effects [
48] and seaweeds’ nutritional values may change notably depending on the species, genera, dose, duration, and other factors [
47].
The beneficial role of phytobiotics, seaweeds, and their products in the health of aquatic animals and their providing of resistance against viral and bacterial infections have been widely reported [
19,
20,
49,
50,
51]. For this purpose, serum and mucus non-specific immune parameters can be considered helpful tools for monitoring the influence of medicinal herbs on fish health status [
52]. Indeed, skin mucus is a significant part of the fish defense system. It consists of numerous antimicrobial components, such as complement systems, lectins, LYZ, immunoglobulins, antimicrobial peptides, and antibodies, which provide a robust physiological barrier against invasive pathogens [
53]. Ig and LYZ are common humoral components of host mucosal and systemic immunity and are usually admitted as biomarkers of immune responses with feed supplements in aquatic species [
54,
55]. Our results highlighted the immunostimulatory effects of
G. oblongata in rainbow trout, characterized by a higher serum (LYZ and total Ig) and mucosal (total Ig) immune response compared to the control. Consistent with our results, a remarkable increase in aquatic animal immunological indices was also reported following the dietary administration of different red macroalgae species [
16,
39,
56,
57].
Additionally,
G. persica powder boosted serum and mucus total Ig and LYZ values in Persian sturgeon compared to the non-supplemented group [
48]. Chen and Zhang [
42] found that grass carp (
Ctenopharyngodon idella) that received
Porphyra yezoensis polysaccharide-containing diets (3 and 5 g kg
−1) showed substantially higher serum LYZ activity. Furthermore, adding
G. gracilis (1%) to the fish diet caused a remarkable increase in the mucosal total Ig levels of zebrafish [
47]. In another study, dietary Spirulina platensis increased serum Ig and LYZ activity in coral trout (
Plectropomus leopardus) [
58]. In addition, increased serum LYZ activity was determined in juvenile black sea bream (
Acanthopagrus schlegelii) after feeding brown algae (
Sargassum hornei) [
41]. However, concerning serum, the maximum immune responses (LYZ and Total Ig) were observed in rainbow trout fed with a lower concentration of
G. oblongata (G1). This study’s findings agree with a previous study on barramundi (
Lates calcarifer), where a significant decline was observed in LYZ and Ig in response to increased dietary levels of
Gracilaria pulvinata [
56]. In line with our results, Ghafarifarsani et al. (2022) reported that adding savory essential oil to roach diets increased Ig and lysozyme activity in mucus and serum. However,
G. oblongata employed in the current study at different doses exhibited no significant differences in the mucus total Ig value. A similar result has been reported for Ig value in the research carried out on zebrafish evaluating the influence of red macroalgae (
Halopithy sincurva) [
19]. Although the exact mechanism responsible for the immunostimulatory effect of seaweeds is still unclear, it is well known that they are rich sources of polysaccharides and polyphenolic compounds with high antibacterial features and a promising influence on the welfare of fish [
12]. However, including dietary
G. oblongata had no marked effects on mucus LYZ activity in this research. Contrary to our finding, a study on the impacts of
G. persica (5 and 10 g kg
−1) in Persian sturgeon showed a promoting effect on skin mucus LYZ activity [
48]. It may, therefore, be concluded from the contradictory results that the immunostimulatory effects of dietary seaweeds on fish may differ from one species to another, and various parameters, e.g., fish and algae species, administration dose and duration, as well as experimental conditions may alter the outputs [
19,
56].
It has been proved that dietary exogenous antioxidant compounds may cooperate with endogenous antioxidants to diminish the overgeneration of reactive oxygen species (ROS) [
19,
59]. Correspondingly, in vitro studies have well exhibited the antioxidant effects of dietary macroalgae on several cultured organisms because of the existence of carotenoids, specific polysaccharides, prebiotics, and phenolic compounds in their chemical profile, scavenging free radicals [
60]. SOD, CAT, and GPx are generally considered appropriate biomarkers of oxidative stress in fish, with an increase in concentration being translated into an increase in antioxidant performance [
61,
62]. Accordingly, in the current study, the potential of
G. oblongata to improve antioxidant capacity is also exhibited through increased serum SOD, CAT, and GPx activity compared to the unsupplemented group. Parallel to our results, Chen, L. and Y. Zhang [
42] reported improved serum SOD, CAT, and GPx activity in grass carp fed with dietary polysaccharides extracted from
P. yezoensis (1, 3, and 5 g kg
−1). MDA is a biomarker of lipid peroxidation and cell and tissue disorders. When the generation of ROS is above its elimination, the MDA levels subsequently increase [
63]. In this study, the observed increase in mucus GPx and SOD levels following macroalgae supplementation, coupled with the absence of elevation in MDA levels, suggests that
G. oblongata could be employed to mitigate oxidative stress in
O. mykiss [
14]. These findings are concurrent with Hoseinifar et al. [
19], who assayed the effects of dietary red macroalgae (
H. incurva) on zebrafish and claimed that this supplement boosted serum and mucus SOD, CAT, GPx, and decreased MDA levels.
It has also been determined that GPx and CAT may play the same role since both are antioxidant enzymes responsible for detoxifying H
2O
2 to H
2O and O
2 [
64]. The existence of the GPx enzyme may have a limiting effect on the CAT enzyme, as featured in the present research just for skin mucus. Similar results were also obtained in the case of serum GPx and CAT activity in rainbow trout fed with red algae (
Laurencia caspica) extract [
65]. Additionally, the results of the present study support the hypothesis that antioxidant enzyme activity may possess contradictory patterns in different tissues [
66].
Many studies aim to confirm the antioxidant and immunostimulatory capabilities of different macroalgae at the molecular level (21, 48, 66). However, the current trial is the first attempt to investigate the molecular-level effectiveness of
G. oblongata on the antioxidant response and immunity of rainbow trout, demonstrating a positive role in specific genes. The up-regulation of hepatic
gpx was shown in the group fed by
G. oblongata-supplemented diet. Based on the documents, it can be suggested that herbal remedies could notably up-regulate hepatic antioxidant gene expressions of aquatic species [
67,
68]. However, in the present trial, no significant variation was found between fish fed
G. oblongata supplemented and no-supplemented diets on
sod gene expression.
Similarly, Hoseinifar et al. [
47] reported that
G. gracilis enriched diet (0.25, 0.5, and 1%) resulted in up-regulation of
cat gene expression. At the same time, no significant difference in
sod value was detected in the whole body of zebrafish. Also, the oral administration of dietary red macroalgae (
H. incurva) (0.25 and 0.5%) in zebrafish [
19] and ulvan in labeorohita (25, 50, and 100 mg kg
−1) [
69] increased
gpx gene expression. However, the up-regulation of an enzyme mRNA would not always lead to increased enzyme synthesis due to numerous causes [
70]. Therefore, the higher
gpx gene expression in the fish liver may suggest promising impacts of
G. oblongata on hepatic antioxidant activity. Nevertheless, further studies are required to support this hypothesis with different genes in subsequent research.
An inflammatory response usually accompanies extra pro-oxidant production [
69,
71]. Additionally, the inflammatory and immune responses of fish are moderated by cytokines [
72]. In this regard,
tnf-α and
il-6 are two important pro-inflammatory cytokines that could trigger inflammatory reactions and play an essential task in modifying the immune strength of the host [
73,
74]. Accordingly, our findings demonstrated that feeding the highest level of
G. oblongata could significantly up-regulate the liver’s
tnf-α and
il-6 gene expression. The up-regulation of
tnf-α and
il-6 as the mediator of inflammation through macroalgae treatments could stimulate antibodies and numerous immune cell production [
75,
76]. In line with our results, dietary glycoprotein extracted from hizikia fusiformis (10 g kg
−1) [
39] and
P. yezoensis (20 g kg−1) [
77] increased hepatic
il-6 gene expression of olive flounder only at the highest dietary inclusion level. In addition, a significant elevation of the hepatic
tnf-α gene expression value was exhibited in zebrafish following feeding by all dietary inclusion levels of
H. incurva (0.25, 0.5, and 1%) [
19]. In an 8-week feeding trial carried out to assess the impacts of an
L. caspica containing diet on rainbow trout, marked increases in the kidney
tnf-α and interleukin-1β (
il-1β) cytokine gene expression were reported after bacterial challenge in fish fed 1.5% of this supplement [
65]. Additionally, an elevation in kidney
tnf-α and
il-6 gene expression was reported in rainbow trout after feeding with dietary
Aloe vera (10 and 15 g kg
−1) [
78]. Finally, in juvenile black sea bream supplemented with
S. hornei (3, 6, and 9%), the liveril-
1β, interleukin-8 (
il-8), and
tnf-α genes expression increased significantly [
41]. Such positive changes in these pro-inflammatory cytokine genes may explain why dietary macroalgae can be considered immunostimulants, which can incite the expression of significant immune genes.