Introduction

Rainbow trout is esteemed as one of the foremost economically vital species in freshwater environments, renowned for its premium flesh quality and adaptability to diverse aquaculture systems (Hajirezaee et al. 2024). In recent years, the increasing demand for seafood products worldwide has led to the development of intensive aquaculture systems. However, under intensive production systems, fish farming compromises the immune systems of fish, resulting in the emergence of infectious diseases that decrease growth and survival rates, ultimately leading to significant economic losses and reduced fish production (Limbu et al. 2021; Tadese et al. 2022). In response to these challenges, antibiotics and synthetic growth promoters have become prevalent in aquaculture practices to stimulate growth, mitigate stress, and control bacterial infections (Ahmadifar et al. 2021a; Hajirezaee et al. 2024). These substances and antibiotics may increase fish growth and illness resistance, but fish and the ecosystem will inevitably suffer as a result (Rashidian et al. 2020a Therefore, the increasing concern about the widespread use of chemicals, especially antibiotics, in fish farming has led to regulatory interventions in many countries worldwide (Van Hai 2015; Santos and Ramos 2018). Although antibiotics generally prove successful in treating diseases, their excessive and improper use leads to the presence of antibiotic residues in aquatic products and the environment (Grenni et al. 2018; Elumalai et al. 2020; Limbu et al. 2021). Furthermore, inappropriate utilization of antibiotics for disease treatment or as growth promoters in aquaculture fosters the escalation of antimicrobial resistance (Awad and Awaad 2017; Tadese et al. 2022). On the other hand, the non-selective effect of antibiotics will adversely affect the gut microorganisms of aquatic animals and disrupt the balance of the microbial ecosystem (Limbu et al. 2021; Dien et al. 2023). Therefore, it is imperative to replace chemicals with alternative options that are more preferable in terms of public health and environmental considerations (Rahimi et al. 2022).

Feed additives are non-nutritive components that play a significant role in enhancing the quality of aquafeed (Mohan et al. 2022). Among the array of potential feed supplements, plant compounds emerge as promising alternatives due to their minimal adverse effects on organisms and the environment, absence of drug resistance, cost-effectiveness, and sustainability (Liu et al. 2021a, b). The utilization of plant components in aquatic species has gained substantial attention for their potential to bolster growth, improve digestibility and nutrient availability, modulate intestinal microbiota, and influence gene expression related to physiological processes (Rashidian et al. 2020a; Liu et al. 2021a, b; Mostafavi et al. 2022; Zhang et al. 2022).

The Asteraceae family genera are renowned for their diverse array of biologically active compounds, showcasing substantial potential for therapeutic applications (Ayad & Akkal 2019). Cyanus depressus (CD), also recognized as “gökbaş” in Türkiye, is a member of the Asteraceae family and is found in Western Asia, Iran, and the Caucasus region. This herbaceous annual plant features striking blue-purple flowers and can reach heights of up to 60 cm (Duman et al. 2022). Within Turkish traditional medicine, Cyanus species, including CD, are extensively utilized due to their recognized antidiarrheal, expectorant, antipyretic, and antidiabetic properties (Duman et al. 2022). Moreover, these species are employed in folk medicine for their purported hepatoprotective, wound healing, antidiabetic, antioxidant, anticarcinogenic, antiviral, anti-inflammatory, and antimicrobial activities, along with their traditional use in treating fever (Khammar & Djeddi 2012; Escher et al. 2018; Fattaheian-Dehkordi et al. 2021; Gawlik-Dziki et al. 2023). Despite this extensive therapeutic potential, as far as our current knowledge extends, no research has yet explored the effects of Cyanus depressus on aquatic species.

Given the recognized benefits of Cyanus depressus, we hypothesize that its incorporation into fish feed as an additive could offer advantages to the aquaculture industry. Such utilization can offer significant revelations regarding the advantageous impacts of Cyanus depressus on growth performance, intestinal microbiota, and gene expression associated with digestion, antioxidative processes, stress management, and immune reactions in rainbow trout.

Considering the economic significance of rainbow trout in the global aquaculture sector and the promising attributes of Cyanus depressus (CD) as a feed additive, this study was undertaken for the first time to evaluate the dietary impact of CD extract on rainbow trout’s growth performance, intestinal microflora composition, and gene expression profiles. The primary objective of this study is to assess the efficacy of Cyanus depressus extract as a dietary supplement in promoting the growth and health of rainbow trout, thereby contributing to the optimization of aquaculture practices. By elucidating the mechanisms underlying the observed effects, this research endeavors to provide valuable insights into the potential use of natural additives in aquafeed formulations, ultimately aiming to improve the sustainability and efficiency of rainbow trout production systems.

Materials and methods

Ethical approval

The Local Ethic Committee of Van Yüzüncü Yıl University approved (protocol no: 2023/06–11), and the experiment was carried out in accordance with standard ethics.

Preparation of CD extract

In May 2023, Cyanus depressus plants were collected from the campus of Van Yüzüncü Yıl University in Van, Turkey. Flower parts were utilized for the study. The lyophilized extract was prepared according to the procedure by Duman et al. (2022), with some modifications. The CD plants were first thoroughly cleaned with distilled water and then allowed to dry for ten days at room temperature in the shade. After that, a blender was used to grind the dried flower pieces into a powder. A total of 40 g of the flower powder sample was then mixed with 1 L of 80% ethanol in a shaker for 24 h. To separate the extract from solid particles, the resulting mixture was first passed through sterile cheesecloth and then subjected to centrifugation at 3500 rpm for 5 min. The filtered solution was further clarified by passing it through Whatman filter paper (Whatman Paper, No. 1). Following the mixture’s filtration, the extract was transferred to a rotary evaporator, where the alcohol was evaporated at 40 °C to obtain a concentrated extract. Finally, 20 ml of concentrated extract was obtained from 100 g of plant powder. After this, the concentrated extract underwent lyophilization in a freeze dryer at − 85 °C and 50 millitorr pressure until it was completely dry. The resulting lyophilized extracts were then stored at − 20 °C until they were utilized for the experimental application.

Phenolic composition of CD extract

At the Eastern Anatolia High Technology Application and Research Center (DAYTAM/Erzurum, Turkey), the concentration of phenolic compounds in the CD extract utilized in the study was measured. Chromatographic separation was carried out using a C18 column (Reversed Phase C18 Column) and an LC–MS/MS system (Agilent 6460 Triple Quad LC–MS/MS with 1290 Infinity UPLC system). The temperature in the column was maintained at 30 °C. Two different mobile phases were used: mobile phase A, which included ultra-pure water and formic acid, and mobile phase B, which included acetonitrile and formic acid. The injection volume was set at 5 μL, and the solvent flow rate was set at 0.4 mL/min.

Preparation of samples, formulation of diets, and rearing conditions

The research was conducted at Van Yüzüncü Yıl University’s Aquatic Creatures Trial Unit in Van, Türkiye. Rainbow trout (O. mykiss) were obtained from a local fish farm and kept for 2 weeks to acclimatize to the experimental conditions. After this period, 240 healthy fish weighing 3.29 ± 0.34 g were randomly distributed into 12 fiberglass tanks (volume of 300 L, 20 fish per tank, in triplicates). In the study, a commercial trout feed (Aquanorm, İzmir/Türkiye) was considered as a basal diet (Table 1). Fish in the control group were fed with the commercial trout feed, while the CD05, CD1, and CD2 groups received the commercial trout feed supplemented with 0.5, 1, and 2 g kg−1 of Cyanus depressus extract, respectively. By spraying, extracts were combined with fish feed (Bilen et al. 2021; Sönmez et al. 2022b). Fish were hand-fed three times at 08.00 am and 1.00 and 6.00 pm until satiation. Throughout the experiment, a central air pump was used to aerate the rearing tanks, and each morning, 20% of the tank’s water was replaced with new water. Regularly siphoned waste and other materials from the tank floor were also done. Every day, measurements were made of the water quality parameters, which included pH (8.13 ± 0.1), dissolved oxygen (8.48 ± 0.16 mg/L), and temperature (13.35 ± 0.25 °C). In addition, a photoperiod of 12 h of light and 12 h of darkness was performed for the study.

Table 1 Composition of the basal diet used in the study
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Growth parameters

The fish were weighed before the feeding trial and at the conclusion of the 60-day period in order to measure growth parameters using the following standard formulae:

Weight gain (WG; g/fish) = final weight (g) − initial weight (g),

Body weight increase (BWI, %) = 100 × (final weight – initial weight)/initial weight,

Specific growth rate (SGR; %/day) = ((ln (final weight) – ln (initial weight))/days) × 100,

Daily weight gain (DWG; g/fish) = (final weight (g) − initial weight (g))/days,

Thermal growth coefficient (TGC) = (((final weight)1/3 – (initial weight)1/3)/temperature in °C × time in days) × 1000,

Feed conversion ratio (FCR) = total feed given (g)/weight gain (g),

Protein efficiency ratio (PER) = Weight gain (g)/protein intake (g),

Survival rate (SR; %) = (final number of fish/initial number of fish) × 100.

Gene expression analysis

In the study, the activities of digestion, antioxidant function, stress response, and immune response were evaluated at the gene level using molecular tests. Fish were anesthetized using clove powder at a concentration of 200 mg/L before sampling (Naderi et al. 2017). Three fish were then randomly selected from each tank (N = 9) following the feeding trial. The liver and intestinal tissues of the selected fish were subsequently stored in RNA later for further examination. Following the manufacturer’s instructions, the RNeasy Plus Mini Kit in a QIACUBE (Qiagen) device was used for total RNA isolation. The quality of the extracted RNAs was assessed at 260 and 280 nm using a thermonanospectrophotometer. Subsequently, isolated RNAs were diluted to a concentration of 1 ng/μl for each sample, and cDNAs were synthesized using the Qiagen RT2 First Strand Kit. Subsequently, qRT-PCR was analyzed using the RT2 SYBRGreen qPCR Master Mix (Qiagen) in the RotorGene Q 9000 (Qiagen) device. In the gene expression analysis, β-actin was chosen as the reference gene, while TRP, LPZ, AML, SOD, CAT, GPX, IL-1β, TNF-α, and HSP70 were designated as target genes. The PCR composition included 12 μL of SybrGreen qPCR Master Mix, 2.5 μL of forward and reverse assay primer, and 4 μL of H2O in a total volume of 21 μL, with 4 μL of cDNA added last. The PCR protocol involved 40 cycles, with initial incubation at 95 °C for 10 min, followed by annealing at 94 °C for 15 s and 60 °C for 30 s. Beta-actin was used as the reference gene to normalize the Ct values obtained from real-time PCR. Analysis of the real-time PCR data was conducted using the ΔΔCT method (Livak and Schmittgen 2001) (Table 2).

Table 2 Primer sequences used to assess rainbow trout's expression of the genes related to immune response, stress, antioxidants, and digestion
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Intestinal microbiota analysis

At the end of the feeding trial, the intestines of 3 fish from each group were collected to obtain a diverse microbial flora (Liu et al. 2021a, b), and the total bacterial genomic DNA was extracted. The QIAamp DNA Extraction Mini Kit (Qiagen) was used for the extraction following the manufacturer’s protocol. The quality and concentration of DNA were assessed using a spectrophotometer (Qiaxpert; Qiagen). For bacterial diversity analysis, the V3–V4 regions of the 16S rRNA gene were amplified using the primer sequences F: 5'-CCTACGGGNGGCWGCAG-3' and R: 5'-GACTACHVGGGTATCTAATCC-3' with the SimpliAmp Thermal Cycler. The PCR conditions included pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 90 s for 35 cycles, and final extension at 72 °C for 10 min. Prior to NGS analysis, purification was performed using the “Qiaseq beads Clean-Up Kit, Cat. No: 180795” from Qiagen. Library preparation for the 16S rRNA V3–V4 amplicons was carried out using Illumina’s “Nextera XT DNA Library Prep Kit, Cat. No.: FC-131–1096,” and indexing was done using the “TG Nextera XT Index Kit v2 Set A (96 Indices, 384 Samples), Cat. No: TG-131–2001.” The concentrations of the libraries were determined using the iQuant™ dsDNA HS Assay Kit (ABP Bioscience, USA). Sequencing was performed on the Illumina iSeq100 platform in paired-end (PE) 2 × 150 mode (Langmead and Salzberg 2012). Raw data reads were classified into OTUs using the Kraken Metagenomics system, which assigns taxonomic labels to short DNA sequences with high accuracy and speed (Wood and Salzberg 2014). The Kraken confidence filter was set at 0.05, minimum hit group at 2, and the RefSeq 2022.02 database was used, which includes archaea, bacteria, fungi, protozoa, and viruses. OTUs were clustered and defined at ≥ 97% identity and 97% similarity, and those representing at least 0.005% of the total reads were retained. Alpha diversity measurements were estimated using the Shannon and Simpson indices. Additionally, beta diversity was determined by principal coordinates analysis (PCoA). A Venn diagram was created using the web tool at https://bioinformatics.psb.ugent.be/webtools/Venn/ to identify and visualize shared and unique OTUs among groups (Rimoldi et al. 2019).

Statistical analysis

Statistical analyses were conducted using the SPSS 20 statistical package program. Before analysis, the data underwent checks for normality and homogeneity of variance. Differences among groups were assessed using one-way analysis of variance (ANOVA). Upon finding significant differences (p < 0.05), means were compared using the Duncan multiple-range test. Results were expressed as means ± standard error.

Results

Phenolic composition of CD extract

Table 3 displays the phenolic component concentration of the CD extract. Thirty-five distinct phenolic compounds were analyzed in the CD extract used in the study. After analyzing the data, it was found that the CD extract contained 15 of the 35 phenolic chemicals.

Table 3 The level of phenolic components within the Cyanus depressus extract
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Growth performance

As delineated in Table 4, the CD1 and CD2 cohorts showcased the utmost concluding weight (FW) and the most minimal feed conversion ratio (FCR). Among the CD treatments, the CD2 group showed the most substantial increase in growth performance. Parameters of WG, BWI, SGR, DWG, TGC, and PER displayed no noteworthy variances between the control and CD05 groups (p > 0.05). Nonetheless, statistically significant differences (p < 0.05) were observed between these groups and the CD1 and CD2 groups. No mortality was recorded among the experimental groups throughout the 60-day feeding trial.

Table 4 Effects of dietary Cyanus depressus extract on growth performance in rainbow trout
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Gene expression related to digestion

Below are the alterations in expression levels of genes that stimulate TRP-LPZ-AML enzymes linked to the digestive system (Fig. 1). According to these results, overexpression of TRP was observed in the CD1 group compared to the control group (p < 0.05). Significant downregulation was observed in the CD05 and CD2 groups compared to the control group (p < 0.05). Regarding LPZ expression results, upregulation was observed in all groups compared to the control group (p < 0.05). While the AML gene expression levels showed upregulation in the CD1 and CD2 groups compared to the control group, the CD05 group showed downregulation (p < 0.05).

Fig. 1
figure 1

The impact of CD extract in the diet on the expression of genes related to digestion (TRP, LPZ, AML) in rainbow trout

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Gene expression associated with antioxidants

The levels of gene expression related to antioxidants, namely SOD, CAT, and GPX, were assessed, and variations in their expression levels are depicted below (Fig. 2). In SOD gene expression levels, overexpression was observed in the CD2 group compared to the control group (p < 0.05). Upregulation was observed in the CD1 group compared to the control group (p < 0.05). In the CD05 group, insignificant downregulation was observed, similar to the control group. CAT results showed overexpression in the CD2 group compared to the control group, while upregulation was observed in the CD1 group (p < 0.05). In the CD05 group, upregulation similar to the control group was observed. In GPx expression levels, overexpression was observed in the CD2 group compared to the control group, and upregulation was observed in the CD1 and CD05 groups compared to the control group (p < 0.05).

Fig. 2
figure 2

The impact of CD extract in the diet on the expression of genes related to antioxidants (SOD, CAT, GPx) in rainbow trout

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Gene expression related to stress and immunity

Expression levels of IL-1β, TNF-α, and HSP70 genes used in the study related to immunity and stress are provided below (Fig. 3). According to these results, overexpression of the IL-1β gene was observed in the CD2 group compared to the control group, and significant upregulation was observed in the CD1 group samples (p < 0.05). The CD05 group was found to be downregulated compared to the control group (p < 0.05). In TNF gene expression results, overexpression was observed in all groups compared to the control group (p < 0.05). In HSP70 gene expression results, overexpression was observed in the CD2 group compared to the control group, downregulation was observed in the CD05 group, and significant upregulation similar to the control group was observed in the CD1 group (p < 0.05).

Fig. 3
figure 3

The impact of CD extract in the diet on the expression of genes related to stress, and immune (IL-1β, TNF-α, HSP70) in rainbow trout

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Effects of Cyanus depressus extract on intestinal microbiota of rainbow trout

16S-rRNA sequencing analysis was utilized to investigate the changes in the gut microbiota of rainbow trout. The average read numbers, observed OTU counts, and alpha diversity indices of the experimental groups are presented in Table 5. In the study, the number of observed OTUs in the rainbow trout gut flora ranged from 172 to 220, with the lowest OTU count observed in the CD2 group and the highest in the CD1 group. An increase in OTU counts was observed in the CD05 and CD1 groups compared to the control group. The read numbers varied between 14.595 and 34.877 among the groups. Additionally, alpha diversity indices based on read numbers were used to measure diversity, revealing a decrease in Shannon and Simpson indices in the other groups compared to the control group (Table 5).

Table 5 The average read numbers, observed OTU counts, and alpha diversity of rainbow trout intestinal microbiota fed on diets containing different concentrations of CD extract for 60 days
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The PCoA analysis revealed a differentiation in the structure of the intestinal microbiota following the application of CD extract. As shown in Fig. 4a, the first principal coordinate (PC1) and second principal coordinate (PC2) accounted for 35% and 32% of the microbial community structure variance, respectively, indicating differences among the groups. The results from the PCoA plot illustrate that the control group was distinct from the groups fed with CD extract, clustering separately. Furthermore, it was observed that the groups fed with CD extract also clustered differently among themselves. According to the Venn diagram (Fig. 4b) illustrating sample similarity and overlap, a total of 687 unique non-singleton OTUs were identified in the rainbow trout gut microbiota. The numbers of unique OTUs in control, CD05, CD1, and CD2 were found to be 133, 168, 178, and 126, respectively.

Fig. 4
figure 4

The PCoA analysis and Venn diagram of rainbow trout intestinal microbiota fed on diets containing different concentrations of CD extract for 60 days

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A general overview of the taxonomic profile is presented in Fig. 5. Overall, the OTUs were assigned to 23 phyla, 37 classes, 83 orders, and 169 families. The most prevalent microbiota phyla and families in the intestine were determined. Proteobacteria and Firmicutes were found to be the most prevalent phyla in the study, constituting between 91.9 and 98.3% of the total gut microbiota (Fig. 5a). The relative abundance of Proteobacteria increased in the CD05 and CD2 groups compared to the control group, while it decreased in the CD1 group. In the CD1 group, Firmicutes was the most abundant phylum. Additionally, the third most abundant phylum, with the highest bacterial density, was Bacteroidota in the control, CD05, and CD1 groups, whereas it was Actinobacteria in the CD2 group at the phylum level. At the family level, Enterobacteriaceae in the control and CD2 groups, Erwiniaceae in the CD05 group, and Streptococcaceae in the CD1 group were found to have the highest relative abundances (Fig. 5b). The abundance of Enterobacteriaceae was observed to increase in the CD2 group compared to the control group.

Fig. 5
figure 5

The relative abundance of the most frequently identified phyla and family in the intestinal microbiota of rainbow trout fed diets containing varying concentrations of CD extract for 60 days

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Discussion

Numerous medicinal plants and their active compounds have been utilized in aquaculture for many years due to their advantageous effects, including promoting growth, stimulating appetite, enhancing immunity, improving antioxidant status, increasing immunity against diseases, and reducing stress in aquatic species (Bilen et al. 2021; Liu et al. 2021a, b; Dadras et al. 2023). To our knowledge, there has been no prior investigation into the impacts of Cyanus depressus on fish. Therefore, this research was designed to assess the growth performance, intestinal microbiota, and gene activity associated with the process of digestion, antioxidants, stress, and immunity in rainbow trout subjected to diets supplemented with Cyanus depressus.

Flavonoids and phenolic acids are significant phytochemicals derived from various plants (Nwozo et al. 2023). In vitro and in vivo pharmacological investigations have demonstrated that these compounds serve as natural growth promoters (Awad and Awaad 2017; Ahmadifar et al. 2021b; Tadese et al. 2022), antioxidants exhibiting potent free radical scavenging properties (Kaurinovic and Vastag 2019), and possess immune-stimulating effects (Tungmunnithum et al. 2018; Elumalai et al. 2020). The LC–MS/MS analysis results of the CD extract (Table 3) indicate the presence of various flavonoids and phenolic acids such as quinic acid, chlorogenic acid, vanillic acid, luteolin, fumaric acid, gallic acid, 4-OH-benzoic acid, and apigenin. Similar flavonoid and phenolic compounds in the CD extract have been reported in previous studies (Mishio et al. 2015; Boğa et al. 2016; Duman et al. 2022). Also, various studies report the positive effects of these compounds on the health and performance of aquatic animals (das Neves et al. 2021; Ghafarifarsani et al. 2023; Jin et al. 2023; Zhang et al. 2023; Lin et al. 2024). These compounds found in the CD extract, as in many plants, have been reported to potentially reduce inflammatory propagation by inhibiting nuclear factor kappa B (NF-κB), Janus kinase (JAK), and mitogen-activated protein kinase (MAPK) pathways, thereby potentially preventing inflammation-associated tissue damage (Vendrame and Klimis-Zacas 2015; Yin et al. 2021; Alharbi et al. 2022; Hamsalakshmi et al. 2022). Additionally, it has been noted that these compounds may contribute to the clearance of excessive cellular free radicals by concurrently upregulating several important antioxidant factors, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1), either dependent or independent of nuclear factor erythroid 2 (NF-E2)–related factor 2 (Nrf2) pathways (Bajpai et al. 2018; Iranshahy et al. 2018; Bhattacharjee and Dashwood 2020). On the other hand, these compounds have been reported to potentially regulate and support metabolic homeostasis of lipids and glucose by modulating glucose release and absorption, as well as lipid synthesis, through activation of the AMP-activated protein kinase (AMPK) pathway (Marín-Aguilar et al. 2017; Herath et al. 2018; Pietrzyk et al. 2021). On the other hand, the growth-promoting effects of flavonoids and phenolic acids observed in animals may be attributed to various mechanisms resulting from the diverse biological activities of phytochemicals (Valenzuela-Grijalva et al. 2017). For instance, it has been reported that various biological activities related to intestinal function, such as increased digestive secretions, nutrient absorption, and intestinal morphology improvement, could enhance the growth performance of animals (Mahfuz et al. 2021; Waqas et al. 2023).

The growth efficiency of fish stands as a critical determinant for successful production in aquaculture (Bilen et al. 2021). The findings from the present study indicate a beneficial impact on growth performance in fish following a 60-day feeding period with diets incorporating varying levels of CD extract. This could be attributed to the bioactive constituents (Table 3), which increased the attractiveness of the feed, leading to increased intake of the diets. Previous studies have reported that compounds such as chlorogenic acid, luteolin, gallic acid, and fumaric acid found in CD extract have positive effects on the growth performance of aquatic animals (das Neves et al. 2021; Ghafarifarsani et al. 2023; Jin et al. 2023; Zhang et al. 2023; Lin et al. 2024). Moreover, these bioactive compounds may enhance the production of digestive enzymes (Fig. 1), consequently improving feed digestibility and nutrient uptake. The enhancements observed in fish growth performance might be attributed to the activation of digestive enzymes and improved nutrient absorption (Jeney et al. 2015; Yousefi et al. 2021). Establishing the suitable dosage of medicinal herbs is a critical aspect of their administration. Typically, guidance on the optimal concentration of medicinal herbs is derived from in vivo experiments, often using a “trial and error” method (Adel et al. 2020). In our investigation, among the assessed concentrations, the CD 2% treatment displayed the most advantageous impact on growth rate, implying that a particular dosage level influences the optimal condition of growth performance. As of now, there is no available data regarding the function of CD as a growth enhancer in the rainbow trout aquaculture sector. Nonetheless, previous research has documented the enhancement of rainbow trout growth performance with the use of various herbal supplements such as Coriandrum sativum (Farsani et al. 2019), Malvae sylvestris (Rashidian et al. 2020b), Polygonum minus (Adel et al. 2020), Plantago lanceolata (Elbesthi et al. 2020), Ziziphora clinopodioides (Oroji et al. 2021), Viscum album (Yousefi et al. 2021), Allium hirtifolium (Ghafarifarsani et al. 2022), Astragalus caudiculosus (Sönmez et al. 2022b), and Taraxacum officinale (Mostafavi et al. 2022). These investigations reported comparable growth-promoting effects to our chosen herb in rainbow trout. Conversely, Sönmez et al. (2022a) noted no influence on the growth performance of rainbow trout when administered extracts from peel (Punica granatum) and Veratrum (Veratrum album). Likewise, Salem et al. (2021) documented no differences in growth performance among rainbow trout supplied with extracts from Taraxacum officinalis and Usnea barbata.

It is well recognized that medicinal plants can boost the function of digestive enzymes, thereby enhancing the digestibility and accessibility of nutrients, ultimately leading to improved food utilization, protein synthesis, and growth rates (Citarasu 2010; Awad et al. 2012; Beltrán et al. 2020). The heightened functionality of digestive enzymes contributes to achieving better fish growth (Bilen et al. 2021). The findings of our study demonstrated that CD extract effectively enhances the digestive capabilities of rainbow trout. In the present study, CD extract notably influenced the expression of genes responsible for promoting digestive enzyme activity. These observations suggest that the CD extract elicits beneficial effects on the appetite of rainbow trout, consequently augmenting digestive efficiency. This result might be explained by the fact that CD extract contains a variety of flavonoids and phenolic acids. Also, bioactive compounds in plants typically serve as tonics for the secretion of digestive enzymes and exert a direct effect on intestinal microflora (Vijayaram et al. 2022). Previous studies have reported that flavonoids and phenolic acids stimulate the activity of digestive enzymes (Ahmadifar et al. 2021a, b; Tadese et al. 2022). Our study aligns with previous research, which has consistently reported improvements in the digestive enzyme activity of rainbow trout through the use of various medicinal plants, including Quercus brantii (Rashidian et al. 2020a), Dioscorea oppositifolia (Wang et al. 2020), Malvae sylvestris (Rashidian et al. 2020b), Viscum album (Yousefi et al. 2021), Ziziphus jujuba (Liu et al. 2021a, b), and Taraxacum officinale (Mostafavi et al. 2022). These findings align closely with our research. The congruence in findings across different studies reinforces the notion that these medicinal plants possess a shared potential to positively impact the digestive capabilities of rainbow trout.

Antioxidants have a crucial function in combating oxidative damage and are often influenced by the conditions of culture (Ashour et al. 2021). The principal antioxidants within the antioxidant defense system encompass SOD, GPx, and CAT. In fish, the activities of antioxidant enzymes are linked to nutritional aspects (Habte-Tsion et al. 2020). In this study, the gene expression profiles of SOD, CAT, and GPx exhibited a significant rise across all groups in comparison to the control group. It is widely acknowledged that the beneficial effects of plant extracts are related to their antioxidant properties (Ghafarifarsani et al. 2022). The noted elevation in SOD, CAT, and GPx levels indicates that CD extract contains bioactive compounds capable of modulating the immunity of rainbow trout. The phenolic compound concentration found in CD extract, as presented in Table 3, provides evidence to support this conclusion. These results indicate that CD extract may enhance the capacity of the antioxidant system and shield organisms from oxidative harm. Additionally, previous studies have reported that compounds such as chlorogenic acid, luteolin, and gallic acid found in CD extract have positive effects on the antioxidant capacity of aquatic animals (Ghafarifarsani et al. 2023; Jin et al. 2023; Zhang et al. 2023; Lin et al. 2024). While there is a lack of prior reports on the effects of CD on fish antioxidant parameters, the current findings align with studies conducted using other herbal substances. Consistent with our findings, Ghafarifarsani et al. (2022) documented a significant rise in SOD, CAT, and GPX activity in rainbow trout fed Allium hirtifolium compared to the control group. In a separate investigation, it was reported that the addition of Ziziphus jujuba extract to diets could increase the expressions of SOD and CAT in rainbow trout (Liu et al. 2021a, b). Similarly, in the study by Mostafavi et al. (2022), it was reported that the addition of Taraxacum officinale, flower extract to the diet of rainbow trout up to 4 g/kg improved the SOD, CAT, and GPx activities. These findings may be linked to the existence of bioactive compounds possessing antioxidant properties identified in medicinal plants (Citarasu 2010).

Pro-inflammatory cytokines such as TNF-α and IL-1β are vital for the modulation of the innate immune response, and thus, several studies proposed that studying their levels of expression could be used to predict changes in immune responses (Hoseinifar et al. 2020, 2022). In our investigation, notable variations were noted in the expression of genes associated with immunity among treatments, with the highest expression levels of TNF-α and IL-1β observed in rainbow trout receiving a diet supplemented with CD extract. Increasing in the TNF-α and IL-1β levels may be attributed to the activity of the compounds in CD extract like flavonoids and antioxidants. Indeed, previous studies have reported that some of the phenolic compounds listed in Table 3 strengthen immunity (Jin et al. 2023; Zhang et al. 2023; Lin et al. 2024). Various studies indicate that the expression of genes related to immunity in rainbow trout is influenced by medicinal plants. For example, Adel et al. (2020) found that incorporating Polygonum minus extract into the diet significantly boosted gene expressions related to immunity and disease resistance in rainbow trout. Similarly, Hoseinifar et al. (2020) documented that adding 2% Aloysia citrodora meal to the diet notably increased the transcriptional levels of IL-1β and TNF-α. Likewise, Shekarabi et al. (2021) noted substantial increases in TNFα and IL-1β cytokine gene expression levels in rainbow trout after a bacterial challenge when provided with a diet incorporating 3-g/kg Taraxacum officinale extract compared to other treatments and the control group. The incorporation of caper extract (Capparis spinosa) into the rainbow trout diet led to heightened transcription of cytokine genes (TNF and IL-1), bolstering the immune response, as detailed by Bilen et al. (2016). Yousefi et al. (2022) also noted the positive impact of dietary Hyssopus officinalis extract inclusion on the expression of IL-1β and TNF-α genes in rainbow trout. In our current study, the elevated levels of selected immune genes indicate the immunostimulatory effect of dietary CD at the molecular level in rainbow trout.

In general, HSP70 serves multiple roles that enhance the immune response and aid in safeguarding cytoplasmic components during diverse stressful situations (Basu et al. 2002). Numerous investigations have shown that medicinal plants upregulate the relative gene expression of HSP70 in species such as caspian roach (Paknejad et al. 2020), rainbow trout (Sheikhzadeh et al. 2019), red swamp crayfish (Liu et al. 2020), Pacific white shrimp (Lei and Zeng 2008; Anirudhan et al. 2021), hybrid grouper (Sun et al. 2018), and golden pompano (Zhou et al. 2015). Additionally, some studies have reported that medicinal plants downregulate (Tan et al. 2017; Rajabiesterabadi et al. 2020; Espinosa et al. 2020; Giri et al. 2020) or do not alter (Giri et al. 2019; Khieokhajonkhet et al. 2023) HSP70 gene expression. In our current study, the increased HSP70 expression in fish fed with 2 g/kg of CD extract may be considered a positive response that enhances the cells’ ability to cope with stress factors. Furthermore, Liu et al. (2021b) reported that upregulated HSP70 expression results in increased immunity. The increased expression of immune-related TNFα and IL-1β genes in our study supports this finding. It is possible that certain chemical compounds in the CD extract contributed to the increased HSP70 expression observed in the fish. Additionally, different application doses of the CD extract in the fish diet could have also played a role. However, additional research is warranted to validate these hypotheses, as there is a lack of information regarding the impacts of dietary plant components on stress and HSP70 pathways and functions in rainbow trout.

Biotic factors such as genotype, physiological status, pathobiology, and lifestyle, along with abiotic factors like environmental conditions, have the potential to impact the composition and diversity of the fish gut microbiota (Yukgehnaish et al. 2020). Several investigations have demonstrated notable impacts of medicinal plants and their extracts on the intestinal microbiota (Ye et al. 2019; Liu et al. 2021a; Zhang et al. 2022; Kamble et al. 2024). In this study, considering the positive results obtained from growth, digestion, antioxidant, and immune parameters, it is believed that the CD extract affects the fish intestinal microbiota. However, when the diversity of the intestinal microbiota obtained in the study was evaluated, it was unexpectedly observed that bacterial diversity narrowed but bacterial density increased an elevation in comparison to the control group. In our investigation, a different result was obtained from other studies reporting an increase in bacterial diversity of the intestinal microbiota with dietary plant supplements (Desai et al. 2012; Xu et al. 2022; Chen et al. 2024; Kamble et al. 2024). However, the lack of an effect of the CD extract used in our study on bacterial diversity could be viewed as a beneficial result, as decreased diversity might entail decreased competition for opportunistic or invasive pathogens. The studies by Apper et al. (2016) and Rimoldi et al. (2018) support our findings.

In this study, Proteobacteria and Firmicutes were found to be the two most common phyla in the intestines of rainbow trout. Despite the observed plasticity in bacterial diversity and composition, most studies resembling our findings have indicated that the microbiome is primarily influenced by these two phyla (Mougin et al. 2023; Singh et al. 2024). However, contrasting results from other investigations have shown these two phyla to be present in considerably lower quantities compared to more prevalent organisms such as Bacteroidota, Actinobacteria, Fusobacteria, and Tenericutes (Wang et al. 2020; Hines et al. 2021; Kamble et al. 2024). Moreover, the ratio of Firmicutes to Bacteroidota (F/B) in the intestinal microbiota is regarded as a key biomarker linked to weight regulation and the maintenance of intestinal balance in humans (Zhao et al. 2023). The changes observed in the abundance of Firmicutes/Bacteroidota in fish fed with CD extract in this study can indicate a potential association with weight gain.

At the family level, it was observed that the dominant families changed in the groups fed with CD extract (especially CD05 and CD1 groups), and their abundances increased compared to the control group. The dominance or increased abundance of different families in each group is thought to be due to the different doses of CD extract affecting the fish microbiota. Erwiniaceae, Enterococcaceae, and Streptococcaceae are bacterial families commonly found in both animal and fish intestinal floras. Similar to our study, various feeding trials in fish have reported that these bacterial families can become dominant (Hartviksen et al. 2014; Araújo et al. 2015; Hines et al. 2022). It is assumed that Erwiniaceae bacteria play a nutritious role by hydrolyzing plant biomass and fixing nitrogen. Additionally, Erwiniaceae bacteria are known to produce antibiotics (Cambronero-Heinrichs et al. 2023). Members of the Streptococcaceae and Enterobacteriaceae families include various bacterial species involved in the anaerobic breakdown of complex carbohydrates. This breakdown yields short-chain fatty acids (SCFAs) as end products, which are readily assimilated by the host and enhance food energy utilization efficiency (Rimoldi et al. 2018). The positive growth data obtained in the CD1 and CD2 groups support this. Furthermore, in all groups (especially CD1 and CD2 groups) compared to the control group, the abundance of the Sphingomonadaceae family increased. Some bacteria in the Sphingomonadaceae family are known to break down aromatic compounds that can have negative effects on fish (Asaf et al. 2020; Hao et al. 2020). Ma et al. (2022) reported that the Vicia faba extract increased the abundance of Sphingomonadaceae in the intestine, promoting the growth of grass carp. Similarly, it is estimated that in our study, the CD extract possibly promoted the growth of rainbow trout by increasing the abundance of Sphingomonadaceae in the intestine. Further studies are needed to evaluate the effects of CD extract on fish.

Conclusion

In conclusion, the findings of this study indicate that Cyanus depressus (CD) extract holds considerable potential for enhancing various aspects of rainbow trout physiology and health. Notably, the dietary supplementation of CD extract demonstrated promising outcomes in promoting growth, enhancing digestive enzyme activity, boosting antioxidant capacity, stimulating the immune response, and influencing the composition of the gut microbiota. Specifically, the supplementation with 2 g/kg of CD extract appeared to be a beneficial strategy for rainbow trout in this study. However, further investigations are warranted to delve deeper into the underlying mechanisms behind these effects and to optimize the utilization of CD extract in aquaculture practices. This study adds to the expanding literature on the utilization of medicinal plants in aquaculture, providing insights into potential strategies for improving fish health and productivity.