Comparative effects of free and nanomicelle silymarin supplementation on growth, antioxidant status, and fatty acid composition in caspian trout (Salmo caspius) larvae.

Comparative effects of free and nanomicelle silymarin supplementation on growth, antioxidant status, and fatty acid composition in caspian trout (Salmo caspius) larvae - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Sci Rep . 2026 Jun 3;16:17142. doi: 10.1038/s41598-026-42377-3 Comparative effects of free and nanomicelle silymarin supplementation on growth, antioxidant status, and fatty acid composition in caspian trout ( Salmo caspius ) larvae Seyed Reza Miri Shaktai 1 , Ashkan Banan 2, ✉ , Seyed Ali Johari 1 , Saeed Keyvanshokooh 3 , Hadis Mansouri Taee 2 Author information Article notes Copyright and License information 1 Department of Fisheries, Faculty of Natural Resources, University of Kurdistan, Sanandaj, Kurdistan Iran 2 Department of Fisheries and Environmental Sciences, Faculty of Natural Resources, Lorestan University, P.O. Box 465, Khorramabad, Lorestan 68151-44316 Iran 3 Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khuzestan Iran ✉ Corresponding author. Received 2025 Sep 27; Accepted 2026 Feb 25; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13234121  PMID: 42236756 Abstract This study evaluated the comparative effects of free silymarin (FS) and nanomicelle silymarin (NS) supplementation on growth, antioxidant defenses, and fatty acid composition in Caspian trout ( Salmo caspius ) larvae. Larvae (0.27 ± 0.01 g) were randomly assigned to triplicate groups and fed either control, FS (200 mg kg⁻¹), or NS (200 mg kg⁻¹) for 60 days. Growth performance indices, antioxidant enzyme activities, fatty acid composition, and leukocyte differentials were assessed. Results showed that NS supplementation significantly improved final weight, weight gain, and specific growth rate compared to both FS and control groups ( p < 0.05), while FS had no significant effect on growth. NS-fed fish exhibited higher glutathione peroxidase (GPx) activity, elevated whole-body polyunsaturated fatty acids (PUFA), and increased deposition of long-chain n-3 fatty acids, particularly EPA and DHA. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, and leukocyte differentials did not differ significantly among treatments. These findings demonstrate that nanomicelle encapsulation can markedly improve the biological performance of silymarin in Caspian trout larvae, supporting improved growth and antioxidant resilience while enriching physiologically important fatty acids. Nanomicelle delivery systems may therefore represent a practical strategy to optimize phytochemical use in salmonid aquaculture, particularly during early life stages when fish are most vulnerable to stress. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-026-42377-3. Keywords: Aquaculture, Fatty acid profile, Nanoencapsulation, Oxidative stress, Silymarin, Caspian trout larvae Subject terms: Biochemistry, Biotechnology, Physiology, Zoology Introduction The Caspian Sea—the world’s largest inland water body—has remained ecologically isolated from the global ocean for millennia, fostering a distinctive community of endemic taxa 1 , 2 . Among its most emblematic inhabitants is the Caspian trout ( Salmo caspius ), an anadromous species that is larger and heavier, and exhibits faster growth than members of the brown trout complex 3 . It is noteworthy that the Caspian trout, referred to here as Salmo caspius Kessler, 1877, is considered in several taxonomic and phylogenetic studies to represent an anadromous ecotype or subspecies ( Salmo trutta caspius ) of the broader brown trout ( Salmo trutta ) complex 4 . For consistency with regional aquaculture and fisheries literature, the name Salmo caspius is employed throughout this manuscript. In Iran, it occupies a dual role as a culturally iconic species and a promising candidate for aquaculture expansion, bridging conservation concerns with opportunities for sustainable production 5 . In recent decades, natural populations of Caspian trout have experienced a sharp decline due to overfishing, dam construction, poaching, drought, pollution, and the destruction of spawning habitats 6 – 8 . Recent large-scale assessments further quantify the severity of these trends: annual catches of economically valuable fish species along the southern Caspian coast have declined by approximately 50% between 2001 and 2021, and the number of native species classified as threatened has increased by about 67% over the same period. Caspian trout is currently listed as endangered, emphasizing the urgency of improving early-life survival 1 . Hatchery-based stocking programs have long been employed to counteract these pressures, yet the sustainability of both wild enhancement and aquaculture production ultimately depends on improving larval survival, growth, and resilience under captive conditions 9 . Early life stages of Caspian trout exhibit limited digestive capacity, elevated metabolic demands, and heightened sensitivity to nutritional and oxidative stress, all of which contribute to substantial larval mortality under hatchery conditions. Developmental investigations—such as those by Najafpour et al. and Pourkhazaei et al. examining S. caspius and its hybrids—underscore the species’ distinctive physiological constraints and reinforce the need for enhanced larval-stage nutritional strategies 3 , 10 . Improving early-life robustness is therefore essential for both restocking programs and aquaculture development. Aquaculture has become a cornerstone of global food security, supplying a high-quality source of animal protein and essential nutrients for human consumption 11 . With the world population projected to reach 9.7 billion by 2050, demand for aquaculture products is expected to rise substantially, driving the rapid expansion of intensive and super-intensive farming systems 12 . While such intensification improves production efficiency, it also imposes significant challenges, including heightened stress, immunosuppression, and increased susceptibility to disease outbreaks, all of which contribute to economic losses 13 . Moreover, the heavy reliance on antibiotics for disease prevention and treatment has accelerated the emergence of antimicrobial resistance, underscoring the urgent need for sustainable alternatives to support fish health and performance 14 . Like many cultured fish, Caspian trout are vulnerable to stressors associated with intensive aquaculture, such as high stocking densities, handling, and suboptimal water quality. These conditions can suppress immunity, elevate oxidative stress, and impair growth 15 . With increasing interest in intensive farming of Caspian trout 16 , feed-based strategies that improve growth, stress resistance, and product quality have become a research priority. Sustainable disease management in aquaculture emphasizes functional feed additives as environmentally friendly alternatives to chemotherapeutics 17 . In recent years, dietary phytochemicals have attracted considerable attention due to their antioxidant, immunomodulatory, and growth-promoting properties 18 . Among medicinal plants, milk thistle ( Silybum marianum ) is notable for its bioactive flavonolignan complex, collectively known as silymarin. Silymarin is widely recognized as a hepatoprotective compound with strong antioxidant and cytoprotective effects in both terrestrial animals and fish 19 , 20 . However, the practical application of silymarin in aquafeeds is limited by its poor water solubility and low bioavailability 21 . Advances in nanotechnology, particularly nanomicelle encapsulation, provide solutions by enhancing intestinal absorption, systemic distribution, and tissue retention of bioactive compounds 22 – 24 . A growing body of evidence highlights the beneficial effects of silymarin supplementation across aquatic and terrestrial species. In fish, dietary silymarin has been shown to enhance growth, antioxidant defenses, and immune function in Nile tilapia ( Oreochromis niloticus ), large yellow croaker ( Larimichthys crocea ), thinlip mullet ( Liza ramada ), European seabass ( Dicentrarchus labrax ), and Hefang crucian carp ( Carassius auratus ) 19 , 25 – 28 . Encapsulation technologies further strengthen these effects by improving silymarin’s bioavailability and physiological impact. For example, Faryadi et al. reported that nano-silymarin supplementation enhanced nutrient digestibility, performance, and egg quality in laying hens compared with powdered or lecithinized forms 29 . Even with conventional formulations, positive outcomes have been reported: Armanini et al. showed that silymarin inclusion in broiler diets increased polyunsaturated fatty acid (PUFA) deposition and improved oxidative stability of meat 30 . These effects were linked to silymarin’s antioxidant and hepatoprotective properties. Beyond growth and immunity, silymarin influences lipid metabolism and tissue fatty acid composition. Yao et al. demonstrated that silymarin supplementation reduced visceral lipid accumulation in large yellow croaker larvae by downregulating lipogenic genes and upregulating lipid oxidation pathways 25 . Shahin et al. similarly found that silymarin increased long-chain n-3 PUFA deposition, particularly EPA and DHA, while reducing saturated fatty acids in D. labrax larvae 26 . In poultry, Armanini et al. reported higher muscle PUFA levels and oxidative stability following silymarin supplementation 30 . Moreover, under stress conditions, Veisi et al. showed that nano-silymarin provided greater protection against oxidative damage in Nile tilapia than free silymarin, underscoring the importance of delivery method 31 . Despite this promising evidence, limited information is available on the comparative effects of free versus nanoencapsulated silymarin in salmonid larvae, particularly Caspian trout. Given the species’ conservation and aquaculture value, and the heightened metabolic demands and vulnerability to stress during early life stages, optimizing dietary strategies is critical. To address this gap, the present study aimed to evaluate the effects of dietary free silymarin (FS) and nanomicelle silymarin (NS) supplementation on growth performance, oxidative status, fatty acid profile, and leukocyte differentials of Caspian trout larvae. By focusing on this sensitive stage, we sought to determine whether nanomicelle technology provides measurable advantages over conventional formulations in supporting growth and physiological resilience in a species of both ecological and aquacultural importance. Materials and methods Ethics statement All experimental procedures were approved by the Research Ethics Committee of the University of Kurdistan, and were conducted in accordance with the institutional guidelines, national regulations, and the ARRIVE guidelines. All fish handling and experimental protocols complied with the principles of the Animal Welfare Act and the Interagency Research Animal Committee 32 . A certificate of ethical approval is available upon request. Experimental fish and rearing conditions Caspian trout ( S. caspius ) larvae were obtained and reared at the research facility of Abzi Exir Kosar Fish Breeding and Rearing Company in Aleshtar, Iran. Throughout the trial, water quality parameters were maintained at dissolved oxygen levels of 8.1 ± 0.3 mg L⁻¹, pH of 7.74 ± 0.06, and a temperature of 11.63 ± 0.05 °C. Total ammonium nitrogen (TAN) was measured 30 min after feeding, daily during the first week and weekly thereafter, and remained low and stable throughout the experiment (0.04 ± 0.01 mg L⁻¹). No signs of water quality deterioration or abnormal fish behavior were observed during the trial. At the start of the experiment, biometric measurements were performed, and larvae had an average initial weight of 0.27 ± 0.01 g. Fish were randomly assigned to three dietary treatments, each with three replicate troughs containing 500 larvae (1500 larvae per treatment). The 60-day feeding trial was conducted in 180 L fiberglass troughs (total capacity: 200 L) supplied with flow-through freshwater, which provided a complete water exchange every hour. Diet preparation and feeding regime The dietary treatments consisted of: Control diet (no silymarin supplementation), Free silymarin diet (FS)—supplemented with 200 mg kg⁻¹ free silymarin powder (Sami Labs, India), Nanomicelle silymarin diet (NS)—supplemented with 200 mg kg⁻¹ nanomicelle silymarin ( Sinalive ® , Exir Nano Sina Co., Iran). The inclusion level of 200 mg kg⁻¹ was selected for both FS and NS based on earlier recommendations by Veisi et al. 31 , Xiao et al. 33 , and Wang et al. 34 , which demonstrated that this dosage reliably induces a strong antioxidant response in fish without adverse physiological effects, and is further supported by larval-stage studies reporting significant improvements in growth, antioxidant capacity, and lipid metabolism at comparable dietary levels (e.g., 150 mg kg⁻¹ in large yellow croaker larvae and ≥ 200 mg kg⁻¹ in seabass larvae) 25 , 26 . The NS used in this study has been previously characterized by Soleimani et al., showing that the formulation consists of spherical nano-micelles with an average particle size of approximately 8–9 nm, a polydispersity index of < 0.25, and more than 3000-fold increased aqueous solubility due to micellar encapsulation, which also protects silymarin from gastric degradation 35 . The formulation is a commercially manufactured product, and no additional batch-specific physicochemical characterization (e.g., particle size distribution or encapsulation efficiency) was performed prior to dietary incorporation; therefore, the present study relied on supplier documentation and published characterization data, and official nanoscale certification by the Iran Nanotechnology Initiative Council (INIC), together with qualitative confirmation of nanoscale morphology by Transmission electron microscopy (TEM). TEM analysis of nanomicelle-encapsulated silymarin was performed using a Philips EM-208 S transmission electron microscope, as shown in Fig. 1 . FS and NS were dissolved in 100 mL ethanol and tepid water, respectively, and uniformly sprayed onto the required amount of basal diet. To ensure homogenous distribution, each solution was applied gradually while the diet was mixed in a stainless-steel ribbon mixer for 10 min. The volume of 100 mL per kg diet was selected to provide complete surface coverage without deforming the pellets. After spraying, the diets were spread as a thin layer on stainless trays and vacuum freeze-dried at − 55 °C and 0.04 mbar for 24 h to remove ethanol or water. Following freeze-drying, the diets were coated with a 10% (w/v) bovine gelatin solution (20 mL per kg diet) using a drum coater to minimize leaching. The coated diets were air-dried at room temperature for 12 h, stored in airtight polyethylene bags, and kept at 4 °C in the dark until use. All diets were prepared weekly, and the control diet underwent identical processing without silymarin. The proximate composition of the basal diet included crude protein (49%), crude fat and oils (15.7%), crude fiber (3%), crude ash (10.6%), and phosphorus (3.65%). The basal diet was further analyzed for fatty acid composition (following the same analytical procedures described in Sect.  2.5), and the results are provided in Table 1 . Fig. 1. Open in a new tab Transmission electron microscopy (TEM) of nanomicelle-encapsulated silymarin (NS). Table 1. Fatty acid composition (% of total fatty acids) of the basal diet and whole-body of Caspian trout larvae fed control (CTRL), free silymarin (FS), or nanomicelle silymarin (NS) diets for 60 days. Parameters Basal diet Experimental groups CTRL FS NS C14:0 4.39 ± 0.11 5.53 ± 0.58 6.36 ± 0.34 7.26 ± 0.17 C16:0 13.21 ± 0.42 20.93 ± 0.49 b 28.03 ± 1.73 a 24.90 ± 0.77 ab C18:0 2.65 ± 0.09 1.70 ± 0.37 ab 0.73 ± 0.08 b 2.06 ± 0.29 a C20:0 0.35 ± 0.26 0.46 ± 0.14 0.33 ± 0.33 0.36 ± 0.08 16:1n-7 4.76 ± 0.17 2.90 ± 0.32 3.36 ± 0.78 4.83 ± 0.20 18:1n-9 28.44 ± 1.04 17.26 ± 0.61 17.40 ± 2.26 21.33 ± 0.40 18:2n-6 12.69 ± 1.12 16.50 ± 0.17 16.73 ± 2.88 19.70 ± 0.36 20:4n-6 0.8 ± 0.62 1.03 ± 0.17 b 1.73 ± 0.42 ab 2.46 ± 0.08 a 18:3n-3 4.43 ± 0.24 2.16 ± 0.08 b 1.50 ± 0.11 c 3.73 ± 0.14 a 20:5n-3 7.12 ± 1.56 1.80 ± 0.05 b 1.66 ± 0.40 b 3.03 ± 0.14 a 22:5n-3 1.33 ± 0.04 0.833 ± 0.04 b 1.80 ± 0.28 a 1.56 ± 0.08 ab 22:6n-3 4.92 ± 0.52 6.90 ± 0.26 7.43 ± 0.71 8.86 ± 0.34 Total SFA 20.6 ± 0.11 28.63 ± 0.56 b 35.46 ± 1.35 a 34.60 ± 0.95 a Total MUFA 33.2 ± 0.04 20.16 ± 0.74 b 20.76 ± 1.80 b 26.16 ± 0.60 a Total PUFA 31.29 ± 0.18 29.23 ± 0.13 b 30.86 ± 1.93 b 39.36 ± 0.87 a n-3 17.80 ± 0.12 11.70 ± 0.33 b 12.40 ± 0.65 b 17.20 ± 0.43 a n-6 13.49 ± 0.07 17.53 ± 0.34 18.46 ± 2.47 22.16 ± 0.44 n3/n6 1.31 ± 0.01 0.66 ± 0.03 0.70 ± 0.11 0.77 ± 0.00 Open in a new tab Values are expressed as mean ± standard error. Different superscript letters within the same row denote statistically significant differences among treatments (one-way ANOVA followed by Tukey’s HSD test, P < 0.05). Fish were fed daily at an average rate of 3.5% body weight, divided into 8–10 short feeding bouts (≤ 15 min each) to limit water exposure and reduce potential leaching. Uneaten feed and waste were removed daily by siphoning. The feeding ration was adjusted weekly based on average body weight measurements from a subsample of 15 larvae per trough. Mortalities were monitored and removed daily. Cumulative mortality was calculated at the end of the 60-day trial. Overall mortality remained below 5% and did not differ significantly among dietary treatments ( P > 0.05). Growth performance assessment Growth parameters, including final weight (FW), weight gain (WG) (Eq. 1 ), weight gain percentage (WGP) (Eq. 2 ), specific growth rate (SGR) (Eq. 3 ), and daily growth rate (DGR) (Eq. 4 ), were calculated on the final day of the trial using the following equations 36 : 1 2 3 4 Fatty acid composition analysis Following blood collection, fish were euthanized by rapid cervical transection following anesthesia with clove oil (120 mg L⁻¹), consistent with the recommendations of Nickum et al. 32 , and whole-body samples were stored at − 80 °C until analysis. Fatty acid composition was analyzed using pooled samples of three larvae per replicate to ensure sufficient tissue mass and minimize individual-level variability. For each trough, two independent pooled samples were prepared and analyzed separately, and the mean of the two analytical measurements was used as the representative value for each trough in statistical analyses. Total lipid content was extracted following the method of Folch et al. using chloroform: methanol (2:1, v/v) 37 . Samples (≤ 1 g) were homogenized in solvent and left to extract lipids for 24 h. The solvent was evaporated under a nitrogen stream in a water bath. Lipid extracts were saponified with 5 mL sodium hydroxide (2%), followed by methylation with 3 mL boron trifluoride–methanol (20%) at room temperature. Fatty acid methyl esters (FAMEs) were recovered using 1 mL hexane and 1 mL sodium chloride (30%) 38 . FAMEs were analyzed via gas chromatography (Philips GC, FID detector, BPX70 column, 30 m × 0.25 mm × 0.22 μm) with helium as the carrier gas. Oven temperature was programmed from 160 °C (5 min) to 180 °C at 20 °C/min, then to 200 °C at 1 °C/min, and finally to 230 °C at 30 °C/min, held for 5 min. Fatty acids were identified by comparison to retention times of standard mixtures. Oxidative and antioxidant status assays Whole-body homogenates were prepared from fed larvae (1 g tissue per pooled sample; two independent pooled samples per trough, each consisting of three larvae) in ice-cold buffer (20 mM phosphate buffer, pH 7.4; 1 mM EDTA; 0.1% Triton X-100). Homogenates were centrifuged at 30,000 × g for 30 min at 4 °C. Supernatants were collected, debris discarded, and samples stored at − 80 °C until analysis. All oxidative and antioxidant status assays were performed using commercial kits (Kiazist, Iran) according to the manufacturer’s instructions. Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined using an adrenaline oxidation assay based on the inhibition of adrenaline autoxidation to adrenochrome at pH 10.2. The increase in absorbance at 480 nm was monitored kinetically for 5 min. The assay sensitivity was approximately 0.5 U/mL, with a linear detection range of 0.5–30 U/mL. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of adrenaline autoxidation under the assay conditions. SOD activity was normalized to total protein content, and expressed as international units (IU) per mg tissue protein 39 . Glutathione peroxidase (GPx; EC 1.11.1.9) activity was assayed based on a coupled enzymatic reaction in which GPx reduces H₂O₂, and the resulting oxidized glutathione is recycled by glutathione reductase with concomitant NADPH oxidation. The decrease in absorbance at 340 nm was monitored kinetically over 5 min. According to the manufacturer, the assay sensitivity is 0.5 mU/mL (equivalent to 0.5 nmol/min/mL). GPx activity was normalized to total protein content, and reported as milliunits (mU) per mg tissue protein, where 1 mU corresponds to 1 nmol of NADPH oxidized per minute under the assay conditions 39 . Malondialdehyde (MDA) level was quantified based on the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions. The pink-colored MDA-TBA adduct was measured colorimetrically at 532–560 nm (peak at 546 nm). The manufacturer-reported sensitivity of the assay is 10 µM, with a dynamic detection range of 20–100 µM. MDA levels were normalized to total protein content, and expressed as nmol per mg tissue protein 39 . Leukocyte differential counts At the end of the feeding trial, blood samples were collected from the caudal vein of anesthetized fish using heparinized syringes. One to two blood smears per fish were prepared immediately, air-dried, and fixed in 95% methanol for 5 min. Slides were stained with Giemsa and examined under light microscopy to determine the relative proportions of lymphocytes, neutrophils, eosinophils, and monocytes (differential white blood cell count) 40 . Statistical analysis The experiment followed a completely randomized design, with each trough ( n = 3 per treatment) considered the experimental unit. Dietary treatments consisted of a basal diet (control) and two additive-supplemented diets, each applied to three replicate troughs. For all analyses, the mean value of each trough was used, ensuring that individual fish were not treated as independent replicates. Data were first tested for normality using the Kolmogorov–Smirnov test and for homogeneity of variances using Levene’s test. When assumptions were met, differences among treatments were assessed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test. All statistical analyses were conducted using SPSS version 27 (IBM Corp., Armonk, NY, USA). Results are expressed as mean ± standard error (SE), and statistical significance was accepted at P < 0.05 41 . Results Growth performance Dietary supplementation with NS had a significant impact on growth performance in Caspian trout larvae over the 60-day feeding period (Fig. 2 ). Fish receiving the NS diet exhibited significantly higher final weight (FW), weight gain (WG), weight gain percentage (WGP), specific growth rate (SGR), and daily growth rate (DGR) compared with both the control and FS groups ( P < 0.05). Specifically, NS increased FW by approximately 20% relative to control (1.02 vs. 0.85 g) and by 24% relative to FS (1.02 vs. 0.82 g). SGR was elevated by about 16% compared with control (2.22 vs. 1.91% day⁻¹) and about 19% compared with FS (2.22 vs. 1.86% day⁻¹), while WGP increased by approximately 37% compared with control (279.6 vs. 215.4%). In contrast, growth indices in the FS group were not significantly different from those of the control ( P > 0.05). Fig. 2. Open in a new tab Growth performance of Caspian trout larvae fed control (CTRL), free silymarin (FS), or nanomicelle silymarin (NS) diets for 60 days, expressed as: A final weight (FW), B weight gain (WG), C weight gain percentage (WGP), D specific growth rate (SGR), and E daily growth rate (DGR). Values are means, with standard errors represented by vertical bars. Different letters above bars indicate significant differences among treatments (one-way ANOVA, Tukey’s HSD, P < 0.05). Fatty acid composition Analysis of whole-body fatty acid composition (Table 1 ) revealed statistically significant differences among treatments for several key fatty acids. Larvae fed the NS diet had significantly elevated levels of nutritionally important fatty acids, including EPA (20:5n-3), which increased by approximately 68% relative to the control (3.03 vs. 1.80%) and by approximately 83% relative to FS (3.03 vs. 1.66%). In addition, α-linolenic acid (18:3n-3) levels were significantly higher in the FS group, increasing by approximately 76% compared with the control (3.73 vs. 2.16%) ( P < 0.05). Total PUFA content was also significantly higher in the NS group (approximately 35% higher than the control; 39.36 vs. 29.23%), accompanied by a marked elevation in total n-3 fatty acids (approximately 47% higher than the control; 17.20 vs. 11.70%) ( P < 0.05). The FS group showed moderate increases in some lipid fractions, including total SFA, but did not exhibit significant enrichment of long-chain n-3 PUFAs relative to the control. Although the n-3/n-6 ratio was numerically highest in NS-fed fish (0.77) compared with FS (0.70) and control (0.66), these differences were not statistically significant ( P > 0.05). Oxidative and antioxidant status GPx activity was significantly enhanced in larvae fed the NS diet compared with both FS and control groups ( P < 0.05; Fig. 3 A). GPx activity in the NS group was more than twofold higher than in the control group (6.98 vs. 3.07 mU mg⁻¹ protein) and approximately 2.5-fold higher than in the FS group (6.98 vs. 2.75 mU mg⁻¹ protein). In contrast, SOD activity and MDA levels did not vary significantly among treatments ( P > 0.05; Fig. 3 B, C). However, FS-fed fish exhibited a slight, non-significant increase in SOD activity relative to controls. Fig. 3. Open in a new tab A Glutathione peroxidase (GPx) activity (mU mg⁻¹ tissue protein), B superoxide dismutase (SOD) activity (IU mg⁻¹ tissue protein), and C malondialdehyde (MDA) concentration (nmol mg⁻¹ tissue protein) in Caspian trout larvae fed control (CTRL), free silymarin (FS), or nanomicelle silymarin (NS) diets for 60 days. Values are means, with standard errors represented by vertical bars. Different letters above bars indicate significant differences among treatments (one-way ANOVA, Tukey’s HSD, P < 0.05). Leukocyte differential counts Hematological assessments (Table 2 ) showed no significant differences in the proportions of lymphocytes, monocytes, neutrophils, or eosinophils among dietary treatments ( P > 0.05). Table 2. Differential leukocyte counts (% of total white blood cells) in Caspian trout larvae fed control (CTRL), free silymarin (FS), or nanomicelle silymarin (NS) diets for 60 days. Parameters Experimental groups CTRL FS NS Lymphocyte (%) 81.67 ± 1.85 82.00 ± 2.51 82.33 ± 1.85 Monocyte (%) 2.00 ± 0.57 3.33 ± 0.66 2.33 ± 0.33 Neutrophil (%) 16.00 ± 2.51 14.00 ± 2.08 14.66 ± 1.85 Eosinophil (%) 0.33 ± 0.33 0.66 ± 0.33 0.66 ± 0.33 Open in a new tab Data are shown as mean ± SE. Superscript letters in the same row indicate significant differences between treatments (ANOVA, Tukey’s HSD test, P < 0.05). Discussion In this study, dietary supplementation with nanomicelle silymarin (NS) resulted in a significant growth advantage in Caspian trout larvae, with final weight and specific growth rate being significantly higher than those observed in both free silymarin (FS) and control diets. These findings underscore the potential of nanoencapsulation technology to enhance the functional efficacy of phytochemicals in aquafeeds, particularly during the early, metabolically demanding life stages of salmonids 42 . This observation aligns with findings in other taxa, such as improved growth and antioxidant status in olive flounder ( Paralichthys olivaceus ) fed micelle-form silymarin 22 and enhanced performance in poultry given nano-silymarin 29 . However, our finding that NS outperformed FS in growth indices contrasts with previous reports in Nile tilapia, where nanoencapsulation primarily enhanced biochemical or stress-resistance markers without clear gains in growth under non-challenged conditions 31 . Several studies, even at elevated silymarin supplementation levels, have failed to demonstrate growth performance enhancements in rainbow trout, grass carp, Nile tilapia, turbot and common carp 33 , 34 , 43 – 45 . The discrepancy likely reflects differences in developmental stage and digestive capacity. Larval salmonids, with immature digestive systems and high metabolic demand, may benefit disproportionately from the enhanced solubility and absorption offered by nanomicelle formulations 46 , 47 . In addition, because the final silymarin content of the experimental diets and potential leaching losses under flow-through conditions were not analytically verified (e.g., by HPLC), some uncertainty remains regarding absolute dietary exposure, particularly for free silymarin. However, identical preparation, coating, storage, and feeding procedures were applied across treatments to minimize differential loss. Also, batch-specific physicochemical characterization of the nanomicelle formulation was not independently performed, and the study relied on prior peer-reviewed characterization, commercial standardization, and qualitative confirmation of nanoscale morphology by TEM. Under these standardized conditions, the superior performance of the NS group reflects improved biological outcomes of the nanoformulation rather than procedural artifacts. Future studies incorporating chemical verification and batch-level characterization would further strengthen mechanistic insight. Silymarin is known for its cytoprotective activity, primarily due to its antioxidant and free radical–scavenging properties 48 . Under routine husbandry or environmental stress, excessive reactive oxygen species (ROS) can accumulate within tissues, initiating lipid peroxidation of cellular membranes and inducing structural and functional alterations in membrane transporters, glycoproteins, and both estrogenic and nuclear receptors 49 . Supplementation with silymarin is proposed to counteract these ROS-mediated injuries, thereby preserving cellular integrity and function 50 , 51 . The enzymes GPx, and SOD play pivotal roles in maintaining cellular antioxidant defense systems and are frequently assessed as broad-spectrum immunological biomarkers in fish 52 . GPx reduces hydrogen peroxide and organic peroxides to harmless products using glutathione 53 , while SOD converts superoxide radicals into less reactive species like oxygen and hydrogen peroxide 54 . The antioxidant status data provide additional context for the observed growth advantages in the NS group. While SOD activity was highest in FS-fed fish, NS supplementation resulted in markedly higher GPx activity compared to both FS and control groups, suggesting enhanced glutathione-dependent peroxide detoxification capacity. MDA content was measured as one of the main markers of ROS-mediated lipid peroxidation 55 . MDA levels did not differ significantly among treatments, indicating that lipid peroxidation status was relatively stable under the trial conditions. This selective elevation of GPx without corresponding changes in SOD or MDA is biologically plausible and has been documented in other larval fish studies using phytogenic or functional dietary antioxidants. Liu et al. observed significantly increased GPx activity in Cynoglossus semilaevis postlarvae fed dietary glutamine, whereas SOD and MDA remained unchanged, suggesting that early life stages may depend more strongly on the glutathione peroxidase system during rapid development 56 . Likewise, Veisi et al. reported that Nile tilapia fed nanoencapsulated silymarin exhibited pronounced GPx elevation under silver nanoparticle–induced oxidative stress, with no significant alteration in SOD at moderate supplementation levels 31 . Collectively, these findings support the idea that antioxidant responses can be pathway-specific and that GPx may serve as a more sensitive biomarker of improved redox balance in larval fish. The elevated GPx activity in NS-fed fish is consistent with the hypothesis that nanoencapsulation can improve bioactive delivery and retention, supporting key antioxidant enzymes even under baseline conditions, when oxidative stress is not overtly present 57 . This is consistent with reports where nano- or micelle-form silymarin improved antioxidant status in olive flounder 22 , poultry 29 , and Nile tilapia under oxidative challenge 31 . These findings imply that nanoencapsulation may strengthen both baseline physiological defenses and ability to cope with acute oxidative stress. At the mechanistic level, the nanoscale structure of NS likely enhances passive diffusion and endocytotic uptake in intestinal epithelial cells, facilitating faster systemic distribution and improved tissue retention of bioactive compounds 23 . Dietary silymarin, particularly in nanoencapsulated form, influenced the whole-body fatty acid profile of Caspian trout larvae. The NS group exhibited significantly higher total PUFA and n-3 PUFA levels compared to both FS and control diets, with notable increases in biologically important long-chain n-3 fatty acids such as EPA (20:5n-3) and DHA (22:6n-3). Elevated n-3 PUFA deposition is particularly relevant in salmonids, given their reliance on these fatty acids for membrane fluidity, neural development, and stress tolerance 58 – 60 . The observed improvement in DHA and EPA status in the NS group coincided with elevated GPx activity, suggesting that reduced oxidative degradation may have contributed to greater retention of these long-chain fatty acids. GPx, together with SOD, plays a critical role in preventing lipid peroxidation, and the observed enzymatic enhancement in NS-fed fish likely contributed to the preservation of long-chain PUFAs from oxidative degradation. Such preservation of n-3 fatty acids aligns with the elevated n-3/n-6 ratio observed in the NS group—a parameter often associated with improved physiological performance and stress resilience in aquaculture species 61 . In contrast, FS supplementation mainly increased total SFA and certain MUFAs (e.g., 16:1n-7), suggesting that the two formulations may differ in how dietary lipids are digested, absorbed, and metabolized. The observed changes in saturated fatty acids, including palmitic acid (C16:0) and stearic acid (C18:0), may reflect formulation-dependent differences in downstream lipid handling, as these fatty acids serve as key intermediates in fatty acid elongation, β-oxidation, and membrane phospholipid remodeling 62 . The superior n-3 HUFA retention in NS-fed larvae aligns with previous findings that nanoencapsulation improves intestinal uptake of lipophilic bioactives and may modulate lipid transport and deposition pathways 22 , 23 . These findings are consistent with reports in other taxa where silymarin supplementation enhanced PUFA deposition. For example, Armanini et al. showed that silymarin inclusion in broiler diets significantly increased muscle PUFA levels, an effect they attributed indirectly to its hepatoprotective properties 30 , which may influence hepatic lipid metabolism and downstream tissue fatty acid composition 19 . Similarly, Shahin et al. reported that D. labrax larvae fed silymarin-enriched weaning diets exhibited increased PUFA content and reduced SFA levels, particularly in DHA and EPA, supporting the role of silymarin in modulating lipid biosynthesis and protecting PUFA from oxidative degradation 26 . Likewise, Xiao et al. found that silymarin supplementation improved hepatic lipid metabolism and promoted PUFA deposition in grass carp 33 , while Wang et al. demonstrated improvements in PUFA/SFA balance in turbot 34 . Overall, the data point to nano-silymarin as a promising tool for improving lipid balance in both aquatic and terrestrial animals. From a mechanistic perspective, these changes may reflect both the antioxidant action of silymarin and its direct fatty acid contribution. The flavonolignan complex in S. marianum is a potent scavenger of reactive oxygen species 63 , 64 , which can prevent PUFA peroxidation and preserve membrane lipid integrity. Furthermore, S. marianum seeds contain appreciable amounts of linoleic acid (~ 39–44%), oleic acid (~ 20–36%), palmitic acid (~ 9–14.6%), and stearic acid (~ 6–7.5%) 65 , 66 , providing potential direct incorporation into tissue lipids. In grass carp, silymarin supplementation upregulated hepatic HMGCR and CYP7A1 expression, promoting lipid synthesis and bile acid production, thereby improving lipid absorption and metabolism 33 . The higher total PUFA and n-3 content observed in our NS-fed larvae may therefore result from a synergistic interaction between improved intestinal absorption (enhanced by the nanomicelle formulation), hepatic metabolic regulation, and protection against oxidative loss. From a nutritional perspective, enriching larval tissue with n-3 PUFA and improving the n-3/n-6 ratio is beneficial for product quality and fish health, as these fatty acids are critical for membrane fluidity, neural development, and stress resilience 58 , 67 . This dual benefit of enhanced antioxidant protection and improved fatty acid deposition could provide larval fish with greater robustness and growth potential. At the molecular level, several mechanisms may explain the enhanced antioxidant status and fatty acid modulation observed in NS-fed larvae. In fish, silymarin and its active flavonolignans have been shown to activate the Nrf2–Keap1 signaling pathway, a central regulator of cellular redox homeostasis, thereby increasing transcription of antioxidant enzymes such as GPx, SOD, and catalase 51 , 68 . Activation of Nrf2 confers improved protection against lipid peroxidation and supports greater preservation of long-chain n-3 PUFAs within membranes. In parallel, studies in teleosts indicate that silymarin can influence lipid metabolism by modulating the expression of key metabolic genes, including upregulation of lipid oxidation regulators (PPARα, CPT-1) and suppression of lipogenic genes such as SREBP-1 and FAS 25 , 33 . These pathways collectively support increased β-oxidation and reduced susceptibility of PUFAs to oxidative degradation. The superior outcomes observed in the NS group may therefore reflect both improved bioavailability and more effective activation of these redox- and lipid-regulatory systems in early-life salmonids. In the present study, neither dietary FS nor NS supplementation significantly altered leukocyte differentials. Lymphocytes accounted for over 80% of leukocytes across treatments, consistent with healthy salmonids under non-challenged conditions 69 , 70 . Monocyte and neutrophil proportions remained within the physiological ranges reported for Caspian trout 69 , indicating that neither FS nor NS induced a strong pro-inflammatory or myelopoietic response in the absence of pathogen or pollutant challenge. This contrasts with some reports in which silymarin supplementation modulated leukocyte profiles under immunological stress. For example, Kim et al. observed enhanced innate immune indices in olive flounder 22 , and Veisi et al. reported increased neutrophil activity in Nile tilapia exposed to silver nanoparticles, particularly in NS-fed fish 31 . In our trial, the stable neutrophil percentages across treatments may reflect the lack of an immune challenge to trigger differential recruitment or activation of phagocytic cells. The eosinophil percentages observed here (< 1%) are typical for Caspian trout and generally associated with low levels of allergic-type or parasitic responses 9 , 71 . The similarity among treatments again supports the interpretation that FS and NS supplementation did not elicit marked alterations in circulating granulocyte profiles in unstressed fish. Although our hematological data indicate limited impact of FS or NS on leukocyte differentials under optimal rearing conditions, previous work in poultry 29 and fish 31 , 72 suggests that silymarin’s immunomodulatory effects may be more evident during challenge scenarios, where enhanced antioxidant status and hepatic protection could indirectly support leukocyte function. In the present study, dietary silymarin supplementation did not significantly affect basal hematological parameters (leukocyte differentials) or oxidative stress indicators such as SOD and MDA. The absence of significant changes in leukocyte differentials, SOD, and MDA under optimal rearing conditions aligns with recent findings in nutritional immunology showing that many plant-derived immunostimulants and antioxidants exert measurable effects primarily when the immune system is activated by pathogens, stressors, or suboptimal conditions, whereas effects on baseline parameters under non-stressed conditions are limited 73 – 75 . The high water quality, absence of disease, and non-stressful environment during our trial likely provided minimal immune or oxidative stress stimuli, thereby limiting observable modulation by silymarin. Moreover, because only three replicate troughs were used per treatment, the statistical power to detect small differences, particularly for SOD, MDA, and leukocyte differentials, may be limited; thus, nonsignificant results for these parameters should be interpreted with caution. Future studies with higher biological replication and/or inclusion of challenge tests or environmental stressors would be necessary to fully evaluate the immunomodulatory and antioxidative potential of nanomicelle silymarin in Caspian trout. While nanomicelle delivery systems can markedly improve stability and bioavailability, their use warrants balanced evaluation due to concerns about long-term toxicity, bioaccumulation, and environmental release 76 . As highlighted by studies such as Veisi et al. 31 , nanoparticles can interact with oxidative pathways and induce physiological alterations, underscoring the need for careful dose optimization and environmental risk assessment. Accordingly, comprehensive evaluations of chronic dietary exposure, tissue accumulation, and nanoparticle fate in aquaculture effluents will be essential before large-scale application of nanomicelle-based feed additives. Conclusion In conclusion, our results provide evidence that nanomicelle silymarin supplementation confers measurable advantages over free silymarin in Caspian trout larvae. NS-fed fish displayed superior growth, enhanced GPx activity, and enriched n-3 PUFA deposition, including EPA and DHA, highlighting both antioxidant and nutritional benefits of nanomicelle delivery. In contrast, free silymarin supplementation produced only marginal effects under the same conditions. These findings suggest that encapsulation technology can overcome the solubility and bioavailability limitations of conventional silymarin, thereby unlocking its full potential as a functional feed additive. Given the ecological and aquacultural importance of Caspian trout, integrating nanomicelle-based phytochemicals into larval diets could strengthen hatchery success, reduce reliance on antibiotics, and support the sustainable expansion of Caspian trout aquaculture. However, because only a single inclusion level of silymarin was evaluated, the present study does not allow determination of optimal or threshold dosages for Caspian trout larvae. Future investigations should therefore incorporate dose–response designs, along with long-term performance assessments and challenge trials, to refine species- and life-stage–specific dietary recommendations and better define practical applications of nanomicelle silymarin in salmonid culture systems. Supplementary Information Below is the link to the electronic supplementary material. Supplementary Material 1 (19.2KB, docx) Acknowledgements The authors gratefully acknowledge the support provided by the University of Kurdistan for the execution of this project. We also thank Abzi Exir Kosar Company for supplying the required fish and providing the facilities for trial implementation. Special thanks are extended to Exir Nano Sina Company for generously providing the nanomicelle silymarin used in this study. Additional support was provided by the Water, Drought, Erosion, and Environment Technology Development Headquarters, affiliated with Iran’s Vice Presidency for Science, Technology, and Knowledge-Based Economy. Author contributions SRMS: Investigation, Formal analysis; AB: Supervision, Methodology, Conceptualization, Project administration, Validation, Writing - original draft; SAJ: Conceptualization, Funding acquisition, Supervision, Methodology, Resources, Project administration, Validation, Writing - review & editing; SK: Conceptualization, Supervision; HMT: Conceptualization, Supervision; BS: Writing - review & editing. Funding This research received no funding. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. 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Supplementary Materials Supplementary Material 1 (19.2KB, docx) Data Availability Statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Articles from Scientific Reports are provided here courtesy of Nature Publishing Group ACTIONS View on publisher site PDF (1.9 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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