Introduction

Tilapia farming is prevalent in Malaysia and holds significant potentials for the country’s economic development, as Malaysia is among Southeast Asia’s largest tilapia producers, with production estimated at 150,000 metric tonnes in 20191. Consequently, with domestic and international demands for tilapia continuously on the rise, the industry is poised for further expansion over the next decade. However, various economic, environmental, and social factors pose challenges to the sustainability and productivity of tilapia farming, as commercial feed is among the most critical in optimising the growth of tilapia aquaculture. Rising production costs, particularly of commercial feed is exerting severe pressure on profit margins within the industry2 .

To align with the National Agrifood Policy 2021–2030 (NAP 2.0), the Malaysian Government plans to improve agricultural productivity, competitiveness, and sustainability through initiatives such as agricultural research and development, and stakeholder collaboration3. Consistent with these national policies, seaweed was identified as a potential aquaculture feed supplement, with emphasis to develop a holistic and sustainable plan for seaweeds as therapeutic products.

Sargassum polycystum is a brown seaweed species that is rich in protein, carbohydrates, vitamins, minerals, and bioactive compounds, offering significant benefits for the health and growth of aquaculture species4. Studies have shown that Sargassum seaweed improves digestibility, lowers feed conversion ratio, and boosts immune response in penaeid shrimp aquaculture5. The abundance of beneficial bioactive compounds in Sargassum sp., including polysaccharides, polyphenols, peptides, and carotenoids, further underscores its huge potentials6. The high polysaccharide content of Sargassum sp. makes it a promising resource as prebiotic for aquatic animals that promotes the growth of beneficial gut bacteria, while inhibiting pathogenic bacteria7.

Inclusion of Sargassum seaweed in the diet for tilapia aquaculture has been studied, where its ability to quantitatively substitute fishmeal was shown to be effective, without compromise to feed utilisation or growth8. Furthermore, feeding Sargassum to fish was also demonstrated to enhance their nutritional composition by increasing omega-3 fatty acid and antioxidant contents9,10. Overall, Sargassum has been shown as a sustainable and cost-effective alternative to traditional feed additives, with potentials to improve both the quality and quantity of aquaculture products.

In a previous study, we reported that S. polycystum offered option as a promising source for functional food due to its high protein, lipid, carbohydrate, and mineral contents, including Na, K, Ca, Mg, Fe, Se, and Mn. The phytochemical analysis of S. polycystum extract revealed the presence of several secondary metabolites4. To utilise seaweed as feed additive would vary, depending on the species targeted and fish culture conditions, emphasizing the need for further research to determine the ideal inclusion levels and processing techniques to consider, in order to integrate Sargassum polycystum seaweed into diets for particular aquaculture species.

Therefore, the present study was aimed to assess the potentials of Sargassum as a dietary component for red tilapia fingerling (RTF), and to evaluate the optimal inclusion levels, and overall impacts on fish growth and nutritional composition. Although the use of Sargassum brown seaweed as a feed supplement in aquaculture holds promise, these additional investigations are warranted in the effort to explore its long-term potentials as sustainable alternative to antibiotics and other chemical additives. In this study, the dried form of Sargassum was selected to reflect practical feed formulation strategies in aquaculture, as it offers greater handling convenience, longer shelf life, and more consistent nutrient content.

Materials and methods

Ethics statement In the present study, we strictly followed the guideline of ARRIVE (Animal Research: Reporting of In Vivo Experiments). All animals used in the experiment were treated humanely, according to approved procedures and guidelines of the Universiti Putra Malaysia Animal Ethics committee guidelines (approval number: UPM/IACUC/AUP-R024/2024). All experiments were followed in accordance with the relevant guidelines and regulations for caring the animals during the experiment.

Sample collection and processing

Sargassum polycystum specimens (Fig. 1) were harvested by hand at low tide (less than 0.09 m) from reef flats, about 100 m offshore at Teluk Kemang, near Port Dickson, Negeri Sembilan, Malaysia. Taxonomic identification involved cross-referencing with books, monographs, and reference herbaria11. The collected seaweed was cleaned with seawater to remove debris such as epiphytes, sand, pebbles, and shells. The cleaned specimens were then, stored in chilled plastic containers, and conveyed to the Aquatic Animal Health and Therapeutics Laboratory at the Institute of Bioscience, Universiti Putra Malaysia.

Fig. 1
figure 1

Sargassum polycystum C. Agardh (Order: Fucales, Class: Phaeophyceae), ID: IBS-SW1011.

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While in the lab, the S. polycystum samples were additionally washed with tap water followed by distilled water. The clean seaweeds were subsequently grouped into three (3), each processed according to the chosen drying method they were subjected to, as explained in subsequent sub-sections.

Drying process

The cleaned S. polycystum divided equally into three (3) groups were labelled as follows; (1) air-drying (AD) at room temperature, (2) freeze-drying (FD), and (3) vacuum oven drying (OD). All samples were dried until they attained constant biomass weight.

In the air-drying (AD) group, the S. polycystum samples were spread evenly on clean paper towels, on table-tops and left to dry at ambient temperature (25 °C) for six days. For vacuum oven drying (OD), the samples were spread evenly on cleaned metalic trays and placed in a vacuum oven (Memmert, USA) maintained constantly at 40 ± 5 °C for 29 h. For freeze-drying (FD), the fresh samples were initially frozen overnight at -80 °C (Thermo Scientific, USA), then lyophilized using a freeze dryer (Labconco FreeZone, USA) until they attained constant biomass weight.

After drying, samples in all groups were ground into fine powder using a laboratory-scale blender and sieved through a 200 μm sieve. The fine powdered seaweed samples were collected separately in screw-capped bottles, appropriately labeled, and stored in a freezer maintained at -80 °C until needed in subsequent evaluations12.

NMR measurement and multivariate data analysis

NMR spectroscopy is a robust and non-destructive analytical technique that is extensively used in metabolomics and natural products research. It allows for the simultaneous detection and quantification of a wide range of primary and secondary metabolites in a single acquisition. This method eliminates the need for prior chromatographic separation, thus providing a comprehensive metabolic fingerprint of the extract13. The 1H-NMR and J-resolved investigations were carried out using a 500 MHz Varian INOVA NMR spectrometer (Varian Inc., Palo Alto, CA, USA), which was operated at a frequency of 499.887 MHz and ambient temperature (25 °C). Samples were prepared according to slight modifications to procedures described by12. The spectra obtained were manually phased and the baseline data was corrected with the Chenomx software (v.5.1, Alberta, Canada), using a consistent setting for all spectra. Spectral intensities were divided into bins of similar width (δ 0.04), corresponding to the range δ 0.50 to 10.00. The regions of δ 4.70–4.90 for water and δ 3.23–3.36 for residual methanol were eliminated. Furthermore, 2D J-resolved was noted to provide additional support for metabolite identification. SIMCA-P software (v. 13.0, Umetrics, Umeå, Sweden) was used to analyse multivariate data by principal component analysis (PCA), with Pareto scaling. The identified metabolite peaks of the S. polycystum brown seaweed were compared to the Chenomx NMR Suite 7.7 library and standard NMR metabolite databases, including the Human Metabolome Database (HMDB; http://www.hmdb.ca).

Preparation of seaweed-supplemented experimental diets

Experimental diets were prepared by mixing milled commercial red tilapia pellet (Cargill, Malaysia) with the designated quantity of powdered freeze-dried S. polycystum to create three distinct formulations: (1) 0% S. polycystum, (2) 2.0% S. polycystum, and (3) 3.0% S. polycystum, as shown in Table 1. The dry powdered components (milled commercial pellet, tapioca-starch binder, and S. polycystum) were accurately weighed and gently mixed using a kitchen mixer. Gradually, water was added to form moist dough, which was then processed into spaghetti-like pellets of approximately 2 mm-size using a meat mincer. The experimental pellets were then dried in a hot, vacuum-air oven maintained at 45 °C overnight. Once dry, the pellets were broken into uniform sizes of approximately 2 mm in length and collected separately in screw-capped sample bottles. The prepared dietary pellets were labelled, and stored separately in a freezer maintained at -20 °C until used during the feeding trial.

Table 1 Ingredients and composition (%, dry matter (DM) basis) of experimental diets and S. polycystum sample.
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Proximate analysis of experimental diets, seaweed, and fish carcass

The S. polycystum powder, the experimental diets and fish carcass were analysed for crude protein (Method 984.13, A-D), crude fat by ether extraction (Method 920.39 A), and crude ash (Method 942.05) using official methods of AOAC14. The content of nitrogen free extract (NFE) was calculated by difference; 100− (crude protein + lipids + fiber + ash).

Fish husbandry and feeding trial

Uniformly aged RTF (Oreochromis spp.) that were hatched in the same batch and measuring approximately 11–12.5 cm in length were purchased from a commercial hatchery located in Puchong, Selangor, Malaysia. The fish were carefully and humanely transported in continuously aerated polyethylene bags inside a van, to the research facility of the Laboratory of Aquatic animal Health, Universiti Putra Malaysia.

Upon arrival at the research facility, the fish were acclimated to the experimental conditions for 7 days: in a continuously aerated, 5000 L capacity fiberglass tank containing filtered water for two weeks, feeding on a commercial diet (2 mm in size, containing approximately 32% crude protein and 4% crude fat).

For the feeding trial, 135 uniformly sized individual fish (mean initial weight of 36.27 ± 2.44 g) were selected and distributed into 9 fiberglass tanks, each of 50 L capacity (15 fish/tank × 3 groups ×3 replicates), and fed with diets formulated with 0%, 2% and 3% of seaweed, respectively. Throughout the 49 days trial, the fish were fed the assigned diets per treatment manually by hand to apparent satiation (3% body weight) two times daily, at 10:00 and 16:00 h. During this period, the experimental facility experienced a natural photoperiod of approximately 12 h light/12 h dark cycles. Feed consumption and fish growth were monitored bi-weekly by measuring the amount of assigned diets consumed and batch-weighing the fish in each tank, respectively. Throughout the feeding trial period, water quality parameters were meticulously maintained at optimal levels for the fish (pH, temperature, and dissolved oxygen, DO ranged between 8.02 and 8.21, 22.21–24.02 °C, and 6.50-7.00 ppt, respectively) in the culture tanks. The water quality of each aquarium was measured twice a week; once in the morning and once in the evening, using a multiparameter water analysis meter (YSI, USA). To maintain water quality parameters within acceptable limits, approximately one-third or 30% of the water in each tank was changed once weekly. Additionally, uneaten diets and accumulated fecal matter at the bottom of the aquariums were siphoned out daily. At the end of the experiment, surviving fish per tank were counted and weighed individually to determine both survival rates, and growth performance at final weight. These monitored parameters were then used to calculate other growth and feed efficiency parameters, including specific growth rate (SGR), protein efficiency ratio (PER), feed consumption (FC), and feed conversion ratio (FCR), respectively. All parameters were calculated using the following formulae, where applicable:

$$\:\mathbf{F}\mathbf{i}\mathbf{s}\mathbf{h}\:\mathbf{s}\mathbf{u}\mathbf{r}\mathbf{v}\mathbf{i}\mathbf{v}\mathbf{a}\mathbf{l}\:\left(\mathbf{\%}\right)\:=\left[(\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{f}\text{i}\text{s}\text{h}\:\text{s}\text{t}\text{o}\text{c}\text{k}\text{e}\text{d}\:-\:\text{F}\text{i}\text{n}\text{a}\text{l}\:\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{i}\text{n}\text{g}\:\text{f}\text{i}\text{s}\text{h})/\:\left(\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{f}\text{i}\text{s}\text{h}\:\text{s}\text{t}\text{o}\text{c}\text{k}\text{e}\text{d}\right)\right]\:x\:100$$
$$\:\mathbf{W}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\:\mathbf{g}\mathbf{a}\mathbf{i}\mathbf{n},\:\mathbf{W}\mathbf{G}\:\left(\mathbf{\%}\right)\:=\left[(\text{m}\text{e}\text{a}\text{n}\:\text{f}\text{i}\text{n}\text{a}\text{l}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:-\:\text{m}\text{e}\text{a}\text{n}\:\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t})\:/\text{m}\text{e}\text{a}\text{n}\:\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\right]\:x\:100$$
$$\:\mathbf{S}\mathbf{p}\mathbf{e}\mathbf{c}\mathbf{i}\mathbf{f}\mathbf{i}\mathbf{c}\:\mathbf{g}\mathbf{r}\mathbf{o}\mathbf{w}\mathbf{t}\mathbf{h}\:\mathbf{R}\mathbf{a}\mathbf{t}\mathbf{e},\:\mathbf{S}\mathbf{G}\mathbf{R}\:\left(\mathbf{\%}\right)\:=\left[(\text{L}\text{n}\text{W}\text{f}\:-\:\text{L}\text{n}\text{W}\text{i})/\text{T}\right]\:x\:100$$
$$\:\mathbf{F}\mathbf{e}\mathbf{e}\mathbf{d}\:\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{v}\mathbf{e}\mathbf{r}\mathbf{s}\mathbf{i}\mathbf{o}\mathbf{n}\:\mathbf{r}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o},\:\mathbf{F}\mathbf{C}\mathbf{R}\hspace{0.17em}=\hspace{0.17em}\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{d}\text{r}\text{y}\:\text{f}\text{e}\text{e}\text{d}\:\text{i}\text{n}\text{t}\text{a}\text{k}\text{e}\:\left(\text{g}\right)/\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{g}\text{a}\text{i}\text{n}\:\left(\text{g}\right)$$
$$\:\mathbf{P}\mathbf{r}\mathbf{o}\mathbf{t}\mathbf{e}\mathbf{i}\mathbf{n}\:\mathbf{E}\mathbf{f}\mathbf{f}\mathbf{i}\mathbf{c}\mathbf{i}\mathbf{e}\mathbf{n}\mathbf{c}\mathbf{y}\:\mathbf{r}\mathbf{a}\mathbf{t}\mathbf{i}\mathbf{o},\:\mathbf{P}\mathbf{E}\mathbf{R}\hspace{0.17em}=\hspace{0.17em}\text{m}\text{e}\text{a}\text{n}\:\text{f}\text{r}\text{e}\text{s}\text{h}\:\text{f}\text{l}\text{e}\text{s}\text{h}\:\text{g}\text{a}\text{i}\text{n}\:\left(\text{g}\right)/\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{i}\text{n}\text{t}\text{a}\text{k}\text{e}\:\left(\text{g}\right)$$
$$\:\mathbf{F}\mathbf{e}\mathbf{e}\mathbf{d}\:\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{s}\mathbf{u}\mathbf{m}\mathbf{p}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n},\:\mathbf{F}\mathbf{C}\:\left(\mathbf{g}\right)\:=\left(\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{f}\text{e}\text{e}\text{d}\:\text{p}\text{r}\text{o}\text{v}\text{i}\text{d}\text{e}\text{d}\:\left(\text{g}\right)\:-\:\text{U}\text{n}\text{e}\text{a}\text{t}\text{e}\text{n}\:\text{f}\text{e}\text{e}\text{d}\:\left(\text{g}\right)\:\right)/\left(\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{f}\text{i}\text{s}\text{h}\:\text{x}\:\text{D}\text{a}\text{y}\text{s}\right)$$

Where, LnWf = natural logarithm of final weight, LnWi = natural logarithm of initial weight and T = number of days of the feeding trial.

The 49-day feeding trial was strategically selected as it provides a scientifically appropriate duration to detect the nutritional and physiological responses of fish to dietary modifications without introducing confounding factors. This timeframe is widely accepted in aquaculture nutrition studies and is sufficient to observe measurable changes in growth performance, feed utilization, and potential Health impacts. Extending the trial beyond 49 days could risk the onset of sexual maturation, environmental stress, or tank limitations that may interfere with the interpretation of dietary effects. Additionally, the chosen duration aligns with similar studies in other aquaculture species15,16,17,18, ensuring consistency with existing literature while optimizing resource use and adhering to animal welfare standards. Thus, the 49-day period represents a balanced, efficient, and scientifically justified approach, sufficient to evaluate the effects of seaweed supplementation in fish diets.

Blood sampling from fish and analytical procedure

At the end of the 49-day feeding trial, fish from each replicate tank were subjected to physiological and biochemical evaluations to assess the potential systemic effects of dietary Sargassum polycystum supplementation. From each treatment group (0%, 2%, and 3% inclusion), three fish per replicate (n = 3 fish × 3 tanks = 9 fish per treatment) were randomly selected. Fish were anesthetized using 200 mg/L of 2-phenoxyethanol (Sigma-Aldrich, USA) to minimize handling stress, as described by Manna et al. (2021)19.

Approximately 2 mL of blood was aseptically collected from the caudal vein of each fish using sterile, non-heparinized syringes and transferred into sterile, labelled tubes. The samples were centrifuged at 3000 rpm for 15 min at 4 °C, to separate the serum. The obtained serum was subjected to blood biochemical analysis, which included determination of amylase (Amy), calcium (Ca), cholesterol (Chol), glucose (Gluc), total protein (TP), and triglycerides (Tgl) concentrations, following the method adapted in Rairat et al. (2021)20. Additionally, the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured calorimetrically at 540 nm, using a modified protocol to that described in Al-Deriny et al. (2020)21.

Proximate analysis of fish carcass

To a second group of randomly pooled fish, their carcass was subjected to proximate analysis. Proximate analysis of crude protein, crude fat, crude ash and the content of NFE, were carried out according to methods described in Section “Proximate analysis of experimental diets, seaweed, and fish carcass”.

For fatty acid analysis, total lipids were extracted, filtered through GF/A filters, washed with 0.5 M ammonium formate solution, lyophilised, and kept at − 20◦C until subjected to fatty acid analysis. The preparation of fatty acid methyl ester (FAME) from total lipid was performed according to a modified method reported by Farahin et al., 202122, with minor modifications. Briefly, filtered samples were extracted using 3 mL chloroform: methanol (1:2, v/v) solution and sonicated for 15 min at 15 °C. The extract was then centrifuged at 4,000 × g for 8 min, and the supernatant was transferred into a new glass tube. The remaining residue was re-extracted three times and extracts obtained were pooled into one tube. To remove impurities in the extract, 10 mL of ultrapure water was added, and the mixture was separated by centrifugation for 10 min at 3,000 × g. Thereafter, 100 µL of internal standard of heneicosane (C21) was added to the extract. Next, the mixture was dried completely under nitrogen gas at 35 °C. For transesterification, 1 mL of methylation mixture (methanol: acetyl chloride, 100:5, v/v) was added to the dry lipid fraction and Heated at 100 °C for 60 min. After cooling to room temperature, 2 mL of Hexane was added and shaken for 5 s. The upper Hexane phase was collected as FAMEs, and dried at 45 °C under a steam of nitrogen gas. The FAMEs were dissolved in Hexane and quantified using an Agilent 6890 gas chromatograph-mass spectrometer (GCMS) (6890 N GC/5973MS, Agilent Technologies, USA) equipped with a capillary column 30 m × 0.25 mm ID × 0.25 μm (Agilent J&W DB-WAX, USA). Helium was used as the carrier gas at a flow rate of 1.3 mL/min and a Head pressure of 75.7 kPa. The injector temperature was programmed at 250 °C and the detector temperature at 255 °C. The column temperature was initially set at 50 °C for 1 min, then programmed to increase to 100 °C at 10 °C/min, Held for 5 min, and finally ramped to 240 °C at a rate of 4 °C/min, where it was Held for 15 min. The identification of individual spectra was performed by comparing the mass spectra with NIST libraries using data analysis software (Agilent MSD Productivity ChemStation). Identification of each fatty acid was conducted by comparing the retention time and mass spectrum of the standard and quantified by comparing the peak area with that of the internal standard.

Statistical analysis

The percentages of FAMEs were subjected to arcsine transformation prior to statistical analysis. A one-way analysis of variance, ANOVA was conducted, followed by multiple comparisons posthoc tests (Tukey) to compare FAME levels between treatments.

Results

Multivariate metabolite data analysis

The identification of metabolite variations among the three drying methods, i.e., air-dried, freeze-dried, and oven-dried S. polycystum samples, was conducted using PCA biplot analysis. A PCA biplot was employed to visualise the effect of drying methods (score) on the variation of metabolites in seaweed samples (loading). Distinct discriminations among air-dried, freeze-dried, and oven-dried seaweed samples were observed (Fig. 2).

Fig. 2
figure 2

A biplot of 1H NMR data obtained from principal component analysis (PCA) describing the metabolites variations among air-dried, freeze-dried, and oven-dried seaweed samples.

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The principal component 1 (PC1) showed the highest variation, followed by PC2. Together, PC1 and PC2 accounted for 79.4% of the total variations. The separation of air-dried, freeze-dried, and oven-dried S. polycystum samples in the score plots was primarily achieved through the combination of PC1 and PC2. The PC1 delineated the separation between freeze-dried samples, air-dried samples, and oven-dried samples, whereas PC2 highlighted the distinction between air-dried, freeze-dried and oven-dried samples. The analysis revealed that the 15 S. polycystum samples clustered into three distinct groups, with R2X cum and Q2 cum values of 0.885 and 0.778, respectively, indicating robust clustering patterns. Furthermore, there were no significant outliers detected within the dataset. The freeze-dried samples exhibited significantly higher (p < 0.05) levels of N-acetylglycine, sarcosine, betaine, choline, dimethylsulfone, and malonic acid compared to the air-dried and oven-dried samples.

S. polycystum content and grade levels in feeds, and effects on dietary composition

Table 1 shows the feed constituents and composition of diets formulated with varying levels of S. polycystum (0%, 2%, and 3%), respectively. Upon examining the composition of seaweed and experimental diets (Table 1), it is evident that the ash content of seaweed was relatively high. Additionally, the S. polycystum used contained appreciable levels of proteins (7.10%), low lipids (1.98%), and moderate carbohydrates (59.34%). Consequently, the addition of graded quantities of S. polycystum to the experimental diets did not significantly (p > 0.05) modify the dietary protein and total carbohydrate (NFE) levels. On the other hand, the total ash contents of the experimental diets were significantly (p < 0.05) altered by the S. polycystum supplementation levels.

Fish survival, growth performance, feed intake and efficiency

The results for growth performance, including final weight, specific growth rate (SGR); and feed efficiency parameters, including protein efficiency ratio (PER), feed consumption (FC), feed conversion ratio (FCR), and survival of RTFs fed diets containing graded levels of powdered S. polycystum for 49 days are presented in Table 2.

Table 2 Feed intake and efficiency, survival, and growth performance of red tilapia fingerlings fed diets supplemented with graded levels of S. polycystum powder for 49 days presented on wet weight basis.
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Average initial weigh of 37.52 ± 0.05 g of RTF was used for this study (Table 2). The highest weight gain (%) was recorded in RTF fed with 3% S. polycystum powder (187%), whereas weight gain in the 2% group (170.67%) was similar to that noted in the control (170.11%). Therefore, diets fed to fish containing 3% S. polycystum powder led to significant improvement (p < 0.05) in weight gain and SGR of the RTF.

Feed consumption rate (FC) was significantly lower (p < 0.05) in fish reared with 3% S. polycystum compared to the control group, which consumed diets at a rate similar to the 2% S. polycystum inclusion group. However, inclusion of 3% S. polycystum powder in the fish diet was noted to have significantly elevated (p < 0.05) FCR value in the fish compared to the group fed the 2% seaweed, in which was noted FCR values similar to the control group. Nonetheless, the PER value in RTF fed with 2% S. polycystum powder was noted as significantly the highest (p < 0.05) compared to the 2 other groups that showed similar values.

No significant difference (p > 0.05) was observed in the survival rates between fish fed the 2% and 3% S. polycystum inclusion dietary treatments, although survival in both groups was significantly (p < 0.05) better than in the control treatment.

Carcass proximate crude protein, lipid, and fatty acid composition

The results of RTF carcass composition, specifically crude protein and lipids following the supplementation of fish diets with graded levels of S. polycystum powder are shown graphically in Fig. 3. Addition of 3% S. polycystum powder to the diet significantly increased (p < 0.05) RTF carcass crude protein, compared to the control and 2% groups, in which were measured similar carcass crude protein contents. For carcass lipids however, progressively increasing the dietary inclusion of S. polycystum powder from 0 to 3% led to no significant impact on fish carcass lipids.

Fig. 3
figure 3

Compositions of crude proteins and lipids in RTF carcass (dry weight), of fish reared with graded levels of dietary-supplemented S. polycystum for 49 days. (Different lowercase letters on the same shaded bars imply significant differences, p < 0.05). The proximate composition of the fish is presented on wet weight basis.

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To determine the fatty acid content of RTF carcass, fish fed with 2% and 3% S. polycystum powder (treatment) and that fed with 0% S. polycystum powder (control) were subjected to further evaluation. The percentage distribution of saturated, monounsaturated, and polyunsaturated fatty acids in the carcass of RTF when fed with graded levels of dietary-supplemented S. polycystum are shown in Fig. 4. From the Figure, it could be observed clearly that supplementation of S. polycystum powder in the fish diet altered the composition of fatty acids in the carcass. Saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) were significantly higher (p < 0.05) in diets of both fish supplemented with S. polycystum powder compared to the control group. In contrast, unsaturated fatty acids (UFA) and monounsaturated fatty acids (MUFA) were significantly higher (p < 0.05) in the control group compared to the carcass of fish fed diets with S. polycystum included. Overall, the composition of the experimental diets fed made notable impacts on the fatty acid profiles of the fish carcasses.

Fig. 4
figure 4

Percentage distribution of saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids in RTF fingerling carcass, of fish reared with graded levels of dietary-supplemented S. polycystum for 49 days. (Different superscript lowercase letters on data per row indicate significant differences, p < 0.05).

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Serum biochemical analysis

The serum biochemical concentrations in blood samples obtained from RTF fed with S. polycystum-supplemented diets (2% and 3% S. polycystum powder) and a commercial diet (control) over 49 days period are shown in Table 3. The inclusion of S. polycystum powder in the commercial diet of RTF resulted in changes in serum biochemical parameters monitored. Fish fed with the control diet displayed lower (or similar) levels of serum ALT, amylase, AST, calcium, cholesterol, total protein, and triglycerides compared to those fed diets containing 2% and 3% S. polycystum powder, except the serum glucose.

Table 3 Blood biochemistry of red tilapia fingerlings-fed diets containing 0% (control), 2% and 3% S. polycystum powder and for 49 days, n = 3.
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Discussion

1H NMR spectra of the differently dried seaweed and metabolite identification

Sargassum seaweed is fit for consumption, albeit necessitating processing prior to ingestion. This study emphasised the significance of different drying methods on S. polycystum seaweed before its incorporation as a feed supplement for RTF. In our previous studies, multivariate data analysis revealed variations in metabolites among different extracts derived from three distinct drying methods i.e., air, freeze, and oven drying12. Therefore, drying method was confirmed to influence the metabolic composition of a seaweed species, a factor of crucial importance when incorporating it into animal feed formulations. The application of inappropriate sample processing technique during the drying process could result in high variability, interference with instrumentation, or degradation of metabolites.

Metabolites generally have higher vapor pressures at higher temperatures and higher rates of chemical conversion of heat-labile metabolites to different compounds at the higher temperature of methods employed during drying. Commonly utilized drying techniques for seaweed include sun drying, oven drying, and freeze drying, which are usually selected for cost-effectiveness23,24,25,26. However, unlike drying processes for other plants, drying of marine seaweed typically requires longer duration due to the need for multiple washing steps to remove salt residues. Therefore, a metabolomics approach is suggested to evaluate and monitor variations in metabolite content under different drying conditions, as suggested by other researchers27,28.

Results of this current study indicated that metabolite degradation can be circumvented through the application of the freeze-drying method, owing to the low temperature involved. Specifically, in oven-dried samples, metabolites such as formic acid, trimethylamine, 3-hydroxyisovaleric acid, propylene glycol, and valporic acid were found to be more abundant. On the other hand, air-dried samples may experience metabolite loss due to degradation caused by air oxidation and thermal effects. Although air drying can inhibit microbial growth and prevent specific biochemical changes, it may introduce other changes affecting the overall sample quality. The consideration of these factors is imperative when selecting a drying method for S. polycystum seaweed, as they could affect the nutritional composition and quality as a feed supplement in animal feed29,30.

In the present study, N-acetylglycine, sarcosine, betaine, choline, dimethylsulfone, and malonic acid were noted as more abundant following freeze drying, as lyophilization seemed to be the generally superior technique, compared to air and oven drying. This is likely as it seemed to preserve the original metabolome of the sample to a higher degree, based on the general observation of it containing more metabolites, or the same metabolites at higher concentrations compared to those in air and oven dried samples. Consequently, the freeze-dried sample was chosen for the subsequent experiments.

Previously, we observed several bioactive metabolites that may be potentially used as functional components in aquaculture feed from the Sargassum polycystum’s GC-MS profile4. The detection of n-hexadecanoic acid (10.35%) and methyl palmitate (5.28%) were expected to function as essential fatty acids that may have boosted energy availability, supported membrane integrity, and promoted larval development in fish31,32. Furthermore, there were traces of antioxidant compounds such as benzenepropanoic acid derivatives (7.03%), phenol, 3,5-bis(1,1-dimethylethyl)- (6.54%), and phytol (6.24%), which are known to reduce oxidative stress, improve liver function, and support immune responses, thereby contributing to overall fish health and enhancing feed stability33,34,35. In addition, compounds such as 1-dodecanol, 3,7,11-trimethyl- (4.26%) and squalene (0.74%), despite being present in low concentrations, have been reported to possess notable antibacterial and immunostimulatory properties36,37,38. These bioactive compounds likely acted synergistically to enhance the nutritional and functional attributes of the experimental feeds evaluated, potentially contributing to improved growth performance, gastrointestinal health, and disease resistance in the cultured fish during the feeding trial to minimize mortality. Overall, the diverse profile of bioactive metabolites identified in Sargassum polycystum supported its potential as a sustainable and functional feed additive in aquaculture diets.

Fish feed intake and efficiency, and growth performance

The economic factors of production, harvesting, and processing of seaweed have a significant impact on its viability as a component of fish feed. Previously, research highlighted that incorporating seaweed into fish diets enhances growth performance. This is as seaweeds are identified to have high nutrients profile, abundance in compliments of vitamins and minerals that are essential for fish growth. Of particular importance for Tilapia and catfish is the calcium content of seaweeds, given the vital role that calcium plays in their growth39. Furthermore, seaweeds contain a spectrum of beneficial vitamins, minerals, antioxidants, and protective pigments that actively support fish development40,41.

The present study aimed to explore the potential use of S. polycystum as a natural dietary supplement in feeds to improve the growth performance of RTF (Oreochromis spp.). Growth performance of RTF was evaluated in a feeding trial via weight gain, FCR, and SGR. Results of the feeding trial revealed that incorporating S. polycystum in the diet of RTF positively influenced growth performance. The experimental groups fed with diets supplemented with the S. polycystum exhibited statistically significant increase (p < 0.05) in weight compared to the control group. In addition, a statistically significant increase (p < 0.05) in the FCR and SGR of RTF fed with the S. polycystum diet was observed, compared to the control group. These results corroborate those of other previous studies reported by other scholars. For instance, a recent study42 (Wassef et al., 2023) demonstrated that inclusion of seaweed into fish diets resulted in significant improvements in final body weight, specific growth rate, and feed conversion ratio across multiple fish species. Studies on other seaweed species, such as Gracilaria sp43. and Ulva lactuca44 supplemented feed, also demonstrated enhanced growth performance in gilthead seabream and catfish, respectively. The improvement was evident in increased body weight, enhanced feed utilisation efficiency, and higher specific growth rates compared to control groups. Similarly, another study highlighted the beneficial effects of incorporating Ascophyllum nodosum into the diet of rainbow trout45. The inclusion of this seaweed species positively influences growth patterns, utilisation of nutrients, and gastrointestinal health in the fish. Collectively, these empirical data highlight the potential utility of seaweed supplements as significant components of fish diets.

Nonetheless, in spite of evidence in the literature revealing the potentials of seaweed as an important dietary component for fish, there remains a gap in understanding the ideal inclusion levels and the overall effects on fish growth and Health. The available body of data indicates that including seaweed into fish diets can have varying effects on growth performance and feed utilisation, depending on both the fish and seaweed species involved. Incorporating seaweeds into fish diets up to a maximum limit of 5% resulted in enhanced body weight, improved feed efficiency, and enhanced protein retention46,47,48. In contrast, another study reported that higher levels of seaweed inclusion were linked to reduced growth performance and decreased feed utilisation efficiency in seabass41. Likewise, the incorporation of numerous seaweed species, including Cystoseira barbata, U. lactuca, U. rigida, and G. cornea, at a rate of 10% led to impaired growth performance and reduced feed utilisation in fish9.

Overall, supplementing the diet with 3.0% Sargassum polycystum was well tolerated by the fish and promoted optimal development and performance. However, this treatment produced a greater FCR than other diets, indicating that the fish grew well but required slightly more feed to do so. This may be attributable to S. polycystum’s unique nutrient content (high ash or mineral content) or metabolic characteristics of RTF, which showed enhanced feed intake, but which did not fully translate it into better feed utilization. Despite leading to higher FCR, the better tolerance and performance of fish fed makes 3.0% S. polycystum a sufficient quantity to be added to the diet.

Carcass proximate protein, lipid, and fatty acid compositions

The examination of carcasses of fish fed the different treatments indicated that the gradual addition of Sargassum to fish diet resulted in similar carcass protein contents between the experimental and the control groups. This observation is consistent with the results previously reported7,49, in which an elevation was observed in the carcass crude protein content when fish was fed diets that contained varying quantities of the seaweeds Glacilaria and P. purpurea. Furthermore, the results of the present investigation also demonstrated that inclusion of Sargassum in fish diets led to a slight decrease in carcass lipids. This observation is consistent with results published by other researchers49, who noted a decrease in lipid content in juvenile European sea bass (Dicentrarchus labrax) that was fed a diet containing 10% Gracilaria compared to a control group.

Seaweeds have been recognized as valuable sources of essential fatty acids, suggesting their potential use as nutraceuticals or as integral components of dietary regimens50. Studies examining the incorporation of Sargassum into the dietary regimen of RTF have predominantly focused on the changes in the carcass fatty acid composition. Fatty acids are essential components of fish oil known for their positive effects on human health, which was noticeable in the fatty acid contents, as dried Sargassum was gradually added to the diet.

Notably, increased inclusion of Sargassum powder in the diet resulted in appreciable increase in MUFA and PUFA in the carcass of RTF. Although the lipid content of the macroalgae was relatively low and ranged between 2 and 10% (similar to that reported in Biris-Dorhoi et al., 2020)51, this clearly suggests that it did not make significant contributions in the diet as energy source. Nonetheless, it is important to highlight that PUFA in seaweeds have been noted to play crucial roles in regulating various physiological processes, including blood pressure, blood clotting, and the optimal development and functioning of the brain and nervous systems52.

Overall, results of the present study suggest that feeding red tilapia with Sargassum improves the fatty acid content of the fish, which may have potential health benefits for the human consumers. However, more research is required to determine the optimal level of inclussion and duration to feed Sargassum to red tilapia, to enable the proper determination of effect on important nutrients, such as carcass protein and amino acid profiles.

Blood composition analysis

The results of the fish blood composition analysis indicated a slight increase in serum ALT, amylase, AST, calcium, cholesterol, total protein, and triglycerides in RTF fed diets supplemented with seaweed, as compared to the control group. It is also important to highlight that the blood enzyme levels recorded in this present study were within the recommended range53,54. However, it is crucial to recognise that various factors, such as the age and health condition of the fish, the type and amount of feed offered, and the current environmental circumstances may have made impact on the results of blood analysis monitored.

Based on previous research, the incorporation of Sargassum into the dietary regimen of fish was observed to modify the blood chemistry. To illustrate this, a study demonstrated that the inclusion of 5% Sargassum in the diet of young tilapia led to alterations in various serum biochemical parameters, specifically affecting liver function, lipid metabolism, and antioxidant status. Further investigations documented alterations in blood glucose, cholesterol, and triglyceride concentrations among rainbow trout that were offered a diet containing Sargassum in abundance.

Significantly, within the confines of the current investigation, it was observed that the glucose level in RTF fed with Sargassum was comparatively lower than those in the control group. This suggests the presence of favourable health markers in the diets offered to the experimental groups55. Increased quantities of glucose in the bloodstream can have detrimental effects on fish, leading to a decrease in their feed consumption and growth rates56. The observation of slight differences in blood cholesterol and triglyceride levels between the control and fish fed seaweed supplemented diets could be related to the ability of fish to metabolise supplementary nutrients found in seaweed, which were not available in the control diet57. Only excessively elevated levels of cholesterol and triglycerides are potentially attributed to heightened oxidative stress or a diet lacking in antioxidants58.

The liver plays a crucial role in the uptake, accumulation, biotransformation, and excretion of toxicants, making it an essential organ during sublethal exposure to these substances. The concentrations of AST and ALT are used as indicators of healthy liver function. The presence of these liver enzymes in circulation is commonly associated with cellular damage in fish experiencing stress, hence serving as stress indicator59,60. Aspartate aminotransferase (AST) has been widely employed as a general indicator of fish health in various research investigations61. It is released into the bloodstream when hepatocytes undergo lysis. In a previous study4, it was documented that the methanolic extract of S. polycystum contained a range of metabolites, such as steroids, phenols, tannins, saponins, flavonoids, terpenoids, and glycosides. The presence of some other metabolites as alkaloids, tannins, and saponins has been found to potentially play a role in safeguarding and maintaining the integrity of cell membranes in the face of toxicants that are facilitated by free radicals62.

Feasibility impact of utilizing sargassum polycystum as a nutritional feed component in RTF diet

The current approach of utilizing Sargassum polycystum as a dietary supplement in tilapia feed demonstrates promising feasibility for broader aquaculture applications. As a widely available and underutilized brown seaweed, Sargassum offers a sustainable and cost-effective alternative to conventional feed ingredients63. Its rich profile of bioactive compounds, minerals, and polysaccharides not only supports fish growth and immune health but may also reduce dependency on synthetic additives and fishmeal64. The abundance of seaweed locally in tropical coastal regions such as Malaysia ensures reliable sourcing with minimal ecological disruption65. Furthermore, preliminary results from this study suggest that low inclusion levels (2–3%) are sufficient to deliver beneficial effects, enhancing the practicality of scaling up for commercial feed production. This aligns well with the increasing global demand for eco-friendly and functional aquafeeds, highlighting the potential of Sargassum-based formulations for large-scale adoption in sustainable tilapia farming systems.

Conclusions

The high nutritional value and bioactive compounds present in S. polycystum can enhance and support the growth performance of RTF. These observations suggest that S. polycystum could be a valuable natural dietary supplement for aquaculture, contributing to sustainable and eco-friendly fish farming practices. Finally, the brown seaweed S. polycystum has the potential to improve the growth performance of RTF. Although its use as a feed supplement in aquaculture yields promising outcomes, further research is necessary to fully explore its potential as a sustainable alternative to antibiotics and other chemical feed additives on the long term. Future investigations should examine the differential effects of fresh versus dried Sargassum, particularly in terms of bioactive compound stability, nutrient availability, and potential impacts on fish growth and health performance. Therefore, these findings underscore the potential of S. polycystum as a valuable natural dietary supplement for aquaculture, aiding in the promotion of sustainable and environmentally friendly fish farming practices.