Functional role of Chlorella sp. and Porphyra sp. as natural feed additives in ornamental goldfish culture

Introduction

The ornamental fish industry represents one of the fastest-growing sectors of global aquaculture, with an estimated annual trade value of approximately USD 15 billion and more than two billion live fish traded worldwide. Asian countries contribute over 60% of global ornamental fish exports, while the major markets are concentrated in the United States, Europe, China, and Japan. In India, ornamental fish farming has emerged as an important disease resistance in several cultured fish species. Similarly, Porphyra sp., a commercially important red macroalga, contains high-quality proteins, β-carotene, phycobiliproteins, taurine, polyunsaturated fatty acids, vitamins, minerals, and sulfated polysaccharides such as porphyran. These bioactive constituents exhibit antioxidant, immunomodulatory, antimicrobial, and pigmentationenhancing properties and have shown positive effects on fish growth and physiological health. Although numerous studies have independently evaluated the effects of microalgae and macroalgae in aquaculture diets, direct comparative information on the functional efficacy of Chlorella sp. and Porphyra sp. in ornamental fish remains limited. Therefore, the present study was undertaken to comparatively evaluate the effects of dietary supplementation with Chlorella sp. and Porphyra sp. on growth performance, feed utilization, pigmentation, hematological responses in goldfish (Carassius auratus), and to identify their optimum dietary inclusion levels for the development of sustainable functional feeds for ornamental aquaculture.

Materials and Methods

Experimental location

The feeding trial was conducted during 2024-2025 at the Wet Laboratory of the Department of Aquaculture, College of Fisheries Science, Kamdhenu University, Veraval, Gujarat, India. All experimental procedures were performed under controlled laboratory conditions for a period of 75 days.

Experimental fish and acclimatization

Healthy goldfish (Carassius auratus Linnaeus, 1758) fingerlings were procured from a local ornamental fish hatchery and transported to the laboratory under continuous aeration. Upon arrival, the fish were acclimatized for 30 days in 500-L fiberglass-reinforced plastic (FRP) tanks supplied with continuous aeration. During the acclimation period, fish were fed a commercial basal diet twice daily and monitored regularly to ensure good health. After acclimatization, healthy fish with a uniform initial body weight of 1.85 ± 0.003 g were randomly selected for the feeding experiment.

Experimental system

The experiment was conducted using twenty-eight rectangular plastic aquaria (60 × 30 × 30 cm; approximately 35 L capacity). Before stocking, all aquaria were thoroughly cleaned, disinfected, and filled with filtered freshwater. Continuous aeration was supplied throughout the experimental period to maintain adequate dissolved oxygen levels. To maintain water quality, uneaten feed and fecal matter were removed daily by siphoning, followed by the replacement of approximately 10% of the tank water with fresh disinfected water.

Experimental design

The experiment followed a completely randomized design (CRD) consisting of seven dietary treatments, each with four replications. The treatments were divided into two groups based on dietary inclusion:

Diet preparation

Feed ingredients, including fish meal, groundnut oil cake, rice bran, wheat flour, tapioca flour, vegetable oil, fish oil, and a vitamin-mineral premix, were obtained from local commercial suppliers. Commercially available powders of Chlorella sp. and Porphyra sp. were used as dietary supplements. The proximate composition of individual feed ingredients was determined according to the standard methods of AOAC (2005) [2] . Moisture content was measured by oven drying, crude protein by the Kjeldahl method, crude lipid by Soxhlet extraction, and ash by incineration in a muffle furnace. Experimental diets were formulated using Pearson's square method to achieve a uniform crude protein level of 30%.

Feeding management

Fish were hand-fed twice daily throughout the experimental period. The daily ration was provided at 5% of the total biomass and was adjusted every 15 days following bulk weighing of fish to account for growth. Fish were carefully observed throughout the trial for feeding behavior, health status, and mortality. Total carotenoids (µg g⁻¹) = (Absorbance × Dilution factor)/(0.25 × Sample weight) 2.9 Hematological analysis At the end of the feeding trial, fish were anesthetized using clove oil (150 mg L⁻¹), and blood samples were collected by cardiac puncture using sterile EDTA-coated syringes. Blood samples were immediately analyzed using an automated hematology analyzer to determine red blood cell count (RBC), white blood cell count (WBC), hemoglobin concentration (Hb), and hematocrit (HCT). These parameters were used to evaluate the physiological and health status of experimental fish.

Growth performance

Statistical analysis

All experimental data were analyzed using the Statistical Package for the Social Sciences (SPSS). Before statistical analysis, the data were examined for normality and homogeneity of variance. Differences among treatment means were evaluated by one-way analysis of variance (ANOVA), followed by Duncan's Multiple Range Test (DMRT) for multiple comparisons. Statistical significance was accepted at p < 0.05. Results are expressed as mean ± standard error (SE).

Results

Proximate composition of experimental diets

The proximate composition of the experimental diets is presented in Table 1. All diets were formulated to be isonitrogenous, containing approximately 30% crude protein on a dry weight basis. Moisture content ranged from 7.27 to 7.89%, while dry matter varied between 91.09 and 91.32%. Crude protein remained consistent among all dietary treatments (30.02-30.10%), indicating successful formulation of nutritionally comparable diets. Similarly, crude fat (7.78-8.09%) and ash (8.43-8.88%) exhibited only minor variations among treatments, confirming that the inclusion of Chlorella sp. and Porphyra sp. did not substantially alter the overall nutritional composition of the experimental diets.

Growth performance and feed utilization

The effects of dietary Chlorella and Porphyra supplementation on growth performance and feed utilization of Carassius auratus after 75 days are summarized in Table 2. No significant differences (p > 0.05) were observed in the initial body weight among treatments, confirming uniformity of fish at the commencement of the experiment. However, dietary supplementation significantly influenced (p < 0.05) all growth and feed utilization parameters at the end of the feeding trial.

Among all treatments, fish fed the 10% Chlorella diet (CT 3 ) exhibited the highest final body weight (5.87 ± 0.003 g), weight gain (4.13 ± 0.005 g), weight gain percentage (223.00 ± 0.772%), and specific growth rate (1.56 ± 0.003% day⁻¹), followed by CT 2 and CT 1 . In contrast, the control group recorded the lowest values for these parameters. Similarly, the 10% Porphyra diet (PT 2 ) produced significantly higher (p < 0.05) final body weight (5.45 ± 0.008 g), weight gain (3.60 ± 0.008 g), weight gain percentage (194.31 ± 0.580%), and specific growth rate (1.43 ± 0.002% day⁻¹) compared with PT 1 , PT 3 , and the control. However, the highest inclusion level of Porphyra (PT 3 ) did not improve growth performance and showed values comparable to the control. Feed utilization was also significantly affected by dietary treatments. Fish receiving CT 3 exhibited the lowest feed conversion ratio (1.36 ± 0.002) together with the highest feed efficiency ratio (0.73 ± 0.001) and protein efficiency ratio (2.08 ± 0.003). Within the Porphyra treatments, PT 2 recorded the lowest feed conversion ratio (1.71 ± 0.007) and the highest feed efficiency ratio (0.58 ± 0.003) and protein efficiency ratio (1.67 ± 0.007). The control and PT 3 groups showed comparatively lower feed utilization efficiency. Overall, dietary supplementation with Chlorella produced greater improvements in growth and feed utilization than Porphyra, with 10% Chlorella representing the most effective dietary inclusion level.

Carotenoid deposition

Dietary supplementation with both algal species significantly affected (p < 0.05) carotenoid deposition in C. auratus (Table 3; Figure 1). The control group exhibited the lowest carotenoid concentration (3.25 ± 0.006 μg g⁻¹). Carotenoid accumulation increased progressively with increasing dietary Chlorella inclusion, reaching the highest value in CT 3 (5.94 ± 0.020 μg g⁻¹), followed by CT 2 (5.75 ± 0.017 μg g⁻¹) and CT 1 (4.86 ± 0.011 μg g⁻¹). Among the Porphyra-supplemented groups, PT 2 recorded the highest carotenoid concentration (5.66 ± 0.011 μg g⁻¹), followed by PT 1 (5.26 ± 0.012 μg g⁻¹). In contrast, PT 3 showed carotenoid levels (3.23 ± 0.009 μg g⁻¹) comparable to those of the control group. Temporal changes in carotenoid accumulation throughout the feeding period are illustrated in Figure 1, demonstrating a gradual increase in pigmentation with increasing dietary supplementation, particularly in the CT 3 and PT 2 treatments.

Hematological responses

The effects of dietary algal supplementation on hematological parameters are presented in Table 3 and Figure 2. Significant differences (p < 0.05) were observed among treatments for red blood cell count (RBC), white blood cell count (WBC), hemoglobin concentration (Hb), and hematocrit (Hct). Within the Chlorella treatments, RBC, Hb, and Hct increased significantly as dietary inclusion increased. Fish fed CT 3 exhibited the highest RBC (1.31 ± 0.037 × 10⁶ μL⁻¹), Hb (8.51 ± 0.012 g dL⁻¹), and Hct (25.52 ± 0.027%), whereas the control group showed the lowest values. Conversely, WBC count declined progressively from 3.32 ± 0.014 × 10³ μL⁻¹ in the control to 2.64 ± 0.013 × 10³ μL⁻¹ in CT 3 . Among the Porphyra treatments, PT 2 exhibited the highest RBC (1.56 ± 0.018 × 10⁶ μL⁻¹), Hb (8.64 ± 0.013 g dL⁻¹), and Hct (25.48 ± 0.025%). The lowest WBC count (2.15 ± 0.585 × 10³ μL⁻¹) was also observed in PT 2 , whereas PT 3 recorded the highest WBC value (3.46 ± 0.014 × 10³ μL⁻¹) among all treatments. The temporal variation in hematological parameters during the experimental period is presented in Figure 2, which shows a progressive improvement in erythrocytic parameters with increasing dietary supplementation of Chlorella and moderate supplementation of Porphyra.

Discussion

The present study demonstrated that dietary supplementation with Chlorella sp. and Porphyra sp. significantly enhanced growth performance, feed utilization, pigmentation, and hematological status of Carassius auratus. Among the tested dietary treatments, supplementation with 10% Chlorella consistently produced the greatest improvements in most evaluated parameters, whereas 10% Porphyra represented the optimum inclusion level among the macroalgal diets. These findings highlight the nutritional and functional potential of algal biomass as a sustainable feed additive for ornamental aquaculture.

Effect of algal supplementation on diet composition

The proximate composition of the experimental diets showed only negligible variations in moisture, crude protein, crude lipid, ash, and dry matter, confirming that all diets were successfully formulated as isonitrogenous. Consequently, the differences observed in fish performance can be attributed primarily to the physiological effects of algal supplementation rather than variations in nutrient composition. Maintaining comparable dietary protein and energy levels is essential in nutritional experiments to accurately evaluate the biological effects of functional ingredients. Similar observations have been reported in studies evaluating microalgae and macroalgae as feed additives, where dietary inclusion of algal biomass did not markedly alter proximate composition but significantly influenced fish growth and physiological responses through their bioactive constituents. Chlorella sp. is recognized as a rich source of highly digestible protein, essential amino acids, chlorophyll, carotenoids, vitamins, minerals, nucleotides, and polyunsaturated fatty acids, whereas Porphyra sp. contains abundant proteins, phycobiliproteins, taurine, carotenoids, sulfated polysaccharides, vitamins, and trace minerals (Holdt & Kraan, 2011) [14] . These bioactive compounds function not only as nutrients but also as physiological modulators capable of improving digestion, metabolism, antioxidant defense, and immune function. Therefore, the beneficial responses observed in the present study are likely associated with the synergistic effects of these biologically active compounds rather than simple nutrient supplementation.

Growth Performance and Feed Utilization

The present study demonstrated that dietary supplementation with Chlorella and Porphyra significantly improved growth performance and feed utilization of Carassius auratus. Among the dietary treatments, fish fed the 10% Chlorella diet exhibited the highest final body weight, weight gain, specific growth rate, feed efficiency ratio, and protein efficiency ratio, together with the lowest feed conversion ratio. Similarly, 10% Porphyra supplementation produced the best growth response among the macroalgal diets, whereas further increasing Porphyra inclusion to 15% resulted in reduced performance. The superior growth performance observed in the Chlorellasupplemented groups may be attributed to its high-quality protein content and abundance of bioactive compounds, including essential amino acids, vitamins, carotenoids, polyunsaturated fatty acids, and nucleotides. These components are known to improve nutrient digestibility, stimulate digestive enzyme activities, and enhance protein synthesis and tissue accretion (Becker, 2013) [3] . Similar improvements in growth and feed utilization following dietary Chlorella supplementation have been reported in gibel carp (Carassius auratus gibelio) by Xu et al. (2014), who observed enhanced specific growth rate and protein efficiency ratio in fish fed diets containing 0.8-1.2% Chlorella. Likewise, Abdel-Tawwab and Ahmad (2009) [1] reported significant improvements in growth and feed conversion efficiency in Nile tilapia fed Chlorella vulgarissupplemented diets. The beneficial effects of Porphyra supplementation may be associated with its highly digestible proteins, taurine, carotenoids, vitamins, and sulfated polysaccharides, particularly porphyran, which have been reported to improve nutrient metabolism and physiological performance (Holdt and Kraan, 2011) [14] . Similar findings have been reported by Kalla et al. (2008) [17] , who observed improved growth and feed utilization in red sea bream fed diets supplemented with Porphyra spheroplasts. Furthermore, Cheung (2021) reported that inclusion of 5-10% Porphyra amplissima significantly improved wet weight and condition factor in Atlantic salmon. However, growth performance declined at the highest inclusion level of Porphyra (15%), suggesting that excessive macroalgal incorporation may adversely affect nutrient utilization. Red macroalgae contain relatively high concentrations of structural polysaccharides and dietary fiber that can reduce nutrient digestibility and limit nutrient absorption when included at excessive levels (Holdt and Kraan, 2011) [14] . Similar reductions in growth performance at higher seaweed inclusion levels have been reported in rainbow trout and gilthead seabream (Soler-Vila et al., 2009) [31] . Collectively, the present findings indicate that moderate dietary inclusion of algal biomass improves growth performance and feed utilization in goldfish, with 10% Chlorella providing the greatest biological response under the present experimental conditions.

Carotenoid Deposition and Pigmentation

Body coloration is one of the principal quality attributes determining the commercial value of ornamental fish. Since fish are unable to synthesize carotenoids de novo, dietary supplementation with natural pigment sources is essential for maintaining desirable body coloration. In the present study, both Chlorella and Porphyra significantly enhanced carotenoid deposition compared with the control diet, with the highest values observed in fish fed 10% Chlorella. The enhanced pigmentation observed in the Chlorella-fed groups can be attributed to the presence of natural pigments such as lutein, β-carotene, zeaxanthin, and chlorophyll derivatives, which serve as precursors for chromatophore pigmentation (Becker, 2013) [3] . Moreover, the presence of essential fatty acids may facilitate intestinal absorption and transport of carotenoids, thereby improving pigment deposition in skin tissues. Similar findings were reported by Teimouri et al. (2013), who observed improved body coloration and carotenoid accumulation in goldfish fed diets supplemented with Spirulina and Chlorella. Likewise, Gouveia et al. (2003) [12] reported significant enhancement of pigmentation in ornamental fish following supplementation with carotenoid-rich microalgae. Dietary Porphyra supplementation also significantly improved carotenoid deposition, particularly at the 10% inclusion level. Red macroalgae contain carotenoids, phycobiliproteins, and antioxidant compounds that contribute to pigmentation enhancement and pigment stability (Holdt and Kraan, 2011) [14] . However, the reduced carotenoid deposition observed at 15% Porphyra inclusion suggests that excessive macroalgal supplementation may reduce carotenoid bioavailability, possibly because of impaired lipid digestion and nutrient absorption associated with higher fiber levels. Similar observations have been reported in marine fish and ornamental species receiving excessive seaweed supplementation (Wan et al., 2019) [35] . The results indicate that moderate supplementation with Chlorella and Porphyra can serve as an effective and sustainable strategy for improving pigmentation and market quality of ornamental goldfish.

Hematological Responses

Hematological parameters are sensitive indicators of nutritional status and physiological health in fish. In the present study, dietary supplementation with both algal species significantly improved erythrocytic indices, including RBC count, hemoglobin concentration, and hematocrit values, with the highest responses observed in fish fed 10% Chlorella and 10% Porphyra. The improvement in hematological parameters following Chlorella supplementation may be attributed to its rich supply of high-quality protein, iron, folic acid, vitamin B₁₂, and chlorophyll, which are essential for erythropoiesis and hemoglobin synthesis (Becker, 2013) [3] . Enhanced erythrocyte production increases oxygen transport to tissues, thereby supporting improved metabolism and growth. Similar improvements in hemoglobin concentration and erythrocyte count following dietary Chlorella supplementation have been reported in Nile tilapia (Abdel-Tawwab and Ahmad, 2009) [1] and gibel carp. The positive hematological responses observed in the Porphyra-supplemented groups may be associated with the presence of bioavailable iron, taurine, essential amino acids, vitamins, and sulfated polysaccharides that support hematopoietic activity and cellular protection (Holdt and Kraan, 2011) [14] . However, the comparatively lower responses observed at 15% inclusion suggest that excessive incorporation of macroalgal biomass may reduce the availability of essential nutrients because of higher levels of indigestible polysaccharides and dietary fiber. The improved erythrocytic parameters obtained in the present study indicate enhanced physiological condition and metabolic efficiency in algal-fed fish. Therefore, dietary supplementation with 10% Chlorella and 10% Porphyra appears to be effective for improving blood physiological status and promoting better overall health in C. auratus.

Conclusion

The present study demonstrated that dietary supplementation with Chlorella sp. and Porphyra sp. significantly improved the growth performance, feed utilization, carotenoid deposition, and hematological status of Carassius auratus. Among the evaluated dietary treatments, 10% Chlorella supplementation consistently produced the most pronounced improvements in final body weight, weight gain, specific growth rate, feed conversion ratio, feed efficiency ratio, protein efficiency ratio, carotenoid accumulation, and erythrocytic parameters. Similarly, 10% Porphyra supplementation enhanced growth performance, pigmentation, and hematological indices; however, its overall effectiveness was comparatively lower than that of Chlorella. In contrast, increasing the dietary inclusion of Porphyra to 15% did not provide additional benefits and resulted in reduced performance, suggesting that excessive macroalgal supplementation may impair nutrient utilization because of its higher fiber and ash contents. The improved performance observed in algal-supplemented fish can be attributed to the rich nutritional profile of both algae, including high-quality proteins, essential amino acids, carotenoids, vitamins, minerals, polyunsaturated fatty acids, chlorophyll, and other bioactive compounds that enhance nutrient utilization and physiological function. The increased carotenoid deposition further demonstrates the effectiveness of these natural algal resources as sustainable pigmentation enhancers for ornamental fish, offering an environmentally friendly alternative to synthetic colorants. Overall, the findings indicate that Chlorella sp. and Porphyra sp. are promising functional feed additives for ornamental aquaculture, with 10% dietary Chlorella identified as the optimum inclusion level under the present experimental conditions. The incorporation of Chlorella at this level can improve growth, feed efficiency, pigmentation, and physiological health, thereby increasing the commercial quality and market value of goldfish. Future investigations should focus on the long-term effects of algal supplementation on reproductive performance, intestinal microbiota, molecular immune responses, and nutrigenomic regulation to further elucidate the mechanisms underlying their beneficial effects and to optimize their application in sustainable ornamental fish production.

Acknowledgments

The authors gratefully acknowledge the College of Fisheries Science, Kamdhenu University, Veraval, Gujarat, India, for providing the laboratory facilities and infrastructure necessary to conduct this research. The authors also express their sincere gratitude to the faculty members and technical staff of the Department of Aquaculture for their valuable assistance during the experimental work.

References

1. response of rainbow trout (Oncorhynchus mykiss). J Appl Phycol. 2021;33:3113-3122.
2. Dawood MA, Koshio S, Esteban MÁ. Beneficial roles of feed additives as immunostimulants in aquaculture: A review. Rev Aquac. 2018;10:950-974.
3. El-Habashi N, Fadl SE, Farag HF, Gad DM, Elsadany AY, El Gohary MS. Effect of using Spirulina and Chlorella as feed additives for elevating immunity status of Nile tilapia experimentally infected with Aeromonas hydrophila. Aquac Res. 2019;50:27692781.
4. Galal AA, Reda RM, Mohamed AAR. Influences of Chlorella vulgaris dietary supplementation on growth performance, hematology, immune response and disease resistance in Oreochromis niloticus exposed to sub-lethal concentrations of penoxsulam herbicide. Fish Shellfish Immunol. 2018;77:445-456.
5. Ghosh A, Mahapatra BK, Datta NC. Ornamental fish farming-Successful small-scale aqua business in India. Aquac Asia. 2003;8:14-16.
6. Gouveia L, Rema P. Effect of microalgal biomass concentration and temperature on ornamental goldfish (Carassius auratus) skin pigmentation. Aquac Nutr. 2005;11:19-23.
7. Gouveia L, Rema P, Pereira O, Empis J. Colouring ornamental fish (Cyprinus carpio and Carassius auratus) with microalgal biomass. Aquac Nutr. 2003;9:123-129.
8. Gupta S, Abu-Ghannam N. Recent developments in the application of seaweeds or seaweed extracts as a means for enhancing the safety and quality attributes of foods. Innov Food Sci Emerg Technol. 2011;12:600-609.
9. Holdt SL, Kraan S. Bioactive compounds in seaweed: Functional food applications and legislation. J Appl Phycol. 2011;23:543-597.
10. Hoseinifar SH, Yousefi S, Capillo G, Paknejad H, Khalili M, Tabarraei A, et al. Mucosal immune parameters, immune and antioxidant defense related genes expression and growth performance of zebrafish (Danio rerio) fed on Gracilaria gracilis powder. Fish Shellfish Immunol. 2018;83:232-237.
11. Huang K, Liu X, Ma R, Wang B, Ho SH, Chen J, et al. Effects of substituting fish meal with Chlorella meal on growth performance, whole-body composition, pigmentation and physiological health of marbled eel (Anguilla marmorata). Algal Res. 2024;80:103523.
12. Kalla A, Yoshimatsu T, Araki T, Zhang DM, Yamamoto T, Sakamoto S. Use of Porphyra spheroplasts as feed additive for red sea bream. Fish Sci. 2008;74:104-108.
13. Khan MND, Yoshimatsu T, Kalla A, Araki T, Sakamoto S. Supplemental effect of Porphyra spheroplasts on the growth and feed utilization of black sea bream. Fish Sci. 2008;74:397-404.
14. Kim WH, Lee S, Ahn TY. Flavihumibacter cheonanensis sp. nov., isolated from sediment of a shallow stream. Int J Syst Evol Microbiol. 2014;64:3235-3239.
15. Lykkesfeldt J. Determination of malondialdehyde as dithiobarbituric acid adduct in biological samples by HPLC with fluorescence detection: Comparison with ultraviolet-visible spectrophotometry. Clin Chem. 2001;47:1725-1727.
16. Mahmoud EA, El-Sayed BM, Mahsoub YH, NeamatAllah AN. Effect of Chlorella vulgaris-enriched diet on growth performance, hemato-immunological responses, antioxidant and transcriptomics profile disorders caused by deltamethrin toxicity in Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol. 2020;102:422-429.
17. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47:469-474.
18. MPEDA. 23rd India International Seafood Show at Chennai Trade Centre, Chennai. Kochi: Marine Products Export Development Authority; 2022.
19. Olsen RE, Hansen AC, Rosenlund G, Hemre GI, Mayhew TM, Knudsen DL, et al. Total replacement of fish meal with plant proteins in diets for Atlantic cod (Gadus morhua L.). II. Health aspects. Aquaculture. 2007;272:612-624.
20. Olson JA. Provitamin A function of carotenoids: The conversion of β-carotene into vitamin A. J Nutr. 1989;119:105-108.
21. Rahimnejad S, Lee SM, Park HG, Choi J. Effects of dietary inclusion of Chlorella vulgaris on growth, blood biochemical parameters and antioxidant enzyme activity in olive flounder, Paralichthys olivaceus. J World Aquac Soc. 2017;48:103-112.
22. Raja K, Aanand P, Padmavathy S, Sampathkumar JS. Present and future market trends in the Indian ornamental fish sector. Int J Fish Aquat Stud. 2019;7:615.
23. Satam SB, Sawant BT, Metar SY, Patil AB, Kumar R. Marine ornamental fish trade: Status, challenges and opportunities in India. Indian J Geo-Mar Sci. 2018;47:1346-1354.
24. Sinha AK. Colorimetric assay of catalase. Anal Biochem. 1972;47:389-394.
25. Snedecor GW, Cochran WG. Statistical Methods. 8th ed. New Delhi: Wiley Blackwell; 2014.
26. Soler-Vila A, Coughlan S, Guiry MD, Kraan S. The red alga Porphyra dioica as a fish-feed ingredient for rainbow trout (Oncorhynchus mykiss): Effects on growth, feed efficiency and carcass composition. J Appl Phycol. 2009;21:617-624.
27. Spolaore P, Joannis-Cassan C, Duran E, Isambert A. Commercial applications of microalgae. J Biosci Bioeng. 2006;101:87-96.
28. Taboada MC, Millán R, Miguez MI. Nutritional value of the marine algae wakame (Undaria pinnatifida) and nori (Porphyra purpurea) as food supplements. J Appl Phycol. 2013;25:1271-1276.
29. Valente LMP, Rema P, Ferraro V, et al. Functional properties of seaweeds in fish nutrition and health. Aquaculture. 2016;458:1-15.
30. Wan AH, Davies SJ, Soler-Vila A, Fitzgerald R, Johnson MP. Macroalgae as a sustainable aquafeed ingredient. Rev Aquac. 2019;11:458-492.
31. Xu W, Gao Z, Qi Z, Qiu M, Peng JQ, Shao R. Effect of dietary Chlorella on the growth performance and physiological parameters of gibel carp, Carassius auratus gibelio. Turk J Fish Aquat Sci. 2014;14:1-8.