Effect of partial replacement of fishmeal with Chlorella spp. and Haematococcus spp. in the diet of Labeo rohita (Hamilton, 1822)

Introduction

Fish is considered as a cheap source of protein (Muruganandam et al., 2019) [22] . Aquatic food systems are diverse, providing environmental, economic, and social benefits. Globally, they are valued for their nutritional importance and role in supporting healthy diets and aquatic biodiversity. In 2022, the world fisheries and aquaculture production was 223.2 million tonnes. About 51% from total production was contributed by aquaculture sector 94.4 million tonnes. Carps dominate freshwater aquaculture in India, with Catla (Catla catla), Rohu (Labeo rohita), and Mrigal (Cirrhinus mrigala) as key species due to their fast growth and consumer preference (Rutaisire et al., 2017; Bais, 2018) [30,6] . L. rohita, a highly demanded food fish in India, is preferred for its taste (Rajikkannu et al., 2015) [28] . Improved growth was observed with algae-supplemented feed compared to the control diet (Mukherjee et al., 2011) [21] . The fish meal act as a main source pf protein due to its high protein content, presence of essential amino acids, easy nutrient digestibility and minimum anti-nutritional factors. Due to decline in capture fisheries and less availability of fish meal is a major problem facing by feed manufacturers. To counter such problem there is a need to find alternative protein sources which can replace fish meal without any adverse effect (Daniel, 2018) [8] . Several protein sources are available which have the potential of replacing fishmeal in aquaculture feeds without affecting the growth performance of fish (Tacon, 2004) [35] . Microalgae are microscopic unicellular organisms which have many advantages (rich in a variety of important bioactive substance-protein, amino acids, polysaccharides, unsaturated fatty acids and cytochrome etc.), which make it become a new source of feed material (source of protein, fatty acids, vitamins, minerals, pigments etc.) (Li et al., 2015) [17] . Chlorella is freshwater, unicellular, green algae which have huge nutritional value as it is enriched with proteins, minerals, lipids, etc. Some research has proved that Chlorella has a range of health benefits (Rani et al., 2018) [29] . It consists of 55-60% protein, 1-4% chlorophyll, 9-18% dietary fibre, vitamins and minerals (Shim et al., 2008) [33] . In the diet of fish dried biomass of Chlorella or their extract are added to improve the quality of cultured fish; for instance, it has been reported that enhancement of resistibility to diseases (Nakagawa et al., 1981) [23] . The high nutritional value of Chlorella has used as dietary protein sources for marine and freshwater fish to improve weight gain and carcass quality (Kim et al., 2002; Badwy et al., 2008; Xu et al., 2014) [16,5,36] . Haematococcus pluvialis, which is also an important microalgae having good amount of natural astaxanthin also considered as "super anti-oxidant". Astaxanthin has important applications in the nutraceuticals, cosmetics, food, and aquaculture industries (Shah et al., 2016) [32] . H. pluvialis is a freshwater species of Chlorophyta (Lorenz and Cysewski, 2000) [20] . Generally, it contains 1.5-6.0% natural astaxanthin. The natural astaxanthin of H. pluvialis is trans, ester form and stable (Zhao et al., 2011) [37] , which can be well utilized by aquatic animals (Gong et al., 2014) [11] . H. pluvialis in dried powder form has been used in feed with specific functions such as for colouration and immune enhancer, because of such functions it is considered to be a potential feed additive for aquaculture feeds (Parisenti et al., 2011) [25] . Many kinds of research have been carried out which shows that astaxanthin in H. pluvialis can protect from free radicals (Ambati et al., 2014) [3] . H. pluvialis has gained acceptance in aquaculture and other markets as a concentrated form of natural astaxanthin (Lorenz and Cysewski, 2000) [20] . Considering the above factors, the present study was conducted to evaluate the effect of partially replacing fish meal with microalgae Chlorella spp. and Haematococcus spp. in the diet of Labeo rohita (Hamilton, 1822) [12] fry.

Materials and Methods

Experimental Setup

The experiment was conducted at Aquaculture Laboratory, College of Fisheries Science, Kamdhenu University, Veraval, for 60 days.

Collection of Feed Ingredients

The required ingredients for the preparation of the experimental diet such as fish meal (FM) and groundnut oil cake (GNOC) as a protein source, wheat flour (WF), fish oil, sunflower oil, tapioca powder (TP) and vitamin & mineral (V&M) for feed formulation were brought from the local market. Chlorella powder (CP) and Haematococcus powder (HP) was brought from Oceanic Seaweed Private Limited, Rajkot.

Proximate analysis of Ingredients

Proximate composition of ingredients (Table 1) was determined by the standard methods (AOAC, 2012). Moisture content was obtained by heating the samples to 105 C for 30 minutes and then drying at 65 C till a constant weight was obtained (Boyd, 2019) [7] . Crude protein values were obtained by the semi-automatic Micro-Kjeldahl digestion and distillation apparatus (Gerhardt, Germany). Crude fat was estimated with the Soxhlet apparatus. The total ash content was determined with the help of a muffle furnace.

Formulation and preparation of feed

The experimental diet was formulated with 30% protein level using locally available ingredients. In five experimental diets, the microalgae Chlorella spp. and Haematococcus spp. was added at the rate of 5%, 10%, 15% and 20%, while control diet was prepared with 0% of microalgae. The required quantity of ingredients was weighed accurately as per feed formula as shown in (Table 2). The dough was prepared by mixing all the ingredients and then it was thermally processed at 121 °C and 15 lbs pressure for 10-15 minutes. After steam cooking of feed mixture, vitamin and mineral mixture was mixed. The dough was pelletized by using hand pelletizer. The pellets were spread on a plastic sheet, exposed to sunlight for sun drying. The dried feed was packed in air-tight plastic jars.

Treatments details

The treatments are selected as follows: T 0 = Diet prepared with 0% level of Chlorella spp. and Haematococcus spp. (Control) T 1 = Diet prepared with 5% level of (2.5% Chlorella spp. + 2.5% Haematococcus spp.) T 2 = Diet prepared with 10% level of (5% Chlorella spp. + 5% Haematococcus spp.) T 3 = Diet prepared with 15% level of (7.5% Chlorella spp. + 7.5% Haematococcus spp.) T 4 = Diet prepared with 20% level of (10% Chlorella spp. + 10% Haematococcus spp.)

Experimental Setup

Labeo rohita fry was procured from a commercial hatchery, were transported to the Aquaculture Laboratory in Veraval. After 10 days of acclimatization in a 500 L plastic tank with continuous aeration, fry weighing approximately 1.065±0.013 g were selected for the experiment. The experiment utilized 20 disinfected rectangular plastic aquaria (size 2x1x1 feet) filled with 30 liters of chlorine-free filtered freshwater. Each tank was stocked with 10 fishes with continuous aeration. Fry were fed twice daily at 10% of their body weight, and tanks were cleaned daily to remove excess feed pellets and faecal matter. Sampling occurred fortnightly to assess juvenile fish body weight and adjust feed quantities. Physiochemical parameters (temperature, pH, alkalinity and dissolved oxygen) were monitored weekly throughout the experimental period.

Evolution Methods

The study involved assessing the bodyweight of fishes at 15-day intervals using an electronic weighing balance. Various growth and efficiency parameters, such as mean weight gain (WG), weight gain percentage (%), specific growth rate (SGR), food conversion ratio (FCR), protein efficiency ratio (PER), and Survival Rate (%), were calculated using specific formulas. Water quality measurements were recorded using a multi-parameter test kit for pH and temperature, total alkalinity was measured using HiMedia alkalinity testing kit, while dissolved oxygen was determined using Winkler's method. The results of partial replacement of fish meal with Chlorella spp. and Haematococcus spp. on the growth performance and survival parameters were statistically analysed using completely randomized design (CRD) and one-way analysis (ANOVA) using IBM® SPSS® version 26.00 software and significant differences (p<0.05) among treatment means were compared using duncan multiple range test (DMRT) (Snedecor and Cochran, 2014) [34] .

Results

Physico-chemical Parameters of Water

The water quality parameters recorded during the experimental period shown in (Table 3). remained within the suitable range for the growth and survival of the experimental fish. The water temperature varied slightly across treatments, with values ranging from 28.85±0.19 to 29.15±0.09 °C. Dissolved oxygen levels were relatively stable, ranging between 5.97±0.13 and 6.14±0.08 ppm, indicating adequate oxygen availability for fish metabolism. The pH values remained within the optimal range, varying from 7.94±0.05 to 8.04±0.09, ensuring a suitable aquatic environment. Total alkalinity, an important parameter influencing buffering capacity, ranged from 157.50±4.43 to 164.38±5.86 ppm, reflecting stable water conditions throughout the study. These findings suggest that the water quality parameters were well-maintained and did not impose any adverse effects on fish growth and health during the experimental period.

Growth Performance

The study assessed the impact of partially replacing fish meal with Chlorella sp. and Haematococcus sp. in the diet of Labeo rohita fry over a 60-day period. Growth parameters, including weight gain, specific growth rate (SGR) and survival rate shown in (Table 4) were statistically analyzed to determine the effects of microalgal supplementation. The initial weight of L. rohita fry across treatments (T 0 , T 1 , T 2 , T 3 , and T 4 ) remained similar, ranging from 1.065±0.006 gm to 1.073±0.005 gm, with no significant difference (p>0.05), indicating uniform stocking conditions. However, at the end of the experimental period (60 days), significant differences (p<0.05) were observed in final weight and total weight gain among treatments. The highest final body weight was recorded in T 2 (3.11±0.063 gm), which was significantly higher (p<0.05) than the other treatments, followed by T 3 (2.80±0.022 gm), T 1 (2.63±0.107 gm), and T 4 (2.66±0.044 gm), while T 0 (2.36±0.056 gm) had the lowest final body weight. While treatments (T 1 , T 3 , and T 4 ) showed statistically similar growth but were still higher than control treatment. Similarly, the mean weight gain varied among treatments, with T 2 showing the highest value of 2.05±0.068 gm, which was significantly different (p<0.05) from the control (T 0 ) and other treatments. Treatments T 1 (1.55±0.111 gm), T 3 (1.73±0.023 gm), and T 4 (1.59±0.039 gm) exhibited intermediate growth, whereas T 0 recorded the lowest mean weight gain (1.30±0.053 gm). The weight gain percentage followed a similar trend, with the highest percentage observed in T 2 (292.37±7.41%), significantly differing (p<0.05) from the other treatments. T 3 (261.70±2.43%), T 1 (244.88±10.89%), and T 4 (248.56±2.89%) showed moderate weight gain percentages, whereas the lowest was in T 0 (221.57±4.67%). The specific growth rate (SGR) was also influenced by dietary treatments, with the highest SGR recorded in T 2 (1.79±0.042%), which was significantly higher (p<0.05) than the other groups. Treatments T 1 (1.49±0.075%), T 3 (1.60±0.016%), and T 4 (1.52±0.019%) exhibited intermediate values, while the lowest SGR was observed in T 0 (1.32±0.035%). These findings indicate that partial replacement of fish meal with Chlorella spp. and Haematococcus spp. had a positive impact on the growth performance of L. rohita fry, with the best results observed in T 2 , suggesting an optimal level of replacement in this treatment.

Feed efficiency

The results of feed conversion ratio (FCR) of Labeo rohita fry at the end of the 60-day experimental period are presented in Table 4. The lowest FCR was recorded in Treatment T 2 (1.78±0.048), indicating better feed efficiency, followed by T 3 (2.04±0.049), T 4 (2.16±0.034) and T 1 (2.22±0.052), and the highest in T 0 (2.57±0.111). The statistical analysis showed significant differences (p<0.05) between all treatments. The protein efficiency ratio (PER) values for different treatments are presented in Table 4. The highest PER was recorded in Treatment T 2 (1.88±0.050), followed by T 4 (1.55±0.025), T 3 (1.63±0.039), T 1 (1.52±0.092), and the lowest in T 0 (1.30±0.052). Statistical analysis indicated significant differences (p<0.05) among all the treatments.

Survival rate

The survival rate of Labeo rohita fry across different treatments is presented in (Table 4). The survival rate was recorded at 100% in all experimental groups (T 0 , T 1 , T 2 , T 3 , and T 4 ) throughout the culture period. The data indicate that there was no mortality in any of the treatment groups, with survival rates remaining consistently at 100%. This finding suggests that all dietary treatments supported optimal health conditions for the fry. Statistical analysis showed there was no significant differences (p>0.05) found between all the treatments.

Proximate composition of fish carcass

At the end of the feeding period, five randomly selected fish samples from each tank underwent proximate analysis, results are shown in (Table 5). The protein content was significantly highest in T 2 (14.71±0.041%) and lowest in T 0 (11.85±0.040%). The fat content was significantly highest in T 3 (3.65±0.026%) and lowest in T 2 (3.11±0.018%). The ash content was significantly highest in T 4 (4.88±0.021%) and lowest in T 0 (2.50±0.029%). The moisture content was highest in T 0 (82.20±0.087%) and lowest in T 4 (77.80±0.058%).

Discussion

The present study evaluated the growth performance of Labeo rohita fry fed with different dietary inclusions of Chlorella spp. and Haematococcus spp. a 60-day experimental period. The results indicated that the final weight gain, percentage weight gain, and specific growth rate (SGR) were significantly higher (p<0.05) in the treatment group T 2 (10% Chlorella spp. and Haematococcus spp.) compared to all other treatments. These findings are consistent with previous studies by Khani et al., (2017) [ 15] , who reported that Cyprinus carpio fed with a 5% Chlorella diet exhibited optimal weight gain. Similarly, Raji et al., (2019) [27] observed enhanced growth parameters in Clarias gariepinus fingerlings when fish meal was partially replaced with Chlorella vulgaris powder. Abdulrahman et al., (2019) [1] also demonstrated that C. carpio exhibited increased growth rate, improved FCR and PER when fed with Chlorella pyrenoidosa-incorporated diets. Furthermore, Galal et al., (2018) [10] reported improved growth in Oreochromis niloticus fed with C. vulgaris, while Enyidi (2017) [9] found that C. gariepinus exhibited significantly better weight gain and SGR with C. vulgaris inclusion. Additionally, Ju et al., (2012) [14] reported that dietary incorporation of Haematococcus pluvialis enhanced growth in large yellow croaker (Pseudosciaena crocea), and Li et al., (2014) [18] observed similar trends in Litopenaeus vannamei when fed defatted H. pluvialis meal. The diet containing Chlorella meal can significantly enhance the digestive enzymes activity of of Carassius auratus gibelio, which in results showed better feed efficacy and growth rate (Xu et al., 2014) [36] . Also according to Lordan et al., (2011) essential amino acids, low molecular weight and rich protein of Chlorella makes Chlorella easily digestible. Pham et al., (2014) observed better feed and protein efficiency in juvenile olive flounder (Paralichthys olivaceus) with Hematococcus pluvialis inclusion in their diet. The carotenoids content of Hematococcus shows beneficial role in metabolic process of aquatic animals (Segner et al., 1989) due to which the utilization of nutrient increases which helps easy digestion and in results shows better growth (Amar et al., 2001). The declining trend in growth at higher inclusion levels (T 3 and T 4 ) suggests that excessive microalgal incorporation may reduce digestibility, alter feed palatability, or introduce anti-nutritional factors. Previous studies indicate that high levels of microalgae can increase fiber content and reduce protein digestibility, leading to suboptimal growth. These results indicate that microalgal supplementation can positively influence growth performance in L. rohita. The observed variations among different studies could be attributed to species-specific responses, diet formulations, and experimental conditions. Due to such quality of this microalgaes they have positively enhanced the growth performance and feed efficacy in L. rohita in this present study. Throughout the experimental period, a 100% survival rate was recorded across all treatment groups, with no observed mortality. This suggests that the environmental conditions were optimal for L. rohita and that the formulated diets did not induce any nutritional deficiencies or incompatibilities. These findings align with Raji et al., (2019) [27] , who reported 100% survival in C. gariepinus fed with diets containing Spirulina platensis and C. vulgaris. Similarly, Pakravan et al., (2018) [24] found that L. vannamei exhibited 100% survival when fed with C. vulgaris-supplemented diets. Jiang et al., (2019) [13] also reported 100% survival in juvenile yellow perch (Perca flavescens) when fed defatted H. pluvialis meal. However, it cannot be conclusively determined that dietary inclusion levels were solely responsible for the observed survival rates. The high survival rates suggest that the experimental diets provided adequate nutrition and did not induce any toxic effects. The proximate analysis results of the experiment indicate that the partial replacement of fish meal with Chlorella spp. and Haematococcus spp. revealed significant differences (p<0.05) among treatments. The significantly higher protein content in T 2 suggests that the inclusion of Chlorella spp. and Haematococcus spp. in the diet improved protein deposition, likely due to its high-quality protein content and bioavailable nutrients. This finding is consistent with Abdulrahman et al., (2019) [1] , who reported increased protein and ash content in Cyprinus carpio fed with Chlorella pyrenoidosa. Conversely, Pakravan et al., (2018) [24] observed a reduction in protein content when Chlorella vulgaris meal was incorporated into the fish diet, possibly due to differences in species-specific protein utilization or diet formulation. The highest fat content in T 3 suggests that Haematococcus spp. contributed to lipid accumulation, likely due to its rich carotenoid and astaxanthin content, which may have influenced lipid metabolism. Similar findings were reported by Li et al., (2014) [18] in large yellow croaker (Pseudosciaena crocea), where diets containing H. pluvialis led to increased lipid deposition. The significantly higher ash content in T 4 indicates increased mineral deposition, which may be attributed to the bioavailable minerals present in microalgae. The lowest moisture content in T 4 suggests a higher deposition of dry matter in the fish body, correlating with higher protein and ash content. However, Jiang et al., (2019) [13] reported no significant changes in whole-body moisture, crude protein, or lipid content in juvenile yellow perch (Perca flavescens) when fed with defatted microalgae meal, highlighting potential variations due to species, diet formulation, and inclusion levels of microalgae.

Conclusion

The present study demonstrated that the partial replacement of fish meal with Chlorella spp. and Haematococcus spp. in the diet of Labeo rohita fry positively influenced growth performance, feed efficiency, and carcass composition. Among the different dietary treatments, T 2 (10% inclusion) showed the highest final weight, weight gain, specific growth rate (SGR), and protein efficiency ratio (PER), along with the best feed conversion ratio (FCR), suggesting that this level provides the most beneficial balance of nutrients and digestibility. The 100% survival rate across all treatments indicates that microalgal supplementation did not induce any adverse effects on fish health, confirming its suitability for aquafeed formulations. Proximate composition analysis revealed higher protein content in T 2 , while T 3 exhibited increased lipid deposition, highlighting the potential of Haematococcus spp. as a source of beneficial carotenoids. However, the declining growth performance at higher inclusion levels (T 3 and T 4 ) suggests that excessive microalgal incorporation may reduce feed palatability or nutrient digestibility. Overall, these findings indicate that Chlorella spp. and Haematococcus spp. can serve as effective alternative protein sources in aquafeeds, reducing dependency on fish meal while maintaining optimal growth and health in L. rohita fry. Future research should focus on optimizing inclusion levels for different life stages and evaluating the long-term effects of microalgal supplementation on fish physiology and immunity.

Acknowledgement

AcknowledgementsThe author expresses gratitude to College of Fisheries Science, Kamdhenu University, Veraval for financial support. Special thanks to Dr. N. H. Joshi for guidance and inspiration during this research work. The author also grateful to the unwavering support and love from parents and family.

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