Effect of partial supplementation of Chlorella sp. on growth performance and survival of Labeo catla (Hamilton, 1822) fry

Introduction

Recently, aquaculture is one of the fastest growing in our world as it has a high level of protein, which is considered the lowest price in comparing with other sources of protein (Abd-El Alim et al., 2018) [2] . The value of aquaculture goes beyond replacing capture fisheries as the main source of seafood; it is one of the most efficient ways of producing protein for human consumption (Lucas et al., 2019) [17] . Fish is an essential dietary animal protein source in human nutrition. Production of sea-going species through freshwater fisheries and aquaculture for protein supply is being energized all through the world. As indicated by nutritionists, fish is an astounding substitute of protein for red meat (Akhil and Akhilesh, 2018) [4] . In India, 80% aquaculture production is composed of carps (Sivagami and Ronald, 2018) [31] . Freshwater aquaculture contributes over 89% of the total aquaculture production in the country and three Indian major carps (Arya et al., 2019) [6] . The Indian major carps Labeo catla, Labeo rohita and Cirrhinus mrigala are the most important commercial fishes in India with a maximum market demand and acceptability as food by the consumers due to their taste and flesh (Kadhar et al., 2012) [16] . Catla can be easily cultured in low input technology system due to its herbivorous feeding habit (Abarna, 2012) [1] . Its higher growth rate and compatibility with other major carps, specific surface feeding habit and consumer preference have increased its popularity in carp polyculture systems and it contributes a major share to the freshwater aquaculture production for the fish farmers in India (Mohanty et al., 2019) [20] . Fish meal has been the protein source of choice in aqua feeds for many reasons, including its high protein content, excellent amino acid profile, high nutrient digestibility, generally lacking in anti-nutrients, relatively low price and its wide availability (Gatlin et al., 2007) [13] . Sustainability of fish meal production from wild fish is questionable.

To reach a sustainable aquaculture, new alternative protein sources including cheaper plant or animal origin proteins are needed to be introduced for stable aqua feed production (Islam et al., 2018) [14] . Among the potential substitutes, plant ingredients appear to be the best candidates. Many plant protein sources can be used to partially or almost totally replace dietary fish meal (Santigosa et al., 2008) [27] . Plant ingredients have ANFs, are deficient in certain EAA, have less nutrient digestibility, have lesser nutrient bioavailability, and less palatability due to high levels of nonsoluble carbohydrates such as fiber and starch (Daniel, 2018) [12] . In recent years, microalgae have emerged as a natural alternative nutritional source for novel bioactive compounds (Christaki et al., 2011) [11] . Chlorella is commonly used as a dietary supplement (Abid et al., 2018) [3] . Chlorella species are single-cell, spherical shaped (~2 to 10 μm in diameter) and photoautotrophic green microalgae with no flagella (Mobin and Alam, 2017) [19]. Chlorella consists of green photosynthetic pigments chlorophyll-a and chlorophyll-b, courtesy of which it is the richest source of chlorophyll availability. Chlorella consists of 55-60% protein, 1-4% chlorophyll, 9-18% dietary fiber, vitamins and minerals (Rani et al., 2018) [26] . Owing to the high nutritional value, Chlorella has been used as a dietary protein sources for marine and freshwater fish to improve weight gain and carcass quality (Shi et al., 2017) [30] . Previous work showed inclusion of Chlorella in fish feed resulted in improved growth, survival and health status. Positive effect of Chlorella on feed utilization, stress and disease resistance of cultured fish has also been documented in earlier works (Rahimnejad et al., 2017) [25] . Looking to the above aspects, the present study was undertaken to evaluate the effect of Chlorella as feed supplement on growth performance, survival, feed utilization and body composition of Catla (Labeo catla) fry.

Materials and Methods

Experimental setup

The experiment was conducted at the Wet Laboratory of Department of Aquaculture, College of Fisheries Science, JAU, Veraval, over a period of 60 days.

Collection of feed ingredient

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 obtained from the local market. Chlorella powder (CP) was obtained from Oceanic Seaweed Private Limited, Rajkot.

Proximate analysis of Ingredients

Proximate composition of ingredients was determined by the standard methods (AOAC, 1995) [5] . Moisture content was obtained by heating the samples to 105 ºC for 30 minutes and then drying at 65 ºC till a constant weight was obtained (Boyd, 1981) [9] . 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 (Table 1.)

Formulation and preparation of feed

The experimental diet was formulated with 30% protein level using locally available ingredients. The feed was formulated by Pearson's square method. The required quantity of ingredients was weighed accurately as per feed formula as shown in Table 1. In five experimental diets, the microalgae Chlorella sp. was added at the rate of 5%, 10%, 15% and 20% of the ingredients of the control diet. Then prepared dough and then mixture of feed 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 feed mixture was pelletized by using hand pelletizer. The pellets were spread on a plastic sheet, exposed to sunlight. Then feed pellets were packed in marked plastic jars

Treatments details

The experimental treatments are selected as follows: T0 = Diet prepared with 0% inclusion of Chlorella sp. (Control) T1 = Diet prepared with 5% inclusion of Chlorella sp. T2 = Diet prepared with 10% inclusion of Chlorella sp. T3 = Diet prepared with 15% inclusion of Chlorella sp. T4 = Diet prepared with 20% inclusion of Chlorella sp.

Experimental Design

Fry of Labeo catla (catla) were brought from Inland Fisheries Research Station, Junagadh Agricultural University, Junagadh (Gujarat) and transported in polythene bags by road to Veraval. The fish were brought to Aquaculture Wet Laboratory at College of Fisheries Science, Veraval and were allowed to remain in the plastic tank (500 L) with continuous aeration and feeding for 10 days Fry with a total weight of 6.50 ± 0.108 to 6.80 ± 0.041 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 experimental treatment had three replicates which were arranged using a completely randomized design (CRD), design. 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 fecal matter. Sampling occurred fortnightly to assess fish body weight and adjust feed quantities. Physiochemical parameters (temperature, pH, alkalinity and dissolved oxygen) were monitored weekly throughout the experimental period.

Growth Indices

At 15 days' interval sampling was performed to assess the growth performance of the fish. Weight was measured using an electric weighing balance respectively. Growth indices were evaluated using the below described formulae.

Mean

The calculated value indicates the average increase (%) of body weight per day Biochemical Composition of Fish Carcass Proximate composition of fish was estimated just after completion of the experiment. Whole meat of the fish was taken for proximate analysis. The fish meat was minced properly. The samples were analyzed for crude protein, crude fat, ash and moisture employing standard methods as explained earlier.

Water Physico-Chemical Parameters

Water quality parameters such as temperature, pH, dissolved oxygen, and total alkalinity were measured on weekly basis throughout the experimental period. The Temperature was measured by using thermometer. The pH was measured by using pH meter. Dissolved oxygen was measured using Winkler's method. Total alkalinity was measured using HiMedia alkalinity testing kit.

Statistical Analysis

The results of partial replacement of fish meal with Chlorella sp. on the growth performance and survival parameters were statistically analyzed using completely randomized design (CRD) and one-way analysis of variance (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) [33] .

Results

Physico-Chemical Water Parameters

The water quality parameters recorded during the experimental period are shown in table 3. Water temperature ranged from 21.4 °C to 24.9 °C during the experimental period. The pH ranged from 7.5 to 7.7 during the experimental period. The data for dissolved oxygen ranged from 6.46 to 7.01 ppm during the experimental period. The alkalinity of water ranged from 156 to 185 ppm during the experiment. All the physico-chemical parameters observed were within the optimum range required for the culture of the fish.

Growth Performance

This experiment was carried out to find the effect of Chlorella sp. supplemented diet on growth and survival of L. catla fry for 60 days of experimental period. The results of growth parameters including mean weight gain and specific growth rate (SGR) are shown in table 4. The initial weight of L. catla fry across treatments (T0, T1, T2, T3, and T4) remained similar, ranging from 6.50 ± 0.108 gm to 6.80 ± 0.041 gm. The final average wet weight recorded were 20.52 ± 0.75 g, 20.58 ± 0.085 g, 22.90 ± 0.238 g, 23.88 ± 0.165 g and 33.50 ± 1.132 g in treatment T0 (Control), T1, T2, T3 and T4 respectively. There was a significant difference in growth observed between treatments and over time. At the end of the experimental period (60 days), significant differences (p < 0.05) were found between the treatments for wet weight of fish. Final body weight was recorded in T4 (33.50 ± 1.132) treatment which was significantly higher (p < 0.05) than the other treatments, followed by T3 (23.88 ± 0.165 gm), T2 (22.90 ± 0.238 gm), and T1 (20.58 ± 0.085 gm), while T0 (20.53 ± 0.075 gm) had the lowest final body weight. There was no significant difference between T2 and T3 treatments, and also between T1 and T0. But all treatments showed higher weight gain, except T0 (control) treatment. Similarly, the total mean weight gain also differed among treatments, with T4 showing the highest (26.70 ± 1.093) value, which was significantly (p < 0.05) different from the control (T0) and other treatments. No significant (p˃0.05) difference among Treatment T2 (16.30 ± 0.158) and T3 (17.38 ± 0.131). While treatment T0 (control) and T1 (13.98 ± 0.103) were at par with each other. Highest SGR was found in T4 diet treatment (2.66 ± 0.046) followed by T3 (2.17 ± 0.022), T2 (2.07 ± 0.010), T0 (control) (1.90 ± 0.018) and T1 (1.87 ± 0.016). Significant difference (p < 0.05) was observed among the treatment diets; however, treatment T0 and T1 were at par with each other and T2 and T3 also at par with each other. However, T4 was found to be higher as compared to the other treatments.

Feed Utilization

This experiment was carried out to find the effect of Chlorella sp. supplemented diet on feed utilization and body composition of L. catla fry for 60 days of experimental period. The results of feed conversion ratio (FCR) and protein efficiency ratio (PER) obtained in the present experiment are given in table 4. Feed conversion ratio was significantly higher (p < 0.05) in T0 diet (2.97 ± 0.023) followed by T1 (2.71 ± 0.022), T2 (2.60 ± 0.020), T3 (2.43 ± 0.028) and lowest in T4 (2.09 ± 0.032) treatments respectively. There was significant difference observed between all the treatment (p < 0.05). The results on protein efficiency ratio (PER) of L. catla fry of all the treatments differed significantly from each other (p < 0.05). The highest PER was found in T4 (1.60 ± 0.025) followed by T3 (1.37 ± 0.016), T2 (1.28 ± 0.010), T1 (1.23 ± 0.010) and the lowest in T0 (control) (1.14 ± 0.009). Statistical analysis showed significant differences (p < 0.05) among all the treatments.

Survival Rate

Survival rate of L. catla fry in the respective treatments are presented in Table 4. Survival rate was observed at 100% among all the treatments (T0, T1, T2, T3 and T4) at 60 days of experimental period. There was no mortality indicated in this experiment throughout the culture period. This finding suggests that all dietary treatments supported optimal health conditions for the L. catla fry. Statistical analysis showed there were no significant differences (p > 0.05) found between all the treatments.

Proximate Analysis of Fish Carcass

At the end of the feeding period, five fish from each tank were collected randomly for proximate analysis of fish carcass. The data for proximate analysis of fish whole body after the experiment (dry weight basis) are given in Table 5. The protein content was significantly higher (16.92 %) in T4 compared to other treatments. The fat content was significantly highest (3.44 %) as compared to the control, with maximum in T4 followed by T1, T0, T3 and T2. The ash content was significantly highest in T4 (4.39 %) and the moisture content was found highest (80.98 %) in T0 as compared to other treatments and lowest found in T4 (75.25%).

Discussion

In the present study, the different percentages of Chlorella were added in the basal diet of L. catla and growth performance, survival and body composition were examined. The results indicated that Chlorella can be an alternative choice for fish diets. Based on the obtained data at the end of the 60 th day experiment, it was observed that the final wet weight gain and SGR were significantly higher in T4, while lower in T0 and T1 treatments. Significantly higher growth was reported in fish fed with the diet containing 20% Chlorella (T4), as compared to all the other experimental diets. Similar results were also observed by Xu et al. (2014) [34] , who observed a significant increase in growth parameters when fed Chlorella-incorporated diets at inclusion levels of up to 1.2% in Gibel carps Carassius auratus gibelio (Bloch). Sergejevova and Masojıdek (2013) [28] also observed positive effects on growth and skin coloration in freshwater sterlet, Acipenser ruthenus L. at 2.5% Chlorella inclusion. Similarly, Khani et al. (2017) [17] reported that dietary supplementation of 5% Chlorella powder significantly enhanced growth (98.06 ± 0.73 g), then control diets (57.8 ± 0.48) in koi fish culture up to 60 days. The similar growth performance in Catla catla fish fed Chlorella diets may be credited to growth promoters, such as adequate amounts of macronutrients and naturally occurring bioactive ingredients (Chlorella growth factor (CGF)) found in Chlorella vulgaris (Badwy et al. 2008) [7] . The growth-promoting effect of dietary Chlorella in fish has also been reported in other aquatic species such as shrimp or prawn. According to Maliwat et al. (2017) [18] 4% Chlorella vulgaris diet allowed the highest growth performance when compared to the control diets and final body weight increased in Macrobrachium rosenbergii post larvae. Radhakrishnan et al. (2015) [24] reported that, the growth and level of digestive enzymatic activities of M. rosenbergii fed C. vulgaris meal up to 50% level increased significantly. The current experiment indicates that inclusion of microalgae can positively influence growth in Catla catla fry. The observed improvements in growth and feed utilization associated with Chlorella powder imply that microalgae contain a range of substances acting as dietary protein, minerals, vitamins, fiber, feeding stimulants, antioxidants, and other unidentified growth-promoting compounds. These bioactive constituents contribute to the widespread use of Chlorella as a feed supplement that promotes enhanced fish growth and feed intake (Mustafa and Nakagawa, 1995) [22]. It was also observed that initially the effect of Chlorella diet was not much significant in terms of weight gain, but as culture days increased, the significant effect of Chlorella diets can be observed. Although the inclusion of Chlorella in this experiment is up to 20%, it can be assumed that if a higher percentage of Chlorella in diets were included, the growth may increase. In the present study the lowest FCR was recorded in T4 and highest in T0. The highest PER was found in T4 and lowest in T0. With respect to FCR and PER, all the treatments were significantly different from each other (p < 0.05). Similarly, Zeinhom (2004) [35] found that inclusion of algae in fish diets significantly improved the FCR, PER and PPV, whereas feed intake was significantly increased. In addition, Badwy et al. (2008) [7] reported that the inclusion of Scenedesmus sp. and Chlorella sp. up to 50% in diets could increase FI and PER, and reduce FCR in Nile tilapia. Similarly, Abid and Koyun (2018) [3] , recorded that Food Conversion Ratio in treatment with 5 g (Chlorella/kg diet) and treatment with 7.5 g (Chlorella/kg diet) were significantly lower than control diet, protein Efficiency ratio was also significantly higher in the same treatment than control. Increased protein efficiency ratio in fish fed chlorella containing diets in this study indicated that inclusion of chlorella improved protein utilization of labeo catla mainly due to abundant amino acids and nucleic acids which have been reported to increase the growth of fish (Cai et al., 2017) [10] . In this experiment, 100% survival was recorded throughout the experimental period. Similarly, Rahimnejad et al. (2017) [25] reported that inclusion of 15% chlorella meal fed to Olive Flounder, Paralichthys olivaceus gave 100% survival. Moosavi and Montajami (2013) [21] also reported 100% survival of angel fish (Ptrophyllum scalare) fed Spirulina plantesis. The higher survival rate may be attributed to the presence of carotenoids in chlorella algae, which enhance fish health and increase resistance to infections by reducing stress and strengthening immune function. In addition, stable water quality parameters, proper feeding management, and the absence of pathogenic challenges throughout the 60-day experimental period likely minimized stress and mortality, resulting in complete survival even without algal supplementation. These excellent survival rates indicated that the experimental diets supplied sufficient nutrition and had no harmful effects. At the end of the 60 th day of the feeding period, whole body protein, fat and ash content were enhanced; however, moisture content decreased along with chlorella supplementation. The protein, fat and ash content was significantly highest in T4 compared to other treatments. The moisture content was found highest in T0 as compared to other treatments. The moisture content of freshwater fish is reported to be between 72.1 and 83.6%, which supports the findings of the present study (Paul et al. 2018) [23] . The treatment T4 exhibited higher protein (40% more than control), which shows that Chlorella has higher acceptability in catla fry. Sivakumar et al. (2011) [32] reported that three live micro algal strains (Chlorella sp., Tetraselmis sp., and Isochrysis sp) and two cyanobacterial (Synechococcus sp. and Phormidium sp) strains fed P. monodan had significant improvement in body composition of protein, lipid and ash etc. In addition, similar results were also obtained by Bai et al. (2001) [8] , who recorded an increase in protein and fat content in Korean rockfish fed with diet inclusion of 2% and 4% chlorella powder as compared to control diets. Physico-chemical parameters of water were found inside the acceptable range in the present study. Temperature, pH and dissolved oxygen and alkalinity were found within the favorable range to enhance the growth of L. catla.

Conclusion

Recently the majority of juveniles of fish and waste fish are used for fishmeal in fishmeal production. Such activities are harmful to sustainable fisheries, so that fishmeal uses as a sources of protein is critical. Micro-algae meal supplementation improved growth, feed consumption, lipid metabolism, body structure, disease tolerance, and carcass consistency of a variety of fish. It is observed that higher growth; higher SGR; higher PER; better FCR and survival in L. catla fry can be obtained by feeding the (T4 treatment) 20% chlorella powder on the basis of the results obtained from the experiment. Apart from these observations, micro alga has significant effect on disease resistance antioxidant potentials, in future the experimental diets can also be utilized to check the efficiency of Chlorella for disease resistance and better survival rate.

Acknowledgement

AcknowledgmentThe authors wishes to acknowledge the College of Fisheries Science, Kamdhenu University, Veraval for its monetary assistance. The author is deeply indebted to Dr. N. H. Joshi for insightful direction and continual inspiration throughout the research endeavor. Profound gratitude is also extended to the author's parents and family members for their steadfast support, care, and encouragement.

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