Efficiency of Black Soldier Fly Larvae Meal (BSFLM) as Partial or Total Replacement of Fishmeal (FM) on Growth Performance,  Nutrient Utilization and Haematological Changes in Walking Catfish Clarias magur (Hamilton, 1822)

Intensification and adoption of feed-based aquaculture practices is developing quickly in North-Eastern region of India where 95% of the population are fish eater (Singh et al., 2017). FM is a major component in aquafeed due to its highly digestible protein as well as good palatability. However, an increasing demand and unstable production of FM has led to an increasing cost of aquaculture production. Therefore, there is a practical interest for partial or total replacement of FM with less expensive and protein-rich animal or plant ingredients has become a focus of research (Ashour et al., 2021; Sharawi, 2024). Among the insect species used as protein source in aquaculture, Black Soldier Fly (Hermetia illucens L. 1758) is one of the most interesting source for its sustainability related to its capacity to convert organic waste material into biomass containing proteins (Caligiani et al., 2018). In terms of protein quality, BSF larvae contain a favourable essential amino acid profile closer to fish meal than that of soybean meal (Anankware et al., 2018) and has been suggested as a promising alternative for the fast-growing feed-based aquaculture sector.
       
Several studies have shown that feeds with FM partially replaced by BSFLM as well as feeds without replacement in terms of growth of several fish species, e.g. Oreochromis aureus (Bondari and Sheppard, 1981) and Scophthalmus maximus (Kroeckel et al., 2012). Additionally, Maranga et al., (2023) and Abd Hamid et al., (2022) reported that BSFLM can replace FM without impairing and affecting performance in growth and survival of Clarias gariepinus. It is also cited that BSFLM is a good ingredient for broiler in their early stage on both nutrient digestibility and growth (Alqazzaz et al., 2019).
       
Clarias magur requires comparatively higher level of dietary animal protein for rapid growth (Mishra and Mukhopadhyay, 1996). Although several studies have been conducted on Clarias magur on efficiency of non-conventional animal protein like vermi meal and chicken viscera meal (Hussain et al., 2021), a very few information is available regarding the use of sustainable animal protein sources in practical diet formulation for Clarias magur on growth performance and health status. Therefore, the present study was designed to determine the suitable proportion of dried BSFLM on growth performance and haematological parameters in Clarias magur as a substitute to FM.

Study area and duration
 
The experiment was conducted at Fisheries Research Centre (Coordinates) lies at latitude 26.72^oS and longitude 94.20^oE and College of Fisheries (Coordinates) lies at latitude 26.22^oS and 92.50^oE, Assam Agricultural University, Jorhat, Assam, India from 31^st July 2023 to 28^th November 2023.
 
Animal and experimental design
 
Healthy and disease free Clarias magur (average wt. 2.13±0.04 g) stocked in experimental tanks at a stocking density of 4 fish per m². Fishes were acclimatized prior the experiment for two weeks in cement cisterns. The water quality parameters were in normal ranges i.e. pH (7.73-8.23), dissolved oxygen (5.3-7.1 mgl^-1), alkalinity (>70 mgl^-1), ammonia (0.23-0.42 mgl^-1) and nitrite (0.007-0.031 mgl^-1) during the entire experiment (APHA, 2023).
 
Experimental diets
 
The six-test supplementary iso-nitrogenous sinking feeds containing 35% crude protein level with a variation of composition level (Table 1). The main ingredient in feed is BSFLM, which is an alternate ingredient source to FM along with soyabean meal (SM), mustard oil cake (MOC) and wheat bran (WB) with different ingredient composition.


       
The mixed ingredients were subjected to pre-conditioning and pelletized in a mechanical pelletizer and were dried to a moisture level of 6-8% in an oven and the dried diets were stored in air tight polythene until used. Proximate analysis of all the dietary ingredients (Table 2) and experimental diets (Table 3) were conducted (AOAC, 2023).




 
Feeding and sampling of the experimental fish
 
Feeding frequency was twice in 24 hrs i.e. during forenoon and evening. The feeding rate varied between 1- 4% of body weight of the total biomass according to the acceptance of the feed. The experimental tank was arranged in completely randomized design (CRD). The growth performance was assessed fortnightly.
 
Growth and nutrient utilization
 
Growth performance of the fish was monitored by the following parameters like

 

 
The feed utilization performance of the fish evaluated by the following parameters like
 

        
Proximate analyses of experimental fish were also carried out after the experiment (AOAC, 2023). Haematological changes of the fish were evaluated by the following parameters like 
 

 

 

Hemoglobin concentration in blood was determined by Sahlis haemometer (Microsil, India) and Packed cell volume (PCV) was measured following modified method of Schaperclaus (1991).
 
Statistical analysis
 
The data from the experiment was subjected to Shapiro-Wilk test for assessment of normality. All the dependent variables were normal except SGR. Different transformation of non-parametric data was done which failed the normality assumption again, therefore Kruskal-Walli’s test, followed by Dunn’s test for comparison of means. One way analysis of variance (ANOVA) was performed for all the parametric variables and Duncan multiple range test was used for post hoc comparison. The significant level was set at 5% level (P<0.05). All the statistical test were performed in IBM SPSS Software 26.0.

Growth, survival and nutrient utilization parameters
 
In general, experimental fish exhibited consistent increase in mean weights during the culture period (Table 4). The highest weight gain observed in T2 followed by T1 and the values were significantly different (P<0.05). Increase in weights of Clarias magur become more evident with a weight of 50.47±0.23 g (T2) followed by 40.46±0.35 g (T1) and lowest in control (19.43±0.20 g). Similarly, the Specific Growth Rate (% day^-1) of Clarias magur are presented in Table 4. The values were significantly different (P<0.05) with highest SGR was found in T2 (0.42±0.002) followed by T1 (0.34±0.003) and lowest in control (0.16±0.002).


       
In the present study, there were no significant differences (P>0.05) in the survival (%) among the treatments. Overall survival of the fish varied from 88% (T5) to 96% (T1) in different treatments (Table 4).
       
The apparent FCR showed significant variation (P<0.05) between different treatments (Table 4). However, the lowest value of FCR (2.10±0.03) was noticed for T2 followed by T3(2.16±0.06) and the FCR (2.62±0.06) was the highest for control (C). PCE also showed significant variation (P<0.05) among treatments. The highest PCE (91.71±0.92) was obtained in treatment T2 followed by that for T3 (90.31±2.59) and the lowest for control (74.94±1.72). Similarly, the apparent PER was the highest for Treatment T2 (1.41±0.02) followed by T3 (1.40±0.04), while the lowest value of apparent PER (1.20±0.02) was obtained for control (P<0.05).
 
Yield parameters
 
There were significant differences (P<0.05) in the final biomass, daily weight gain and NFY (Table 5). Notably the highest net gain in biomass (755.13±27.60 g) was observed in T2 as compared to other treatments and lowest was found in control (275.10±5.59 g).


 
Proximate composition of fish
 
The crude protein, crude lipid and NFE content showed significant variations (P<0.05) among different treatments (Table 6) whereas crude ash and moisture content did not show significant variation (P>0.05). The highest and lowest crude ash content was found in control (8.73±0.19) and T2 (8.03±0.24) respectively. The highest protein content was found in T2 (64.83±0.32%) followed by T1 (64.54±0.28%) and lowest in control (62.57±0.47 %). Similarly, lipid content was highest in T4 (21.10±0.38%) followed by T3 (20.95±0.27%) and lowest in control (19.00±0.25%).


 
Haematological parameters
 
Haematological (RBC, WBC, Hb and PCV) parameters showed significant differences (P<0.05) among the treatments (Table 7). Fish in the T2, recorded the highest RBC (2.10±0.01 million/m³) followed by T3 (1.92±0.02 million/m³) whereas, the lowest RBC count was found in control (1.35±0.02 million/m³). Similarly, haemoglobin content was highest in T2 (8.2±0.05 g/dl) followed by T1 (7.89±0.02 g/dl) and PCV was also highest in T2 (24.63±0.05%) followed by T1 (23.36±0.01%). On the contrary, WBC level was found highest in treatment T5 (49.64±0.71 million/m³) followed by T4 (48.17±0.16 million/m³) and lowest (33.36±0.01 million/m³) in control (P<0.05).


       
In the present study, Clarias magur juveniles fed with 50% BSFLM (T2) exhibited highest growth performance and feed/nutrient utilization. Similar results were found by Fawole et al., (2020), where feeding African catfish, Clarias gariepinus with 50% partially defatted BSFLM increased the growth performance. The results were in accordance with a study on Atlantic salmon (Salmo salar) showed a good growth response when fed with BSFLM (Belghit et al., 2018) and also in loach (Misgurnus anguillicaudatus) where superior growth performance achieved while BSFLM incorporation level was 50% (Nguyen and Tran, 2025). Similarly, 50% replacement of FM with BSFLM in the diet of tilapia (Oreochromis niloticus) also showed positive effects on growth and feed utilization parameters (Muin et al., 2017).
       
The knowledge of the body composition of fish and factors affecting, a tool to know about fish health, determination of efficiency of transfer of nutrients from the food to the fish made it possible to predictably modify carcass composition (Al-Ogaily et al., 1996). In the present study crude protein, crude lipid and NFE showed significant variations (P<0.05) among different treatments and superior proximate composition (higher crude protein and lower crude lipid) was observed in T2. The findings of the present study were in contrast to previous studies in Jian carp (Zhou et al., 2018) and Atlantic salmon (Belghit et al., 2019) where BSFLM incorporated diet showed no significant impact on flesh quality. This phenomenon may be due to the fact that Clarias magur is a carnivore fish and thus, is probably more capable of digesting insect larvae which contains chitin than the other fish species mentioned above.
       
Haematological indices are essential for evaluating formulated feed efficacy, physiological stress and fish health and growth performance (Dawood et al., 2020). The Haemoglobin, RBC and PCV is a function of oxygen supply, absorption and transportation within a living cell and depletion in the count may cause anaemia and lead to death in fish. In the present study, highest RBC, Hb and PCV values were noticed in T2 group of fish as compared to the other treatments (P<0.05) and the values were within the normal ranges recommended for Clarias gariepinus (Adedeji, 2009), a congeneric species of Clarias magur. This finding is supported by a study where dietary supplement of BSFLM (even at 100%) did not have deleterious effect on health status of Clarias gariepinus (Ayodeji et al., 2020).
       
WBC count increases due to metabolic stress (Misra et  al., 2006). The WBC count obtained in this study have not been associated with any detrimental health effect in Magur. This explained the efficacy of BSFLM as a protein source in maintaining good and healthy condition in Magur. In this study, comparatively lower WBC values were noticed in T2 group of fish as compared to the other treatments which may be due to lower metabolic stress in T2 group of fishes. However, future research is required to study the inclusion level of BSFLM on metabolic stress in Clarias magur.

Our study demonstrated that Clarias magur can be fed pelleted feed containing BSFLM up to 50% combined with 50% FM which has the higher growth performance, nutrient utilization and overall health status of Magur as compared to other inclusion level. Thus, it suggests that BSLF could be considered a promising protein source for Clarias magur in aquafeed industry contributing to low-cost and healthy fish feed production. However, further research efforts are necessary to find out the Chitin composition of BSFL and its effect on metabolic and physiological activity in Clarias magur.

The authors would like to express their deepest gratitude to the Dean, College of Fisheries, Assam Agricultural University, Raha and Head, Department of Aquaculture, College of Fisheries, Raha, Nagaon, Assam for providing all the necessary facilities and support for conducting this work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for Animal care and handling techniques were approved by the Institutional Ethics Committee of College of Fisheries, Assam Agricultural University, Raha, Nagaon, India. The animals used in this study were not endangered or protected species. The study was conducted with compliance to the provisions of the guidelines of the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests, Government of India, for care and use of animals for scientific experiments.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.