Evaluation of Silkworm Pupae Meal and Fish Protein Hydrolysate as a Sustainable Fish Meal Replacement in Striped Murrel (Channa Striata) Diet: Impact on Growth Performance, Enzyme Activity and IGF-1 Gene Expression

The SWP and FPH were sourced from M/s Silvermine Pvt. ltd, Udumalaipet, Tamil Nadu, India, and M/s Janatha Fishmeal and Oil Products, Pvt. Ltd., Karnataka, India, respectively. Five isonitrogenous (44% crude protein), isolipidic (11% crude lipid), and isocaloric (18 MJ/kg gross energy) experimental diets were formulated and prepared. The experimental diets were labeled as control (35% FM), 25 SWP (replacing 25% FM with SWP), 50 SWP (replacing 50% FM with SWP), 25 SWP+FPH (replacing 25% FM with a combination of SWP and 3.5% FPH), and 50 SWP+FPH (replacing 50% FM with a combination of SWP and 3.5% FPH). Table 1 presents the ingredient and nutrient compositions of SWP- and FPH-based experimental diets.

To prepare the feed, all dry feed ingredients were ground into a fine powder using a pulverizer and then sieved through a 180-micron mesh. After weighing the powdered ingredients in accordance with the formulation, the appropriate volume of water was added and mixed well with a vertical feed mixer. The blended ingredients were then extruded using a 2-mm die after being run through a twin-screw extruder (Jinan Sunpring Machinery, China). The extruder was maintained at a temperature of 90–95°C, a pressure of 2–3 bars, and a moisture level of 15–17% during the extrusion process. The extruded pellets were then dried using a horizontal dryer at 60°C for 15 mins to achieve approximately a 10% moisture level. After drying, the extruded pellets were coated with fish oil and palm oil using a Pegasus^® vacuum coater (KK Life Sciences, Chennai, India). Lastly, before being used, the extruded pellets were kept in plastic containers at 4°C.

The striped murrel (C. striata) fingerlings, weighing 2.43±0.35 g on average, were purchased from M/s Maria Integrated Fish farm located in the Thiruvannamalai district of Tamil Nadu, India. The striped murrel fingerlings were initially placed in a nursery tank that was 4 meters long, 4 meters wide, and 1.5 meters deep. Then, they were acclimated to the experimental setup for a duration of 30 days. To reduce cannibalistic activity during the acclimatization period, the fish were fed a commercial diet consisting of 44% crude protein and 10% crude lipid (Growel Feeds Pvt. Ltd., Andhra Pradesh, India) five times a day until satiation. After completing the acclimatization period, a total number of 450 striped murrel juveniles (initial weight: 9.99±0.15 g) were randomly stocked into 15 experimental cages (1 m long × 1 m wide × 1.5 m high). The volume of water maintained in each experimental net cage was 700 liters, and the stocking density per cage was 30 fish. The experimental cages were installed in a cement tank (4 m long × 4 m wide × 1.5 m high). A total of 15 experimental cages were utilized for this study, with each treatment performed in triplicate (n=3). The fish were fed experimental diets until they reached satiety three times daily at 08:00, 13:00, and 18:00 hours for a duration of 60 days.

During the feeding trial, the optimum water quality parameters such as temperature (29.88±0.37°C), dissolved oxygen (5.69±0.34 mg/L) (Lutron DO-5519 Dissolved Oxygen Meter, Instrukart Pvt. Ltd., Hyderabad, India), pH (8.15±0.19) (KLPHM-119 pH Meter, Kinglab Instruments Pvt. Ltd., Chennai, India), ammonia-N (0.67±0.36 mg/L), nitrite-N (0.43±0.35 mg/L), hardness (181±13 mg/L), and total alkalinity (133±6.0 mg/L) were maintained and checked every three days. All parameters were analyzed according to standard protocols outlined by APHA (2012).

Following the 60-day feeding trial, all fish from each replicate cage was not fed for 24 h. The total weight of fish in each replicate cage was then measured (MINI 20KG REAR weighing scale, Activa Corporation, Chennai, India), and the number of fish was counted to assess the somatic growth performances and nutrient utilization efficiencies. The parameters assessed included weight gain (WG), feed intake (FI), average daily growth (ADG), survival rate (SR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), lipid efficiency ratio (LER), protein retention rate (PRR), and lipid retention rate (LRR).

The growth performance and nutrient utilization of fish in this feeding trial were calculated as follows:

WG(g)=Final wet weight (g)−Initial​ wet weight (g),ADG(g)=(Final wet weight (g)−Initial wet weight (g))/Days of culture,SR(%)=(Number of fish survived /Number fish stocked)×100,SGR(% da y−1)=[(LN (final weight)−(LN (initial weight)/Number of days)]×100,FCR=Total feed consumption (g)/Wet weight gain (g),PER=Wet weight gain of fish (g)/Protein intake (g),LER=Wet weight gain of fish (g)/Lipid intake (g),PRR(%)=100×[(Final body weight× Final body protein)−(Initial body weight× Initial body protein)]/Protein consumed,LRR(%)=100×[(Final body weight×Final body lipid)−(Initial body weight×Initial body lipid)]/Lipid consumed. $$\matrix{ {WG\textit{(g) = Final}\,\textit{wet}\,\textit{weight}\,\textit{(g)} - Initial\,wet\,weight\,\textit{(}g\textit{),}} \cr {ADG\textit{(}g\textit{) = (}Final\,wet\,weight\,\textit{(}g\textit{)} - Initial\,wet\,weight\,\textit{(}g\textit{))}/Days\,of\,culture,} \cr {SR\textit{(}\% \textit{)} = \textit{(}Number\,of\,fish\,survived\,/Number\,fish\,stocked\textit{)} \times \textit{100},} \cr {SGR\textit{(}\% \,day^{ - 1} \textit{)} = \textit{[(}LN\,\textit{(}final\,weight\textit{)} - \textit{(}LN\,\textit{(}initial\,weight\textit{)}/Number\,of\,days\textit{)]} \times \textit{100},} \cr {FCR = Total\,feed\,consumption\,\textit{(}g\textit{)}/Wet\,weight\,gain\,\textit{(}g\textit{)},} \cr {PER = Wet\,weight\,gain\,of\,fish\,\textit{(}g\textit{)}/Protein\,intake\,\textit{(}g\textit{)},} \cr {LER = Wet\,weight\,gain\,of\,fish\,(g)/Lipid\,intake\,\textit{(}g\textit{),}} \cr {\textit{PRR(\% ) = 100} \times \textit{[(}Final\,body\,weight \times \,Final\,body\,protein\textit{)} - \textit{(}Initial\,body\,weight \times \,Initial\,body\,protein\textit{)]}/Protein\,consumed,} \cr {LRR\textit{(}\% \textit{)} = \textit{100} \times \textit{[(}Final\,body\,weight \times Final\,body\,lipid\textit{)} - \textit{(}Initial\,body\,weight \times Initial\,body\,lipid\textit{)]}/Lipid\,consumed.} \cr } $$

To assess the hepatosomatic index (HIS) and visceral somatic index (VSI), five fish from each replicate cage were anesthetized using tricaine methanesulfonate (MS-222, 200 mg/l, Sigma-Aldrich Inc.) (IACUC, 2014) and sacrificed. The visceral organs were carefully dissected and weighed to calculate the VSI. The liver was then separated from the visceral organs and weighed to determine the HSI. The indices were calculated using the following formulas:

HSI(%)=[Weight of liver(g)/Total weight of fish (g)]×100,VSI(%)=[Weight of visceral (g)/Total weight of fish (g)]×100. $$\matrix{ {HSI\textit{(}\% \textit{)} = \textit{[}Weight\,of\,liver\textit{(}g\textit{)}/Total\,weight\,of\,fish\,\textit{(}g\textit{)]} \times \textit{100},} \cr {VSI\textit{(}\% \textit{)} = \textit{[}Weight\,of\,visceral\,\textit{(}g\textit{)}/Total\,weight\,of\,fish\,\textit{(}g\textit{)]} \times \textit{100}.} \cr } $$

After acclimation, a pooled twenty numbers of initial fish were ice killed, and the whole-body samples were collected for estimation of initial whole-body proximate composition. Following the 60-day feeding trial, six fish from each replicate cage were ice killed, and the samples were taken for final whole-body proximate composition. The proximate composition of both the experimental diets and the whole-body carcass samples was determined using standard protocols established by AOAC (2010). Moisture content was assessed using a hot air oven (HTLP-013, Hi-Tech Lab Solutions, Mumbai, India) at 105°C for 5 h. The crude protein content was evaluated using the Kjel-dahl method (Kelplus-Distyl Em Ba, Pelican Equipment, Chennai, Tamil Nadu, India), while the crude lipid was analyzed via the Soxhlet method (Socsplus – SCS 04 AS DLSTS, Pelican Equipment, Chennai, Tamil Nadu, India). Crude fiber levels in the experimental diets were measured using the fibercap method (FIBRAPLUS FES 04, Pelican Equipment, Chennai, Tamil Nadu, India). The total ash content of both the diet and whole-body samples was estimated using a muffle furnace (HT-MF900-4.25S/G, Hi-Tech Lab Solutions, Mumbai, India) at 550°C for 6 hours. The gross energy content of the experimental diets was determined using a bomb calorimeter (IKA-C 6000, IKA^® India Private Limited, Bangaluru, India). The proximate composition of the experimental diets and whole-body proximate composition are summarized in Table 1 and Table 6, respectively.

Ultra-pressure liquid chromatography (UPLC) (Waters ACQUITY-UPLC, Waters (India) Private Limited, Bangaluru, India) was used to ascertain the amino acid composition of the experimental diets in accordance with the approach outlined by Ishida et al. (1981). Briefly, 50 mg of dried samples were placed in a nitrogen-sealed Borosil glass ampule and hydrolyzed with 6 N HCl for 24 hours at 110°C. After hydrolysis, the samples were neutralized and filtered through a 0.2 μm PTFE filter. The hydrolyzed samples were then derivatized using the AccQ-Tag Ultra Derivatization Kit and separated on a Waters ACQUITY UPLC equipped with a 2.1 × 100 mm column with a 1.7 μm pore size (AccQ-Tag Ultra C18), utilizing a stepwise gradient elution. Calibration was carried out with Amino Acid Standard H (Product no: WAT088122), and amino acids were quantified based on absorbance readings at 260 nm, as measured by a tunable UV detector, and analyzed using Empower 2 Software. Tryptophan levels were assessed following alkaline digestion of the samples with lithium hydroxide. Table 2 presents the essential and non-essential amino acid composition of experimental diets.

At the conclusion of the feeding trial, all fish were starved for 24 h. Three fish from each replicate were then anesthetized with tricaine methanesulfonate (MS-222, 200 mg/L, Sigma-Aldrich Inc.), dissected, and sampled. Intestinal samples were collected for digestive enzyme analysis, while liver samples were taken for antioxidant enzyme analysis. After sample collection, a 5% tissue homogenate of the intestine and liver was prepared using a chilled 0.25 M sucrose solution with a Teflon-coated mechanical homogenizer (REMI Equipments, Mumbai, India). This entire process was conducted under ice-cold conditions. The homogenates were then centrifuged at 4000 rpm for 5 minutes at 4°C, and the supernatant was collected and stored at −20°C until the enzyme activities were analyzed.

The casein hydrolysis method was used to measure protease activity in the tissues of the intestine and liver (Drapeau, 1976). One unit of enzyme activity was defined as the amount of enzyme required to release acid-soluble fragments corresponding to Δ0.001A280 per minute at 37°C and pH 7.8. Amylase activity was assessed using the 3,5-dinitrosalicylic acid (DNS) method (Rick and Stegbauer, 1974), with starch serving as the substrate. Amylase activity was expressed as the amount of maltose produced from starch per minute at 37°C. The titration method developed by Cherry and Crandall (1932) was used to estimate lipase activity. This method measures the quantity of fatty acids produced during the enzymatic hydrolysis of triglycerides present in a stabilized olive oil emulsion. Lipase activity was expressed in enzyme units (U) per milligram of protein. The Bradford (1976) technique was used to quantify the total protein content of the samples. The basis of the Bradford assay is the binding of Coomassie Blue G250 dye to proteins.

Catalase (CAT) activity was measured following Aebi (1984) method. The assay was based on the decomposition of hydrogen peroxide (H₂O₂) and monitored spectrophotometrically at 240 nm with a 1 cm path length. One unit of catalase activity was defined as the amount of enzyme required to decompose 1.0 µmol of H₂O₂ per minute at pH 7.0 and 25°C. Super-oxide dismutase activity (SOD) was assessed following the method of Beauchamp and Fridovich (1971), which measures the enzyme’s ability to inhibit the photoreduction of nitro blue tetrazolium (NBT). One unit of SOD activity was defined as the amount of enzyme needed to inhibit 50% of NBT reduction under assay conditions at pH 7.8 and 25°C. Glutathione peroxidase (GPx) activity was determined using Wendel (1980) method, where one unit of enzyme activity was defined as the amount required to oxidize 1.0 µmol of reduced glutathione to oxidized glutathione per minute, using H₂O₂ as a substrate at pH 7.0 and 25°C. The malate dehydrogenase (MDH) was determined by Ochoa (1955). Malate dehydrogenase catalyzes the reversible conversion of L-malate to oxaloacetate with NAD⁺ as a cofactor. The enzyme activity is measured by monitoring the decrease in absorbance at 340 nm due to NADH oxidation. One unit of MDH activity is defined as the enzyme amount required to convert 1 µmol of NADH and oxaloacetate to L-malate and NAD⁺ per minute at 25°C and pH 7.5. Glucose-6-phosphate dehydrogenase (G-6-PD) was determined by the methods of Morales et al. (1990). Glucose 6-phosphate dehydrogenase catalyzes the oxidation of D-glucose to D-glucono-1, 4-lactone, using NADP⁺ as a cofactor. Activity is measured by monitoring the increase in absorbance at 340 nm due to NADPH formation. One unit of G6PDH activity oxidizes 1 µmol of glucose per minute at pH 7.0 and 37°C.

At the conclusion of the feeding trial, one fish from each replicate was randomly euthanized using tricaine methanesulfonate (MS-222, 200 mg/l, Sigma-Aldrich Inc.) to collect mid-intestine samples for histological evaluation. The intestinal samples were immediately fixed in 10% neutral buffered formalin. Subsequently, the samples underwent dehydration through a series of ethanol concentrations, were cleared in xylene, and were embedded in paraffin blocks. The blocks were then sectioned into 4−5 μm thick slices using an RM2125RTS rotary microtome (Leica), mounted on glass slides, and stained with hematoxylin and eosin (H&E). Finally, the slides were examined with a DM750 light microscope (Leica), and digital images were captured using a Leica EC3 camera with the Leica Application Suite Version 2.0.0. Villi length (VL) and villi width (VW) were measured on three slides per sample using a micrometer, and mean values were calculated.

At the conclusion of the feeding trial, three fish from each replicate were randomly euthanized using tricaine methanesulfonate (MS-222, 75 mg/l, Sigma-Aldrich Inc.) to collect the blood samples. A 1-ml heparinized syringe was used to obtain both heparinized and non-heparinized blood samples from the caudal vein of fish. The samples were then promptly placed on ice after being properly moved into tubes that had been heparinized and those that had not. The heparinized samples were analyzed for various hematological parameters, including hemoglobin (Hb), leukocyte (Leuk) counts, erythrocyte (Ery) counts, hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) using a fully automated hematology analyzer (Zybio Z3 Inc., China). The nitroblue tetrazolium (NBT) assay, or respiratory burst activity, was measured in accordance with the protocol established by Anderson and Siwicki (1995).

The non-heparinized blood samples were subjected to clot for 2 h at 4°C. After clotting, serum was extracted alone by centrifuging the clotted blood at 3500 g for 25 minutes at 4°C using a refrigerated centrifuge (Eppendorf Centrifuge 5804R). Serum biochemical parameters, such as glucose (GLU), cholesterol (CHO), triglycerides (TRY), total protein (TP), albumin (ALB), globulin (GLB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), were examined using commercial kits (Pathozyme Diagnostics Pvt. Ltd., Maharashtra, India) along with a semi-automatic biochemistry analyzer (Cytokine SK3002B, Cytokine Healthcare Pvt. Ltd., Chennai, Tamil Nadu, India). Total antiprotease in the serum was found using the method which was explained in Zuo and Woo (1997). Additionally, lysozyme activity was measured turbidimetrically according to the method of Sankaran and Gurnani (1972), using lyophilized hen egg white lysozyme (HEWL; Sigma) as the standard.

The relative mRNA expression of immune-related genes was estimated using a C1000 Touch thermal cycler-CFX96 Real-Time PCR system (Bio-Rad). Total RNA for the TGF-β1 and NF-κ B genes was extracted from fish kidney samples with PureZOL™ RNA Isolation Reagent (Takara Bio Inc.). Subsequently, 2 μg of total RNA was converted into first-strand complementary DNA (cDNA) using the first-strand cDNA synthesis kit (Thermo Scientific). Each quantitative real-time polymerase chain reaction (qRT-PCR) had a total volume of 20 μL, containing 10 μM of each primer (forward and reverse), 20 ng of cDNA template, and 1× SYBR^® Green Master Mix. The initial denaturation step was performed at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60–62°C (depending on the target gene) for 30 seconds, and extension at 72°C for 30 seconds. The cycling ended with a dissociation curve analysis. The 2^−ΔΔCt technique (Livak and Schmittgen, 2001) was utilized to determine the relative expression level of each individual gene. The relative mRNA expression levels of the TGF-β1 and NF-κ B genes were compared with β-actin (housekeeping gene). Primer sequences (Table 3) and relative gene expression procedures were analyzed according to Siddaiah et al. (2022).

All the observed data were assessed for normality using the Shapiro-Wilk test and homogeneity of variance with Levene’s test. Prior to the statistical analysis, percentage data underwent arcsine transformation. One-way ANOVA was used to statistically analyze all data, followed by Duncan’s multiple range test (Duncan, 1955) to identify significant differences (P<0.05) among treatments. The analysis was performed using SPSS 24.0 software (SPSS Inc., USA) for Windows. Results are presented as mean values ± standard deviation (SD) (n=3).

The growth performance and survival of striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets are shown in Table 4. Significant differences (P<0.05) were observed in growth performance indicators, including FW, WG, ADG, and SGR in striped murrel fed different levels of SWP, both individually and also in combination with FPH. However, no significant differences (P>0.05) were found in SR, HSI, and VSI. Among the dietary groups, fish fed the control, 50SWP+FPH, and 25SWP+FPH diets exhibited significantly higher (P<0.05) FW than other diets. Similarly, fish fed the control, 50SWP+FPH, and 25SWP+FPH diets showed significantly higher (P<0.05) WG, ADG, and SGR than other diets.

The feed conversion, nutrient utilization, and retention of striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets are shown in Table 5. Significant differences (P<0.05) were noted in FCR, PER, LER, PRR, and LRR among striped murrel fed varying inclusion levels of SWP alone and also supplemented with FPH. Fish fed the control and 50SWP+FPH diets exhibited a lower FCR than other diets. Similarly, fish fed the control and 50SWP+FPH diets showed significantly higher (P<0.05) PER, LER, PRR, and LRR than other groups.

The whole-body proximate composition of striped murrel, including moisture, crude protein, crude lipid, and total ash, remained unaffected by different dietary inclusion levels of SWP alone or in combination with FPH. Statistical analysis showed no significant differences (P>0.05) among treatments, indicating that replacing FM with SWP, either independently or supplemented with FPH, did not alter the overall body composition of the fish. Table 6 provides a detailed comparison of the whole-body proximate composition of striped murrel fed diets containing varying levels of SWP and SWP supplemented with FPH.

The digestive enzyme activities of striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets are presented in Figure 1. Significant differences (P<0.05) were noted in the digestive enzymes, including amylase, protease, and lipase in striped murrel fed different inclusion levels of SWP alone and also SWP supplemented with FPH. Fish fed with control and 50SWP+FPH diets showed significantly higher (P<0.05) amylase and protease, but it was not significantly different from those fish fed with 25 SWP+FPH diet. Similarly, significantly higher (P<0.05) lipase activity was noted in fish fed the control diet, but it was not significantly different from that of the 50SWP+FPH diet.

Figure 1.

Digestive enzyme activities (U mg⁻¹ protein) of C. striata fed varying inclusion levels of SWP and SWP supplementation with FPH diets

The activities of hepatic antioxidant and metabolic enzymes, including CAT, SOD, GPx, G-6-PDH, and MDH in striped murrel fed varying inclusion levels of SWP alone and also SWP supplemented with FPH diets are shown in Figure 2. Significant differences (P>0.05) were noted in CAT and SOD activities among striped murrel fed varying inclusion levels of SWP alone and also with SWP supplemented with FPH. Fish fed the control and 50SWP+FPH diets showed significantly higher (P<0.05) CAT activity than other diets, although this was not significantly different from the 25SWP+FPH diet. Similarly, significantly higher (P<0.05) SOD activity was observed in fish fed the 50SWP+FPH diet, but it was not significantly different from those fed the 25SWP+FPH and control diets. The activities of GPx, G-6-PDH, and MDH were not significantly affected (P>0.05) by the different inclusion levels of SWP alone and also SWP supplemented with FPH diets in striped murrel.

Figure 2.

Antioxidant and metabolic enzyme activities (U mg⁻¹ protein) of C. striata fed varying inclusion levels of SWP and SWP supplementation with FPH diets

The intestinal VL and VW of striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets are shown in Table 7, and the intestine morphology of striped murrel is shown in Figure 3. Significant differences (P<0.05) were found in both VL and VW among striped murrel fed varying inclusion levels of SWP alone and also SWP supplemented with FPH. Fish fed the control diet showed significantly higher (P<0.05) VL compared to other diets, although it was not significantly different from the 50SWP+FPH diet. Similarly, fish fed the 50SWP+FPH and control diets showed significantly higher (P<0.05) VW, but these values were not significantly different from the 25SWP+FPH diet. The intestine morphology of striped murrel fed varying inclusion levels of SWP alone and also SWP supplemented with FPH diets are displayed in Figure 3.

Figure 3.

Transverse sections of the mid intestine from C. striata fed varying inclusion levels of SWP and SWP supplementation with FPH diets; The sections were stained in H & E to enhance the contrast (100× magnification; scale bar with 20 μm); (A, 35% FM (control); B, 25% FM replaced with SWP (25 SWP); C, 50% FM replaced with SWP (50 SWP); D, 25% FM replaced with a combination of SWP and 3.5% FPH (25 SWP+FPH); E, 50% FM replaced with a combination of SWP and 3.5% FPH) (VL, Villi length; VW, Villi width and MT, Muscular thickness)

The hemato-biochemical profile of striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets is shown in Table 8. No significant differences (P>0.05) were found in hematological parameters, including Hb, Leuk, Ery, Ht, MCV, MCH, MCHC, and NBT values among striped murrel fed varying inclusion levels of SWP alone and also SWP in combination with FPH.

Similarly, the serum biochemical parameters, including GLU, TRY, CHO, TP, ALB, GLB, ALT, AST, ALP, APA, and LYZ values, were not significantly (P>0.05) affected by varying inclusion levels of SWP alone and also SWP in combination with FPH.

The IGF-1 gene expression in striped murrel fed varying inclusion levels of SWP and SWP supplemented with FPH diets are displayed in Figure 4. Fish fed the control and 50SWP+FPH diets exhibited significantly higher (P<0.05) relative mRNA expression of IGF-1 compared to other diets.

Figure 4.

Relative mRNA expression of IGF-I in the liver of C. striata. Bars with different superscripts indicate significant differences determined by Duncan’s test (P<0.05)