Effect of deoiled silkworm pupae meal (Bombyx mori) as a replacement of fish meal in the diet of juveniles of Silver pompano, Trachinotus blochii

doi:10.21203/rs.3.rs-2740866/v1

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Effect of deoiled silkworm pupae meal (Bombyx mori) as a replacement of fish meal in the diet of juveniles of Silver pompano, Trachinotus blochii

https://doi.org/10.21203/rs.3.rs-2740866/v1

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A ninety days growth trial and four weeks of digestibility study were conducted to evaluate the effect of replacing fish meal (FM) with deoiled silkworm pupae meal (DSWP) in the diet of juvenile silver pompano. Silver pompano seeds (0.93 ± 0.07 g) were randomly distributed into 15 FRP tanks (120 × 55 × 38cm) each consisting of 20 fish. Fish were fed thrice a day until apparent satiation with five experimental diets (RD, 25FMODSWP, 50FMODSWP, 75FMODSWP and 100 FMODSWP) which was prepared by replacing fishmeal at 0, 25, 50, 75 and 100% with DSWP respectively. The replacement of fishmeal with DSWP at 25% (p < 0.05) improved the final body weight, specific growth rate, feed conversion ratio and protein efficiency ratio. Apparent digestibility coefficient (ADCs) values of crude protein and fat was found to be higher in the RD and 25FMODSWP groups. Protease activity was significantly (p < 0.05) higher in fish fed the 25FMODSWP diet while amylase and lipase activities were higher in the 75FMODSWP and 100FMODSWP, respectively. The replacement of fishmeal by 75FMODSWP resulted in lower (p < 0.05) fat deposition in the whole-body tissue. The dietary inclusion of DSWP significantly (p < 0.05) improved overall blood profile until 50% FM protein replacement. The replacement of fishmeal with DSWP beyond 50% affects the intestinal structure due to necrotic lesions in the microvilli and detachment of surface epithelial cells from lamina propria. The results of this study suggested that DSWP can be used to replace 30.5% of FM protein which accounts 9.5% of SWP in the diet of silver pompano without any adverse effect on fish.

Silver pompano

deoiled silkworm pupae

growth performance

digestibility and digestive enzyme.

Fish meal is the major protein source in fish feed with an excellent nutritional profile. The high use of fish meal in aqua feed has increased the demand and cost of this product (Olsen and Hasan 2012). The effective utilization is not only the well-known conventional feed ingredients but also the identification and introduction of new and lesser-known plant and animal-based feed ingredients that are not in direct conflict with humans will be the only chance of providing billions of animal protein. In this context, aquaculture species have benefited greatly from the addition of plant-based protein in their diets, but there are still certain persistent issues, such as the presence of anti-nutritional factors and an unbalanced amino acid profile (Francis et al. 2001). With increasing pressure on fish meal, novel feed ingredients such as insects offer new avenues for growth and an effort to reduce the demand of fish meal. Insects are a promising alternative material for fish feed due to their nutritional and zootechnical benefits, including their amino acid composition, digestibility, immune-boosting properties, and animal health. It could be applied to the developmental stages of fish or in its infancy (Nogales-Merida et al. 2019). The primary benefits of employing insects are their low cost, quick reproduction, and high feed conversion efficiency (Katayama et al. 2008). In addition, Insects are highly sustainable and has a highly nutritious composition (Tran et al. 2015). In comparison, the annual protein yield of soybean crops was 0.12kg/m² and average Insect protein yields (accounting for feedstock inputs) have been reported at 2.18, 4.06, and up to 7.93 kg/m² for cricket, mealworms, and Black solider fly (BSF) larvae (Koutsos et al. 2019). Greater insect protein yields are due to the short duration of growth cycles annually that are yet unattainable with crops (Koutsos et al. 2019). Insect yields require significantly less water, feed and land while producing much less CO₂ (Van Huis et al. 2017). There is a potential to use insects as a fish meal replacement, including black soldier fly larvae, house fly larvae, house fly maggots, mealworm, grasshoppers, crickets and silkworm meal (Makkar et al. 2014; Nogales-Mérida et al. 2019).

Though there is number of insects, silkworm (Bombyx mori) is one of the economically important and harmless group of insect. It is the larvae or caterpillar of a silkmoth, classified under the Bombycidae family. Silkworms are raised on mulberry leaves and used to make silk thread from the pupae after killed by heat and the residual pupae can be used as feed ingredients (Nisha et al. 2014). According to the Bureau of Indian standards (1971), silkworm pupae (SWP) are composed of 50% crude proteins, 25% crude fat, 50% moisture, 3% crude fiber and 5% ash. Presently India is the second largest producer of silk and the world's largest consumer of silk. The total silk production in India reached 33770 MT in the year 2020-21 (Central Silk Board 2022). Silkworm pupae have been proven to be suitable as livestock feed, particularly for monogastric species including fish but also for ruminants due to their high production potential and high digestibile protein content (Trivedy et al. 2008). Due to the high moisture content, fresh wasted silkworm pupae are highly perishable, so they are often sundried and pulverized (Usub et al. 2008). Instead of using fresh or undefatted SWP meal, defatted silkworm pupae meal was used in the experiment due to the good qualities like less perishable and more protein content and avoiding saturated fat rich oil (Blair 2008).

The experiment was carried out in silver pompano, Trachinotus blochii. Silver pompano is a tropical marine fish abundant in the Indo-Pacific region (Kapoor et al. 2002) with several good attributes such as good quality white meat, less spines, rapid growth rate and tolerance to wide range of salinity (Kalidas et al. 2012). It is caught occasionally in the commercial fishery, hence demand can only be met through aquaculture. By considering all these points, the experiment was designed in silver pompano with various inclusion of deoiled silkworm pupae meal (DSWP).

2.1. ETHICAL STATEMENTS:

This study was carried out with the agreement of the Tamil Nadu Dr. J. Jayalalithaa Fisheries University ethical committee in Nagapattinam, Tamil Nadu, India.

2.2. Experimental system

The experiment was conducted for 90 days at the Marine Research Farm Facility, Fisheries College and Research Institute, Thoothukudi (India) using 250-L rectangular FRP tank (120 × 55 × 38cm) with adequate oxygen levels in tanks were maintained for fish rearing. Silver pompano seeds (0.35 ± 0.06 g) were procured from the ICAR-Vizhinjam regional center of Central Marine Fisheries Research Institute, India and acclimatized for 4 week with 50 % crude protein commercial diet (Avanti Feeds Ltd., India). After the acclimatization, three hundred seeds were randomly distributed into 15 FRP tanks each consisting of 20 fish and all the fish were bulk weighed to determine the mean weight of a fish (0.93 ± 0.07 g).

2.3. Chemical analysis

Chemical analysis of ingredients, experimental diets, fish and feces were examined in triplicates using standard protocols Association of Official Analytical Chemists (AOAC 2005) as follows: moisture content was determined by drying the sample in the oven at 105 ⁰C for 24 h; Crude protein (N × 6.25) was determined by Kjeldahl method with FOSS 8400 analyzer (Tecator systems, Hoganas, Sweden); Crude lipid was determined by Soxhlet extraction using ether in Soxhlet apparatus (FOSS soxtec2043) and crude fiber was determined by using Fibertec system. Total ash content was measured by combustion of the sample at 600 ⁰C for 6 h in a muffle furnace. The gross energy content of the sample was determined using an adiabatic bomb calorimeter (Lab-X, India). Chromium (Cr) content in the diet and feces sample were determined by an inductive coupled plasma atomic emission spectrometer (Arcos-SOP, Spectro Analytical Instruments GmbH, and Kleve, DE). At the final sampling, fish from each tank were randomly taken for carcass composition analysis.

2.4. Experimental diets

Dried silkworm pupae was obtained from a silk processor (Silver mine silk processors, Erode, India) and ground into powder. The powdered meal was deoiled by the solvent extraction method (using n-hexane and ethanol) and sieved through a 500-micron test sieve. Five isonitrogenous (42%) and isocaloric (4000 kcal/kg) experimental diets ((RD, 25FMODSWP, 50FMODSWP, 75FMODSWP and 100 FMODSWP) were prepared by substituting fish meal with DSWP at 0, 25, 50, 75 and 100 % levels. The feed ingredients were obtained from the local shop were finely powdered and sieved using 200 micron sieve. The proximate composition of major ingredients were analyzed for the preparation of experimental feed formulae (Table 1). In all the experimental diets, 1% chromic oxide was added as an external digestibility marker. Feeds were pelletized using pelletizer, dried in hot air oven at 45⁰C and stored until use.

2.5. Feeding and sampling of fish

The fish were hand-fed to apparent satiation three times daily (09:30, 12:30 and 15:30h) with the respective experimental feeds. Initially 1 mm dia feed was given to the fish and later increased the feed size to 1.2 and 1.4 mm according to the mouth size of the fish during the course of experiment. Water quality parameters such as dissolved oxygen (DO), water temperature, salinity, pH, hardness, alkalinity, ammonia, nitrite and nitrate were measured for each tank by using standard procedures American Public Health Association (APHA 2005) and about 90 % of water was exchanged daily. Fish were weighed (bulk) for every 15 days to assess the mean body weight (g) using a digital electronic balance (Shimadzu Corporation, Philippines). At the end of growth trial, fish in each tank were weighed (bulk). The growth performance was assessed using the following standard formulae for different growth parameters and body indices.

2.6. Digestibility trial design

The digestibility trial was conducted in the circular experimental troughs (100 L) for 4 weeks with one reference and four groups in triplicate as described by Manikandan and Felix (2021). After the completion of growth study, fish with a size (11.28± 2.57 g) were stocked at a density of 10 fish per trough. Experimental troughs were closed using a net to prevent leaping out of fish. Fishes were hand-fed to apparent satiation three times daily (9:30, 12:30 and 15:30h). Water exchange was carried out daily morning (08:00h) and fish were fed with 1.8mm size feed without feed fines. After 30 minutes of first feeding, uneaten feed was siphoned out and discarded. Two hours after the first feeding, the first feces collection was done by using plankton net. The collected feces in the plankton net were dried and stored in the freezer as described by Cruz-suarez et al. (2009). The protocol was repeated after the second and third feeding of fishes and feces were collected 3 times in a day. For analysis, the feces from 4 consecutive weeks were pooled. Thus, a total of five samples (1 sample/ treatment) of feces were obtained and each sample was subjected to triplicate analyses. The Apparent Digestibility Coefficient (ADCs) was assessed by following standard formulae

2.7. Sample collection

At the end of the growth trial, fish from each tank in a group were randomly collected, anesthetized with 0.10 mL clove oil / L water (Hamackova et al., 2006) and used for body indices analysis, digestive enzyme analysis, hematological analysis, serum biochemical analysis and histopathological analysis (in duplicates).

Body indices analysis

For body indices analysis, the liver and intestine were dissected from three anesthetized fish in each group, weighed and measured by using the following standard formula.

Hematological and serum biochemical analysis

After anesthetizing fish using clove oil (0.10 mL/L), blood samples were collected by caudal vein puncture (Prabu et al., 2016). Three blood samples were collected from each group using a 1 mL disposable syringe fitted with a 26-gauge needle rinsed with 2.75% EDTA. The collected samples were immediately transferred from the syringe to a separate heparin-coated vial placed on crushed ice for hematological analysis. Then another three blood samples were taken without an anticoagulant, transferred to plain vials and used for serum biochemical analysis. Hematological parameters were determined by placing the whole blood samples in an automated hematology analyzer (Medonic M-series, Boule, India). Serum cholesterol was estimated by the cholesterol oxidase/peroxidase (CHOD/PAP) method (Meiattini et al., 1978) using a cholesterol kit (Coral Clinical Systems, Goa, India). Serum triglyceride was determined by the glycerol phosphate oxidase/peroxidase (GPO/PAP) method (Fossati and Prencipe, 1982) using a triglyceride kit (Coral clinical systems, Goa, India). Lipoproteins (HDL and LDL) were analyzed using commercial kits (Coral clinical systems, Goa, India) and Serum very low-density lipoprotein (VLDL) level was calculated as per the methods of Friedewald et al. (1972),.VLDL = Triglycerides/5. Serum total protein was determined by the Biuret method (Reinhold, 1953) using a protein estimation kit (Coral clinical systems, Goa, India). Albumin was estimated by the Bromocresol green binding method (Doumas et al., 1971) using the kit (Coral clinical systems, Goa, India), whereas globulin level was determined by deducting the values of serum albumin from serum protein.

Digestive enzyme analysis

The liver and intestine samples were collected from the fish in each replicate tank. Immediately after collection of liver and intestine, homogenized with 0.25 M sucrose buffer by taking 5% (w/v) and centrifuged at 10000 rpm for 30 min. The supernatant was collected in tube (15 mL) and stored at -20°C. The supernatant was used as an enzyme extract. Protease activity was determined by the casein digestion method (Drapeau 1976). The α-amylase activity on carbohydrates was estimated by the dinitro-salicylic acid method (Rick and Stegbauer 1974) and lipase activity was determined by following the method described by Zamani et al. (2009).

Histopathological analysis

One anaesthetized fish from each tank (3 fish/ group) were randomly taken for histopathological analysis. The intestine of fish was dissected and collected in 10 % neutral buffered formalin fixative solution. The method described by Luna (1968) was followed to analyze histopathological observation of intestine. A light microscope was used to observe any abnormalities on the intestine of experimental fishes.

2.8. Statistical analysis

The results are presented as means ± SD. Parameter-wise data sets were statistically analyzed using analysis of variance (ANOVA) followed by Tukey's test. Statistical significance was tested at p<0.05. All the statistical tests were performed using IBM SPSS statistical software (Version 26.0). Second-order polynomial regression analysis was carried out to optimize the level of inclusion of DSWP to replace FM in the diet of silver pompano juveniles. All the statistical tests were performed using IBM SPSS statistical software (Version 26.0).

3.1 Water quality

The dissolved oxygen (DO), water temperature, alkalinity, hardness, salinity, pH, ammonia, nitrite and nitrate were not significantly (p>0.05) affected by the addition of DSWP in the silver pompano diets (Table 2).

3.2. Growth performance and feed utilization

The growth performance and feed utilization of silver pompano fed experimental diets are presented in the Table 3. The survival, final mean body weight, weight gain (g), body mass gain (%), specific growth rate (SGR), feed intake (g), feed conversion ratio (FCR), protein efficiency ratio (PER) and average daily weight gain (g) were significantly affected (P<0.05) by dietary DSWP. Survival of fish fed 100 FMODSWP diet was reduced (80%) compared to fish that fed other experimental diets (>90%). The final body weight, weight gain, body mass gain (%), SGR, FCR, PER and average daily weight gain were observed insignificant (p>0.05) among the fish fed RD and 25FMODSWP diet. However, 25FMODSWP showed higher growth performance than other groups (RD, 50FMODSWP, 75FMODSWP and 100 FMODSWP). The lowest and better FCR value was recorded in 25FMODSWP whereas the highest value was in 100 FMODSWP diet.

3.3 Apparent digestibility of nutrients

The apparent digestibility of silver pompano fed experimental diets are presented in the Figure 1. Inclusion of DSWP in experimental diets had a significant (p<0.05) effect on apparent dry matter digestibility, crude protein digestibility and crude fat digestibility. Dry matter digestibility showed insignificant result between the experimental diets 25FMODSWP, 50FMODSWP, 75FMODSWP and RD but these diets were significant with 100 FMODSWP (p<0.05). The protein digestibility showed higher value in 25FMODSWP and lower value was recorded in 100FMODSWP. In addition to that, higher lipid digestibility value was recorded in RD and 25FMODSWP, while lower value was recorded in 100 FMODSWP. Altogether, increase in the addition of DSWP significantly (p<0.05) reduced the apparent digestibility of silver pompano.

3.4. Digestive enzyme activity

Digestive enzyme activities in the liver and intestine were significantly affected by the dietary inclusion of DSWP (p<0.05) (Table 4). The activity of protease in both liver and intestine was observed maximum in fish fed 25FMODSWP diet followed by the RD group. Higher activity of amylase was recorded in both liver and intestine of the , 75FMODSWP group. In the case of lipase, the highest activity was recorded in the liver and intestine of 75FMODSWP and 100 FMODSWP group. Whereas in intestine, the both lipase and amylase activity had an inverse relationship with protease activity.

3.5. Body composition

The effect of DSWP on body composition was shown in the Table 5. The result of carcass composition revealed a significant reduction (p<0.05) in lipid content in 75FMODSWP group compared to other groups. Fish fed with RD obtained highest body lipid (p<0.05) when compared to other groups. Other than RD, the highest lipid deposition in the carcass was recorded in the group of fish reared on diet 25FMODSWP. However, the inclusion of DSWP in the diets did not show any changes in the results of moisture, protein, total ash, crude fiber, Nitrogen Free Extract (NFE) and gross energy of experimental fish at any level of inclusion (p>0.05).

3.6 Body indices

Body indices of fish fed experimental diets were presented in the Figure 2. Both the Hepatosomatic index (HSI) and Visceral somatic index (VSI) of fish were significantly different in all groups (p<0.05). Among the four DSWP-included diets, the 100% replacement of fish meal with DSWP (100 FMODSWP) resulted in the greatest HSI and VSI. However, the HSI of the 100 FMODSWP group was lower than the RD group and showed no significant difference between the diets 25 FMODSWP, 50 FMODSWP and 75 FMODSWP. The VSI values were noticeably reduced in the fish fed 50FMODSWP diet and showed no significant difference between the 25FMODSWP and 75FMODSWP.

3.7. Hematological analysis

The dietary inclusion of DSWP on hematological parameters of silver pompano was summarized in the table 6. Hematological parameters varied significantly (p<0.05) among all the experimental animals. Fish fed with 50FMODSWP and 100 FMODSWP had the highest RBC (Red Blood Cells), hemoglobin (Hgb), and hematocrit (Hct) levels compared to other groups. Noticeable reduction (p<0.05) of mean corpuscular volume (MCV) was observed in fish fed 25FMODSWP diet but showed higher WBC (White blood cells) and erythrocyte sedimentation rate (ESR) compared to other group of experimental animals. It was also observed that replacement of fish meal by 50FMODSWP resulted in highest thrombocyte count (TC).

3.8. Serum biochemical analysis

The results of serum biochemical analysis are presented in the Table 7. The dietary inclusion of DSWP at 50 % level significantly (P<0.05) increased the serum lipid and protein indices. Noticeable reduction of serum cholesterol, triglycerides, high density-lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) and serum proteins were observed in the fish fed 75FMODSWP and 100FMODSWP diets. Up to 50% inclusion of DSWP in diets resulted in higher serum protein, albumin and globulin. The serum biochemical indices of the 0FMODSWP group were observed higher, while the 100 FMODSWP group had lower index.

3.9. Histomorphological observation

The intestinal morphology of silver pompano from different treatments showed variations among the groups (Fig. 3). The RD group showed the microvilli with intact intestinal epithelial cells and very less undigested feed materials at the time of sampling after 12 hours of starvation. The intestinal morphology of 25FMODSWP group revealed that the microvilli and lamina propria (LP) are looking apparently healthy with no feed remnants. In the 50FMODSWP group the lamina propria and microvillai are normal and the intestinal section noticed with moderately higher amount of undigested feed materials. Intestinal structure of , 75FMODSWP group noticed with necrosis (N) in some of the microvilli and detachment of lamina propria from the microvilli was observed. The intestinal construction of 100FMODSWP group showed necrotic lesions (N) in the microvilli observed by detachment of surface epithelial cells from lamina propria. In this group more amount of undigested feed was observed in the intestine at the same time of sampling. These findings revealed that substituting DSWP for more than 50% of fish meal will lead to necrosis in the microvilli, detachment of the lamina propria from the microvilli and also the presence of feed remnants may be an indication of a digestive issue.

3.10. Dose optimization

The second-order polynomial regression analysis of weight gain percentage (Fig. 4) in snubnose pompano fed with the fish meal replaced using DSWP in the diet revealed that the DSWP can effectively replace 30.5% of FM protein in the diet without any adverse effect in growth.

Asian countries have been feeding silkworm meal to ruminant and monogastric species for many decades. Due to its affordability, silkworm meal was used in aquaculture as a source of protein. According to the reports, the meal made from silkworm pupae contains 34.4% fat and 51.1% crude protein (Pereira et al. 2003). The fatty acid composition of silkworm pupae contains 8.57% of linoleic acid (omega 6), 24.4% of linolenic acid (omega 3), and 32.52% of saturated fatty acids (Pereira et al. 2003). Transforming animal by-products into feed ingredients helps alleviate the environmental problems caused by the processing of animal by-products and it is also beneficial for the feed sector. Therefore, the present study focused on the effective utilization of sericulture by-products in aquatic feed.

The results of the present study revealed that the fish meal can be partially replaced with DSWP for the better growth. Among the four DSWP incorporated diets, 25 % inclusion of DSWP resulted in highest weight gain, as well as improved SGR, PER and FCR. These results were consistent with the findings of Raja et al. (2020), who reported that 30% inclusion of SWP meal in rainbow shark (Epalzeorhyrchos frenatum) resulted in better growth and feed utilization compare to other SWP included diets. Furthur, Jayaram and Shetty (1980) in common carp and Nandeesha et al. (1989) in Catla also reported similar results when fed with different level of SWP meal in diet.

The dietary inclusion of DSWP resulted in significant difference among the growth performance of silver pompano. These results were not in agreement with Rahimnejad et al. (2019) who reported that the growth performance of white leg shrimp were not significantly affected when shrimp were fed diets supplemented with DSWP. In the present study, the highest SGR value was obtained with diet D25 having 25 % DSWP and the range of SGR was recorded between 2.56 to 2.95% day^-1. While, Kurbanav et al. (2015) recorded better SGR (4.0% day^-1) in African cat fish when fed with 50 % inclusion of SWP meal in diet. Moreover, Ji et al. (2015) in Jian carp reported that the replacement of fish meal above 50% with SWP meal resulted in reduced SGR values (2.10 to 2.32).

The lower FCR values improves the feeding results of experimental fishes (De silva et al. 2002). In the present investigation, the range of FCR values were 1.33 to 1.95 and these values were similar to the findings of Kurbanav et al. (2015) who recorded the FCR values of 1.2 to 1.8 in Clarius garipinnus fed with Silkworm pupa-based diet. However, higher FCR values (>1.9) were observed by Nandeesha et al. (1990) in common carp and Oso and Iwalya (2014) in African cat fish when fed with SWP incorporated diets.

Higher inclusion of DSWP in diets showed a significant reduction of PER. The PER values were observed in the range of 1.22 to 1.76 and fish fed with 25% DSWP had highest PER when compared to other experimental diets. Similarly, Raja et al. (2020) observed PER value of 1 to 1.5 in rainbow shark fed diets containing different levels of Silkworm pupae. In addition, Salem et al. (2008) reported PER of 1.57 to 2.18 in Nile tilapia fed with various levels of SWP meal incorporated diets.

The complete replacement of fish meal leads to reduced growth, PER and higher FCR. These results confirmed the findings of Salem et al. (2008) who reported that dietary inclusion of 100% SWP meal resulted in lower growth, PER and higher FCR. The higher growth performance of silver pompano observed at 25% inclusion of DSWP could be due to maximum protein utilization whereas reduced growth performance of silver pompano at higher inclusion (>25%) of DSWP could be due to reduced digestibility of diets (Kroeckel et al. 2012).

In the present study, replacing fishmeal with DSWP resulted in reduced digestibility of silver pompano in a significant manner (p<0.05). This results was agreed with the findings of Jintasataporn et al. (2011) who reported the higher inclusion of SWP meal in diets had reduced the protein digestibility of fish. On the otherhand, Hossain et al. (1997) observed higher levels of protein digestibility in rohu fed with non-defatted and defatted SWP meal than those of fish meal. In the current study, silver pompano exhibited higher protein and fat digestibility at inclusion levels of 0 to 25% in FMODSWP and declined at higher inclusion levels (beyond 25% FM protein replacement). The results were consistent with Gangadhar et al. (2017) who reported that 10–30% inclusion level of silkworm pupa increased the digestibility of protein and fat in common carp. The reason for the reduced digestibility of DSWP at higher inclusion may be due to chitin, which was the primary component in the exoskeleton of silkworm pupae (Suresh et al. 2012). In this study, inclusion of 100 % FMODSWP resulted in lower growth of silver pompano which may be due to poor digestibility. The growth performance and digestibility of silver pompano followed the same pattern among different diets, this suggests a vital role for digestive and absorptive processes in influencing the growth and nutrient utilization of fish. The findings were comparable with Tibaldi et al. (2006) who documented that the growth performance of sea bass followed the same trend as diet-dependent digestibility values.

The dietary components will affect the activity of digestive enzymes (Gangadhara et al. 1997; Fountoulaki et al. 2005) and this enzymes are essential for feed utilization and digestion (Dabrowski and Glogowski 1977). In the present investigation, the dietary inclusion of DSWP showed a significant difference in the activity of digestive enzymes. In terms of deoiled black solider fly meal addition, Li et al. (2017) observed that Jian carp showed no significant difference in the digestive enzyme activity. The digestive enzyme activity in the intestine was observed higher than the liver (Kumar et al. 2006). A study by Gangadhar et al. (2017) revealed that protease activity was found to be higher in carps at 10–30% SWP incorporation levels when compared to the RD. Similarly, in present study the protease activity in liver and intestine was observed higher at 25% inclusion and showing a declining trend at higher levels. The high protein digestibility in the RD and 25FMODSWP may led to increase in protease activity of the experimental fish. Similarly, Rapatsa et al. (2017) found that high levels of protein digestibility in the mopane worm meal incorporated diet may have resulted in increased protease activity of tilapia (Oreochromis mossambicus). The higher amylase activity in liver and intestine of 75FMODSWP and 100 FMODSWP group may be connected to the higher composition of carbohydrates when compared to other diets (Correa et al. 2007; Keshavanath et al. 2002) and also the diet composition may affect the digestive enzyme activity (Bakke et al. 2010). In this study, the lipase activity was significantly affected by the dietary inclusion of DSWP. On the otherhand, replacement of fish meal with fermented meal mixture of silkworm pupae, rapeseed and wheat have shown no significant difference in lipase activity (Zhou et al. 2017). However in the current study, there was no noticeable trend in lipase activity. This is in line with the finding of Nida et al. (2013) and Rapatsa et al. (2017) who stated that there is no evident connection between fish lipase secretion and diet. In addition, Sabat et al. (1999) reported that dietary regulation of digestive enzyme activity in vertebrates are common but not universal.

In the present study, the fat in fish body was affected by higher inclusion levels of DSWP. However, the inclusion of DSWP in the diets not showed any significant changes in the body moisture, protein, ash, fiber and gross energy at any level of inclusion. The protein results are consistent with the finding of Salem et al. (2008) who reported that the dietary SWP meal did not affect the body protein of Nile tilapia. Similarly, Seema et al. (2011) revealed that Kaira river prawn (macrobrachium dayanum) fed SWP meal diet showed no differences in body protein. Noticeably, fish fed diets containing higher inclusion of DSWP had lesser carcass fat levels compared to RD and 25FMODSW group, which was similar to the result of Zhou et al. (2017) who observed the increasing replacement of FM with fermented meal mixture of silkworm pupae, rapeseed and wheat had reduced body fat in juvenile mirror carp (Zhou et al. 2017). According to Elangovan and Shim (2000), the reason for the higher fat composition in RD and D25 group may be due to better protein utilization that was transferred into lipids and stored in the body.

The Hepatostomatic index of fish fed DSWP diets were lower than the fish meal based diet, but the highest Viscerostomatic index were observed in fish fed diet containing 100 FMODSWP. In terms of other insect meal addition, Mapanao et al. (2021) found that the inclusion of black soldier fly meal in the diet of climbing perch (Anabas testudineus) increased both visceral somatic and hepatosomatic indices. In comparison with other DSWP incorporated diets, the reason for higher HSI and VSI in D100 may be due to higher dietary carbohydrate levels on experimental diets that was converted into fat and stored in the liver as a result of the extra carbohydrate (Ahmad et al. 2012; Gumus and Ikizand 2009).

Fish hematological features plays a crucial role in determining the state of health (De Pedro et al. 2005; Martins et al. 2008). The results of the present study showed that the inclusion of DSWP in the diet considerably enhanced the levels of RBC and Hgb. The present results are not in agreement with Shakoori et al. (2015) who observed that the values of RBC and Hgb were considerably reduced with the inclusion of silkworm pupae in the diet. But the increased levels of MCV and WBC were consistent with the findings of Shakoori et al. (2015). Noticeable reduction in erythrocyte sedimentation rate and TC were observed with higher inclusion of DSWP in diets. The elevated white blood cell levels are only associated with immunity, (Shen et al. 2018). The reason for elevated levels of WBC and RBC was not clear. However, it is suggested that some components in silkworm pupae may be a reason for higher WBC that stimulate an immune response (Ijayva and Eko 2009). Thrombocytes were mainly to form a protective barrier (Magnadottir 2006) and reduced number of thrombocytes with higher inclusion of DSWP could be related to various nutritional differences (Romano et al. 2017). The variation in ESR of experimental fish may be due to alteration in blood plasma or due to stress conditions (Joseph john 2007).

The triglycerides, cholesterol, and total protein levels are considered as main indicators of teleost health (Zhou et al. 2009) and fish feed create an impact on the serum biochemistry (Jawad et al. 2014). In the present study, the inclusion of DSWP above 50% in the diets significantly reduced the serum triglyceride, cholesterol, and lipoprotein levels (HDL, LDL and VLDL) showing a hypolipidemic effect and also reduced the serum protein levels of silver pompano. These results were not consistent with the findings of Xu et al. (2018) who found that juvenile mirror carp fed diets with enzymatic hydrolysates of defatted silkworm pupae had shown no significant difference in HDL, LDL, total serum protein and albumin. In current study, increasing the dietary intake of DSWP up to 100% replacement of FM significantly reduced the triglyceride levels. This may be due to the presence of chitin, which involved in the hydrolysis of triglycerides (Zhang et al. 2008). The higher level of serum total protein, globulin and albumin in the D50 group reflects the superior health condition and immune status of fish (Kumar et al. 2007; Fawole et al. 2016) and confirmed by the higher survival rate when compared to other groups. However, the cause of the reduction in cholesterol, lipoproteins and serum proteins were not known and this requires additional study to understand the effects of B.mori on silver pompano.

Intestine is the primary site for feed digestion and optimum utilization of dietary nutrients depends on their functional effectiveness (Caballero et al. 2003). Dietary protein and lipid content can adversely affect the intestinal microvilli structure in several fish species including gilthead sea bream (Caballero et al. 2003), common carp (Ostaszewska et al. 2010) and Jian carp (Ji et al. 2015). Diets deficient in essential amino acids especially lysine may adversely affect the morphological structure of fish liver and intestinal epithelium (Ostaszewska et al. 2010). In the present study, higher inclusion level of the deoiled silkworm pupae meal (50 DSWP-100 DSWP groups) affects the microvilli structure in the juvenile snubnose pompano. This can be evident from reduced protease activity as the inclusion level of DSWP in the diet. Similar response was reported by Ji et al. (2015) in Cyprinus carpio (var. Jian), fed with the diet containing SWP meal. However, Chen et al. (2017) suggested that silkworm chrysalis oil could be incorporated in the diet of Jian carp, without altering the morphological structure in the mid-intestine. The reason for the abnormalities in intestinal morphology at higher replacement levels of fish meal could be due to the presence of more chitin in the diets which has an ability to cause histological changes. (Elia et al., 2018).

In conclusion, DSWP may replace 30.5% of fishmeal protein which accounts 9.51% inclusion of DSWP in the diets for silver pompano, Trachinotus blochii without having adverse effects on growth, nutrient digestibility, carcass composition, blood profile and gut health.

ACKNOWLEDGEMENTS

The author would like to thank Tamil Nadu Dr. J. Jayalalithaa Fisheries University for providing the facilities and support necessary to conduct this research as part of the postgraduate thesis research work.

Ethical approval:

This study was carried out with the agreement of the Tamil Nadu Dr. J. Jayalalithaa Fisheries University’s ethical committee in Nagapattinam, Tamil Nadu, India. (Human and animal ethics)

Consent to participate:

This study was conducted based on the approval by the ethical committee of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India.

Consent for publication:

The authors agree to publish the article under the springer publication.

Competing interests:

The authors state they have no material competing interests, either financial or otherwise.

Author’s contribution:

S. Mithun Kumar: The growth trial, digestibility, digestive enzyme, hematological and serum biochemical studies were conducted; data analyzed for statistical analysis and drafted the manuscript.

J. Jaculine Pereira: Conceptualized, technical support and corrected the manuscript.

S. Athithan: Corrected the manuscript.

P. Padmavathy: Digestibility trial designed.

D. Linga Prabu: Carried out the histopathological analysis and corrected the manuscript.

K.S. Vijay Amirtharaj: Growth trial designed.

Funding:

Tamil Nadu Dr. J. Jayalalithaa Fisheries University (TNJFU).

Availability of data and materials:

The data that support the findings of this study are available within the article.

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Table 1: Feed formula and proximate composition of the major ingredients and experimental diets (% as as-is basis) FMODSWP

Ingredients	Fish meal	Deoiled SWP meal	RD (0%)	25FMODSWP (25%)	50FMODSWP (50%)	75FMODSWP (75%)	100FMODSWP (100%)
De-oiled SWP			0	7.8	15.6	23.4	31.2
Soyabean meal			32.15	31.65	31.35	30.4	29.2
Rice bran			4	5.35	6.5	8.1	9.9
Groundnut oil cake			5	5	5	5	5
Fish meal			35	26.25	17.5	8.75	0
wheat powder			16	16	16	16	16
Sunflower oil			1.2	1.2	1.2	1.25	1.25
Fish oil			1	1	1	1	1
Vit & min premix^a			3	3	3	3	3
BHT^b			0.1	0.1	0.1	0.1	0.1
SMB^c			0.1	0.1	0.1	0.1	0.1
Methionine			0.2	0.3	0.3	0.35	0.5
Lysine			0	0	0.1	0.3	0.5
Vitamin C			0.25	0.25	0.25	0.25	0.25
Long pepper leaf			0.5	0.5	0.5	0.5	0.5
MCP^d			0.5	0.5	0.5	0.5	0.5
Chromic oxide			1	1	1	1	1
Proximate composition (%)
Moisture	6.23	4.03	7.67	7.30	7.55	8.31	7.81
Crude Protein	58.81	67.82	42.71	43.06	42.67	42.31	42.34
Crude Lipid	7.58	9.83	7.62	7.88	7.34	7.80	8.24
Total ash	24.20	5.05	16.80	14.91	12.29	11.23	10.11
Crude fiber	0.70	6.31	5.13	5.44	5.88	5.69	5.39
NFE^e	2.47	6.96	20.07	21.41	24.30	24.66	26.11
Gross energy (Kcal/kg)	4296.90	5211.96	3907.79	3952.39	3999.85	4038.23	4030.46

RD, 25FMODSWP, 50 FMODSWP, 75FMODSWP and 100 FMODSWP, diets which silver pompano fed with deoiled SWP meal included at 0, 25, 50, 75 and 100 % by replacing fish meal.

^aVitamin and mineral premix (Agrimin forte, India), each 1 kg contains: vitamin A (700000 IU), vitamin D₃(70,000 IU), Vitamin E (250 mg), Zn (9600 mg), Mg (6000 mg), Mn (1500 mg), Cu (1200mg), I (325 mg), DL-Methionine (1000mg), Co (150mg), K (100 mg), Na (5.9 mg), Ca (25.5%), P (12.75%), S (0.72%); ^b Butylated hydroxy toluene; ^c Sodium metabisulphite; ^dMonocalcium phosphate; ^e Nitrogen free extract (NFE) = 100- (moisture + crude protein + lipid + fiber + ash content).

Table 2: Effect of deoiled SWP inclusion on water quality in experimental tank

	RD (0%)	D25 (25%)	D50 (50%)	D75 (75%)	D100 (100%)	p-value
Dissolved oxygen (mg/L)	6.86±1.12	6.51±0.66	6.15±0.18	7.02±0.12	6.40±0.43	0.46
Temperature)	29.76±1.16	30.10±1.01	30.46±0.35	30.5. ±2.17	29.33±1.04	0.78
Phenolphthalein Alkalinity(mg/L)	27.33±4.16	30.66±6.11	33.33±6.11	25.33±3.05	28.66±3.05	0.34
Total Alkalinity(mg/L)	89.33±20.52	81.33±8.32	94.66±4.61	104.00±6.92	101.33±7.57	0.16
Salinity (g/L)	27.33±1.52	27.66±2.51	28.33±1.52	27.66±1.52	27.33±2.51	0.96
pH	8.01±0.20	7.99±0.14	8.05±0.15	7.82±0.17	7.75±0.086	0.14
Ammonia (mg/L)	0.23±0.01	0.30±0.03	0.26±0.04	0.29±0.03	0.28±0.06	0.42
Nitrite (mg/L)	0.057±0.009	0.072±0.009	0.067±0.010	0.065±0.010	0.068±0.013	0.52
Nitrate (mg/L)	0.146±0.026	0.144±0.014	0.142±0.026	0.149±0.019	0.169±0.010	0.52

Data are mean value ± Standard deviation (n=3). Mean values with no superscripts in the same row were insignificant (p>0.05).

Table 3: Growth performance and feed utilization of silver pompano juvenile fed experimental diets

Parameter	RD (0%)	D25 (25%)	D50 (50%)	D75 (75%)	D100 (100%)	P-Value
Initial weight (g)	0.96 ± 0.08	0.94 ± 0.12	0.93 ± 0.03	0.93 ± 0.06	0.92 ± 0.10	0.98
Final weight (g)	13.10 ± 0.80^a	13.53 ± 0.92^a	10.86 ± 0.47^b	9.73 ± 0.60^b	9.30 ± 0.78^b	0.001
weight gain (g)	12.14 ± 0.83^a	12.58 ± 0.84^a	9.93 ± 0.44^b	8.80 ± 0.63^b	8.37 ± 0.68^b	0.001
Body mass gain (%)	1272.22 ± 124.47^ab	1339.89 ± 138.25^a	1060.03 ± 26.85^bc	951.26 ± 115.78^c	905.78 ± 33.74^c	0.001
SGR¹ ( % d^-1)	2.89 ± 0.09^ab	2.95 ± 0.10^a	2.69 ± 0.04^bc	2.60 ± 0.11^c	2.56 ± 0.03^c	0.001
FCR²	1.33 ± 0.09^a	1.27 ± 0.12^a	1.36 ± 0.10^a	1.61 ± 0.08^ab	1.95 ± 0.26^b	0.001
PER³	1.76 ± 0.13^a	1.83 ± 0.17^a	1.71 ± 0.13^a	1.46 ± 0.07^ab	1.22 ± 0.15^b	0.002
Average daily weight gain (g)	0.134 ± 0.009^a	0.139 ± 0.009^a	0.109 ± 0.004^b	0.097 ± 0.006^b	0.096 ± 0.011^b	0.001
Survival (%)	90.19 ± 3.39^a	92.15 ± 3.90^a	96.07 ± 3.40^a	90.19 ± 3.39^a	90.48 ± 3.23^b	0.001

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

SGR¹= specific growth rate;

FCR²= feed conversion ratio;

PER³ = protein efficiency ratio.

Table 4: Nutrient digestibility of silver pompano fed experimental diets.

Digestibility

(0%)

D25

(25%)

D50

(50%)

D75

(75%)

D100

(100%)

p-value

Dry matter digestibility

65.80 ± 0.36^a

64.31 ± 1.06^a

63.94 ± 1.06^a

62.19 ± 0.17^a

58.33 ± 1.65^b

0.001

Protein digestibility

91.89 ± 1.42^a

92.30 ± 3.30^a

90.71 ± 0.52^a

85.33 ± 0.70^b

82.61 ± 1.21^b

0.001

Lipid digestibility

96.91 ± 0.58^a

96.67 ± 0.65^a

95.34 ± 0.42^b

92.72 ± 0.48^c

90.07 ± 0.37^d

0.001

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

Table 5: Digestive enzyme activity in the liver and intestine of silver pompano fed experimental diets.

Liver

(0%)

D25

(25%)

D50

(50%)

D75

(75%)

D100

(100%)

p-value

Protease

(micromoles/mL/minute)

0.414 ± 0.039^b

0.556 ± 0.033^a

0.280 ± 0.021^c

0.189 ± 0.014^d

0.178 ± 0.019^d

0.001

α-amylase

(micromoles/mL/minute)

0.042 ± 0.001^c

0.047 ± 0.003^c

0.053 ± 0.006^c

0.068 ± 0.004^a

0.056 ± 0.006^b

0.001

Lipase (U/mL)

8.50 ± 0.51^c

9.16 ± 0.83^bc

10.83 ± 0.95^abc

13.33 ± 1.6^a

11.50 ± 1.10^ab

0.002

Intestine

Protease

(micromoles/mL/minute)

3.337 ± 0.347^ab

3.551 ± 0.311^a

3.039 ± 0.264^abc

2.738 ± 0.247^bc

2.435 ± 0.201^c

0.004

α-amylase

(micromoles/mL/minute)

0.070 ± 0.005^b

0.072 ± 0.007^ab

0.086 ± 0.012^ab

0.106 ± 0.023^a

0.092 ±0.008^ab

0.032

Lipase (U/mL)

9.16 ± 0.75^bc

8.33 ± 0.56^c

10.85 ± 0.62^ab

11.66 ± 0.67^a

12.50 ± 1.05^a

0.001

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

Table 6: Carcass composition of silver pompano fed experimental diets.

	RD (0%)	D25 (25%)	D50 (50%)	D75 (75%)	D100 (100%)	P value
Moisture	69.64±2.23	69.28±1.57	71.12±1.67	70.96±1.23	70.21±1.18	0.58
Crude protein	18.40±0.32	18.75±0.39	17.71±0.42	18.68±0.49	18.24±0.38	0.06
Crude fat	7.51±0.38^a	7.24±0.44^ab	6.17±0.49^bc	5.72±0.48^c	6.67±0.15^abc	0.02
Total ash	3.42±0.32	3.61±0.26	3.98±0.22	3.62±0.21	3.87±0.27	0.13
Crude fiber	0.02±0.01	0.04±0.01	0.03±0.01	0.03±0.01	0.04±0.01	0.15
NFE¹	1.01±0.33	1.06±0.51	1.02±0.47	0.99±0.29	0.97±0.38	0.99
Gross energy (kcal/g)	5.608±0.47	5.602±0.23	5.391±0.44	5.526±0.25	5.552±0.31	0.94

Data are mean value ± Standard deviation (n=3). Mean values with same superscripts and without superscripts in the same row were insignificant (p>0.05).

NFE ¹(Nitrogen free extract) = 100- (moisture + crude protein + lipid + fiber + ash content).

Table 7: Body indices of silver pompano fed experimental diets.

(0%)

D25

(25%)

D50

(50%)

D75

(75%)

D100

(100%)

P value

HSI¹

1.68±0.10^a

0.85±0.09^c

0.97±0.06^c

0.85±0.05^c

1.41±0.04^b

0.001

VSI²

7.15±0.30^b

7.09±0.01^bc

5.43±0.22^d

6.19±0.40^cd

8.49±0.58^a

0.001

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

HSI¹= Hepatosomatic index;

VSI² = Viscerosomatic index.

Table 8: Hematological parameters of silver pompano fed experimental diets.

	RD (0%)	D25 (25%)	D50 (50%)	D75 (75%)	D100 (100%)	Pvalue
RBC¹(×10⁶/µl)	0.59±0.03^c	0.86±0.07^b	1.57±0.11^a	0.91±0.05^b	1.57±0.06^a	0.001
WBC²(×10³/µl)	1.84± 0.16^c	3.79± 0.27^a	2.17± 0.18^c	1.83± 0.14^c	2.94± 0.11^b	0.001
Hgb³ (g/dl)	3.94± 0.17^c	5.23±0.33^b	6.58±0.51^a	2.75± 0.15^d	6.53±0.52^a	0.001
Hct⁴ (%)	6.59± 0.46^b	5.45± 0.35^bc	13.63±0.80^a	4.74± 0.29^c	12.23± 0.66^a	0.001
ESR⁵ (mm/hr)	10.15± 0.79^a	10.21± 0.58^a	8.46± 0.18^b	6.27± 0.31^c	5.32± 0.27^c	0.001
MCV⁶ (fl)	76.64±2.86^a	59.89±4.48^b	86.92±4.44^a	80.26±3.35^a	78.11± 4.29^a	0.001
TC⁷ (×10³/µl)	363.35±10.90^a	350.58± 21.35^a	374.26± 17.96^a	247.78± 19.26^b	211.39± 12.68^b	0.001

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

RBC¹= Red blood cells;

WBC² = White blood cells;

Hgb³ = Hemoglobin;

Hct⁴ = Hematocrit;

ESR⁵ = Erythrocyte sedimentation rate;

MCV⁶ = Mean corpuscular volume;

TC⁷ = Thrombocyte count.

Table 9: Serum biochemical indices of silver pompano fed experimental diets.

	RD (0%)	D25 (25%)	D50 (50%)	D75 (75%)	D100 (100%)	P Value
Serum Cholesterol(mg/dL)	165.53±6.62^b	164.15±10.95^b	201.47±15.45^a	91.46±6.74^c	79.42±5.93^c	0.001
Serum Triglyceride(mg/dL)	138.77±4.95^b	146.29±6.74^b	322.21±31.51^a	118.72±3.41^bc	90.59±3.56^c	0.001
HDL¹ (mg/dL)	49.66±3.42^ab	51.40±4.28^a	51.37±4.85^a	48.76±3.59^ab	39.62±3.20^c	0.02
LDL² (mg/dL)	99.14±7.28^a	84.125±7.69^a	85.48±7.42^a	19.65±1.64^b	12.76±1.19^b	0.001
VLDL³ (mg/dL)	28.17 ±0.84^bc	29.56± 2.1^b	65.14± 2.67^a	24.30± 1.72^c	18.29± 0.62^d	0.001
Total serum protein (g/dL)	6.37±0.27^ab	6.14±0.29^ab	6.69±0.38^a	5.53±0.33^b	5.62±0.35^b	0.006
Albumin(g/dL)	3.25±0.15^b	3.35±0.20^ab	3.87±0.22^a	3.08±0.27^b	3.15±0.22^b	0.008
Globulin(g/dL)	2.84±0.18^ab	3.05±0.15^a	3.17±0.21^a	2.56±0.19^b	2.39±0.17^b	0.002

Data are mean value ± Standard deviation (n=3). Means with different superscript letters in the same row are significantly different (p<0.05).

HDL¹= High density lipoprotein (HDL); LDL² = Low density lipoprotein (LDL); VLDL³ = Very low density lipoprotein (VLDL).

No competing interests reported.

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Effect of deoiled silkworm pupae meal (Bombyx mori) as a replacement of fish meal in the diet of juveniles of Silver pompano, Trachinotus blochii

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1. Introduction

2. Materials And Methods

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