1 s2.0 S0377840123001256 Main

Animal Feed Science and Technology 303 (2023) 115691
Contents lists available at ScienceDirect
Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci
Valorization of the insect waste as a source of dietary protein in

replacing the fishmeal protein for the cage reared Pangasianodon
hypophthalmus: An approach to search the alternate
non-conventional feed resource of animal origin
Rahul Das a, Basanta Kumar Das a, *, Md. Abul Hassan a, Gopal Krishna b,
Narinder Kumar Chadha b, Kiran Dube Rawat b, Karmabeer Jena c
a
ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, India
b
ICAR-Central Institute of Fisheries Education, Mumbai 400061, India
c
Central Tasar Research and Training Institute, Ranchi 835303, India
A R T I C L E I N F O A B S T R A C T
Keywords: A ninety days experiment was carried-out to evaluate the effect of dietary replacement of fishmeal
Silkworm pupae (FM) (locally available dried non-defatted Coilia dussumieri V.) by graded inclusion levels of non-
Fishmeal protein replacement defatted non-mulberry Indian tropical tasar silkworm spent pupae (Antheraea mylitta D.) on
Growth
growth performance, feed consumption, digestive enzyme response, body chemical composition,
Nutrient utilization
Pangasianodon hypophthalmus
and intestinal cellular micro-morphological status of the reared striped catfish (Pangasianodon
hypophthalmus S.). Five iso-nitrogenous (280 g protein kg-1) and iso-energic (19 MJ kg-1) extruded
floating feeds were formulated by gradually replacing the FM with NDSWP (non-defatted silk
worm pupae) at 0%, 25%, 50%, 75%, and 100% (on protein to protein basis) levels and the diets
are designated as NDSWP0, NDSWP25, NDSWP50, NDSWP75, and NDSWP100, respectively. The
contribution of calculated pupae crude protein was 0%, 4.75%, 9.50%, 14.25%, and 19% out of
the total protein (28%) content in the respective diets. The experiment was executed in the
floating net-cage located at the Maithon reservoir, India which is a field research station of ICAR-
CIFRI, Barrackpore, Kolkata, India. The designated diets were randomly allocated to 10 numbers
of experimental cages (each 5 ×5 ×3 m), in triplicates stocked with 2475 numbers (stocking
density at 33 numbers per m3) of striped catfish in each replication and fed daily twice at 08.00 h
and 16.00 h. The initial stocking size of the striped catfish fingerling was 16.56 ± 0.08 g. At the
termination of the feeding experiment, the final body weight (g), individual weight gain (g),
weight gained (%), feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency
ratio (PER) found to be non-significantly different (p < 0.05) up to 50% protein replacement level
(NDSWP50) in comparison to the control (NDSWP0). Among the biological indices, hep
atosomatic index (HSI) exhibited a non-significant difference stretching to NDSWP50 treatment
Abbreviations: IBW, Initial body weight; FBW, Final body weight; IWG, Individual weight gain; WG, Weight gained; FCR, Feed conversion ratio;
SGR, Specific growth rate; PER, Protein efficiency ratio; ANPU, Apparent net protein utilization; Fulton’s CF, Fulton’s condition factor; VSI, Viscero-
somatic index; HSI, Hepatosomatic index; S, Survival; BHT, Butylated hydroxytoluene; MJ, Mega joule; Kg, Kilogram; P:E, Protein energy ratio; mg,
Milligram; ₹, Indian rupee; %, Percentage; h, Hour; g, Gram; ◦ C, Degree centigrade; rpm, rotation per minute; mm, Millimeter; m, Meter; µl,
Microliter; L, Liter; M, Mole; µm, Micrometer.
* Corresponding author.
E-mail address: basantakumard@gmail.com (B. Kumar Das).
https://doi.org/10.1016/j.anifeedsci.2023.115691
Received 17 October 2022; Received in revised form 21 May 2023; Accepted 25 May 2023
Available online 27 May 2023
0377-8401/© 2023 Elsevier B.V. All rights reserved.
R. Das et al. Animal Feed Science and Technology 303 (2023) 115691
group with a distinct significant difference between NDSWP25 and NDSWP100 (p < 0.05), when,
an inverse trend was recorded in relation to increasing pupae meal in the diets. Among the
digestive enzymes, relatively higher protease enzyme activity was observed in the diet fed at
NDSWP50 compared to the rest of the treatment groups, although, it was statistically non-
significant (p > 0.05). Amylase and lipase enzyme activities recorded a significant difference
among the treatment groups (p < 0.05). A higher amount of fish muscle crude protein was noticed
in the NDSWP50 treated group than in the rest of the treatment groups. The lipid content of fish
muscle indicated its higher content over the initial and exhibited an inverse relationship with
moisture and ash content at the end of the experiment. Histo-morphology of gut tissue confirms
the suitability of feeding pupae protein incorporated diet with the gradual replacement of fish
meal protein. The findings of the current feeding trial suggests that the inclusion of non-defatted
non-mulberry silkworm pupae protein at 50% level of fishmeal protein substitution without
compromising the growth performance, nutrient utilization, conversion of feed, and other bio
logical indices, and economics of diet.
1. Introduction
At present, India is the third largest fish producing country in the world accounting for 7.96% of the global production. Globally,
the country has witnessed the all-time highest inland water capture fish production recorded at 1.8 million tonnes, contributing 16% to
the world’s inland capture fish production basket in 2020 with a journey of 0.50 million tonnes in the 1980 s (FAO, 2022). Fishmeal
protein is mostly considered one of the main ingredients in the fish diet for its well-balanced amino acid, fatty acid composition and
high digestibility with better acceptability to the fish which makes it useful to augment the protein uptake with better nutrient
digestion to absorption and assimilation (Zhou et al., 2018). Further, the wild-caught small size fishes with high value protein have
rapidly declined during the recent past decades and the rapid rise in aquaculture fish diet demand emanates the increasing pressure on
the wild-caught fish protein (Cottrell et al., 2020). The cost of the regular main protein source is also a major downside to farm
production attracting greater requirement for a blueprint to partially replace such ingredients in the fish feed (Phumee et al., 2011).
Thus, the competitive nature of using a fishmeal protein in the fish feed sector always envisages hunting for a strategic alternative
source of relatively cheap similar quality protein to make intensive aquaculture systems more profitable and sustainable (Jayant et al.,
2018). In this context, many insect proteins have rigorously tried to replace the fishmeal protein (Makkar et al., 2014; Tran et al.,
2015). The mulberry silkworm pupae (Bombyx mori) were extensively incorporated as an alternate ingredient into the fish diet to
carefully replace the fishmeal protein to the possible extent without deteriorating the growth and flesh quality involving Indian major
carp (Gangadhar et al., 2017; Hossain et al., 1997; Rangacharyulu et al., 2003), common carp (Cyprinus carpio var. Communis)
(Nandeesha et al., 1990), Jian carp (Cyprinus carpio var. Jian) (Chen et al., 2017; Ji et al., 2015), mirror carp (Cyprinus carpio var.
Specularis) (Wan et al., 2017; Xu et al., 2018; Zhou et al., 2017), tilapia (Feng et al., 2021; Sathishkumar et al., 2021), shrimp (Hodar,
2022; Rahimnejad et al., 2019), African catfish (Kurbanov et al., 2015; Olaniyi and Babasanmi, 2013; Oso and Iwalaye, 2014),
respectively. The use of non-defatted silkworm pupae of non-mulberry Indian tropical tasar (Antheraea mylitta D.) has great promise in
formulating fish diet as it is rich in nutritive value containing 510–590 g crude protein kg-1 and 140–221 g ether extract kg-1 pupae on
dry weight basis in comparison to the non-defatted mulberry silkworm pupae (Ghosh et al., 2020; Tripathi et al., 1990) which is
comparable to that of fishmeal, although, slightly higher than soybean meal (Makkar et al., 2014) or black soldier fly larvae meal (Li
et al., 2017). Furthermore, it has a better amino acid index (such as lysine) (Ghosh et al., 2020), rich in polyunsaturated fatty acids
notably α-linolenic with a suitable n-3/n-6 ratio, which could be a better alternative for fishmeal or soybean meal. India is producing
around 20,775 metric tonnes of tasar cocoon yearly of which nearly 60% of this cocoon biomass encounters silkworm pupae waste
(Altomare et al., 2020; CSB, 2021). Apparently, in India annually on average 12,465 metric tonnes of tasar silkworm spent pupae
produced as nutrient waste during the silk de-reeling process. The striped catfish (Pangasianodon hypophthalmus S.) is a catfish,
omnivore in feeding habits (Da et al., 2013; Phumee et al., 2009), and grows at a faster rate with the estimated average annual
production added to 0.70 million tonnes (Singh and Lakra, 2012). Sixty-two percent of the worldwide aquaculture production by 2030
will be comprised of freshwater carp species and Pangas catfish (FAO, 2020). In this scenario, India is accounting for 14,018 numbers
of floating net cages being installed with dominated culture operations using striped catfish existing in the identified geographical
region (Pandit et al., 2021). The bibliographic survey elucidates the scanty study reports on feeding trials executing either the mul
berry or non-mulberry silkworm pupae incorporation in partial replacement of fishmeal protein in the experimental diet of striped
catfish. Hence, in the present investigation, the non-mulberry silkworm spent pupae (Antheraea mylitta D.) meal was employed in its
practical diet at different inclusion levels to find the extent of suitable gradual replacement with the fishmeal protein at protein to
protein contribution level forming five isonitrogenous diets, rendering effective utilization of this tasar silkworm spent pupae waste. At
the end of 90 days feeding trial conducted in outdoor floating net cages, the growth performance indices, biological parameters, muscle
nutrient quality, and diet economics were analyzed to evaluate the effect of partial replacement of fishmeal protein with the
non-defatted non-mulberry silkworm spent pupae protein.
2
Table 1
The nutritive value of ingredients (% dry matter basis) utilized during the experimental diet formulation.
Nutritional facts Fishmeal Non-defatted Ground nut cake Deoiled mustard cake Maize powder
silkworm pupae meal
Crude protein 58.85 ± 0.28 65.01 ± 0.10 44.79 ± 0.43 39.34 ± 0.17 7.60 ± 0.07
Ether extract 8.70 ± 0.18 15.44 ± 0.06 7.59 ± 0.06 0.93 ± 0.04 5.28 ± 0.02
Crude fibre 0.47 ± 0.05 6.98 ± 0.17 10.68 ± 0.09 15.83 ± 0.20 4.80 ± 0.05
Ash content 18.77 ± 0.24 7.50 ± 0.06 6.46 ± 0.02 7.09 ± 0.02 2.42 ± 0.06
Nitrogen free extract 13.21 ± 0.18 5.07 ± 0.23 30.48 ± 0.15 36.81 ± 0.20 79.90 ± 0.24
Values in mean ± standard error, n = 3.
Table 2
The experimental practical diet formulation and its nutritional composition (% dry matter basis).
Ingredient NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100
Fishmeal 32.29 24.21 16.14 8.07 0

Non-defatted silkworm pupae meal 0 7.31 14.61 21.92 29.23
Ground nut cake 6.65 6.65 6.65 6.65 6.65
Deoiled mustard cake 7.04 7.55 7.04 7.04 7.04
Maize powder 51.00 51.26 53.38 54.28 54.91
Soybean oil 1 1 0.15 0.12 0.15
Vitamin-minerala 2 2 2 2 2
BHT 0.02 0.02 0.02 0.02 0.02
Total 100 100 100 100 100
Nutritional composition (% dry matter basis) of the experimental diet
Moisture content 8.42 ± 0.05 8.02 ± 0.06 6.70 ± 0.11 7.73 ± 0.12 11.54 ± 0.01
Crude protein 28.72 ± 0.01 28.89 ± 0.01 28.85 ± 0.03 28.90 ± 0.02 28.92 ± 0.01
Ether extract 7.12 ± 0.05 7.60 ± 0.03 7.32 ± 0.05 7.70 ± 0.02 8.11 ± 0.04
Crude fibre 4.57 ± 0.12 5.04 ± 0.15 5.75 ± 0.18 6.23 ± 0.13 6.75 ± 0.12
Ash content 10.58 ± 0.14 9.25 ± 0.14 7.80 ± 0.19 6.36 ± 0.02 5.11 ± 0.12
Nitrogen free extract 49.63 ± 0.15 49.33 ± 0.12 50.28 ± 0.14 50.81 ± 0.1 51.11 ± 0.11
Gross energy (MJ Kg-1)b 18.89 19.17 19.26 19.52 19.79
c
P:E ratio 75.99 75.84 75.69 74.96 74.39
a
Constituents of vitamin-mineral mixture premix (quantity kg-1): Vitamin A-700000 IU, vitamin D3-70000 IU, Zn-9.6 g, Mg-6 g, Mn-1.5 g, Fe-1.5 g,
Cu-1.2 g, nicotinamide-1 g, I-0.325 g, vitamin E-0.25 g, DL-methionine-1 g, Co-0.15 g, K-0.1 g, Na-0.0059 g, Ca-25.5%, P-12.75%, S-0.72%.
b
Gross energy (MJ kg-1) was figured out as per the gross calorific value of crude protein, ether extract, and total carbohydrate (236, 395, and 172
MJ kg-1 (Brett, 1973).
c
P:E ratio: Crude protein to digestible energy ratio, whereas, digestible energy = 4 (crude protein %) + 9 (ether extract %) + 4 (nitrogen free
extract %) (Halver, 1976). Nutritional composition values in mean ± standard error, n = 3.
2. Material and methods
2.1. The experimental facility site
The experimental feeding trial was carried out in the ICAR-Central Inland Fisheries Research Institute galvanized iron frame-made
floating net cage installed at the Maithon reservoir (Latitude 23◦ 47’29’’E, Longitude 86◦ 49’17’’N) located at the Barakar River in the
state of Jharkhand, India. The experiment was conducted for ninety days from the month of August to October 2021. The cages in
triplicate were utilized against each feeding treatment group with the 75 m3 effective rearing water volume.
2.2. Feed ingredient
The nutritional composition of ingredients was analyzed as specified in Table 1. The fishmeal (Coilia dussumieri V.) was purchased
from the local fish market. The non-defatted Indian tropical tasar silkworm spent pupae in sun-dried crumble form was procured from
the Central Tasar Research Training Institute situated in the state of Ranchi, India. The fishmeal protein was partially replaced by the
incorporation of pupae meal protein, thus both ingredients served as the main protein source holding a nearly equal amount of crude
protein content individually, utilized during the experimental diet formulation and preparation. Among the other utilized ingredients,
groundnut cake, deoiled mustard cake, and maize powder were obtained from the local market. The ingredients were hot air oven
dried at 50 ◦ C overnight, thereafter, the ingredients were pulverized involving a pulverizer to obtain fine meal powder.
2.3. Diet formulation and preparation
The experimental extruded floating feeds were formulated in such a way that the crude protein contribution of basal ingredients
was kept nearly constant to observe the effect of protein to protein replacement between two major protein contributors namely
3
R. Das et al.
Table 3
Amino acid profile of non-deoiled NDSWP meal (A. mylitta), fishmeal (C. dussumieri) and diets (g 100 g-1 dry sample).
Amino acids Feedstuff Diets with graded level of NDSWP meal Requirement reference*
A. mylitta C. dussumieri NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100 C. gariepinus P. hypophthalmus
Arginine 0.60 ± 0.02 1.30 ± 0.02 0.89 ± 0.02 0.81 ± 0.02 1.50 ± 0.02 0.34 ± 0.01 1.19 ± 0.02 1.00–1.20 1.32–1.48
Histidine 0.21 ± 0.02 0.38 ± 0.02 0.16 ± 0.01 0.17 ± 0.01 0.35 ± 0.02 0.19 ± 0.00 0.18 ± 0.00 0.40–0.42 0.32–0.64
Isoleucine 0.53 ± 0.04 1.10 ± 0.04 0.46 ± 0.01 0.44 ± 0.00 0.75 ± 0.01 0.45 ± 0.01 0.43 ± 0.02 0.60–0.73 0.90–1.02
Leucine 0.94 ± 0.02 1.80 ± 0.08 0.82 ± 0.02 0.78 ± 0.01 1.27 ± 0.02 0.76 ± 0.01 0.72 ± 0.02 0.80–0.98 1.60–1.76
Lysine 0.76 ± 0.02 1.68 ± 0.10 0.72 ± 0.02 0.63 ± 0.02 0.91 ± 0.01 1.01 ± 0.02 0.42 ± 0.01 1.20–1.43 0.66–0.93
Methionine 0.14 ± 0.01 0.77 ± 0.02 0.24 ± 0.01 0.21 ± 0.00 0.27 ± 0.02 0.17 ± 0.00 0.15 ± 0.01 0.60–0.64 0.40–0.59
Phenylalanine 0.55 ± 0.04 1.12 ± 0.04 0.43 ± 0.01 0.37 ± 0.01 0.71 ± 0.02 0.46 ± 0.01 0.46 ± 0.00 1.20–1.40 0.91–1.06
Threonine 0.74 ± 0.00 1.58 ± 0.06 0.97 ± 0.02 0.38 ± 0.01 0.68 ± 0.02 0.60 ± 0.02 0.62 ± 0.02 0.50–0.56 0.57–0.77
Valine 0.60 ± 0.02 1.25 ± 0.02 0.55 ± 0.02 0.53 ± 0.00 0.90 ± 0.04 0.54 ± 0.02 0.54 ± 0.01 0.71–0.84 0.95–1.05
ΣEAAa 5.07 ± 0.04 10.98 ± 1.04 4.44 ± 0.02 4.32 ± 0.04 7.34 ± 0.02 4.52 ± 0.02 4.71 ± 0.01
Alanine 0.55 ± 0.02 1.54 ± 0.02 0.69 ± 0.01 0.62 ± 0.02 0.99 ± 0.02 0.61 ± 0.01 0.55 ± 0.00
4
Aspartic acid 0.64 ± 0.01 2.13 ± 0.08 1.07 ± 0.08 1.05 ± 0.01 1.60 ± 0.02 1.01 ± 0.02 1.08 ± 0.04
Cysteine 0.19 ± 0.01 0.09 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 0.12 ± 0.00 0.07 ± 0.00 0.07 ± 0.00
Glutamic acid 0.70 ± 0.02 3.77 ± 0.08 2.06 ± 0.04 1.93 ± 0.02 3.12 ± 0.04 1.77 ± 0.02 1.61 ± 0.01
Glycine 0.44 ± 0.02 2.12 ± 0.04 0.72 ± 0.01 0.62 ± 0.01 1.07 ± 0.02 0.60 ± 0.01 0.53 ± 0.01
Proline 0.72 ± 0.04 1.26 ± 0.02 0.55 ± 0.02 0.52 ± 0.00 0.81 ± 0.02 0.49 ± 0.00 0.48 ± 0.00
Serine 0.50 ± 0.02 0.93 ± 0.08 0.45 ± 0.01 0.42 ± 0.01 0.82 ± 0.01 0.50 ± 0.01 0.49 ± 0.02
Tyrosine 1.00 ± 0.04 0.95 ± 0.04 0.38 ± 0.01 0.37 ± 0.00 0.78 ± 0.01 0.49 ± 0.01 0.50 ± 0.02
ΣNEAAb 3.74 ± 0.08 12.79 ± 1.02 5.97 ± 0.08 5.59 ± 0.02 9.31 ± 0.02 5.54 ± 0.02 5.31 ± 0.02
EAA + NEAA 8.81 ± 0.12 23.77 ± 1.14 10.41 ± 1.02 9.91 ± 0.08 16.65 ± 1.02 10.06 ± 1.04 10.02 ± 0.08
Animal Feed Science and Technology 303 (2023) 115691

ΣEAA: ΣNEAA 1.36 ± 0.04 0.86 ± 0.02 0.74 ± 0.01 0.77 ± 0.01 0.79 ± 0.01 0.82 ± 0.02 0.89 ± 0.01
ΣEAAa: Sum of essential amino acids

ΣNEAAb: Sum of non-essential amino acids
Values in mean ± standard error, n = 3, ND, denote non-detected. Tryptophan was not determined.
*Amino acid requirement (g 100 g-1 diet) for Clarias gariepinus (Djissou et al., 2016; Kari et al., 2022; NRC, 1993, 2011) and Pangasianodon hypophthalmus in fed (Da et al., 2012).
Table 4
Fatty acid profile of non-deoiled NDSWP meal (A. mylitta), fishmeal (C. dussumieri) and diets (mg 100 g-1 dry sample).
Fatty acid A. mylitta C. dussumieri NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100
C13:0 6.11 ± 0.14 172.76 ± 4.56 58.01 ± 1.87 50.78 ± 1.28 20.40 ± 1.45 5.90 ± 0.04 2.98 ± 0.46
C14:0 ND 2.55 ± 0.10 0.94 ± 0.12 0.89 ± 0.02 0.34 ± 0.04 0.17 ± 0.04 0.12 ± 0.04
C15:0 0.84 ± 0.12 4.41 ± 0.12 1.40 ± 0.45 1.58 ± 0.21 0.56 ± 0.02 0.27 ± 0.08 0.12 ± 0.02
C16:0 12.98 ± 1.26 31.01 ± 1.18 16.85 ± 1.14 20.03 ± 1.14 7.32 ± 0.42 5.00 ± 0.57 2.57 ± 0.36
C17:0 4.10 ± 0.14 10.11 ± 1.16 2.20 ± 0.65 4.23 ± 0.89 1.45 ± 0.02 0.62 ± 0.02 0.34 ± 0.02
C18:0 6.95 ± 0.10 19.74 ± 1.18 7.50 ± 0.84 44.46 ± 1.27 2.96 ± 0.10 1.73 ± 0.12 0.85 ± 0.01
C20:0 1.20 ± 0.35 4.00 ± 0.11 ND ND ND ND ND
C21:0 ND ND ND ND ND ND 0.57 ± 0.02
C23:0 ND 27.33 ± 1.16 2.04 ± 0.76 ND 0.52 ± 0.04 ND ND
C24:0 ND 8.69 ± 0.22 13.51 ± 1.18 5.37 ± 0.10 2.61 ± 0.12 1.13 ± 0.24 0.79 ± 0.04
ΣSFA 32.18 ± 1.24 280.6 ± 4.35 102.45 ± 2.45 127.34 ± 2.84 36.16 ± 1.42 14.82 ± 1.12 8.34 ± 1.10
C16:1 1.66 ± 0.25 42.27 ± 1.14 18.78 ± 1.24 17.97 ± 1.08 6.38 ± 1.06 2.12 ± 0.98 0.54 ± 0.02
C18:1 13.52 ± 1.14 50.84 ± 0.98 48.04 ± 1.24 8.04 ± 0.89 20.87 ± 1.12 11.95 ± 1.07 7.29 ± 0.04
C20:1 ND 6.87 ± 0.89 0.57 ± 0.02 14.45 ± 1.04 2.41 ± 0.87 2.67 ± 0.04 1.66 ± 0.27
ΣMUFA 15.18 ± 1.12 99.98 ± 1.18 67.39 ± 1.27 40.46 ± 1.05 29.66 ± 1.14 16.74 ± 1.15 9.49 ± 1.02
C18:2 (n-6) 23.12 ± 1.10 20.98 ± 1.12 61.49 ± 1.25 86.42 ± 1.37 34.31 ± 1.08 19.89 ± 1.24 13.06 ± 0.98
C18:3 (n-3) 30.78 ± 1.15 8.19 ± 0.89 7.15 ± 0.89 11.01 ± 1.06 6.58 ± 0.04 7.36 ± 1.17 5.54 ± 0.64
C20:4 (n-6) ND ND ND 11.03 ± 1.10 2.78 ± 0.78 1.22 ± 0.64 ND
C20:5 (n-3) 2.63 ± 0.24 8.39 ± 1.10 7.31 ± 0.08 29.27 ± 1.04 11.25 ± 1.10 4.83 ± 1.13 2.21 ± 0.85
C22:6 (n-3) 8.87 ± 0.12 133.07 ± 4.26 37.93 ± 1.04 58.77 ± 1.16 21.08 ± 1.21 4.21 ± 0.87 0.88 ± 0.04
ΣPUFA 65.40 ± 1.23 170.63 ± 3.56 113.88 ± 2.47 196.50 ± 1.67 76.00 ± 1.25 37.51 ± 1.21 21.69 ± 1.04
n-3 42.28 ± 1.13 149.65 ± 2.79 52.39 ± 1.05 99.05 ± 1.06 38.91 ± 1.08 16.40 ± 1.24 8.63 ± 0.54
n-6 23.12 ± 1.22 20.98 ± 1.14 61.49 ± 1.02 97.45 ± 1.15 37.09 ± 1.07 21.11 ± 1.30 13.06 ± 1.42
n-3 to n-6 ratio 1.83 ± 0.02 7.13 ± 0.23 0.85 ± 0.04 1.02 ± 0.08 1.05 ± 0.08 0.78 ± 0.06 0.66 ± 0.04
ΣSFA, sum of saturated fatty acid; ΣMUFA, sum of monounsaturated fatty acid; ΣPUFA, sum of polyunsaturated fatty acid; ND (not detected), values
in mean ± standard error, n = 3.
fishmeal and pupae meal to form five iso-nitrogenous (280 g protein kg-1 diet) and iso-energetic (19 MJ kg-1 diet) feeds at 0, 73, 146,
219, 292 g pupae meal kg-1 diet inclusion level (pupae contributes protein at 0%, 4.75%, 9.50%, 14.25%, and 19% of total protein in
the diet) to gradually replace the fishmeal protein at 322, 242, 161, 80, 0 g fishmeal powder kg-1 diet inclusion level (fishmeal
contributes protein at 19%, 14.25%, 9.50%, 4.75%, and 0% of total protein in the diet), the remaining 9% protein was contributed by
the basal ingredient (groundnut cake, deoiled mustard cake, and maize powder). Apparently, among 19% of protein contributors, the
replacement of protein to protein concept was in place in such a way that the protein and lipid composition of the diets was 287 – 289 g
kg-1, and 71 – 81 g kg-1, respectively. The actual level of protein to protein gradual replacement was maintained between two major
protein contributors namely fishmeal and silkworm pupae, and the level of inclusion was back-calculated. The designated ingredient
powder meals were mixed as per the specified inclusion level kg-1 diet (NDSWP0, NDSWP25, NDSWP50, NDSWP75, and NDSWP100),
and moisture was added to the mixture at 20% of the total quantity of diet to be extruded. Subsequently, the lumps formed during
moisture addition were immediately fragmented through hand rubbing bringing uniformity to the ingredient followed by the sieving
utilizing a 1 mm mesh net to remove course granules covering overnight for softening the mixture. At the same time, the required
quantity of vitamin-mineral premix along with BHT (butylated hydroxytoluene) powder was blended into the feedstuff mixture. The
uniform blended mix was fed to the extruder and passed through twin-screw sheer pressure created at 1188 rpm at 120 ◦ C. The pellet
size ranging from 2 to 4 mm floating diets were prepared with 2 h of water floatability. The pellets were dried at room temperature
coated with vegetative oil involving an oil sprayer and packed in an airtight container stacked in ambient condition. The treatment diet
formulation accompanying its nutritional composition is presented in Table 2.
2.4. Amino acid profile of NDSWP meal, fishmeal, diets, and treated fish body muscle
The amino acids were identified and measured with slight modification using a HPLC (High-Performance Liquid Chromatography)
equipped with an ion-exchange column (Ishida et al., 1981). In brief, 50 mg samples were weighed and hydrolyzed using 5 ml of 6 (N)
HCl in presence of inert nitrogen gas at 110 ◦ C for 24 h in a hot-air oven. After cooling at room temperature, 500 µl of digested sample
was neutralized with 6 (N) NaOH and filtered using Whatman™ filter paper No. 1. Exactly, 5 µl of neutralized aliquot was derivatized
using 10 µl of AccQ-Fluor reagent (WAT052880, Waters) and 35 µl of borate buffer. Accurately, 5 µl of the derivatized sample was
injected into HPLC (1525, Waters) equipped with C18 reverse phase (RP) column in presence of a fluorescence detector (2475, Wa
ters). The amino acids were identified and quantified by comparing the retention time and peak areas with that of amino acid standard
(WAT088122, Waters) with the help of software Empower™ 3 database version 7.10.00.00. The amino acid profile of pupae meal,
fishmeal and prepared diets are shown in Table 3.
2.5. Fatty acid profile of NDSWP meal, fishmeal, diets, and treated fish body muscle
Around 30 g sample was homogenized in chilled mixture of chloroform-methanol (2:1 ratio) and filtered through Whatman™ filter
paper No. 1. The chloroform-lipid fraction was dried using rotary evaporator, weighed, dissolved in 10 ml chloroform, and stored. One
5
ml of aliquot was taken from it in a pre-weighed petridish and dried in a hot air oven (2 h, 55 ◦ C) to measure the weight of fat residue in
1 ml extraction. Under fatty acid methyl-ester (FAME) preparation, about 150 mg of sample oil was refluxed for 5 min with 4 ml 0.5 (N)
methanolic NaOH in presence of inert N2 gas. Later, fraction was refluxed with 5 ml BF3-CH3OH solution for 5 min. Saturated 16 ml
NaCl was added to float the methyl-esters into a separating funnel and 20 ml petroleum benzene was added vigorously with repetition
of lower layer collection, twice. The collected petroleum benzene fraction was passed through anhydrous Na2SO4, evaporated
completely, and dissolved with 5 ml HPLC graded hexane. The hexane fraction was filtered through 0.2 µm syringe filter paper,
injecting 1 µl (30:1 split ratio) into GC-MS. The fatty acids were detected in a GC (Trace GC Ultra, Thermo Scientific) equipped with a
capillary column (TR-FAME, 30 m × 0.25 mm, 0.25 µm film thickness) and a MS (ITQ 900, Thermo Scientific) at the ionization voltage
of 70 eV and mass range of 45–600. Helium was a carrier gas with a flow rate of 1.0 ml min-1. The individual constituents showed by GC
were identified and quantified by comparing the retention time and peak area corresponding to standards (ME-14-KT and ME-19-KT,
SUPELCO Analytical) (Folch et al., 1957; Metcalfe et al., 1966; Mohanty et al., 2012). The fatty acid profile of pupae meal, fishmeal and
prepared diets are displayed in Table 4.
2.6. Experimental fish stocking, ration/culture management
The striped catfish (16.56 ± 0.08 g) fingerling was purchased from the local fish farm and carried to the cage site in oxygenated
condition, upon reaching at cages the fishes were exposed to dip treatment with potassium permanganate solution (2 mg L-1) for 10 s to
treat the lesion or parasite occurrence (Peter et al., 2022). Eventually, fishes were randomly stocked at 33 numbers m-3 cage water
volume equal to the designated cages under each diet treatment group. Prior to the commencement of the experimental feeding trial,
12 numbers of fishes were anesthetized by dipping in clove oil-water solution (50 µl L-1), subsequently, the fishes were sacrificed for
initial digestive enzyme analysis, hepatosomatic index, viscero-somatic index, and fish muscle nutrient composition parameters
(AOAC, 2005).
The fingerlings were fed through hand broadcasting to satiation at 5% of body mass for the first month daily and subsequently at
3% of body mass for the rest two months daily, both at 08.00 h and 16.00 h. The ration size was adjusted during monthly sampling
based on accounted biomass. The duration of the feeding trial was ninety days. During husbandry, periodical cage net blockage
clearance and daily fish mortality records were maintained.
2.7. Nutritional composition analysis of ingredients, diets, and fish muscle
The proximate composition of feedstuff samples used in experimental diets along with treated fish flesh was analyzed involving the
standard methods (AOAC, 2005). The analysis of dry matter was assessed by drying the samples in triplicates kept inside the hot air
oven at 105 ◦ C for 24 h unless persistent mass was obtained. The amount of crude protein (N × 6.25) was ascertained through the
determination of nitrogen (N) content in the sample following the Kjeldahl method (Jacobs, 1951), where, samples were digested in
concentrated sulphuric acid (H2SO4) in presence of digestion mixture (K2SO4 and CuSO4 at 9:1 ratio) in a digester (LABQUEST by
BOROSIL® KBD06, India) accompanying sodium hydroxide and boric acid auto distillation engaging a Kjeldahl auto distillation
system (LABQUEST by BOROSIL® KDI040, India) along with 0.1 (N) H2SO4 titration. A Soxhlet apparatus system was involved during
the ether extract analysis, where, lipid fractions were extracted in the heated petroleum ether (40–60 ◦ C). A muffle furnace was
employed to incinerate the crucible and placed dried samples into ashes at 600 ◦ C for 4 h to determine the total ash content. To
determine the crude fibre, fat-free samples were hydrolyzed in 30 min of boiling water containing 25 ml 10% H2SO4 following 25 ml
10% sodium hydroxide for acid-alkali digestion along with subsequent drying inside the hot air oven (105 ◦ C for overnight) to obtain
constant mass trailed with muffle furnace incineration (600 ◦ C for 3 h) (Nduko et al., 2018).
2.8. Growth accomplishment and nutritional utilization indicators
The growth accomplishment and feed nutrient utilization indicators were figured out under the specified equations:
IWG (individual weight gain, g) = final body weight - initial body weight
Feed fed (g) fish-1 = total quantity feed given / final number of fish
Protein intake (g) fish-1 = (feed fed fish-1 × crude protein in feed) / 100
WG (weight gain, %) = {(final body weight - initial body weight) / initial body weight} × 100
FCR (feed conversion ratio) = feed fed fish-1 (dry weight) / individual weight gain (wet weight)
SGR (specific growth rate, % day-1) = {(ln final weight - ln initial weight) / feeding days} × 100
PER (protein efficiency ratio) = individual weight gain (wet weight) / quantity of protein fed (dry weight)
ANPU (apparent net protein utilization, %) = {(final muscle crude protein - initial muscle crude protein) / quantity of protein fed} × 100
S (survival rate, %) = (final number of fish / initial number of fish) × 100
6
2.9. Determination of biological implications
The determination of biological attributes were figured out employing following calculation:
Fulton’s CF (condition factor) = {final body weight (g) / final body length3 (cm)} × 100
VSI (viscero-somatic index, %) = {viscera weight (g) / body weight (g)} × 100
HSI (hepatosomatic index, %) = {liver weight (g) / body weight (g)} × 100
2.10. Digestive enzyme activity determination
At the cage site, the gut tissue samples were removed from the dissected fishes and collected into 0.25 M sucrose solution kept at
4 ◦ C. Eventually, tissues were lysed homogenously within the sucrose solution using a chilled tissue lyser QIAGEN® TissueLyser II
(Qiagen, Germany) (Upadhyay et al., 2021), and centrifuged at 12000 rpm at 4 ◦ C for 12 min to collect the supernatant preserved at −
20 ◦ C to study enzyme activity. The gut tissue sample protein quantification was obtained through the slight moderation involving
alkaline copper sulfate along with diluted Folin-Ciocalteu’s reagent measurement method (Lowry and Randall, 1951), keeping bovine
serum albumin as standard. The gut amylase enzyme activity was determined colorimetrically by measuring brown colour nitro
aminosalicylic acid formed during the reaction between 3, 5-dinitrosalicylic acid and fragments of reducing groups produced through
hydrolysis interaction among starch and α-amylase, in presence of maltose as standard (Rick and Stegbauer, 1974). The gut protease
enzyme activity was analyzed colorimetrically involving 1% casein (pH 7.8) digestion at 37 ◦ C accompanying the reaction termination
by adding 10% trichloroacetic acid solution (Drapeau, 1976). The gut lipase activity was measured by the titrimetric method under 0.1
M sodium phosphate buffer (pH 7.0) and olive oil emulsion incubated for 24 h subsequently added 95% ethanol titrated with 0.05 (N)
sodium hydroxide solution in presence of phenolphthalein indicator (Cherry and Crandall, 1932).
2.11. Gut histo-morphological observation
A gut micro-graphical study was conducted in adherence to the Institutional Animal Ethics Committee of ICAR-Central Inland
Fisheries Research Institute, Barrackpore, Kolkata, India. Upon termination of the feeding trial, two fishes were randomly selected
from each treatment and anesthetized with clove oil (50 µl L-1) following dissection of the gut tissue samples kept preserved in 10%
neutral buffered formalin (NBF) at the cage site for investigating histo-morphological alterations occurred if any to the proximal and
mid portion of the intestine. The samples were subsequently processed for block preparation keeping the cassette containing tissue
samples (4 × 5 mm size) in drop-by-drop running tap water overnight to remove the preservation solution. The cassette containing
hydrated tissue samples was subjected to dehydration dipped through increasing order of ethanol concentration (30% for one h, 50%
for one h, 70% for one h, 90% for one h, 100% - I and II for 30 min each), xylene - I and II for 30 min each, and melted paraffin wax
(58–60 ◦ C) - I and II for one h each inside an automatic programmable tissue processor (Leica® TP1020, India). Subsequently, the
cassette containing tissues was placed inside the chamber filled with melted paraffin wax, tissues oriented desirably embedded into the
wax block under a tissue embedding instrument coupled with a cold plate (Leica® HistoCore Arcadia Embedding Center, India).
Microtome tissue sectioning (4–5 µm thickness), placement of tissue section ribbons from the water bath to the glass slide, staining
process following standard histological hematoxylin-eosin Y staining procedure, DPX mounting with a coverslip, and digital images
were gathered with annotation using compound microscope coupled with NIS-Elements D3.1 software.
2.12. Economical investigation
The experimental diet kg-1 cost and its profit-return indicators were calculated based on available local market retail price during
the feeding trial period, to bring conformity in the price of the prepared practical diet, the cost incurred during transport, feedstuff
process, feed preparation, floating net-cage installation were disregarded.
2.13. Statistical computation
The data normality as well as homogeneity of variances were tested using Shapiro-Wilk and Levene’s test. The comparison of means
of attributes under dietary groups was analyzed using one-way ANOVA followed by Tukey’s HSD post hoc test to identify the sta
tistically significant difference among the mean values at p < 0.05. The linear regression was applied among WG, SGR and FCR to the
replacement level. The statistical computation of parameters was conducted operating IBM® SPSS® Statistics version 22, and
GraphPad Prism® 8.0 for Windows.
7
Table 5
The effect of replacing fishmeal protein with NDSWP protein on growth accomplishment, feed nutrient utilization, and biological indices of striped
catfish (P. hypophthalmus) fingerlings reared in cages at the termination of experiment.
Indicators Diet treatment groups p-value
NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100
IBW (g) 16.36 ± 1.26 16.38 ± 0.94 16.68 ± 1.07 16.73 ± 1.45 16.68 ± 0.72 0.482
FBW (g) 43.63a ± 1.60 42.77a ± 1.58 41.88a ± 1.56 38.74ab ± 1.19 36.20b ± 1.09 0.009
IWG (g) 27.27a ± 0.52 26.39ab ± 0.49 25.21ab ± 1.80 22.01bc ± 0.32 19.52c ± 0.61 0.008
Feed fed (g) fish-1 34.59a ± 0.01 34.60a ± 0.02 34.59a ± 0.02 34.67a ± 0.03 34.81b ± 0.07 0.025
Protein intake (g) fish-1 9.69a ± 0.01 9.69a ± 0.01 9.69a ± 0.01 9.71ab ± 0.01 9.75b ± 0.02 0.018
WG (%) 166.72a ± 1.09 161.17ab ± 5.52 151.15ab ± 10.72 131.56bc ± 2.91 117.12c ± 4.96 0.009
SGR (% day-1) 1.09a ± 0.00 1.07a ± 0.03 1.02ab ± 0.05 0.93ab ± 0.02 0.86b ± 0.03 0.011
FCR 1.27a ± 0.02 1.31a ± 0.03 1.38a ± 0.10 1.58ab ± 0.03 1.78b ± 0.06 0.005
PER 2.82a ± 0.06 2.72ab ± 0.05 2.61ab ± 0.19 2.27bc ± 0.04 2.01c ± 0.07 0.008
ANPU (%) 36.43 ± 0.45 30.18 ± 2.5 31.75 ± 1.44 28.04 ± 2.11 24.20 ± 3.51 0.078
Fulton’s CF 0.74 ± 0.01 0.74 ± 0.01 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.01 0.404
VSI (%) 9.18 ± 0.46 8.49 ± 0.60 8.20 ± 0.67 7.80 ± 0.38 7.81 ± 0.46 0.582
HSI (%) 2.39ab ± 0.13 2.67a ± 0.17 2.32ab ± 0.17 2.13ab ± 0.12 2.07b ± 0.10 0.036
S (%) 99.82a ± 0.02 99.80a ± 0.04 99.82a ± 0.06 99.60ab ± 0.08 99.21b ± 0.18 0.022
Facts indicated as mean ± standard error, n = 3; Mean values holding in the same column labeled with different superscript vary remarkably at alpha
value < 0.05. IBW- Initial body weight (n = 60), FBW- Final body weight (n = 60), IWG - Individual weight gain, WG - Weight gained, FCR - Feed
conversion ratio, SGR - Specific growth rate, PER - Protein efficiency ratio, ANPU - Apparent net protein utilization, Fulton’s CF - Fulton’s condition
factor, VSI - Viscero-somatic index, HSI - Hepatosomatic index, S – Survival.
3. Results and discussion
The present investigation, a counter relationship among individual weight gain, percentage of weight gain, feed conversion ratio,
specific growth rate, and protein efficiency ratio were observed under the growth accomplishment and nutritional utilization matrices,
as the non-mulberry non-defatted silkworm pupae protein gradually replaces fishmeal protein with its increasing level of inclusion in
the diets. The protein content of the present experimental diets (28% crude protein) was within the range (19–30% crude protein)
required for normal growth rate in Pangasius fingerling, but relatively lower than the protein required (27–32% crude protein) for
achieving maximum growth rate (Da et al., 2013; Hung et al., 2002). On the other hand, lipid content of the experimental diets (8%
crude lipid) was close to the optimum dietary lipid requirement of 9.0–10.1% for striped catfish fingerling (Sivaramakrishnan et al.,
2017). In the present study, striped catfish fingerlings were stocked in cages with a stocking density of 33 numbers per m3, selected
based on the previous study for optimum growth in cages using Asian river catfish (Pangasius bocourti) (Jiwyam, 2011) and striped
catfish (P. hypophthalmus) fingerling (Kumar et al., 2015). Based on the results of the current investigation, the incorporation of
non-defatted non-mulberry silkworm pupae protein can effectively replace the fishmeal protein up to the level of 50% in the diet of
striped catfish in cages without compromising the final body weight (g), individual weight gain (g), weight gained (%), feed conversion
ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER). The results obtained in the present experiment are in line
with the observation noticed in omnivorous juvenile Jian carp, Cyprinus carpio var. Jian (Ji et al., 2015), common carp, Cyprinus carpio
var. Communis (Nandeesha et al., 1990), and GIFT tilapia (Sathishkumar et al., 2021) when fed with non-defatted silkworm pupae.
The similar trend of growth performance of striped catfish may be attributed to their omnivore nature having similar enzyme profiles
and digestion efficiency. The present study revealed that the non-defatted silkworm pupae have the potential to replace the fish meal
inclusion level by 50% (silkworm pupae inclusion level at 146.1 g kg-1) in the diet of striped catfish without affecting the growth
performance and feed utilization indicators.
The individual weight gain in fishes fed with the mid-range of protein to protein replacement level (0%, 25%, and 50%) was equally
better in comparison to each other and reflected statistically non-significant differences with similar superscripts. However, as the
protein to protein replacement level increased at 100%, the individual weight gain in fish declined at a significant level (p < 0.05) in
comparison to the rest of the diet treatment groups. In addition, individual weight gain followed a declining trend among the dietary
groups ranging from 27.27 to 19.52 g as the pupae protein inclusion level increased gradually beyond 50% as a major protein
contributor in the diet. A similar observation of the declining trend in individual weight gain was noted in Jian carp (Ji et al., 2015),
when pupae meal inclusion level was increased beyond 50% in its diet. The quantity of feed fed per fish and protein intake per fish fed
to the fishes in the treatment was achieved at the elevated trend from 34.59 to 34.81 g (p < 0.05), and 9.69 – 9.75 g (p < 0.05),
respectively. A significant increasing trend in the feed fed (g) per fish, as well as protein intake (g) per fish, were recorded at 100%
pupae included diet (p < 0.05) in contrast to the rest of the treatment diet groups, a similar impression was also reported beyond 50%
silkworm pupae containing diet (Ji et al., 2015). However, protein intake did not contribute towards the bio-growth of the treated
fishes in the experiment, which might be due to the problem associated with insect meal diet palatability or nutrient unavailability
caused by anti-nutritional factors (Francis et al., 2001; Wan et al., 2017) which get bio-sequestered from leaves on which insect fed
(Finke, 2002). Likewise, up to 66.67% of dietary silkworm pupae meal inclusion level revealed optimum growth performance in
Oreochromis niloticus (Salem et al., 2008; Sathishkumar et al., 2021), 50% in common carp (Nandeesha et al., 2000) and African catfish
(C. gariepinus) (Kurbanov et al., 2015).
The percentage of weight gain and specific growth rate observed remarkably higher (p < 0.05) when fishes were fed at 50%
8
Table 6
The proximate composition (% wet weight basis) of striped catfish (P. hypophthalmus) body muscle under the treatment groups at the conclusion of
experiment.
Parameter Initial facts Diet treatment groups p-value
Moisture content (%) 82.57 ± 0.06 81.01 ± 0.11 79.71 ± 0.54 80.32 ± 0.09 79.58 ± 0.57 79.78 ± 0.47 0.226
Crude protein (%) 12.93 ± 0.00 16.47 ± 0.05 15.86 ± 0.24 16.01 ± 0.14 15.66 ± 0.21 15.30 ± 0.35 0.084
Ether extract (%) 0.67 ± 0.07 1.28 ± 0.13 2.70 ± 0.28 1.95 ± 0.02 3.27 ± 0.43 3.00 ± 0.70 0.069
Ash content (%) 1.00 ± 0.02 1.04 ± 0.00 1.00 ± 0.03 1.03 ± 0.01 1.00 ± 0.01 0.95 ± 0.04 0.125
Value represented as mean ± standard error, n = 3; Mean values of treatment groups did not vary remarkably (p > 0.05).
silkworm protein to protein substitution level in contrast to other diet treatment groups (100% silkworm pupae protein inclusions),
whereas, there is an identical fall in the weight gain and specific growth rate both for 75% and 100% pupae protein inclusion level with
a non-significant difference (p > 0.05). Further, both percentage of weight gain and specific growth rate observed under the treatment
groups 50% and 75% pupae levels were similar with statistically non-significant differences (p > 0.05). Overall, the percentage of
weight gain indicated a declining trend from 166.72% to 117.12% (p < 0.05) along with a reduction of SGR from 1.09% to 0.86% day-1
(p < 0.05) as the pupae protein inclusion level raised. In this regard, a similar cognitive declining trend in specific growth rates was
reported in Jian carp (Ji et al., 2015) and African catfish (Kurbanov et al., 2015) fed with more than 50% pupae included diet.
A similar trend was also observed under the feed utilization indicator such as FCR, where, it admitted an increase in trend ranging
from 1.27 to 1.78 as the protein to protein replacement took place from 0 (control diet) to 100% pupae inclusion level among the
dietary groups, found statistically remarkable difference at p < 0.05 level (Table 5). Further, it has been noticed that the FCR was in the
range of 1.27–1.38 for fishes fed at dietary 0 (control diet) to 50% replacement level with a statistically non-significant difference, as
reflected with similar superscript (p < 0.05). However, a statistically non-significant difference in FCR attribute was distinguished in
the diet containing 75% and 100% fishmeal protein substitution by the non-defatted silkworm pupae protein inclusions, as shown with
a similar superscript. Similarly, an increasing trend in FCR was also recorded when dietary pupae meal was incorporated beyond the
50% level fed to Jian carp (Ji et al., 2015), Nile tilapia (Salem et al., 2008) and African catfish (Kurbanov et al., 2015).
Among the nutrient utilization indices (protein efficiency ratio and apparent net protein utilization), a statistically significant (p <
0.05) lower PER was observed ranging from 2.82 to 2.01, as the protein to protein replaced progressed gradually. The protein effi
ciency ratio between 0%, 25%, and 50% replacement levels was statistically similar reflecting the stability in growth was up to 50%
pupae protein inclusion level, while, undifferentiated PER was recorded between 75% and 100% group. Additionally, similar PER
between the 50% and 75% group was observed, whereas, significantly lower PER was encountered in 75% and 100% pupae protein
groups in comparison to the control group (p < 0.05). Overall, in the present study, a declining trend in PER was recorded beyond the
pupae incorporation level of 50% in the diet, and found in agreement with the measurement perceived in Jian carp (Ji et al., 2015).
Similarly, greater growth rate and nutrient utilization attributes were reported in African catfish (Clarias gariepinus) fingerling fed with
a dietary 50% silkworm pupae meal, whereas, it was lower in 100% pupae meal or fishmeal diet with a remarkable difference in weight
gain, growth rate and protein efficiency ratio (Kurbanov et al., 2015). Similarly, a curtailment in ANPU was perceived during the
experiment at a statistically non-significant level (p > 0.05) extending 36.43 – 24.20%, while, a similar decreasing trend in ANPU was
reported in the case of Labeo rohita (Begum et al., 1994) and Oncorhynchus mykiss (Shakoori et al., 2016), when, those were fed with
dietary pupae meal inclusion level exceeding 50% and 10%, respectively. The result of the present study has underlined the evidence of
feed indigestibility at a higher dietary inclusion level of pupae protein, thus, ingestion of a higher amount of dietary non-defatted
pupae protein did not favour better growth in the striped catfish. This phenomenon may be attributed to the inherent inability of
this fish to digest, absorb, and metabolize the higher amount of insect protein. Moreover, the results of the present feeding trial
elucidate a non-significant effect on the other biological matrices except hepatosomatic index (p < 0.05), which is contrary to the
findings of Jian carp fed with pupae meal (Ji et al., 2015). The Fulton’s condition factor was observed to raise gradually from 0.74
(control diet group) to 0.76 (50% pupae diet group) followed by a fall to 0.75 in the 100% pupae protein diet group (p > 0.05).
Similarly, a decrement in the condition factor was reported in rainbow trout, Oncorhynchus mykiss fed exceeding 10% dietary silkworm
pupae meal (Shakoori et al., 2016). Besides, the viscero-somatic index under increasing levels of substitution among the treatments
revealed a relatively declining trend (9.18 – 7.81%, at p > 0.05). Furthermore, the status of hepatic condition in terms of the hep
atosomatic index deteriorated significantly (2.67 – 2.07%, p < 0.05) among the dietary treatment groups (25–100%) compared to
control (2.39%) as protein to protein replacement takes place by the incorporation of silkworm pupae which find supports with the
similar observation reported in case of Jian carp (Ji et al., 2015) and Nile tilapia (Salem et al., 2008) feeding with fishmeal replaced
pupae meal containing diet. This declining nature of the hepatic condition index indicates the possible metabolic problems encoun
tered and/or any other hepatic cellular complications that might have occurred due to a possible rise in anti-nutrient levels during the
increasing inclusion level of pupae protein in the diet which might have impaired metabolism. Further, a relatively highest percentage
of survival rate was obtained in the treatment groups fed up to 50% pupae protein inclusion level in comparison to the rest of the group,
especially in the 100% pupae meal incorporated diet group with the lowest survival rate was recorded (p < 0.05). Similarly, a 94–98%
survival rate was reported in GIFT tilapia (Sathishkumar et al., 2021) and mirror carp (Zhou et al., 2017) fed with dietary silkworm
pupae.
The proximate composition analysis of treated fish muscle indicated the crude protein (% wet weight basis) content gradually
escalated up to 50% pupae meal containing diet group that eventually diminished through the group fed with 75% and 100% pupae
9
Table 7
Amino acid profile (g 100 g-1 sample on wet weight basis) of the treated fish body muscle at the end of experiment.
Amino acids Status of fish muscle against each dietary group
Initial facts NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100 p-value
Arginine 0.34 ± 0.02 0.54 ± 0.02 0.39 ± 0.02 0.75 ± 0.01 0.63 ± 0.02 0.49 ± 0.02
Histidine 0.08 ± 0.00 0.13 ± 0.00 0.08 ± 0.00 0.18 ± 0.01 0.14 ± 0.01 0.03 ± 0.00
Isoleucine 0.17 ± 0.01 0.26 ± 0.02 0.17 ± 0.01 0.35 ± 0.02 0.30 ± 0.02 0.25 ± 0.01
Leucine 0.34 ± 0.02 0.54 ± 0.01 0.35 ± 0.02 0.70 ± 0.01 0.60 ± 0.02 0.52 ± 0.02
Lysine 0.50 ± 0.02 0.82a ± 0.01 0.51b ± 0.02 1.12c ± 0.04 0.91d ± 0.01 0.81a ± 0.03 0.000
Methionine 0.09 ± 0.00 0.13a ± 0.02 0.10a ± 0.00 0.24c ± 0.02 0.19bc ± 0.01 0.15ab ± 0.01 0.001
Phenylalanine 0.17 ± 0.02 0.26 ± 0.02 0.18 ± 0.01 0.33 ± 0.02 0.28 ± 0.02 0.24 ± 0.01
Threonine 0.19 ± 0.02 0.31 ± 0.01 0.22 ± 0.01 0.42 ± 0.01 0.34 ± 0.01 0.38 ± 0.02
Valine 0.17 ± 0.01 0.27 ± 0.01 0.19 ± 0.01 0.35 ± 0.02 0.30 ± 0.00 0.26 ± 0.01
ΣEAAa 2.05 ± 0.02 3.26 ± 0.04 2.19 ± 0.02 4.44 ± 0.04 3.69 ± 0.06 3.13 ± 0.04
Alanine 0.29 ± 0.01 0.50 ± 0.02 0.31 ± 0.01 0.57 ± 0.02 0.47 ± 0.01 0.44 ± 0.02
Aspartic acid 0.54 ± 0.02 0.85 ± 0.02 0.57 ± 0.02 1.18 ± 0.02 0.91 ± 0.02 0.85 ± 0.02
Cysteine 0.07 ± 0.00 0.12 ± 0.00 0.04 ± 0.00 0.15 ± 0.01 0.12 ± 0.01 0.13 ± 0.00
Glutamic acid 0.85 ± 0.02 1.39 ± 0.02 0.94 ± 0.01 1.92 ± 0.02 1.54 ± 0.03 1.44 ± 0.02
Glycine 0.28 ± 0.02 0.38 ± 0.01 0.27 ± 0.01 0.48 ± 0.01 0.37 ± 0.01 0.32 ± 0.01
Proline 0.19 ± 0.01 0.19 ± 0.00 0.18 ± 0.01 0.29 ± 0.01 0.24 ± 0.00 0.19 ± 0.01
Serine 0.21 ± 0.01 0.36 ± 0.02 0.23 ± 0.02 0.49 ± 0.02 0.38 ± 0.02 0.35 ± 0.01
Tyrosine 0.16 ± 0.00 0.28 ± 0.01 0.17 ± 0.02 0.37 ± 0.01 0.30 ± 0.02 0.26 ± 0.02
ΣNEAAb 2.59 ± 0.04 4.07 ± 0.02 2.71 ± 0.04 5.45 ± 0.02 4.33 ± 0.04 3.98 ± 0.02
EAA + NEAA 4.64 ± 0.12 7.33 ± 0.10 4.90 ± 0.08 9.89 ± 0.04 8.02 ± 0.02 7.11 ± 0.04
ΣEAA: ΣNEAA 0.79 ± 0.02 0.80 ± 0.02 0.81 ± 0.01 0.81 ± 0.02 0.85 ± 0.02 0.79 ± 0.01
ΣEAAa: Sum of essential amino acids

ΣNEAAb: Sum of non-essential amino acids
Values in mean ± standard error, n = 3. Tryptophan was not determined. Mean value with same column labeled with different superscript vary
significantly at p < 0.05.
meal incorporated diet (p > 0.05) (Table 6). A relatively equivalent amount of muscle crude protein content was evident when
silkworm pupae protein diet was fed to Jian carp replacing the fishmeal protein at a 50% incorporation level (Ji et al., 2015), Indian
major carp (Labeo rohita) fed with mulberry silkworm meal (Bombyx mori) (Begum et al., 1994), and in case of Cyprinus carpio var.
Communis fed with non-defatted Bombyx mori up to 30% pupae meal inclusion level in the diet (Nandeesha et al., 1990). However,
Sathishkumar et al. (2021) also reported a non-significant body chemical composition of GIFT tilapia fed with a 66.67% untreated
silkworm pupae meal diet. Crude lipid content elicited an increasing trend in the treated fish muscle under specified diet treatment (p
> 0.05), which is in contrast to the result perceived when the fish meal was substituted by silkworm pupae in Jian carp (Ji et al., 2015)
and GIFT tilapia (Sathishkumar et al., 2021). A similar trend of increasing crude lipid was also noted, as replacement level increases in
the case of Labeo rohita, Labeo catla, Cirrhinus mrigala, Hypophthalmichthys molitrix fed with non-defatted Bombyx mori (Begum et al.,
1994; Rangacharyulu et al., 2003) and in case of Cyprinus carpio var. Communis fed with 30% pupae incorporation (Nandeesha et al.,
1990). Moreover, a distinct decrement in the ash content of the treated fish muscle under different diet treatment regimes was recorded
(p > 0.05), a similar trend was also observed during the feeding trial with the increasing order of silkworm pupae inclusion levels in the
diet of juvenile Jian carp (Ji et al., 2015) and Labeo rohita (Begum et al., 1994), whereas, the muscle ash content in the present study
was found in contrary to the replacement study results observed in Cyprinus carpio var. Communis using non-defatted Bombyx mori
(Nandeesha et al., 1990).
The analyzed amino acid profile of treated fish body muscle (fillet) is presented in Table 7. The lysine and methionine content of
non-deoiled tasar (A. mylitta) dried spent pupae meal was recorded to be 0.76 ± 0.02 and 0.14 ± 0.01 g 100 g-1 dry sample,
respectively. In the current study, amino acid content (expressed in g 100 g-1 protein, at crude protein 65.01%) in non-deoiled tasar
spent pupae meal was detected with arginine (0.93), histidine (0.32), isoleucine (0.82), leucine (1.45), lysine (1.17), methionine
(0.22), phenylalanine (0.85), threonine (1.14), valine (0.93), alanine (0.85), aspartic acid (0.99), cysteine (0.29), glutamic acid (1.07),
glycine (0.68), proline (1.11), serine (0.77), and tyrosine (1.54). Whereas, fishmeal (C. dussumieri) revealed the amino acid content
(expressed in g 100 g-1 protein, at crude protein 58.85%) with arginine (2.21), histidine (0.64), isoleucine (1.87), leucine (3.06), lysine
(2.86), methionine (1.31), phenylalanine (1.91), threonine (2.69), valine (2.12), alanine (2.62), aspartic acid (3.63), cysteine (0.16),
glutamic acid (6.41), glycine (3.60), proline (2.15), serine (1.58), and tyrosine (1.61). In this regard, it was reported that insect meals
are good sources of lysine and methionine ranging from 1.68 to 4.49 and 0.47–4.03 g 100 g-1 dry samples (Nogales-Mérida et al.,
2019), respectively. However, both the lysine and methionine content of A. mylitta pupae meal was comparable to that of eri silkworm
pupae (Samia ricini) (Gangopadhyay et al., 2022), Chinese tasar silkworm pupae (Antheraea pernyi) (Zhou and Han, 2006), mulberry
silkworm pupae (Bombyx mori) (Rao, 1994), black soldier fly pupae (Hermetia illucens) (Park et al., 2013) and mealworm larvae
(T. molitor) (Ravzanaadii et al., 2012). The non-deoiled tasar (A. mylitta) spent dried pupae meal revealed an indispensable to
dispensable amino acid ratio of 1.36 ± 0.04. In general, insect meals have the indispensable to dispensable amino acid ratio between
0.78 and 1.12, whereas, in fishmeal, it is reported to be highest at 1.01, and the lowest value is 0.74 for soybean meal compared to
other meals (Makkar et al., 2014; Nogales-Mérida et al., 2019). Whenever, a meal possesses a ratio close to 1.0 (0.92 in the case of
non-deoiled B. mori mulberry silkworm pupae meal as per Rao, 1994), it is considered to be nutritionally balanced, but when it is below
10
Table 8
Fatty acid profile (mg 100 g-1 sample in wet weight basis) of the treated fish body muscle at the end of experiment.
Fatty acid Initial NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100 p-value
C13:0 234.96 ± 5.42 97.71 ± 1.05 197.43 ± 2.21 68.83 ± 1.21 78.72 ± 1.08 59.75 ± 1.12
C14:0 1.57 ± 0.04 0.62 ± 0.04 1.28 ± 0.75 0.33 ± 0.04 0.32 ± 0.04 0.31 ± 0.02
C15:0 1.73 ± 0.06 0.66 ± 0.06 1.67 ± 0.64 0.42 ± 0.02 0.36 ± 0.02 0.27 ± 0.04
C16:0 41.45 ± 1.10 16.26 ± 1.02 33.26 ± 1.10 13.94 ± 0.67 13.41 ± 1.02 9.44 ± 1.02
C17:0 2.82 ± 0.12 1.16 ± 0.64 3.32 ± 0.24 0.97 ± 0.04 0.82 ± 0.02 0.68 ± 0.02
C18:0 4.10 ± 0.08 6.34 ± 0.87 17.47 ± 1.02 7.08 ± 0.87 1.82 ± 0.21 6.78 ± 0.87
C20:0 ND ND ND ND ND ND
C21:0 ND ND 4.23 ± 0.75 ND ND ND
C23:0 8.60 ± 0.75 3.23 ± 0.54 1.19 ± 0.68 0.82 ± 0.02 2.89 ± 0.75 2.27 ± 0.75
C24:0 10.68 ± 1.04 3.89 ± 0.86 12.50 ± 1.12 3.16 ± 0.08 6.01 ± 1.02 5.43 ± 0.67
ΣSFA 305.91 ± 5.65 129.87 ± 2.12 272.35 ± 2.27 95.55 ± 1.04 104.35 ± 1.12 84.93 ± 1.24
C16:1 14.23 ± 1.05 7.99 ± 1.02 18.44 ± 1.10 5.50 ± 0.12 5.06 ± 0.24 3.09 ± 1.02
C18:1 93.00 ± 1.02 29.63 ± 1.24 60.08 ± 1.04 23.02 ± 1.16 40.85 ± 1.14 19.21 ± 1.12
C20:1 18.21 ± 1.12 1.88 ± 0.08 3.64 ± 0.87 5.00 ± 0.46 4.56 ± 0.87 0.80 ± 0.04
ΣMUFA 125.44 ± 2.24 39.50 ± 1.08 82.16 ± 1.04 33.52 ± 1.10 50.47 ± 1.36 23.10 ± 1.21
C18:2 (n-6) 56.66 ± 1.06 23.83 ± 1.02 71.06 ± 1.02 18.93 ± 1.12 23.59 ± 1.20 23.24 ± 1.14
C18:3 (n-3) 10.88 ± 1.04 2.61 ± 0.54 8.19 ± 1.02 2.84 ± 0.75 5.74 ± 1.02 5.02 ± 1.08
C20:4 (n-6) ND ND 16.99 ± 1.23 ND ND ND
C20:5 (n-3) 12.97 ± 1.02 8.95 ± 1.06 12.37 ± 1.14 3.91 ± 0.58 17.35 ± 1.04 14.52 ± 1.12
C22:6 (n-3) 80.59 ± 1.12 35.62 ± 1.14 47.24 ± 1.08 29.87 ± 1.21 39.10 ± 1.12 26.28 ± 1.06
ΣPUFA 161.10 ± 2.21 71.01a ± 1.07 155.85b ± 2.23 55.55c ± 1.42 85.78d ± 1.34 69.06a ± 1.24 0.000
n-3 104.44 ± 1.87 47.18 ± 0.98 67.80 ± 1.06 36.62 ± 1.08 62.19 ± 1.57 45.82 ± 1.32
n-6 56.66 ± 1.14 23.83 ± 1.04 88.05 ± 1.12 18.93 ± 1.02 23.59 ± 1.04 23.24 ± 1.02
n-3 to n-6 ratio 1.84 ± 0.16 1.98 ± 0.21 0.77 ± 0.02 1.93 ± 0.12 2.64 ± 0.87 1.97 ± 0.08
ΣSFA, sum of saturated fatty acid; ΣMUFA, sum of monounsaturated fatty acid; ΣPUFA, sum of polyunsaturated fatty acid; ND (not detected), values
in mean ± standard error, n = 3. Mean value with same column labeled with different superscript differs significantly at p < 0.05.
1.0 (0.83 in soybean meal), it specifies an imbalance condition. In general, amino alkanoic acid profiles of the diets reflect the rise in
protein levels in the treated fish body tissues (Kari et al., 2022). An imbalanced amino acid profile in the fish diet can often lead to
decreased protein retention efficiency and poor growth performance (Kaushik and Seiliez, 2010). The amino acid requirement of
striped catfish was duly fulfilled with arginine (0.89–1.50), histidine (0.16–0.35), isoleucine (0.43–0.75), leucine (0.72–1.27), lysine
(0.42–1.01), methionine (0.15–0.27), phenylalanine (0.37–0.71), threonine (0.38–0.97), valine (0.53–0.90) in the diets (g 100 g-1
sample, dry weight basis) in comparison to the requirement reference of Clarias gariepinus (Djissou et al., 2016; Kari et al., 2022; NRC,
1993, 2011) and Pangasianodon hypophthalmus (Da et al., 2012). In the present study, amino acid levels appeared to be influenced by
dietary intake, and a decrement in amino acid deposition occurred among the body muscle tissues fed at higher inclusion levels of
dietary non-deoiled tasar pupae protein. A decreased level of lysine and methionine content was investigated in the treated fish body
muscle at different levels of insect meal in the diets, ranging from 0.51 to 1.12 and 0.10–0.24 g 100 g-1, respectively. In the treated
striped catfish body muscle, a significantly high value of lysine and methionine content was investigated in the group fed with
NDSWP50 diet (p < 0.05), which could be due to better digestibility, absorption, utilization, and bioavailability of those amino acids
available in insect meal in comparison to fishmeal, which, indicates that the dietary inclusion of insect meal (A. mylitta) could be
suitable to the extent of 50%. The total amino acid content of muscle along with the individual amino acids was reported to be
decreased with increasing dietary silkworm pupae (B. mori) levels, when it was fed to Jian carp (Cyprinus carpio var. Jian) (Ji et al.,
2015). In contrast to the present study, non-significant differences were reported in GIFT tilapia body muscle fed with graded levels of
B. mori bioprocessed silkworm pupae and its non-deoiled pupae meal included diet (Sathishkumar et al., 2021). Among other insect
meals, a significantly higher whole body amino acid content was reported, when Hermetia illucens larvae meal was fed to rainbow trout
(Oncorhynchus mykiss) (Hossain et al., 2021). Table 8 shows the fatty acid composition of the treated striped catfish body muscle
(fillet). In the current study, fish muscle fatty acid composition revealed a change fed at increasing levels of non-defatted insect meal
protein to the catfish, replacing fishmeal. Among the dietary treatment groups, a decline in trend was observed from NDSWP25 on
wards in the content of fillet saturated and monounsaturated fatty acid, as fishmeal replacement level increased. The downtrend in
total SFA and total PUFA content in fillets was due to the lower saturated fatty acid and polyunsaturated fatty acid content in the insect
meal (A. mylitta). However, lower omega-3 fatty acid content in fillets beyond the 25% replacement level (p < 0.05) could become
problematic for consumers as they desire high content of polyunsaturated fatty acid. Although, a good amount of α-linolenic acid
(C18:3n-3) was investigated at 30.78 ± 1.15 mg 100 g-1 sample of A. mylitta dried pupae meal contributing 27.49% out of the total
fatty acid content. A relatively fair amount of n-3/n-6 ratio was also found with 1.83 ± 0.02 in non-deoiled tasar pupae meal, which
was slightly lower than B. mori meal reported with 4.97 (Tomotake et al., 2010), in comparison to black soldier fly pre-pupae (1.05),
and remaining other insect meals (0–0.50) (Nogales-Mérida et al., 2019). Modification in the diet of A. mylitta silkworm (feeds pri
marily on Terminalia arjuna leaves, a main host plant) may help to improve the long-chain fatty acid content of pupae. For instance,
Sealey et al. (2011) and St-Hilaire et al. (2007) suggested the substrate refinement for black soldier fly larvae meal (BSFLM) to achieve
its better fatty acid profile, which, would reflect on the treated fish.
The digestive enzyme activity analysis revealed that there was a significantly (p < 0.05) elevated amount of amylase enzyme
activity (4.84 Unit mg-1 protein) in NDSWP25 fed fishes compared to activity observed in the fish gut fed with other diet treatment
11
Table 9
The status of digestive enzymes activity parameter at the end of experimental feeding trial.
Parameter Initial facts Diet treatment groups p-value
Amylase 3.00 ± 0.66 0.22a ± 0.22 4.84b ± 1.55 0.20a ± 0.06 0.31a ± 0.04 0.15a ± 0.03 0.003
Protease 68.95 ± 33.03 8.86 ± 4.23 6.37 ± 1.61 42.09 ± 14.04 29.90 ± 18.34 29.73 ± 3.49 0.167
Lipase 0.18 ± 0.03 0.26ab ± 0.04 0.19a ± 0.01 0.37ab ± 0.06 0.42b ± 0.01 0.32ab ± 0.06 0.026
Value represented as mean ± standard error, n = 3; Mean values of amylase enzyme acitivity (micromole maltose released minute-1 mg-1 protein),
protease acitivity (unit minute-1 mg-1 protein), and lipase activity (unit hour-1 mg-1 protein) holding in the same column labeled with different
superscript vary remarkably at alpha value < 0.05.
Fig. 1. The graphical representations elicits the digestive enzyme activity (amylase, protease and lipase) of the treated fish (Pangasianodon hypo
phthalmus) fed with graded levels of dietary inclusions of pupae meal replacing the fishmeal protein, at the end of experiment. Perpendicular bars
indicates standard error of mean, n = 3, and the different superscript symbolizes the significant differences exists between the dietary groups at
alpha value < 0.05.
groups (0, 50, 75, and 100 NDSWP), as its activity showed a declining trend with increased inclusion level of pupae protein. The
present observation was found complementary with the results of a feeding trial conducted in carnivorous turbot (Scophthalmus
maximus L.) (Wang et al., 2016), when fishmeal protein was replaced by maggot meal insect protein in the diet. The gut protease
enzyme activity was raised in the NDSWP50 pupae fed treatment group (42.09 Unit mg-1 protein) in comparison with the control group
and subsequently declined albeit insignificant (p > 0.05) (Table 9). A similar observation of higher protease enzyme activity was noted
in turbot (Scophthalmus maximus L.) when fishmeal protein was replaced by maggot meal protein although there was no concern for
anti-nutrient factors (Wang et al., 2016). The Jian carp (Cyprinus carpio var. Jian) juvenile also exhibited a similar trend when the
fishmeal was replaced up to 50% by the silkworm pupae meal in the diet compared to the inclusion level of 80% (Ji et al., 2015). The
lipase enzyme activity of the gut was recorded to be raised towards the NDSWP75 diet treatment group (0.42 Unit mg-1 protein) in
contrast to 0 (control), 25%, and 50% pupae included diet treatment, and eventually, its activity comparatively declined (p < 0.05).
The present study shows a disparity with the gut lipase activity of turbot (Scophthalmus maximus L.), where, no influence between
treatment groups was noticed when the fish meal was substituted in the diet by maggot meal (Wang et al., 2016). A graphical pre
sentation of the digestive enzyme (amylase, protease, and lipase) activity is displayed in Fig. 1.
The histo-micrograph of the fore and midgut of striped catfish in the control group (NDSWP0) revealed the normal size of villi, the
normal pattern of the mucosal region with digestive and absorptive epithelial cells with well-defined brush border, normal
arrangement of goblet cells, distinct narrow lamina propria, and submucosal region, as presented in Fig. 2A and B. The treated fishes,
when fed beyond 50% pupae meal incorporated protein implicates the signs of inflammation (Fig. 2C, D, E, and F), characterized by the
occurrence of intensified immune cell (lymphocyte) infiltration within the lamina propria and submucosa observed in fore and midgut
of fishes fed with NDSWP75 and NDSWP100 pupae containing diet (Fig. 2 G, H, I, and J). In general, shortening of midgut villi (in size),
immune cell infiltration into the lamina propria of the midgut intensifies as the protein to protein replacement level increased to 100%
with pupae as a protein source (Fig. 2B, D, F, H, and J). In the overall observation, widening of lamina propria was observed in both
fore and midgut sections as protein replacement took place at a higher incorporation level (Fig. 2 A to J). Thus, a clear inflammatory
response featured a noticeable rise in the lamina propria cellularity and submucosal region occurred due to infiltration made by a
mixed leucocyte population mainly lymphocytes and eosinophilic granular cells (EGCs), as a result, the mucosa becomes hypertrophic
and hyperaemic which were observed when non-defatted silkworm pupae meal protein inclusion level in the diet was increased to
replace the fishmeal protein. In brief, an indication of enteritis was perceived under the observations of the histological section of the
fish gut when fed at the higher inclusion level of non-defatted non-mulberry silkworm pupae. The vital nutrients such as protein and
lipids in the diet, in particular, can affect the integration of intestinal mucosal villi. The high level of substitution of fishmeal protein
with the silkworm pupae meal in the diet impairs the microvilli of the gut as observed in Jian carp (Cyprinus carpio var. Jian) (Ji et al.,
2015). A remarkable effect on villi height of the anterior gut was recorded when mannan oligosaccharide was supplemented in the diet
of juvenile striped catfish (Akter et al., 2016), on the contrary, the use of black soldier fly (Hermetia illucens) meal along with its oil
incorporation in the diet averts the soybean meal instigated gut enteritis in the rainbow trout (Kumar et al., 2021).
12
13 (caption on next page)

Fig. 2. The histo-micrograph of fore (A) and midgut (B) in control (NDSWP0) diet fed group, arrow head indicating brush boarder (BB), lamina
propria (LP), submucosa (SM), mucosal region with epithelial cell (M). The cell-morphology of fore (C) and midgut (D) in NDSWP25 diet fed group,
arrow head denoting the Goblet cell (GC), submucosa (SM), lamina propria (LP), and bi-direction of arrow shows the distribution of inner circular
muscularis (CM) with the outer longitudinal muscularis (LM). The histomorphology of fore (E) and midgut (F) in NDSWP50 diet fed group, arrow
head depicts the Goblet cell arrangements (GC), and infiltration of immune cell (lymphocyte) starts entering through the submucosal (SM) region
towards the relatively widened lamina propria (LP). The cellular morphology of fore (G) and midgut (H) in NDSWP75 diet fed group, arrow shows
the eosinophilic granular cells (EGCs), and arrow heads indicate the moderately intensified immune cells (lymphocyte) through the submucosa (SM)
to the lamina propria (LP) region. The micro-histograph of fore (I) and midgut (J) in NDSWP100 diet fed group, arrow head indicate the Goblet cell
(GC), other arrow heads shown inside the connective tissue of lamina propria (LP) indicates the intensification of immune cell (lymphocyte)
infiltration entering through the submucosal (SM) region, and external layer of serosa (S). Scale bar: 50 µm.
Fig. 3. The linear regression between the weight gain (%) and replacement level (%) of fishmeal by NDSWP revealed a negative relationship at P
value = 0.0034.
The linear regression among weight gain and specific growth rate revealed a negative relationship with the replacement level
(p < 0.05), whereas, the feed conversion ratio exhibited a positive relationship with the replacement level (p < 0.05) (Figs. 3, 4 and 5).
In order to carry out the economic analysis of the present feeding trial, the cost of kg-1 experimental diets under each treatment
group was calculated (based on the prevailing retail price of all the ingredients/items utilized during diet formulation and preparation)
at USD 0.74 (NDSWP0), USD 0.62 (NDSWP25), USD 0.48 (NDSWP50), USD 0.36 (NDSWP75), and USD 0.23 (NDSWP100) respectively
(Table 10). In the present investigation, the cost-profit return against each treatment diet containing the designated protein to protein
replacement level was based on the satisfactory performance of growth attributes and dietary nutritional implication indicators ob
tained during the feeding trial. At the 50% pupae meal protein inclusion groups substituting the fishmeal protein displayed a
remarkable abate in the feed cost by USD 0.26 kg-1 (35% trimming in the feed cost) along with the benefit-cost ratio of 1.38. Thus, it
improves incredible net-return to the fishers compared to the fishmeal protein-based diet, such kind of economic analysis has been
carried out highlighting the feed cost for other agricultural wastes (Hassan et al., 2016).
Fig. 4. The linear regression between the specific growth rate (% day-1) and replacement level (%) of fishmeal by NDSWP revealed a negative
relationship at P value = 0.0042.
14
Fig. 5. The linear regression between the feed conversion ratio and replacement level (%) of fishmeal by NDSWP revealed a positive relationship at
P value = 0.0107.
Table 10
The analysis of profit and economic parameters of rearing the Pangasianodon hypophthalmus fingerlings for 90 days in outdoor floating net cages using
non-defatted silkworm pupae (A. myllita) as experimental diet at designated replacement level.
Indices p-
value
A. Capital cost for 10 numbers of cages (75 m3 water volume) Rate/ Unit Cost
Unit
Cost of cage net along with cover net complete set with stitching including 53.85 10 538.54
transportation (USD)
Total fixed cost (FC) (USD) 538.54
B. Variable cost
Parameter NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100
Initial stocking (33 no. fishes cage-1 unit-1) (kg) 36.5 36.5 36.5 36.5 36.5
Initial number cage-1 2475 2475 2475 2475 2475
Cost of fingerling including transportation (USD 0.07 unit-1) 173 173 173 173 173
Cost of diet (USD kg-1) 0.74 0.62 0.48 0.36 0.23
Labour cost (during stocking, growth monitoring and harvesting) (USD) 1.25 1.25 1.25 1.25 1.25
Feed quantity (kg) 85.46 85.46 85.46 85.46 85.46
Feed cost (USD) 63 53 41 31 20
Total variable cost (VC) (USD) 238 227 216 205 194
C. Cost and return indicators
Total fixed cost (FC) (USD) 53.85 53.85 53.85 53.85 53.85
Total variable cost (VC) (USD) 238 227 216 205 194
Total cost (TC=FC+VC) (USD) 292 281 269 259 248
Advanced fingerling produced (P) (kg cage-1 90 days-1 96.25 92.2 84.25 85.15 81.3
Advanced fingerling produced (P) (number cage-1 90 days-1) 2471 2470 2471 2465 2456
Market price of advanced fingerling (MP) (USD 0.15 no.-1) 0.15 0.15 0.15 0.15 0.15
Gross revenue (GR=P × MP) (USD cage-1 90 days-1) 371 371 371 370 368
Net return (NR=GR-TC) ( (USD cage-1 90 days-1) 79 89 101 111 120
Income above variable cost (IVC=GR-VC)(USD cage-1 90 days-1) 133 143 155 164 174
Benefit-cost ratio (BCR=GR/TC) 1.27a 1.32b 1.38c 1.43d 1.49e 0.000
Rate of farm income (%) (RFI=NR/GR×100) 21.31 24.07 27.31 29.92 32.67
Break-even production (BEP=TC/MP) (kg cage-1 90 days-1) 1944 1876 1796 1727 1653
Break-even price (BEP=TC/P) (USD kg-1) 3.03 3.05 3.20 3.04 3.05
Conversion rate of Indian Rupee (₹) to USD using ₹ 79.85 = USD 1. Mean value with same column labeled with different superscript differs
significantly at p < 0.05.
4. Conclusion
In nutshell, as the replacement level moves beyond 50% non-defatted pupae protein inclusion (NDSWP50) to substitute the
fishmeal protein, the growth of fishes under treatment groups was remarkably inferior in comparison to the control diet (NDSWP0).
Therefore, the present outdoor cage feeding trial has displayed that 50% substitution of the fishmeal protein in the striped catfish diet
15
with non-defatted non-mulberry silkworm pupae protein is attainable without adverse effect on the growth-nutritional accountability,
biological indicators of the fishes, and promises a remarkable reduction in the feed cost by USD 0.26 kg-1 (35% trimming in the feed
cost) along with the best benefit-cost ratio of 1.38. Additionally, this first-hand study using non-mulberry silkworm pupae in the fish
diet will encourage researchers to study the feeding trial using the same for other available valuable diversified candidate fish species.
It enlightens the usefulness of converting the sericulture bio-waste into valuable fish protein with the concept of waste to wealth for
cleaner production.
CRediT authorship contribution statement
Mr. Rahul Das has designed the methodology, diet formulation, diet preparation, chemical analysis (ingredients, diet, and treated
fish), conducting feeding trials at the cage, periodic sampling of fishes, digestive enzyme analysis, treated fish gut micro-morphology
with its analysis, economic-statistical analysis along with its computation, results interpretation, and wrote the original manuscript,
Dr. Basanta Kumar Das has conceptualized the experiment, contributed to the manuscript review, corrections, and final manuscript
preparation, procurement of raw materials, chemicals required, and overall supervision of the experiment, Dr. Md. Abul Hassan has
contributed to the diet formulation, and procurement of raw materials, and helped to provide the necessary instrument facility for
chemical analysis, and overall monitoring of the experiment, Dr. Gopal Krishna, Dr. Narinder Kumar Chadha, Dr. Kiran Dube Rawat,
and Dr. Karmabeer Jena have contributed to the manuscript review and editing.
Declaration of Competing Interest
All the concerned authors mentioned here declare no conflict of interest in this manuscript.
Acknowledgment
The author, Mr. Rahul Das expressed his sincere gratitude to the Director, ICAR-Central Inland Fisheries Research Institute, Bar
rackpore for constant support in providing feed laboratory research facility, outdoor cage facility, procurement of raw materials/
chemicals for analysis, and kind guidance during persuasion of the doctoral research program.
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Animal Feed Science and Technology 303 (2023) 115691

Contents lists available at ScienceDirect

Animal Feed Science and Technology

Valorization of the insect waste as a source of dietary protein in

Values in mean ± standard error, n = 3.

Fishmeal 32.29 24.21 16.14 8.07 0

2. Material and methods

2.1. The experimental facility site

2.2. Feed ingredient

2.3. Diet formulation and preparation

A. mylitta C. dussumieri NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100 C. gariepinus P. hypophthalmus

Animal Feed Science and Technology 303 (2023) 115691

ΣEAAa: Sum of essential amino acids

2.6. Experimental fish stocking, ration/culture management

2.7. Nutritional composition analysis of ingredients, diets, and fish muscle

2.8. Growth accomplishment and nutritional utilization indicators

S (survival rate, %) = (final number of fish / initial number of fish) × 100

2.9. Determination of biological implications

2.10. Digestive enzyme activity determination

2.11. Gut histo-morphological observation

2.12. Economical investigation

2.13. Statistical computation

NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100

3. Results and discussion

NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100

Initial facts NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100 p-value

ΣEAAa: Sum of essential amino acids

NDSWP0 NDSWP25 NDSWP50 NDSWP75 NDSWP100

13 (caption on next page)

CRediT authorship contribution statement

Declaration of Competing Interest

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