Aquaculture 561 (2022) 738618

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Replacement of fish meal by black soldier fly larvae meal in diet for goldfish
Carassius auratus: Growth performance, hematology, histology, total
carotenoids, and coloration
Anurak Khieokhajonkhet a, *, Phusanisa Uanlam a, Khanitta Ruttarattanamongkol a,
Niran Aeksiri a, Pattaraporn Tatsapong a, Gen Kaneko b
a
Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand
b
College of Natural and Applied Science, University of Houston-Victoria, 3007 N. Ben Wilson, Victoria, TX 77901, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Black soldier fly (Hermetia illucens) larvae meal (BSFLM) is a promising animal feed ingredient because of its high
Hermetia illucens protein content and sustainable nature in the aquaculture sector. For the potential application of this ingredient
Insect meal in goldfish culture, five isonitrogenous (42% of crude protein) and isolipidic (9% of crude fat) diets were pre­
Liver integrity
pared to replace 0, 43, 84, 145, and 210 g kg− 1 fish meal (FM) with BSFLM (termed BSFLM0 – BSFLM210).
Pigmentation
Triplicate groups of 20 goldfish (approximately 9.35 ± 0.02 g fish− 1) per tank were randomly assigned one of the
Ornamental fish
experimental diets and fed three times daily to satiation for 10 weeks. Dietary inclusion of BSFLM linearly
increased final body weight, weight gain, and specific growth rate (P < 0.001), and linearly and quadratically
improved feed conversion ratio, protein efficiency ratio, and protein productive value (P < 0.05). The highest
weight gain was observed in the BSFLM145 (15.61 g fish− 1) group without a significant difference (P > 0.05)
with BSFLM210 (14.76 g fish–1). Organosomatic indexes were similar among dietary treatments (P > 0.05).
Whole-body moisture and ash contents were not significantly affected (P > 0.05), but crude protein and fat
content were affected by BSFLM inclusion (P < 0.05). Regarding muscle fatty acid composition, C12:0 and C14:0
increased linearly and quadratically with BSFLM levels, resulting in an increase in total saturated fatty acids with
linear and quadratic effects (P < 0.05). In contrast, C18:3n-3, C20:5n-3, C22:6n-3, total polyunsaturated fatty
acids, total n-3, and n-3/n-6 ratio decreased with increasing levels of BSFLM. The color index (a* value) was
linearly and quadratically decreased in the abdominal region by BSFLM inclusion, similar to the decrease in L*
and a* values in the head region. Total carotenoid contents also decreased in fin, skin, and serum by BSFLM
inclusions (P < 0.05). BSFLM inclusion decreased aspartate aminotransferase, alanine aminotransferase, total
cholesterol, and low-density lipoprotein cholesterol with linear and quadratic effects (P < 0.05). A mild degree of
vacuolization with swollen hepatocellular was observed in the BSFLM84 group, being more abundant in
BSFLM145 and BSFLM210 groups. Overall, dietary BSFLM inclusion improved growth performance, feed utili­
zation, and blood biochemistries in goldfish, but it exerted a negative effect on carotenoid content, skin color­
ation and liver histological integrity.

1. Introduction balanced amino acids, as well as it has high digestibility, palatability,

Fish meal (FM) is a common source of animal protein and fat in
aquaculture. This is because FM provides high amounts of vital nutrients
that support fish development and health such as high-quality protein,
long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA), and
and a low amount of anti-nutritional factors (Gatlin et al., 2007; NRC,
2011; Tocher, 2015). In 2030, however, the price of fishmeal is pre­
dicted to rise by 30% compared to 2018 in nominal terms due to the
increasing worldwide demand. While aquaculture production will also
increase by 42% (FAO, 2020), the high cost of FM will cause a significant

* Corresponding author at: Department of Agricultural Science, Faculty of Agriculture, Natural Resources, and Environment, Naresuan University, 99 M. 1, T.
Thapo, A. Muang, Phitsanulok 65000, Thailand.
E-mail addresses: anurakk@nu.ac (A. Khieokhajonkhet), phusanisau61@nu.ac.th (P. Uanlam), khanittar@nu.ac.th (K. Ruttarattanamongkol), nirana@nu.ac.th
(N. Aeksiri), pattarapornt@nu.ac.th (P. Tatsapong), kanekoG@uhv.edu (G. Kaneko).

https://doi.org/10.1016/j.aquaculture.2022.738618
Received 27 May 2022; Received in revised form 6 July 2022; Accepted 11 July 2022
Available online 14 July 2022
0044-8486/© 2022 Published by Elsevier B.V.
A. Khieokhajonkhet et al. Aquaculture 561 (2022) 738618

economic burden to the aquaculture sector. This also poses a danger to at 60 ◦ C for 48 h and ground into a fine powder. The nutritional
wild fish stock reserves. As the scarcity of FM has become more widely composition (dry matter basis) of the BSFLM used in this study was dry
concerned, researchers and feed makers have made significant efforts to matter content 39.10%, crude protein 51.46%, crude fat 13.31%, ash
find other protein sources in order to reduce reliance on FM while still 12.91%, and 9.26% chitin. Five isonitrogenous (42% crude protein) and
providing appropriate nutrition for fish growth (FAO, 2012, 2020). isolipidic (9% crude fat) diets with different levels of BSFLM were pre­
In aquaculture and animal husbandry, insects are becoming a viable pared using 0 g kg–1, 43 g kg–1, 84 g kg–1, 145 g kg–1, and 210 g kg–1
alternative to terrestrial plant and animal protein sources. Entomoph­ BSFLM to replace FM. The basal feed ingredients were purchased from a
agy, the consumption of edible insects, indeed has the potential to be commercial feed supplier (Phraepan Co, Ltd., Phitsanulok, Thailand),
one of the most environmentally friendly, cost-effective, and long-term ground, and passed through mesh sieve. According to each feed formula
solutions for animal and fish nutrition (DeFoliart, 1989; Koutsos et al., (Table 1), all dry feed ingredients were thoroughly homogenized with
2019). Black soldier fly (BSF, Hermetia illucens) is an especially fish oil and lecithin using a kitchen blender (CKI family Co. Ltd., Non­
intriguing protein source since it provides a cost-effective way to thaburi, Thailand) and then added with distilled water at 350 g kg–1 to
transform biological waste into high-quality protein, and as a result, form the feeding dough. The mixture was pelleted with approximately
industrial-scale production has increased dramatically over the last 2.5 mm diameter using a meat mincer (CKI family Co, Ltd., Nonthaburi,
decade (Silva and Hesselberg, 2020). The BSF larval meal (BSFLM) is Thailand), air-dried at 70 ◦ C for 48 h, kept in airtight polyethylene bags,
typically used as a feed ingredient in aquaculture due to its favorable and stored at − 20 ◦ C until used. The proximate composition of experi­
nutritional composition. On a dry matter basis, BSFLM roughly contains mental diets is depicted in Table 1.
42–60% of crude protein and 10–35% of crude lipid that depends on the
bedding substrate (Diener et al., 2009; Newton et al., 1977; NRC, 2011).
The essential amino acid profile of FM is more similar to that of BSFLM Table 1
Formulation and proximate composition of the experimental diets (as fed basis).
than the soybean meal, a commonly used plant protein source (Diener
et al., 2009; NRC, 2011). Moreover, BSFLM is also abundant in minerals, Ingredients Control BSFLM43 BSFLM84 BSFLM145 BSFLM210
provitamin A carotenoids, and bioactive compounds having nutraceu­ − 1
Feed formula (g kg )
tical qualities, such as chitin, lauric acid, and antimicrobial peptides, all Fish meala 350 315 280 230 177
of which can positively manipulate the fish’s health (Borel et al., 2021). Black soldier 0 43 84 145 210
fly meal
Many studies have been conducted on the use of BSFLM as aquafeeds Soybean meal b
310 310 310 310 310
using a variety of fish species, including Atlantic salmon, Salmo salar Squid mealc 80 80 80 80 80
(Bruni et al., 2020; Lock et al., 2016); rainbow trout, Oncorhynchus Broken rice
mykiss (Dumas et al., 2018; Renna et al., 2017); Nile tilapia (Devic et al., meal 60 60 60 60 60
Wheat flour 101 97 96 90 83
2018; Tippayadara et al., 2021); European seabass, Dicentrarchus labrax d
Fish oil 64 60 55 50 45
(Abdel-Tawwab et al., 2020); African catfish, Clarias gariepinus (Adeoye Vitamin pre-
et al., 2020); yellow catfish, Pelteobagrus fulvidraco (Xiao et al., 2018); mix e
10 10 10 10 10
Siberian sturgeon, Acipenser baerii Brandt (Caimi et al., 2020); large­ Mineral pre-
mouth bass, Micropterus salmoides (Fischer et al., 2022); Jian carp, mixf 10 10 10 10 10
Methionine 7 7 7 7 7
Cyprinus carpio var. Jian (Li et al., 2017); grass carp, Ctenopharyngodon
Lysine 5 5 5 5 5
idellus (Lu et al., 2020); Dusky Kob, Argyrosomus japonicus (Madibana Lecithin 3 3 3 3 3
et al., 2020), and Japanese seabass, Lateolabrax japonicus (Wang et al., Total 1000 1000 1000 1000 1000
2019). Due to the species-specific differences in the required quality of
the larvae feed, the optimal dietary replacement levels of FM with Composition
BSFLM vary significantly between these studies, ranging from 25 − Crude protein
100%. However, the above list of species highlighted a problem in the (%) 42.08 42.13 41.96 42.05 42.21
Crude fat (%) 9.53 9.64 9.70 9.99 9.58
current BSFLM study - the target species has been largely limited to
Ash (%) 9.28 9.26 9.98 10.31 10.53
edible fish, and the information on ornamental fish is quite scarce. Total
Farming of ornamental fish is one of the fastest-growing sectors in carbohydrate
aquaculture (Pandey and Mandal, 2017; Saengsitthisak et al., 2020), and (%) 60.89 61.03 61.64 62.35 62.32
food security is also an important issue for ornamental fish. In contrast to Dry matter (%) 91.02 91.01 90.93 91.09 90.67
Gross energy
edible fish, coloration is a major determinant of their market value, (MJ kg− 1)g 24.16 24.24 24.33 24.59 24.46
which is largely regulated by pigments known as carotenoids. To
maintain their pigmentation in the skin and muscle, ornamental fish are Abbreviation: BSFLM, black soldier fly larvae meal.
a
Sardine fish meal, Phraepan Animal Feed Distributor, Phitsanulok, Thailand
primarily fed compound feeds enriched with carotenoids (Chatzifotis
(crude protein, 62.69%; crude fat, 6.12%).
et al., 2005). To the best of our knowledge, the effects of BSFLM on b
Soy protein concentrate, Thai Vegetable oil PCL Co, Ltd., Nakorn Pathom,
carotenoids and pigmentation in ornamental fish species have not been Thailand (crude protein, 42.59%; crude fat, 0.69%).
investigated. The aim of the present study was to investigate the effect of c
Squid meal, Pro-Squid Co, Ltd., Bangkok, Thailand (crude protein, 45.75%;
dietary replacement of fish meal by BSFLM on growth performance, crude fat, 5.72%).
hematology, histology, total carotenoids, and coloration in an orna­ d
Fish oil, The Origin Nature, Norway.
mental fish. Goldfish, Carassius auratus, was used in this study since it is e
Vitamin mixture (mg or IU kg− 1 diet): Retinol, 20,000 IU; Cholecalciferol-
a common freshwater ornamental fish with a high market value. D3, 1000 IU; tocopherol, 500 mg; Phytonadione, 20 mg; Thiamin, 25 mg;
Riboflavin, 40 mg; Pyridoxine, 10 mg; Cyanocobalamin, 100 mg; Inositol, 10
2. Materials and methods mg; Pantothenate, 30 mg; Niacin, 30 mg; ascorbic acid, 500 mg; Folate, 3 mg;
Vitamin B7, 1 mg.
f
Mineral mixture (mg kg− 1 diet); Calcium lactate, 1000; Calcium phosphate,
2.1. Experimental diets preparation
800; Cobalt carbonate, 2; Copper sulphate, 120; Ferrous sulphate, 12; Manga­
nese oxide, 12; Magnesium chloride, 22; Potassium chloride, 2; Potassium
BSFLM was kindly provided from The Thai Ento Food Co, LTD., iodine, 8; Sodium selenite, 10; Zinc oxide, 16.
Phitsanulok, Thailand. The BSFLM were made from larvae fed with a g
Gross energy value was calculated by following the combustion values (NRC,
combination of insect commercial feed (21% crude protein, 4% fat, 5% 2011) including 39.5 kJ g− 1 fat, 23.6 kJ g− 1 protein, and 17.2 kJ g− 1
ash, and 10% moisture) and food waste. The BSFLM was then air-dried carbohydrate.

2
A. Khieokhajonkhet et al.
2.2. Experimental fish and ethics statement
Goldfish were purchased from a commercial nursery farm in Cha­
tuchak market, Bangkok, Thailand, and transported in plastic bags to
The Aquatic Animal Feed Laboratory, Faculty of Agriculture Natural
Resources and Environment, Naresuan University, Phitsanulok,
Thailand. Fish were acclimated in 500 L plastic indoor tanks with a
recirculation system for a month and fed with a commercial feed (400 g
kg–1 crude protein, 70 g kg–1 crude fat, and 40 g kg–1 crude fiber) until
satiation three times daily. A total of 300 goldfish were fasted for 24 h,
anesthetized with 50 mg mL− 1 clove oil solution (ethanol: clove oil, 9:
1), and measured for the initial body weight (IBW, 9.35 ± 0.02 g fish− 1).
Fish were then randomly distributed in 15 indoor glass tanks with a
water capacity of 150 L (3 tanks per diet) at 20 fish per tank for the
initial stocking. To account for any tank position impacts, diets were
distributed to tanks using a completely randomized approach. Fish were
manually fed with one of the five experimental diets until apparent
satiation for 10 weeks (three times a day at 8:30, 12:00, and 16:30). The
feeding trail was conducted under natural photoperiod (approximately
12 h dark: 12 h light). Tanks were cleaned daily and replenished for two-
thirds of dechlorinated water. The total feed used was recorded, and fish
mortality was daily monitored and recorded throughout the feeding
trial. Water temperature ranged between 26.5 − 28.2 ◦ C, and continuous
aeration was maintained using submerged air stones to obtain the oxy­
gen level of 4.8 − 6.3 mg L− 1 throughout the feeding trial.
The present study was performed according to the guidelines for
experimental fish studies approved by the Committee of Animal Ethics
of the Faculty of Agriculture Natural Resources and Environments,
Naresuan University (Approved protocol number 0001/2565). In addi­
tion, all experiments were carried out in accordance with the guidelines
of the Institute of Animals for Scientific Purpose Development (IAD), the
National Research Council of Thailand’s Ethic of Animal Experimenta­
tion (License number, U1/00704/2558).
2.3. Sample collection
At the end of the feeding trial, fish were starved for 24 h and anes­
thetized using clove oil solution. Fish of each tank were bulk weighed
and counted to determine growth parameters and survival rate,
respectively. Five fish from each tank were randomly selected for the
colorimetric analysis. Three fish per tank were individually measured
for the total length and weight to calculate the condition factor (K
value). After measurement of the physical and color parameters, fish
were euthanized using overdose clove oil solution and dissected to
obtain the liver and visceral organs to calculate the hepatosomatic index
(HSI) and viscerosomatic index (VSI), respectively. For whole-body
composition, nine individuals from each treatment were randomly
euthanized using an overdose of clove oil solution on the termination
day and stored at − 20 ◦ C until used for proximate analysis. From the
caudal vein of another five fish per tank (pooled 5 fish per tank / n = 3
per treatment), blood samples were withdrawn using non-heparinized
syringes and subjected to centrifugation at 1500 ×g for 15 min at
4 ◦ C. The serum supernatant was used to determine blood biochemistry
parameters. From another five fish per tank (pooled 5 fish per tank),
blood samples were collected with heparinized syringes to determine
red blood cell count (RBC), white blood cell count (WBC), hematocrit,
and hemoglobin content. The remaining fish (three fish per tank) were
dissected to determine the total carotenoid content (fin, muscle, and
skin) and to conduct histological experiments (liver).
2.4. Analytical methods
2.4.1. Calculation of growth performance and feed utilization, survival
rate, and organosomatic indexes
Growth performance and feed utilization of goldfish fed varying level
of BSFLM were determined by following equations: weight gain (WG, g
3
Aquaculture 561 (2022) 738618
fish− 1) = (final body weight – initial body weight); specific growth rate
(SGR, % day− 1) = [(Ln final body weight − Ln initial body weight) /
days] × 100; feed conversion ratio (FCR) = individual feed intake (g)/
individual weight gain (g); protein efficiency ratio (PER) = wet weight
gain (g) / protein intake (g); protein productive value (PPV, %) = pro­
tein gain (g) / protein intake (g) × 100; survival rate (SR, %) = 100 ×
[(final number of fish) / (initial number of fish)]; condition factor (K, g /
cm3) = 100 × [(final body weight, g) / (final body length, cm)3]; hep­
atosomatic index (HSI, %) = 100 × [(liver weight, g) / (whole body
weight, g)]; and viscerosomatic index (VSI, %) = 100 × [(viscera
weight, g) / (whole body weight, g)].
2.4.2. Chemical scrutiny
Ingredients, feed samples, initial fish samples (15 fish, data not
shown), and fish after the feeding trial (3 fish per tank) were analyzed by
following procedures (AOAC, 1990): moisture (Method 950.46), crude
protein (Method 984.13, N × 6.52), crude fat (Method 920.85), and ash
(Method 920.153). Crude fiber content was determined by following
Yasumaru and Lemos (2014). Gross energy was calculated (NRC, 2011)
using the equation: Gross energy (MJ kg− 1) = [(% crude protein × 23.6)
+ (% crude fat × 39.5) + (% NFE × 17.2)]. Chitin content of BSFLM was
determined as described by Kaya et al. (2015). Briefly, 100 g of dried
BSFLM powder was demineralized using 4 M HCl at 75 ◦ C for 2 h. After
washing with distilled water, the sample residue was subjected to
deproteinization with 4 M NaOH at 150 ◦ C for 20 h. After washed with
distilled water, samples were air-dried at 60 ◦ C for 24 h, and dried
weight of chitin was calculated. To determine fatty acid profiles, total fat
was extracted from experimental diets and skeletal muscle using
chloroform-methanol (2:1, v/v) (Folch et al., 1957). Fatty acids methyl
esters (FAMEs) were trans-esterified with anhydrous methanol con­
taining 2% H2SO4 at 50 ◦ C for 16 h under nitrogen gas. FAMEs were
separated by gas chromatography with flame ionization detection (GC-
FID) on a GC-2010 gas chromatograph (Shimadzu, Tokyo, Japan). The
fatty acid composition of the experimental diet is shown in Table 2.
2.4.3. Colorimetric measurement
After bulk weighed, three different parts of fish (5 fish per tank) were
collected: (1) head; (2) trunk region close to the lateral line of the dorsal
section; and (3) tail. These parts were analyzed for the coloration on the
left side of the goldfish using a Hunter Lab Scan MiniScan EZ 4500 L
(Hunter Associates, USA), with 63.5 mm diffuse /8◦ model sphere
diameter. The port diameter and view diameters were of 31.8 mm for
illumination and 25.4 mm for measuring, respectively. The spectral
range was 400–700 nm. Tissue samples were calibrated with both dark
and white plate references prior to analysis. The obtained L*, a*, and b*
values were used for presenting lightness, redness or greenness chro­
maticity, and blueness or yellowness chromaticity, respectively (Hunter
and Harold, 1987), as described by the international commission on
Illumination (Robertson, 1977).
2.4.4. Total carotenoid analysis
Fin, muscle, and skin (three fish per tank) were used to determine the
total carotenoid content by following the protocol described by Torris­
sen and Naevdal (1988) with minor modifications. After the termination
of the experimental fish, tissue samples of approximately 1 g were
immediately minced with 5 ml acetone and 1 g of anhydrous Na2SO4.
The extraction was repeated with the same volume until exhausted. The
solution mixtures were centrifuged at 4 ◦ C with 3500 ×g for 5 min. The
supernatant was collected and subjected to determine a spectropho­
tometer at 450 nm (UV-1800, Shimadzu, Kyoto, Japan). Blood carot­
enoid content was analyzed as described by Barbosa et al. (1999).
Briefly, 200 μL of blood serum was mixed with a hexane - ethanol (1: 4 v/
v) solution. The mixture solutions were centrifuged at 4500 rpm for 10
min. The supernatant was subjected to spectrophotometer measurement
at 450 nm against hexane. Total carotenoid content was calculated and
expressed as μg carotenoid g− 1 of sample by following equation:
A. Khieokhajonkhet et al. Aquaculture 561 (2022) 738618

Table 2 and Tukey’s post hoc test. In addition, orthogonal polynomial contrasts
Fatty acid profile of experimental diets (% of total fatty acids). were calculated to detect linear and quadratic effects of different levels
Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 of BSFLM. All statistical analysis was carried out using the statistical
program SPSS for Windows software (Version 17.0, Chicago, IL, USA).
SFA
C10:0 0.04 0.06 0.01 0.19 0.25
C12:0 0.25 1.78 3.69 5.56 7.67 3. Results
C13:0 0.01 0.02 0.02 0.02 0.02
C14:0 2.37 2.81 3.36 3.74 4.25 3.1. Growth performance, feed utilization, and organosomatic indexes
C15:0 0.26 0.29 0.27 0.27 0.27
C16:0 14.02 14.40 14.57 14.30 14.46
C17:0 0.46 0.51 0.50 0.52 0.47 During the feeding trial, fish promptly accepted all experimental
C18:0 6.04 6.25 6.31 6.15 6.06 diets. In addition, the SR was not linearly or quadratically affected by
C20:0 0.49 0.48 0.43 0.38 0.35 the BSFLM replacement in diet (P > 0.05, Table 3). The orthogonal
C22:0 0.17 0.17 0.15 0.16 0.14
polynomial contrasts showed that dietary BSFLM inclusion increased
growth parameters including FBW, WG, and SGR with positive linear
MUFA effects (P < 0.001). The highest FBW (24.96 g fish− 1), WG (15.61 g
C14:1 0.15 0.17 0.16 0.18 0.20
fish− 1), and SGR (1.27% day− 1) were observed in fish fed the BSFLM145
C16:1 3.12 3.35 3.13 3.18 2.81
C18:1n-9 32.91 31.20 29.87 28.82 27.32 diet with no significant difference in fish fed with BSFLM210 (P > 0.05).
C20:1n-9 2.84 2.45 2.42 2.17 2.27 In addition, increasing levels of BSFLM inclusion in diet linearly and
C22:1n-9 1.68 1.57 1.33 1.07 1.03 quadratically decreased FCR (P < 0.001, P < 0.005), but linearly and
C24:1 0.21 0.22 0.20 0.19 0.14
quadratically increased PER and PPV (P < 0.001). In regard to orga­
nosomatic indices, there were no linear or quadratic effects in K, HSI,
PUFA and VSI values (P > 0.05, Table 3). Second-order polynomial regression
C18:2n-6 17.30 17.28 17.42 17.62 18.20
analysis predicted that the highest WG and PER are achieved at inclu­
C20:2n-6 0.87 0.82 0.76 0.68 0.61
C18:3n-3 4.81 4.61 4.44 4.32 4.10 sion levels of 211.5 g kg− 1 (y = − 0.0022x2 + 0.9108x + 69.565, R2 =
C20:3n-6 0.24 0.21 0.20 0.18 0.16 0.9501) and 151.5 g kg− 1 (y = – 0.00004x2 + 0.0121x + 1.1867, R2 =
C20:3n-3 0.34 0.30 0.27 0.26 0.22 0.9845), respectively (Fig. 1A, C). The lowest FCR can be achieved at
C20:4n-6 0.32 0.30 0.27 0.25 0.21 149.0 g kg− 1 (y = 0.00004x2 − 0.012x+ 1.9696, Fig. 1B).
C22:5 0.85 0.78 0.70 0.65 0.59
C20:5n-3 2.20 2.08 1.88 1.76 1.67
C22:6n-3 2.81 2.61 2.41 2.31 2.10 3.2. Chemical composition of whole-body fish
Sum of SFA 24.11 26.77 29.31 31.29 33.94
Sum of MUFA 40.92 38.97 37.12 35.64 33.78 Dietary BSFLM inclusion significantly affected crude protein and
Sum of PUFA 29.76 28.99 28.35 28.03 27.86
crude fat contents (P < 0.05), but not moisture and ash contents (P >
Sum n-3 10.16 9.60 9.00 8.65 8.09
Sum of n-6 18.73 18.61 18.65 18.73 19.18 0.05) (Table 4). It is noted that the moisture content showed a negative
Sum of n-9 37.43 35.22 33.62 32.06 30.62 linear response with increasing BSFLM inclusion levels close to statis­
n-3/n-6 FA ratio 0.54 0.51 0.48 0.46 0.42 tical significance (P = 0.051) with the highest value was observed in the
Abbreviations: BSFLM, black soldier fly larvae meal; SFA, saturated fatty acid; fish fed the control diet (72.86%). Crude protein content in fish fed the
MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; FA, fatty BSFLM84 diet (11.00%) was higher than those of the other dietary
acid. groups with a significant linear effect (P < 0.026, Table 4). A significant
linear and quadratic effects were observed for crude fat content, which
carotenoid content (μg g− 1) = 10,000 × volume of the extraction solu­ was the highest in the BSFLM145 group (13.63%) and the lowest in the
tion × absorbance / weight of sample × E1%, 1cm (2500). control group (12.45%, P = 0.001, P = 0.013).

2.4.5. Hematological and biochemical indices
RBC and WBC were determined using a Neubauer hemocytometer
(Hesser, 1960). Total hemoglobin (Hb) was determined using Drabkin’s
colorimetric kit and the absorbance at 540 nm. The Hb was calculated
using a standardized method. Hematocrit (Htc) was performed accord­
ing to Reitman and Frankel (1957) method. The second part of blood
sample was determined using the colorimetric method according to Coz-
Rakovac et al. (2008). Blood biochemical analyses were carried out with
an automatic biochemical analyzer (Hitachi 7180, Tokyo, Japan).
2.4.6. Histological studies
The liver was dissected into approximately 0.5 cm3 and immediately
fixed in 4% neutral formalin fixative containing 0.9% NaCl and 1.2%
Na2HPO4 at 4 ◦ C overnight. Tissues were processed by following our
previous method described in Khieokhajonkhet et al. (2021). Tissue
sections were stained with hematoxylin and eosin (H&E staining) and
photographed using a light microscope (Olympus, Tokyo, Japan).
2.5. Statistical analysis
All values are presented as means ± standard error of the mean
(SEM), and the differences with P values <5% were considered signifi­
cant. Data were analyzed using one-way analysis of variance (ANOVA)
3.3. Fatty acid composition in the muscle of goldfish
The fatty acid compositions in the muscle of goldfish were clearly
affected by dietary BSFLM replacement (Table 5). The content of C12:0
and C14:0 was significantly increased (P < 0.05) with the increment of
BSFLM inclusion with linear and quadratic effects together with the sum
of saturated fatty acid (SFA) (P < 0.001, P = 0.008). Total poly­
unsaturated fatty acids (PUFA), such as C18:3n-3, C20:5n-3, and
C22:6n-3, as well as total n-3 and n-3/n-6 fatty acid ratio, significantly
decreased by the dietary treatment (P < 0.05, Table 5). The total n-6
fatty acids content was not linearly or quadratically affected (P > 0.05).
3.4. Color parameters
The color analysis demonstrated that dietary inclusion of BSFLM
affected the pigmentation of the head and abdomen of goldfish
(Table 6). The head color showed a significant linear and quadratic
decrease in L* and a* with the increase in dietary BSFLM replacement (P
< 0.05). The highest L* (48.37) and a* (21.35) values in the head of the
goldfish were observed in the control group. Similarly, abdominal L*,
a*, and b* values were linearly decreased by dietary BSFLM inclusion,
while a* value decreased with significant linear and quadratic effects (P
< 0.001, P = 0.030). However, the tail color of the goldfish was not
significantly affected by the BSFLM replacement with no linear or

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A. Khieokhajonkhet et al. Aquaculture 561 (2022) 738618

Table 3
Growth performance, nutrient utilization, survival rate, and organosomatic indexes of goldfish fed graded levels of black soldier fly larvae meal for 10 weeks.
Parameters Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic

Growth parameters
IBW (g fish− 1) 9.36 ± 0.03 9.36 ± 0.02 9.35 ± 0.02 9.35 ± 0.03 9.36 ± 0.02 0.001 0.818 0.408
FBW (g fish− 1) 16.43 ± 1.09d 18.41 ± 1.24cd 21.01 ± 1.75bc 24.96 ± 1.22a 24.12 ± 1.29ab 1.789 < 0.001 0.168
WG (g fish− 1) 7.06 ± 1.12d 9.05 ± 1.25cd 11.66 ± 1.74bc 15.61 ± 1.23a 14.76 ± 1.29ab 1.807 < 0.001 0.166
SGR (% day− 1) 0.80 ± 0.10c 0.88 ± 0.09bc 1.05 ± 0.11ab 1.27 ± 0.06a 1.23 ± 0.07a 0.008 < 0.001 0.359
FCR 1.98 ± 0.31b 1.54 ± 0.18ab 1.19 ± 0.12b 1.17 ± 0.08b 1.27 ± 0.10b 0.033 < 0.001 0.005
PER 1.22 ± 0.14c 1.56 ± 0.15b 1.93 ± 0.17a 2.09 ± 0.06a 1.87 ± 0.10a 2.225 < 0.001 < 0.001
PPV (%) 20.12 ± 1.01c 24.34 ± 2.05b 29.27 ± 1.70a 30.39 ± 2.74a 26.49 ± 1.15ab 3.389 < 0.001 < 0.001
SR (%) 100.00 ± 0.00 97.78 ± 3.85 95.55 ± 3.85 100.00 ± 0.00 100.00 ± 0.00 5.932 0.628 0.061

Organosomatic indexes
K 12.47 ± 2.31 13.10 ± 1.76 14.08 ± 1.76 14.84 ± 1.88 13.95 ± 1.96 3.764 0.483 0.210
HSI (%) 2.84 ± 1.49 2.71 ± 0.65 2.11 ± 0.70 2.21 ± 0.71 2.42 ± 0.79 0.861 0.207 0.275
VSI (%) 17.37 ± 2.87 16.53 ± 1.64 16.46 ± 1.52 15.87 ± 1.58 16.66 ± 2.61 4.535 0.390 0.737

Growth indices and feed utilization are mean values of three replicate tanks per treatment (twenty fish of each tank). Organosomatic indexes are mean values from
three replicate treatments (three fish per replicate tank) Abbreviations: BSFLM, black soldier fly larvae meal; IBW, initial body weight; FBW, final body weight; WG,
weight gain; SGR, specific growth rate; FCR, feed conversion ratio; PER, protein efficiency ratio; SR, survival rate; K, condition factor; HSI, hepatosomatic index; VSI,
viscerosomatic index.

Fig. 1. Second-order polynomial relationship of weight gain (A), feed conversion ratio (B), and protein efficiency ratio (C) to dietary fish meal replacement by black
soldier fly meal levels.

Table 4
Whole-body composition of goldfish fed different levels of black soldier fly larvae meal for 10 weeks (dry matter basis).
Composition Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic

Moisture (%) 72.86 ± 0.24 72.57 ± 1.16 71.73 ± 2.77 71.50 ± 1.90 71.30 ± 0.56 1.363 0.051 0.928
Crude protein (%) 10.80 ± 0.21ab 10.64 ± 0.18b 11.00 ± 0.13a 10.78 ± 0.09ab 10.71 ± 0.12ab 0.014 0.026 0.182
Crude fat (%) 12.45 ± 0.09c 12.58 ± 0.15bc 13.18 ± 0.03ab 13.63 ± 0.17a 13.10 ± 0.23ab 0.024 0.001 0.013
Ash (%) 2.41 ± 0.01ab 2.49 ± 0.04ab 2.40 ± 0.07c 2.34 ± 0.06bc 2.56 ± 0.17a 0.008 0.343 0.153

Data are represented as mean values from 3 replicate tanks per treatment (Three fish per replicate tank were pooled). Abbreviation: BSFLM, black soldier fly larvae
meal.

quadratic effects (P > 0.05, Table 6), although all color parameter levels
were slightly decreased compared to the control group.
3.5. Total carotenoids content
Total carotenoid contents in fin, skin, and serum were significantly
influenced by dietary BSFLM replacement levels (P < 0.05), but this was
not the case for muscle total carotenoid content (P > 0.05, Table 7).
Dietary inclusion of BSFLM linearly decreased total carotenoid content
in fin with the highest content observed in the control group (105.62 μg
g− 1, BSFLM0). Similarly, total carotenoid contents of skin and serum
were linearly decreased by dietary BSFLM inclusions (P < 0.001; P =
0.002, Table 7).
3.6. Blood hematology and biochemistry
Blood hematological parameters including RBC, WBC, Htc, and Hb
were not linearly or quadratically influenced by dietary BSFLM inclusion
levels (P > 0.05, Table 8). On the other hand, dietary BSFLM inclusions
linearly and quadratically decreased AST (P < 0.001, P = 0.041), ALT (P
= 0.002, P < 0.001), total cholesterol (P < 0.001, P < 0.001), and LDL-C
(P = 0.005, P = 0.049). However, no linear or quadratic responses (P >
0.05) were observed in the other blood biochemical parameters as
shown in Table 8.
3.7. Histological assessments

Liver histological appraisal appeared normal in the fish fed control

and BSFLM43 diets based on the observation of sinusoids without
congestion, no hepatocyte vacuolization, centrally located nuclei, and

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A. Khieokhajonkhet et al. Aquaculture 561 (2022) 738618

Table 5
Fatty acids profiles in muscle of goldfish (% of total fatty acids).
Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic

SFA
C10:0 0.01 0.01 0.02 0.02 0.02 <0.001 0.496 0.690
C12:0 0.42d 0.67d 1.49c 2.28b 2.93a 0.008 <0.001 0.023
C13:0 0.02 0.01 0.02 0.02 0.02 <0.001 0.216 1.000
C14:0 2.14d 2.17d 2.46c 2.66b 2.94a 0.002 <0.001 0.018
C15:0 0.57a 0.44ab 0.42b 0.42b 0.40b 0.001 0.005 0.035
C16:0 13.19a 13.09a 12.48b 12.29b 12.47b 0.009 0.001 0.001
C17:0 0.53 0.50 0.49 0.51 0.51 <0.001 0.767 0.155
C18:0 4.06c 4.06c 4.50abc 4.61ab 4.76a 0.025 0.002 0.857
C20:0 3.26a 3.22a 3.17a 3.10a 2.82b 0.004 0.001 0.026
C21:0 0.35b 0.38ab 0.36b 0.42ab 0.52a 0.001 0.005 0.088

MUFA
C14:1 0.26 0.29 0.30 0.29 0.32 <0.001 0.703 0.927
C16:1 6.08 6.06 5.34 5.45 5.27 0.057 0.302 0.464
C18:1n-9 32.30b 35.78a 35.45a 34.41a 34.76a 0.162 0.011 0.001
C20:1n-9 0.57 0.62 0.60 0.76 0.75 0.005 0.557 0.760
C22:1n-9 0.22b 0.50a 0.62a 0.50a 0.55a 0.003 0.002 0.040

PUFA
C18:2n-6 14.57 14.22 14.60 14.21 14.47 0.042 0.657 0.429
C20:2n-6 0.87 0.87 0.86 0.82 0.74 0.002 0.347 0.100
C18:3n-6 0.47b 0.60ab 0.59ab 0.62ab 0.64a 0.002 0.011 0.145
C18:3n-3 3.32a 3.25a 3.31a 3.06ab 2.77b 0.009 0.002 0.029
C20:3n-6 0.93b 1.00ab 1.01ab 1.10a 1.10a 0.001 0.003 0.560
C20:3n-3 0.30 0.33 0.34 0.34 0.31 <0.001 0.400 0.123
C20:4n-6 1.19 1.08 0.96 1.00 1.04 0.004 0.164 0.693
C20:5n-3 1.07a 1.03ab 0.98ab 0.87bc 0.77c 0.002 0.001 0.205
C22:6n-3 3.93a 3.54ab 3.39bc 3.09bc 3.16c 0.011 < 0.001 0.043
C22:5 0.57 0.55 0.55 0.54 0.55 0.001 0.695 0.605
Sum of SFA 24.55d 24.55d 25.41bc 26.33b 27.39a 0.068 <0.001 0.008
Sum of MUFA 39.43b 43.25ab 42.31a 41.41a 41.65a 0.267 0.004 0.003
Sum of PUFA 27.22a 26.47ab 26.59ab 25.65b 25.55b 0.135 0.004 0.842
Sum n-3 8.62a 8.15ab 8.02b 7.36c 7.01c 0.014 <0.001 0.373
Sum of n-6 18.03 17.77 18.02 17.75 17.99 0.063 0.852 0.518
Sum of n-9 33.09b 36.90a 36.67a 35.67a 36.06a 0.183 0.004 0.001
n-3/n-6 FA ratio 0.47a 0.45b 0.44c 0.41d 0.38e 0.001 < 0.001 0.019

Fatty acid contents are mean values of each replicate tanks per treatment (3 fish per replicate tank were pooled). Abbreviations: BSFLM, black soldier fly larvae meal;
SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; FA, fatty acid.

Table 6
Color parameters of goldfish fed different levels of black soldier fly larvae meal for 10 weeks.
Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic

Head
Luminosity (L*) 48.37 ± 1.22a 48.33 ± 2.82a 43.88 ± 3.47a 42.27 ± 4.96a 34.24 ± 2.47b 2.064 < 0.001 0.025
Redness (a*) 21.35 ± 1.39a 17.20 ± 2.19b 14.76 ± 0.49b 15.44 ± 1.01b 13.73 ± 1.90b 1.914 < 0.001 0.040
Yellowness (b*) 39.35 ± 1.98 42.05 ± 3.26 37.11 ± 3.51 36.83 ± 2.61 35.03 ± 5.70 1.992 0.317 0.484

Abdomen
Luminosity (L*) 51.20 ± 1.41abc 51.69 ± 1.37a 51.36 ± 1.75ab 49.75 ± 1.76bc 49.31 ± 0.48c 10.478 0.001 0.086
Redness (a*) 19.42 ± 1.03a 19.41 ± 1.29a 17.72 ± 1.83a 15.47 ± 1.59b 17.68 ± 0.67a 4.721 < 0.001 0.030
Yellowness (b*) 40.21 ± 1.04 40.77 ± 2.17 40.99 ± 1.00 37.70 ± 1.16 40.19 ± 1.33 13.279 0.043 0.844

Tail
Luminosity (L*) 49.07 ± 4.08 42.31 ± 4.36 45.45 ± 4.79 43.54 ± 4.84 42.93 ± 3.26 18.578 0.084 0.327
Redness (a*) 17.92 ± 3.56 15.40 ± 2.15 15.88 ± 3.82 16.12 ± 4.14 13.17 ± 3.16 11.838 0.086 0.853
Yellowness (b*) 37.34 ± 2.90 30.69 ± 2.43 35.89 ± 4.98 33.21 ± 5.63 34.21 ± 4.42 18.117 0.540 0.310

Values are mean values from 3 replicate tanks per treatment (five fish per replicate tank). Abbreviation: BSFLM, black soldier fly larvae meal.

no evidence of inflammation (Fig. 2A, B). However, there was a mild
degree of vacuolization with swollen hepatocytes in the fish fed
BSFLM84 group (Fig. 2C). These histological abnormalities were more
abundant in BSFLM145 and BSFLM210 groups with intracytoplasmic
accumulation, enlargement of the cytoplasmic vacuole, irregular shape
of hepatocyte cells and nuclei, nuclear atrophy, and disappearance or
reduction of nuclear hepatocyte cells (Fig. 2D, E, 2Ea). The relative areas
of hepatocyte abnormalities were markedly increased with increasing
dietary BSFLM inclusion.
4. Discussion
Black soldier fly larvae have been a potential animal protein source
for aquatic animals, but little information is available about the impact
of BSFLM on growth and health in ornamental fish. The present study is
the first report to evaluate the effect of dietary replacement of FM by

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A. Khieokhajonkhet et al. Aquaculture 561 (2022) 738618

Table 7
Total carotenoids content in various parts of goldfish fed dietary inclusion of black solider fly larvae meal.
Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic
− 1
Tissue (μg g )
Fin 105.62 ± 2.71a 100.90 ± 7.07ab 84.44 ± 10.83b 80.23 ± 5.48b 81.18 ± 3.34b 43.279 < 0.001 0.054
Muscle 43.99 ± 7.52 41.03 ± 4.81 40.64 ± 7.18 43.96 ± 7.47 44.04 ± 6.28 15.336 0.810 0.516
Skin 139.63 ± 6.05a 127.92 ± 3.65ab 112.05 ± 13.02b 105.76 ± 12.49b 106.35 ± 8.00b 87.990 < 0.001 0.123

Blood (μg mL− 1)

Serum 1.23 ± 0.14a 1.10 ± 0.06ab 0.96 ± 0.15ab 0.89 ± 0.05c 0.91 ± 0.04c 0.012 0.002 0.138

Total carotenoid content in tissues are mean values from 3 replicate tanks per treatment (three fish per replicate tank). Total carotenoid content in serum are mean
values from 3 replicate tanks per treatment (five fish per replicate tank). Abbreviation: BSFLM, black soldier fly larvae meal.

Table 8
Hematological characteristics of goldfish fed different levels of black soldier fly larvae meal for 10 weeks.
Blood parameters Control BSFLM43 BSFLM84 BSFLM145 BSFLM210 SEM Linear Quadratic

Hematological parameters
RBC (×106) 1.07 ± 0.31 1.40 ± 0.26 1.53 ± 0.39 1.38 ± 0.29 1.48 ± 0.33 0.103 0.136 0.246
WBC (×103) 1.21 ± 1.43 1.35 ± 1.14 1.54 ± 2.04 1.53 ± 1.74 1.44 ± 2.08 0.299 0.254 0.064
Htc 28.13 ± 1.67 27.87 ± 0.97 29.09 ± 2.66 29.94 ± 3.93 28.60 ± 3.22 7.357 0.332 0.486
Hb 15.10 ± 1.66 14.50 ± 1.99 15.14 ± 2.07 14.80 ± 1.24 14.75 ± 1.58 3.023 0.856 0.964

Biochemical parameters
Total protein (mg L− 1) 2.81 ± 0.46 2.70 ± 0.10 2.89 ± 0.17 3.02 ± 0.16 2.76 ± 0.15 0.060 0.633 0.519
Albumin (mg L− 1) 1.23 ± 0.21 1.16 ± 0.05 1.13 ± 0.11 1.29 ± 0.10 1.11 ± 0.10 0.016 0.642 0.914
Globulin (mg L− 1) 1.56 ± 0.25 1.60 ± 0.10 1.76 ± 0.05 1.80 ± 0.10 1.66 ± 0.05 0.018 0.137 0.136
AST (UL− 1) 59.10 ± 3.80a 45.71 ± 4.08b 41.06 ± 5.39b 37.28 ± 4.34b 35.84 ± 6.26b 8.642 < 0.001 0.041
ALT (UL− 1) 19.58 ± 0.52a 14.10 ± 0.78c 6.57 ± 1.27c 8.48 ± 1.78c 18.25 ± 0.43a 1.170 0.002 < 0.001
ALP (UL− 1) 35.48 ± 4.52 36.57 ± 0.51 37.44 ± 4.87 36.16 ± 1.04 37.85 ± 4.54 13.124 0.530 0.906
Total cholesterol (mg dL− 1) 274.23 ± 5.18a 199.73 ± 2.53c 205.91 ± 4.44c 230.60 ± 6.32b 201.60 ± 7.66c 30.307 < 0.001 < 0.001
Triglycerides (mg dL− 1) 236.52 ± 11.76 264.97 ± 13.74 250.58 ± 9.98 256.96 ± 11.77 254.59 ± 7.40 23.994 0.196 0.120
HDL-C (mg dL− 1) 26.98 ± 1.75 25.62 ± 3.97 22.16 ± 2.02 26.90 ± 3.50 26.41 ± 1.27 7.358 0.980 0.121
LDL-C (mg dL− 1) 150.38 ± 7.31a 128.96 ± 8.54b 136.68 ± 6.18ab 129.49 ± 3.07b 129.66 ± 5.02b 39.822 0.005 0.049

Blood parameters are mean from three replicate tanks per treatment (five fish per replicate tank were pooled). Abbreviations: BSFLM, black soldier fly larvae meal;
RBC, red blood cell; WBC, white blood cell; Htc, hematocrit; Hb, hemoglobin; AST, aspartate aminotransferase; ALT, alanine transaminase; ALP, alkaline phosphatase;
HDL-C, high density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol.

Fig. 2. Liver histological image (H&E staining, original magnification × 40) of goldfish fed dietary replacement of fish meal by black soldier fly larvae meal with a
control diet (0 g kg− 1, A), 43 g kg− 1(B), 84 g kg− 1 (C), 145 g kg− 1 (D), and 210 g kg− 1 (E) diets for 10 weeks. Figure Ea showed an area of the enlargement from the
framed part in fig. E. The green arrows indicate the enlargement of vacuolization at the cytoplasm. The yellow arrows indicate irregular of nuclear size and shape and
nuclear atrophy. The arrowheads show the disappearance or reduction of nuclear hepatocyte cells. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)

7
A. Khieokhajonkhet et al.
BSFLM in goldfish (C. auratus), in which improved growth performance
and feed utilization were observed by up to 145–210 g kg–1 replacement.
Previous studies have also identified positive impacts of dietary BSF on
growth and feed utilization with species-specific replacement rates: up
to 400 g kg–1 in salmonid diets (Renna et al., 2017), 127.8 g kg–1 in
European seabass (Magalhães et al., 2017), 105–140 g kg–1 in Jian carp
(Zhou et al., 2018), and 223 g kg–1 in yellow catfish (Xiao et al., 2018).
Dietary inclusion of BSFLM at 165–400 g kg–1 sometimes impaired
growth performance in several aquatic species (Cummins et al., 2017;
Kroeckel et al., 2012; St-Hilaire et al., 2007), possibly due to low
palatability and high content of chitin in BSFLM, which may impede
feed intake and nutrient digestibility (Kroeckel et al., 2012; St-Hilaire
et al., 2007). The present study added the knowledge from goldfish to
the literature, enhancing our understanding of the impact of BSFLM
levels on fish growth, which should be related to several factors such as
species, source of BSF, fish stages, and the processing methods of BSFLM
(Lu et al., 2020).
Organosomatic indices of goldfish were not significantly affected by
dietary inclusion of BSFLM (P > 0.05). These results are consistent with
Zhou et al. (2018), Wang et al. (2019), and Abdel-Tawwab et al. (2020)
who found no significant difference in K, VSI, HSI, intraperitoneal fat
index, kidney index, and spleen index after feeding BSFLM to Jian carp,
juvenile Japanese seabass, and European seabass, respectively.
In the present study, dietary inclusion of BSFLM did not show any
significant effects on whole-body moisture and ash contents in goldfish
(Table 4), in contrast to Japanese seabass and rainbow trout, Onco­
rhynchus mykiss, fed BSFLM with significantly decreased ash content
(Renna et al., 2017; Wang et al., 2019). This effect could also be species-
specific since our results are consistent with several previous reports in
channel catfish, Ictalurus punctatus (Yildirim-Aksoy et al., 2020), Nile
tilapia (Devic et al., 2018), Jian carp (Zhou et al., 2018), European sea
bass (Abdel-Tawwab et al., 2020), meagre juvenile, Argyrosomus regius
(Guerreiro et al., 2020), and Pacific white shrimp (Cummins et al.,
2017). The discrepancies could be caused by variations in the size of the
experimental animals and the life stage of BSFL that affects their
nutritional makeup (Aniebo and Owen, 2010; Huis et al., 2013). On the
other hand, dietary BSFLM affected whole-body crude protein and crude
fat contents (P < 0.05) with significant linear and quadratic effects,
respectively. In a study conducted on Siberian sturgeon, a significant
increase in whole-body crude fat content was observed in fish fed BSFML
inclusion up to 375 g kg− 1 (Caimi et al., 2020). In contrast, turbot (Psetta
maxima) showed a significant decrease in whole-body crude fat content
(Kroeckel et al., 2012). In regard to the whole-body protein content,
BSFLM had a significant negative linear effect especially including
>145 g kg− 1 (P < 0.001). In line with this result, striped catfish, Pan­
gasianodon hypophthalmus, fed 174 g kg− 1 BSFLM showed a higher
protein content than those fed other diets, and inclusion higher than this
level significantly decreased (P < 0.05) the whole-body protein (Sudha
et al., 2022). While both whole-body protein and fat content were not
affected by dietary BSFLM inclusion for red seabream, Hypsibarbus
wetmorei × Barbonymus gonionotus (Takakuwa et al., 2022), and negative
effects were observed in lemon fin barb hybrid fingerlings (Kamarudin
et al., 2021). The variations in diet formulas and/or differences in ani­
mal species may have caused the inconsistencies between studies.
In the present study, BSFLM predominantly increased muscle C12:0,
C14:0, C18:n-9, and total SFA, significantly decreasing PUFAs including
C18:3n-3, C20:5n-3, and C22:6n-3, and total n-3 fatty acids and n-3/n-6
ratio. Such increase reflected the high C12:0 and C14:0 content in
BSFLM (Guerreiro et al., 2020; Hoc et al., 2020; Renna et al., 2017) and
experimental diets of the present study (Table 2). This is consistent with
the FA profiles for this feed ingredient described in other research
(Belghit et al., 2019; Guerreiro et al., 2020; Li et al., 2016; Zhou et al.,
2018). Previous studies suggest that the negative effect of replacing FM
with insect meals in aquafeeds on the FA profile of fillets is a major
concern. This could be overcome by including more fish oil in BSFLM-
supplemented diets to increase the PUFAs levels, especially EPA and
8
Aquaculture 561 (2022) 738618
DHA content (Belghit et al., 2019; Guerreiro et al., 2020).
The degree of deposited carotenoids in the tissue affects the beautiful
coloration of ornamental fish, which is highly desired by fish buyers/
owners. Fish are unable to synthesize carotenoids and many other pig­
ments that give them their coloration (Ebeneezar et al., 2020). Fish
feeding thus has a significant impact on coloration along with a variety
of other fillet quality metrics (Tibaldi et al., 2015). In the present study,
dietary inclusion of BSFLM decreased a* and b* values in the head and
L*, a*, and b* values in the abdomen (Table 6). In addition, total
carotenoid content also linearly and/or quadratically decreased in fin,
serum, and skin (Table 7). To our knowledge, there is only one study that
investigated the effect of the replacement of FM by BSF on color pa­
rameters. In rainbow trout fillet, a* value was negatively affected by
dietary BSFLM replacement (P > 0.05) in all test groups (Renna et al.,
2017). A similar tendency has been observed for other insect meals. In
gilthead seabream, Sparus aurata L., partial replacement of plant protein
with BSF pupae meal resulted in poor yellow pigmentation on the lateral
side and opercular region using yellow pixels analysis (Pulcini et al.,
2020). Iaconisi et al. (2017) reported that blackspot sea bream, Pagellus
bogaraceo, fed dietary replacement of FM by Tenebrio molitor larvae meal
decreased a a* value in skin dorsal region and decreased in L* and a*
values of skin ventral region. Panini et al. (2017) replaced FM by
mealworm meal, and the a* value was decreased in Pacific white shrimp
fed 305 g kg− 1 of mealworm meal. These results suggest that insect meal
may contain a lower amount of carotenoid. While the BSFLM includes
β-carotenes, lutein, and zeaxanthin, the content is lower than 1.28 mg
kg− 1 each and 2.15 mg kg− 1 of total carotenoid content (Finke, 2013;
Secci et al., 2018), which is lower than cereal grains e.g., corn, maize,
and wheat (approximately 57 mg kg− 1 of corn, 1.77–6.50 mg kg− 1 of
maize, and 24.61 mg kg− 1 of wheat, respectively) (Beta and Hwang,
2018; Kean et al., 2008; Klempova et al., 2020). The optimal carotenoid
in the diet for goldfish is approximately 45–50 mg kg− 1 (Besen et al.,
2019; Gouveia and Rema, 2005).
The biochemical and hematological indices are critical indicators for
assessing the general health state and physiological stress in fish e.g.,
stress, pollutions, and nutrition conditions (Dawood et al., 2020;
Khieokhajonkhet et al., 2022a; Khieokhajonkhet et al., 2022b). In the
present study, dietary inclusions of BSFLM did not significantly affect
hematological parameters in accordance with several previous studies in
hybrid tilapia, O. niloticus x O. mozambique, European seabass, and Af­
rican catfish (Abdel-Tawwab et al., 2020; Adeoye et al., 2020; Tip­
payadara et al., 2021; Yildirim-Aksoy et al., 2020). Similarly, total
protein, albumin, globulin, ALP, triglycerides, and HDL-C were not
significantly affected by dietary BSFLM inclusions (P > 0.05) as also
reported in juvenile dusky kob, grass carp, and European seabass (Abdel-
Tawwab et al., 2020; Lu et al., 2020; Madibana et al., 2020). Overall,
these results suggest that dietary BFS did not significantly affect the
health condition or immune system in goldfish.
Blood AST and ALT activities, indicators of liver function, were lin­
early and quadratically decreased by dietary replacement of BSFLM
levels. Xu et al. (2021) reported that dietary inclusion of BSF oil de­
creases these enzyme activities in mirror carp (Cyprinus carpio var.
specularis). A similar finding was observed in Jian carp (Li et al., 2016).
Although several studies indicated that dietary inclusion of BSFLM did
not significantly affect AST and ALT levels, these tended to decrease
(Belghit et al., 2019). These results denote that BSFLM inclusion in diets
might not cause hepatic injury or damage. The serum total cholesterol
and LDL-C levels linearly and quadratically decreased with increasing
dietary BSFLM inclusion. A recent study also found that BSFLM inclusion
in diet could reduce LDL-C levels (Xu et al., 2021) and total cholesterol
levels (Wang et al., 2019). These results suggest that BSFLM inclusion in
diets could improve serum lipid metabolism and health in fish.
The hepatic cells are highly sensitive to fish’s nutritional state as well
as the quality of the diet (Brusle and Anadon, 1996). The chitin content
in BSFLM is one of the drawbacks of using it in fish feed that is able to
affect histological abnormalities in the gut and liver, particularly, liver
A. Khieokhajonkhet et al.
lipid accumulation and intestinal inflammation (Elia et al., 2018; Li
et al., 2017; Zarantoniello et al., 2018). Histological examination
showed mild hepatic vacuolization, swollen on the fish fed higher than
43 g kg–1 replacement in goldfish. These results are in agreement with
mild hepatic fat deposition, necrosis, and apoptosis observed in Jian
carp fed BSF larvae meal higher than 79 g kg–1 (Li et al., 2017), zebra­
fish, Danio rerio, fed full-fat BSF prepupae higher than 105 g kg–1 (Zar­
antoniello et al., 2018), and rainbow trout fed BSFLM higher than 200 g
kg–1 (Elia et al., 2018). Another recent study showed severe lesion,
localized inflammation, necrosis, and hemorrhage of hepatic cells in
largemouth bass fed 98.3 g kg–1 BSF prepupae (Fischer et al., 2022). The
authors hypothesized that the observed effects were due to the presence
of chitin and its derivatives in the exoskeleton of insects (Li et al., 2021;
Zarantoniello et al., 2018), which may induce inflammation and
reduction in growth and welfare.
5. Conclusions
This study evaluated the effects of fish meal replacement by black
soldier fly larvae meal on growth performance, hematology, histology,
and pigmentation of goldfish Carassius auratus. The results showed that
black soldier fly larvae replacement at 145 g kg− 1 of fish meal improved
growth performance, feed utilization, and blood biochemistry of gold­
fish. In addition, the inclusion of 210 g kg− 1 of black soldier fly larvae
did not affect growth performance and feed utilization, but slightly
impaired hepatic cell integrity. The results obtained on color and
pigmentation showed that goldfish fed with dietary inclusion of black
soldier fly larvae had pale color in fin, head, skin, abdominal region, and
serum carotenoid compared with the control diet. It is, therefore, rec­
ommended to use black soldier fly larvae meal with carotenoid sup­
plementation for goldfish diet.
Data availability statement
The data that support the finding of this study are available from the
corresponding author upon reasonable request.
Declaration of Competing Interest
None of the authors have any potential conflict of interest.
Data availability
No data was used for the research described in the article.
Acknowledgments
This research study was partially supported by The Fundamental
Fund in 2022 from National Research Council of Thailand (NRCT),
Bangkok, Thailand (grant number: R2565B013/R2565B005).
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