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1. Evaluating Artemia biomass and gut weed (Ulva intestinalis) meal as a
dietary protein source for black tiger shrimp (Penaeus monodon)
postlarvae
Nguyen Thi Ngoc Anh a,⇑
, Huynh Thanh Toi b
, Nguyen Van Hoa a
a
Department of Coastal Aquaculture, College of Aquaculture and Fisheries, Can Tho University, Xuan Khanh Ward, Ninh Kieu District, Can Tho 900000, Viet Nam
b
Department of Applied Hydrobiology, College of Aquaculture and Fisheries, Can Tho University, Xuan Khanh Ward, Ninh Kieu District, Can Tho 900000, Viet Nam
a r t i c l e i n f o
Article history:
Received 18 May 2022
Revised 1 November 2022
Accepted 6 November 2022
Available online 17 November 2022
Keywords:
Black tiger shrimp
Artemia biomass
Gut weed
Growth
Feed efficiency
Stress resistance
a b s t r a c t
A 45-day feeding trial was conducted to assess the influence of Artemia (Artemia franciscana Vinh Chau)
biomass and gut weed (Ulva intestinalis) meal as replacement protein sources for fishmeal (FM) and soy-
bean (SB) protein on the performance of black tiger shrimp (Penaeus monodon) postlarvae (PL). The con-
trol diet did not include Artemia biomass or gut weed meal, and the experimental feeds were formulated
to replace 20%, 40%, 60%, and 80% of the FM protein with Artemia biomass meal, combined with the
replacement of 15%, 30%, 45%, and 60% of SB protein with gut weed meal. All the experimental feeds were
similar in crude protein (40%) and lipid (7%) content, with three replicates per treatment. Shrimp PL with
an average weight of 0.019 ± 0.001 g and length of 1.17 ± 0.02 cm were reared at a salinity of 10 ppt. The
results showed that the survival of the shrimp was not significantly influenced by the feeding treatments
(P > 0.05) and ranged from 81.1% to 86.7%. Interestingly, the growth rates in terms of the weight, feed
efficiency, and resistance to formalin shock of the shrimp that were fed Artemia biomass and gut weed
meal were superior to those that received the control feed. The study findings demonstrated that
Artemia biomass combined with gut weed meal can replace up to 80% FM protein and 60% SB protein
in the P. monodon PL diet. Notably, optimal shrimp performance was obtained using a 40% Artemia bio-
mass and 30% gut weed protein replacement diet.
Ó 2022 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
The high cost of shrimp feed has led to a search for inexpensive
or local alternative protein sources, such as seaweeds, aquatic
plants, and fishery and aquaculture by-products, which could
decrease the need for imported aquafeed ingredients (Boyd,
2015; Ayisi et al., 2017). Among the alternative animal protein
sources, Artemia biomass (adult Artemia) has an excellent nutri-
tional composition, which included 50 to 60% protein, abundant
essential amino acids (EAAs), and unsaturated fatty acids. There-
fore, it is an ideal ingredient for replacing fishmeal (FM) protein
in shrimp feeds (Castro et al., 2009; Hoa et al., 2020;
Zadehmohseni et al., 2020). Artemia biomass by-products from
Artemia cyst culture ponds could be harvested at a rate of 0.2 to
0.3 tons/ha after the completion of the production cycle in the
coastal region of the Mekong delta of Vietnam (Hoa et al., 2020).
The genus Ulva (synonym: Enteromorpha), a green seaweed
(Chlorophyta), is commonly found along ocean coasts and in brack-
ish waters (Messyasz & Rybak, 2009). Gut weed (Ulva intestinalis)
grows naturally in brackish water areas of the Mekong Delta in
Vietnam (Anh et al., 2013) and produces high biomass. Numerous
studies reported that Ulva is a good source of protein (Haroon et al.,
2018), EAAs, fatty acids, minerals, pigments, and bioactive com-
pounds (Aguilera-Morales et al., 2005) and is useful in direct feed
or as an ingredient in the diets of shrimp and fish (Madibana
et al., 2017; Haroon et al., 2018; Anh et al., 2020).
In practice, establishing the high-quality rearing of postlarval
(PL) shrimp during the nursery phase is one of the most essential
biosecurity aspects to improve the early culture stages and con-
tribute to an effective shrimp farming grow-out phase
(Rodríguez-Olague et al., 2021). Specifically, the shrimp PL stage
requires a highly nutritious diet during nursery rearing that
employs high-quality, cost-effective marine ingredients (Ayisi
et al., 2017). Moreover, evaluating the effects of feeding trials on
the performance of PL shrimp includes not only their survival
https://doi.org/10.1016/j.ejar.2022.11.003
1687-4285/Ó 2022 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer review under responsibility of National Institute of Oceanography and
Fisheries.
⇑ Corresponding author.
E-mail address: ntnanh@ctu.edu.vn (N. Thi Ngoc Anh).
Egyptian Journal of Aquatic Research 49 (2023) 97–103
Contents lists available at ScienceDirect
Egyptian Journal of Aquatic Research
journal homepage: www.sciencedirect.com/locate/ejar

2. and growth but also their quality through a stress test (Anh et al.,
2018; Mirzaeia et al., 2021). Among the stress test methods, the
assessment of resistance to formalin shock has proven to be an
effective tool for evaluating the quality of penaeid PL. This method
is the most widely used approach in commercial hatcheries since it
is a simple, inexpensive procedure that produces rapid results
(Mirzaeia et al., 2021).
In the present study, we investigated the utilization of local
ingredients from coastal areas in shrimp feed, such as the replace-
ment of FM protein with Artemia biomass or the replacement of
soybean (SB) protein with gut weed (U. intestinalis) in the diet of
black tiger shrimp (Penaeus monodon) PL (Anh et al., 2020). We also
evaluated the possibility of combining the replacement of FM and
SB meal protein in the diet for this species as part of a research pro-
ject to identify the influence on the growth, feed efficiency, and
stress resistance of shrimp PL. These results could encourage the
utilization of by-products from local aquaculture operations, which
would reduce feed costs and increase profitability for shrimp
farmers.
Materials and methods
Source of experimental shrimp, ingredients, formulation, and diet
preparation
Black tiger shrimp PL12 were purchased from a commercial
hatchery in the Bac Lieu province, Vietnam and stocked in a 2 m3
tank for 3 days to allow them to adapt to the experimental salinity
(from 20 to 10 ppt). The Artemia (Artemia franciscana Vinh Chau)
biomass by-product from the Artemia cyst production in the Bac
Lieu province was obtained at the termination of the cycle and
dried under natural sunlight for 1 day. Gut weed was collected dur-
ing semi-intensive shrimp farming (during the resting phase
between two runs) in the Bac Lieu province. Thin layers of gut
weed were air-dried in the shade for 3 days. Both dried products
were then ground into a powder and stored at 15 °C for later
use. The Kien Giang FM that was used was a commercially avail-
able product manufactured by the Kien Giang Fish Meal Limited
Company in the Kien Giang province. The other components con-
sisted of SB meal, cassava powder, squid oil, lecithin, rice bran,
gelatin, and premixed vitamins and were provided by aquaculture
ingredient dealers. The proximate composition of the feed compo-
nents was examined prior to formulating the experimental diet
(Table 1). The amino acid (AA) profiles of the FM, Artemia meal,
SB meal, and gut weed meal included in the experimental diets
were determined (Table 2).
Five experimental feeds were created to be approximately isoli-
pidic, isonitrogenous, and isoenergetic (40% dietary protein and 7%
lipid) using SOLVER software in Microsoft Excel 2010 (Table 3). The
test feeds were created using various particle sizes (500, 700, and
1000 lm) for shrimp PL at various growth stages and kept in boxes
at 15 °C for later use.
Biochemical analysis
The AOAC 950.46 method was used to determine the following
approximate composition parameters of the dietary ingredients
and experimental feeds: moisture, crude protein, total lipid, fiber,
and ash. The nitrogen-free extract (NFE) was calculated on dry
matter by subtracting the proportions of crude protein, lipids,
crude fiber, and ash from 100%. The AA composition of the FM,
Artemia meal, SB meal, and gut weed meal was determined using
Ref. TCVN 8764:2012. All the samples were analyzed by the
National Agro-Forestry-Fisheries Quality Assurance Department-
Branch 6, Can Tho City, Vietnam.
Experimental design and tank management
Five experimental feeds were developed. The control feed did
not contain Artemia biomass or gut weed meal. The other four
feeds were formulated by replacing 20%, 40%, 60%, and 80% of
the FM protein with Artemia biomass (A) meal combined with
the replacement of 15%, 30%, 45%, and 60% of SB protein with gut
weed (E) meal. The treatments were abbreviated as follows:
20A + 15E, 40A + 30E, 60A + 45E, and 80A + 60E.
The experiment was conducted for 45 days at the experimental
hatchery of the College of Aquaculture and Fisheries, Can Tho
University, Vietnam. The treatments were randomly assigned to
culture tanks, with triplicates for each treatment. The 150 L tank
contained 120 L of seawater at a salinity of 10 ppt, and the tanks
received constant aeration to maintain the dissolved oxygen levels
between 5 and 6 mg/L. A natural photoperiod was maintained dur-
ing the experiment. Feeding trays were placed in each tank to man-
age the feed residue for each feeding.
Sixty shrimp PL with an average weight of 0.019 ± 0.001 g and
length of 1.17 ± 0.02 cm were distributed into each tank. Feeding
was performed four times per day (at 6:00 h, 11:00 h, 16:00 h,
and 21:00 h) at an initial feeding rate of approximately 15% of
the body weight daily, and the feed rations were regulated every
day by observing the amount of feed that remained on the feeding
tray. A water volume exchange (approximately 50%) was con-
ducted every week.
Water quality evaluation
The temperature and pH in the rearing tanks were measured
daily at 7:00 h and 14:00 h using a Thermo pH meter (YSI 60 Model
pH meter, HANNA instruments, Mauritius). The alkalinity was
recorded weekly using test kits (Sera, Germany). The total ammo-
nia nitrogen (TAN) and NO2
–
were determined weekly using a HI-
83303 Aquaculture Photometer kit. Samplings were conducted
prior to the water exchange to maintain an appropriate level for
the normal development of the experimental shrimp. The parame-
ters were as follows: the mean daily water temperature and pH
ranged from 26.0 to 27.2 °C and 7.9 to 8.2, respectively; the aver-
age alkalinity ranged from 102 and 105 mg CaCO3/L; and the aver-
Table 1
Proximate composition (dry matter, g/kg) of the ingredients used for the formulation of experimental diets.
Ingredient Moisture Protein Lipid Ash Fiber NFE*
Fishmeal 110.8 581.4 91.7 213.6 5.6 107.7
Soybean meal 104.3 443.2 22.3 82.5 12.7 439.3
Artemia meal 87.2 584.5 103.5 197.1 3.7 110.2
Gut weed meal 61.9 254.4 21.6 241.7 21.4 460.9
Rice bran 98.6 85.2 81.5 213.2 23.3 596.8
Cassava powder 108.7 51.4 17.7 6.9 8.7 915.3
*NFE: nitrogen free extract.
N. Thi Ngoc Anh, H. Thanh Toi and N. Van Hoa Egyptian Journal of Aquatic Research 49 (2023) 97–103
98

3. age TAN and NO2
–
levels ranged from 0.38 to 0.54 mg/L and 0.78 to
1.07 mg/L, respectively.
Shrimp performance
The initial body weight of 40 randomly sampled from the con-
ditioning tank, individual shrimp PL was determined using a
0.001 g accuracy balance, and the total body length of the shrimp
was measured from the tip of the rostrum to the tip of the telson
using a caliper. The final individual weight and total length of
the shrimp were recorded, and counted to determine the survival
at the termination of the feeding trial. The growth performance
of the experimental shrimp, such as the daily weight gain
(DWG), specific growth rate (SGRW), total feed intake (FI), feed con-
version ratio (FCR), protein efficiency ratio (PER), and survival was
computed as follows:
DWG (g/day) = (final weight - initial weight)/Days of culture.
SGRW (%/day) = (final weight - initial weight)/Days of culture 100.
FI (g/ind) = Total feed supply (DW)/
½ðInitialnumberofshrimp þ FinalnumberofshrimpÞ=2:
FCR = Feed intake (dry weight)/Weight gain (wet weight).
PER = Weight gain/protein intake.
Survival (%) = Final number of shrimp/Initial number of shrimp 100.
The quality of the shrimp was investigated after 45 days of the
feeding trial by evaluating how well the shrimp responded to for-
malin shock, according to the procedure described by Anh et al.
(2018). Specifically, fifteen experimental shrimp were randomly
collected from each rearing tank and placed in a 10 L glass flask
containing a 250 ppm formalin solution at ambient temperature,
with slight aeration and salinity of 10 ppt. The deceased shrimp
were observed every 10 min for a total of 60 min. The cumulative
mortality index (CMI) was determined by summing all of the
deceased animals that occurred during the observation period.
Table 2
Amino acid profiles (dry matter, g/kg) of fishmeal, Artemia meal, soybean meal, and gut weed meal used in the experimental diets.
Amino acid profiles Kien Giang fishmeal Artemia biomass meal Soybean meal Gut weed meal
Total essential amino acids 232.8 346.4 165.7 97.1
Arginine 31.2 48.2 29.4 11.4
Histidine 13.3 27.4 10.2 7.2
Isoleucine 21.4 86.1 17.4 9.7
Leucine 30.7 31.2 26.4 12.2
Lysine 41.3 44.3 27.3 11.6
Methionine 17.8 16.2 4.9 5.3
Phenylalanine 19.8 25.4 18.7 12.1
Threonine 24.4 19.5 14.1 14.1
Valine 32.9 63.9 17.3 13.5
Total non-Essential amino acids 286.7 207.9 186.7 114.5
Alanine 64.6 23.6 18.4 16.2
Aspartic acid 43.2 26.8 42.2 16.9
Cysteine 4.9 6.3 3.2 3.8
Glycine 35.7 23.4 18.6 18.5
Glutamic acid 74.3 78.2 51.1 31.1
Proline 24.1 25.1 21.8 9.7
Serine 22.5 9.2 17.9 9.4
Tyrosine 17.4 15.3 13.5 8.9
Total amino acids 519.5 554.3 352.4 211.6
Table 3
Formulation (dry matter, g/kg) and proximate composition of the five experimental diets.
Ingredients Control 20A + 15E 40A + 30E 60A + 45E 80A + 60E
Fishmeal 445.0 356.1 266.9 178.0 88.9
Artemia biomass meal 0 88.5 177.1 265.6 354.2
Soybean meal 291.9 248.2 204.4 160.6 116.6
E. intestinalis meal 0 76.3 152.6 228.6 305.1
Rice bran 38.0 78.8 93.5 80.0 46.9
Cassava powder 168.5 99.3 56.7 39.3 36.8
Squid oil 5.8 3.9 1.9 1.5 3.3
Soybean oil 5.8 3.9 1.9 1.5 3.3
Lecithin 5.0 5.0 5.0 5.0 5.0
Premix-vitamin1
20.0 20.0 20.0 20.0 20.0
Gelatin 20.0 20.0 20.0 20.0 20.0
Proximate composition (g/kg of dry matter)
Dry matter 895.4 890.3 889.2 896.2 889.6
Protein 406.8 400.4 399.7 400.3 399.2
Lipid 69.8 70.7 71.1 69.7 69.4
Ash 142.8 156.4 164.6 179.8 189.7
Fiber 29.2 27.8 31.2 32.4 32.9
NFE 351.3 344.7 333.4 317.8 308.9
Calcium 21.7 25.1 24.9 26.1 26.3
Phosphorus 13.2 11.2 11.9 13.4 13.8
Gross energy (kgcal/g) 4.43 4.38 4.33 4.25 4.21
N. Thi Ngoc Anh, H. Thanh Toi and N. Van Hoa Egyptian Journal of Aquatic Research 49 (2023) 97–103
99

4. CMI = Nx1 + Nx2 + Nx3 + . . .Nx6, where N is the number of
deceased individuals at time x1, x2, x3 . . . x6.
Statistical analyses
The homogeneity of variance was assessed using Levene’s test,
and the percentage data were transformed to arcsine prior to con-
ducting the statistical analyses. A one-way ANOVA was used to
compare variations in the feed treatments. The Duncan post hoc
test in one-way ANOVA was applied to identify significant differ-
ences among the feed treatments at a value of P 0.05 (SPSS for
Windows, Version 16.0).
Results
Survival, growth rate, and feed efficiency of the shrimp
The survival and growth rate of the shrimp that were adminis-
tered the experimental feeds at day 45 are presented in Table 4.
The results show that the average shrimp survival ranged from
81.1% to 86.7%, and no statistical difference (P 0.05) was observed
among the feeding treatments. Similarly, the final lengths of the
shrimp, which ranged from 4.52 to 4.57 cm, were not influenced
by the test feeds (P 0.05). The average final weights of the shrimp
ranged from 0.97 to 1.08 g, which were equivalent to a DWG of
0.0211 to 0.0235 g/day and an SGRW of 8.62 to 8.85%/day. The
growth rate of the shrimp in the control group was lower than that
of the other groups; however, a significant difference (P 0.05) was
only observed for the 20A + 15E and 40A + 30E groups. When the
four FM and SB protein substitution groups were compared, a ten-
dency for the growth rate of the shrimp to decrease with increasing
inclusion of Artemia biomass and gut weed protein was observed
(the 60A + 45E and 80A + 60E treatments); however, no statistical
differences (P 0.05) were observed.
No significant differences (P 0.05) were observed among the
test feeds regarding the total feed intake (FI) of the shrimp, which
varied from 1.29 to 1.32 g/shrimp. The average feed conversion
ratio (FCR) ranged from 1.22 to 1.37, which corresponded to a pro-
tein efficiency ratio (PER) of 2.06 to 1.79. The control feed showed
the highest FCR and lowest PER values and differed considerably
(P 0.05) compared to the other feed treatments. Furthermore,
among the substitution feed treatments, the 40A + 30E group
showed the best FCR and PER but was not statistically different
(P 0.05) from the 20A + 15E group (Table 5).
Stress resistance of the shrimp that were administered the
experimental feeds
Fig. 1 shows the cumulative mortality index (CMI) of the shrimp
that were subjected to formalin stress. It was observed that the
stress index of the shrimp that received the control feed was signif-
icantly higher (P 0.05) than those that received the feeds contain-
ing Artemia biomass and gut weed protein. This indicates that the
shrimp that were administered the control treatment were more
susceptible to formalin shock than those that were administered
the other treatments. Although the CMI value of the shrimp in
the 40A + 30E group was lower than that of the shrimp in the
20A + 15E, 60A + 45E, and 80A + 60E groups, no significant differ-
ence (P 0.05) was observed.
Discussion
Effects of dietary FM and SB protein replacement on the survival,
growth, and feed efficiency of the shrimp
The new approach in this study is the use of a combination of
Artemia biomass and dried marine algae as substitutes for fishmeal
and soybean.
The new approach in this study is the use of a combination of
Artemia biomass and dried marine algae as substitutes for fishmeal
and soybean. Most previous studies focused solely on replacing FM
protein with other animal or plant protein sources or using plant
protein sources to replace SB protein in the practical diet for fish
and crustaceans.
Early studies evaluated the inclusion of seaweed as a feed ingre-
dient in shrimp and prawn diets. For example, Da Silva and Barbosa
(2009) revealed that the use of red seaweed (Hypnea cervicornis
and Cryptonemia crenulata) powder in the diet of Litopenaeus van-
namei shrimp at 13%, 26%, and 36% had no significant influence
on the survival and growth rate of the shrimp. According to
Serrano et al. (2015), replacing 15% of the SB protein with Ulva lac-
tuca powder resulted in a similar growth rate; however, shrimp
that were administered with a 30% SB protein replacement feed
showed a significantly slower growth rate than those that received
the control feed. The authors observed no statistical differences
among the feed treatments in terms of survival, feed intake, FCR,
PER, protein and lipid deposition, and body composition. In a study
on the freshwater prawn, Macrobrachium rosenbergii, the incorpo-
ration of 5% of gut weed meal into the diet resulted in an improve-
ment in the growth rate, yield, and feed efficiency (Mondal et al.,
2014). Furthermore, prawns that were fed a formulated feed con-
taining 30% gut weed showed noticeably enhanced survival,
growth, and feed efficiency compared with prawns that were fed
commercial feed (Ghosh Mitra, 2015). When FM protein was
substituted with green seaweed (Cladophora sp.) protein for P.
monodon PL, the growth rates, FCR, and PER of the shrimp that
ingested feed with 10%, 20%, or 30% substitution levels were better
or equivalent to that of the shrimp that consumed the control feed
(Anh et al., 2018). The dietary supplementation of Caulerpa sp.
powder at 4% for whiteleg shrimp improved their growth and feed
efficiency after 30 days of culture (Nasmia et al., 2022).
Additionally, other studies assessed the use of Artemia biomass
as a protein source or as a feed ingredient in crustacean diets.
Naegel and Rodriguez-Astudillo (2004) observed that L. vannamei
PL that were fed dried Artemia biomass meal showed a more rapid
growth rate than those that were administered three commercial
Table 4
Survival and growth rate of P. monodon postlarvae fed different experimental diets for 45 days.
Treatment Control 20A + 15E 40A + 30E 60A + 45E 80A + 60E
Survival (%) 84.4 ± 5.1 83.3 ± 5.8 86.7 ± 3.3 81.1 ± 5.1 82.2 ± 3.8
Initial length (cm) 1.17 ± 0.02 1.17 ± 0.02 1.17 ± 0.02 1.17 ± 0.02 1.17 ± 0.02
Final length (cm) 4.52 ± 0.06 4.56 ± 0.07 4.57 ± 0.04 4.54 ± 0.05 4.53 ± 0.06
Initial weight (g) 0.019 ± 0.001 0.019 ± 0.001 0.019 ± 0.001 0.019 ± 0.001 0.019 ± 0.001
Final weight (g) 0.97 ± 0.04a
1.07 ± 0.03b
1.08 ± 0.06b
1.03 ± 0.03ab
1.04 ± 0.03ab
DWG (g/day) 0.021 ± 0.001a
0.023 ± 0.001b
0.024 ± 0.002b
0.023 ± 0.002ab
0.023 ± 0.002ab
SGR (%/day) 8.62 ± 0.10a
8.83 ± 0.06b
8.85 ± 0.11b
8.76 ± 0.05ab
8.78 ± 0.06b
Data are mean ± SD (n = 3), and means in the same row with different superscripts are significantly different (P 0.05).
N. Thi Ngoc Anh, H. Thanh Toi and N. Van Hoa Egyptian Journal of Aquatic Research 49 (2023) 97–103
100

5. feeds and the crustacean meals. Similarly, during the rearing of lar-
val P. monodon, supplementation with 50% commercial feed and
50% Artemia-based feed resulted in a significantly larger PL15 size
and equivalent survival rates compared to those that were admin-
istered commercial feed alone (Anh et al., 2011). For PL of the
freshwater prawn, M. rosenbergii, the replacement of different
rations of dietary FM protein with dried or frozen Artemia meal
(25%, 50%, 75%, and 100%) showed a gradual improvement in the
growth performance of the prawns (as the proportion of Artemia
protein in the diet increased), and the survival was noticeably
higher than that of the prawns that consumed feed without the
addition of Artemia meal (Anh et al., 2009). In a P. monodon PL nurs-
ery, various levels of dietary FM protein were substituted for dried
Artemia meal (20%, 40%, 60%, and 80%). The feeding treatments
showed no effect on the shrimp survival, while the growth rate
steadily increased with increasing Artemia protein inclusion, and
the FCR progressively improved at higher substitution levels com-
pared to those in the FM control diet (Anh et al., 2020).
The results of the present study showed that, in terms of the
weight and feed efficiency, the growth rate of the shrimp was
higher in the Artemia-gut weed-based feeds than in the control
feeds that did not contain Artemia and gut weed meal. The growth
of the shrimp improved significantly when the test feeds included
Artemia meal at rates of 20% and 40% as a replacement for FM pro-
tein, combined with gut weed meal at rates of 15% and 30% as a
replacement for SB protein (the 20A + 15E and 40A + 30E treat-
ments, Table 4). Notably, the 40A + 30E diet (the replacement of
40% of Artemia protein with FM protein, combined with the
replacement of SB protein with 30% of gut weed protein) showed
the best FCR (1.22) and PER (2.06) values (Table 5), indicating that
this feed combination provided the optimum nutrition for P. mon-
odon PL. Additionally, a comparison of the four experimental feeds
used in this trial showed a tendency for the shrimp performance to
decrease when higher levels of Artemia biomass and gut weed pro-
tein were included in the diet (the 60A + 45E and 80A + 60E treat-
ments). Anh et al. (2009) demonstrated that an increase in the
amount of Artemia protein in the diet resulted in more rapid prawn
growth, while a separate study by Anh et al. (2020) showed that P.
monodon PL that ingested feeds containing the replacement of 45%
and 60% SB meal protein with gut weed protein showed poor per-
formance compared with those that were administered 15% and
30% gut weed feeds. Similarly, Qiu et al. (2018) revealed that white
leg shrimp that received formulated feeds with 19.0% and 25.4%
Ulva sp. as a dietary replacement for FM showed a considerably
lower growth rate and higher FCR than shrimp that consumed feed
containing 12.7% Ulva. The authors theorized that the poor shrimp
performance at higher Ulva levels in the formulated feed may have
been due to low nutrient digestibility and excessive mineral levels.
The effect of the nutritional profiles of the Artemia biomass and gut
weed meal included in the test feeds on the performance of the
shrimp, which is discussed further in the section on stress resis-
tance below.
Effects of dietary FM and SB protein replacement on the stress
resistance of the shrimp
In this trial, the impact of the feeding treatments on the forma-
lin stress test followed the same trend as the growth rate, with the
shrimp that ingested feed containing Artemia and gut weed meal
showing greater formalin tolerance than the shrimp that con-
sumed the control feed. The shrimp that were fed the 40% FM pro-
tein replacement with the Artemia protein combined with the 30%
SB protein replacement with the gut weed protein showed the
lowest CMI of all the groups. This finding is in accordance with pre-
vious studies that reported that low levels of seaweed meal in for-
mulated feed improved shrimp growth, feed efficiency, disease
resistance, and stress tolerance (Elizondo-Reyna et al., 2016; Qiu
et al., 2018). Similarly, Anh et al. (2018) observed that replacing
10% and 20% of FM protein in the P. monodon PL diet with green
seaweed (Cladophora sp.) significantly increased the growth, feed
efficiency, and tolerance to formalin shock.
From a nutritional viewpoint, Artemia biomass has excellent
nutritional composition due to its high protein content (50–60%),
which is rich in EAAs, and its lipid content (8–12%), which is high
in unsaturated fatty acids (Castro et al., 2009; Zadehmohseni et al.,
2020). Artemia biomass can be utilized as a direct feed or as a com-
ponent in formulated feeds for aquaculture species in larviculture
and nursery rearing (Anh et al., 2009; Castro et al., 2009;
Zadehmohseni et al., 2020). Furthermore, gut weed has a high pro-
tein content (Haroon et al., 2018) and high levels of essential AAs
and unsaturated fatty acids (Aguilera-Morales et al., 2005), as well
as high protein digestibility, making it a suitable feed for fish and
shrimp (Aguilera-Morales et al., 2005; Haroon et al., 2018). Addi-
tionally, green seaweeds, including Ulva, are rich in chlorophyll a,
b-carotene, lutein, astaxanthin, antheraxanthin, zeaxanthin, and
neoxanthin; these natural compounds benefit species with a high
tolerance for a stressful environment (Sirbu et al., 2019;
Eismanna et al., 2020). These natural compounds show excellent
antioxidant activity and, when green seaweeds are incorporated
into aquafeeds, benefit species with a high tolerance to stressful
environments (Cruz-Suárez et al., 2008; Morais et al., 2020).
According to Miki (1991), astaxanthin plays a crucial role in the
cellular antioxidant function of animals because it contains various
Table 5
Feed efficiency of P. monodon postlarvae fed different experimental diets over 45 days.
Treatment Control 20A + 15E 40A + 30E 60A + 45E 80A + 60E
FI (g/shrimp) 1.30 ± 0.04 1.31 ± 0.04 1.29 ± 0.02 1.32 ± 0.04 1.31 ± 0.03
FCR 1.37 ± 0.03a
1.25 ± 0.02bc
1.22 ± 0.05b
1.30 ± 0.02c
1.29 ± 0.01c
PER 1.79 ± 0.04a
1.99 ± 0.05bc
2.06 ± 0.08c
1.92 ± 0.04b
1.95 ± 0.02b
FI: Feed intake, FCR: Feed conversion ratio, PER: Protein efficiency ratio.
Data are mean ± SD (n = 3), and means in the same row with different superscripts are significantly different (P 0.05).
Fig. 1. Cumulative mortality index of P. monodon PL fed experimental diets after
60 min of exposure to 250 ppm formalin solution. Each bar indicates the average
value and standard deviation. Data with different superscripts indicate statistical
differences (P 0.05) among treatments.
N. Thi Ngoc Anh, H. Thanh Toi and N. Van Hoa Egyptian Journal of Aquatic Research 49 (2023) 97–103
101

6. antioxidants and free radical scavengers, inhibits lipid peroxida-
tion, and reduces oxidative stress induced by environmental fac-
tors. Therefore, the addition of astaxanthin to the diet of shrimp
has been commonly applied to support and regulate the immune
system, enhancing the immune response and stress resistance
against ammonia stress (Pan et al., 2003), hypoxia stress (Chien
and Shiau 2005), and salinity stress (Flores et al., 2007). A recent
study (Zhao et al., 2022) reported that the dietary supplementation
of astaxanthin in L. vannamei shrimp (between 80 and 160 mg/kg)
enhanced the growth performance, feed efficiency, and coloration
as well as the antioxidant ability of the shrimp after hypoxia and
ammonia stress.
The observed differences in the performance of the shrimp
could be attributed to variations in the nutritional profiles of the
dietary ingredients utilized in the present feeding trial. The slower
growth and low-stress resistance of the control group compared
with the groups that were fed an Artemia-gut weed-based diet
are most likely due to the feed quality. For example, the Artemia
biomass meal showed higher levels of EAAs and total AAs (364.4
and 554.3 g/kg dry matter, respectively) than the FM (232.8 and
519.5 g/kg dry matter for the EAAs and total AAs, respectively). Iso-
leucine was the most dominant EAA in the Artemia biomass meal,
followed by valine, arginine, and lysine, all of which were at higher
levels in the Artemia biomass meal than in the FM. All the AAs were
at a higher level in the Artemia biomass meal except for methion-
ine, which showed a lower level in Artemia biomass meal than in
the FM. In contrast, the gut weed meal showed lower levels of EAAs
and total AAs (97.1 and 211.6 g/kg of dry matter, respectively) than
the SB meal (165.7 and 352.4 g/kg of dry matter, respectively);
however, the gut weed meal showed a slightly higher methionine
level (Table 2). Consequently, the Artemia meal was incorporated
into the formulated feed, which was rich in essential nutrients
and may have compensated for the EAA deficit in the gut weed
meal, resulting in improved shrimp performance compared to ani-
mals that were administered the control feed.
A previous study revealed that minerals play various physiolog-
ical roles in crustaceans, including acid-base balance and osmoreg-
ulation. Among the key minerals, calcium (Ca) and magnesium
(Mg) are crucial for molting and shell formation (Davis Gatlin
III, 1996). Regarding marine crustaceans, excessive mineral intake,
whether dietary or environmental, can be toxic, whereas a mineral
deficiency can affect immunity, rendering the animals more vul-
nerable to disease and stressful conditions (Piedad-Pascual, 1989;
Davis Gatlin III, 1996). The minerals that have been most exten-
sively studied are calcium (Ca) and phosphorus (P), and issues with
soft-shelling in P. monodon have been linked to these two minerals
(Piedad-Pascual, 1989). High levels of P supplementation (2.0–
2.5%) in the P. monodon diet have been shown to reduce the growth
rate and enhance the FCR (Ambasankar et al., 2006). A similar find-
ing was obtained by Truong et al. (2020), who demonstrated that
shrimp growth was adversely impacted by the addition of
macro-minerals such as potassium (K) or Ca + P to the diet. More-
over, the authors showed that shrimp survival was negatively
impacted by Ca + P and zinc (Zn). The previously mentioned factors
may explain why, in the present study, the shrimp that were fed
higher levels of the tested feed ingredients showed lower growth
rates, feed efficiency, and resistance to formalin shock compared
with those that were administered lower levels of the tested
ingredients.
According to Fox et al. (2006), shrimp consume dietary protein
to sustain a steady supply of EAAs for their normal growth, such as
methionine, arginine, threonine, tryptophan, histidine, isoleucine,
leucine, lysine, valine, and phenylalanine. As a result, EAAs must
be included in shrimp feed in appropriate quantities and of suit-
able quality to obtain a well-balanced diet. Due to the low EAA
content in plant protein, it has been suggested that plant meal
could be used in shrimp diets at a low (5%) or high (75%) ratio
depending on the plant source and cultured species, whereas ani-
mal meal can be incorporated into the diet of shrimp from 15% to
100% (Ayisi et al., 2017).
Conclusions
The growth rate, feed efficiency, and stress resistance of shrimp
that were administered feed containing the replacement of 40% of
FM protein with Artemia biomass meal (177.1 g/kg) and 30% of SB
protein with gut weed meal (152.6 g/kg) resulted in the optimal
performance, suggesting that these proportions of substituted
ingredients in the shrimp feed were the most effective. The
Artemia-gut weed-based feed offers excellent prospects for the uti-
lization of locally available Artemia biomass and gut weed and
reduces the requirement for FM and SB meal in aquafeed, con-
tributing to sustainable aquaculture production.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgments
This study was granted by the Can Tho University, Vietnam for the
scientific research programs. The authors appreciate Tran Huu Le
and Tran Nguyen Hai Nam for their help in collecting Artemia bio-
mass and gut weed. Special thanks to Duong Hoang Anh and Ta
Xuan Duy for their assistance with conducting experiments.
Ethical approval
The ethical committee of the College of Aquaculture Fisheries,
Can Tho University, Vietnam, approved animal care in this feeding
trial. This study was conducted for aquaculture purpose.
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