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APPLIED STUDIES
Effect of feeding strategy on survival, growth,
intestine development, and liver status of maraena
whitefish Coregonus maraena larvae
Vlastimil Stejskal
1
| Tatyana Gebauer
1
| Roman Sebesta
1
|
Joanna Nowosad
2
| Mateusz Sikora
2
| Mateusz Biegaj
2
|
Dariusz Kucharczyk
2
1
Faculty of Fisheries and Protection of
Waters, University of South Bohemia in
Ceske Budejovice, South Bohemian Research
Center of Aquaculture and Biodiversity of
Hydrocenoses, Institute of Aquaculture and
Protection of Waters, České Budeˇjovice,
Czech Republic
2
Faculty of Animal Bioengineering,
Department of Ichthyology and Aquaculture,
University of Warmia and Mazury, Olsztyn,
Poland
Correspondence
Vlastimil Stejskal, Faculty of Fisheries and
Protection of Waters, University of South
Bohemia in Ceske Budejovice, South
Bohemian Research Center of Aquaculture
and Biodiversity of Hydrocenoses, Institute of
Aquaculture and Protection of Waters, Na
S
adk
ach 1780, 370 05 České Budeˇjovice,
Czech Republic.
Email: stejskal@frov.jcu.cz
Funding information
Ministry of Agriculture of the Czech Republic,
Grant/Award Number: QK1810296
Abstract
Optimizing larval rearing protocols is critical to successful
intensive fish culture. We compared the efficacy of feeding
strategies for larvae of maraena whitefish Coregonus maraena,a
promising candidate for intensive aquaculture. Survival, growth
indicators, intestine development, and liver status were com-
pared in larvae fed live feed, commercial dried feed, and
weaned from live to dried feed at 5, 10, 15, 20, or 25 days post
hatching (dph). Seven experimental groups in three repetitions
used 5,250 larvae (2 dph, initial body weight =7.4±0.1 mg;
initial total length =13.0 ± 0.1 mm). This 30-day trial showed
initial weaning from live feed (Artemia sp.) to artificial diet after
15 days to be the optimal, with beneficial effects on growth,
body weight, and larva yield. No differences in survival rate, size
heterogeneity, and or condition factor were observed among
groups. Live feed and weaning to artificial diet at the appropri-
ate time was beneficial to intestine development, while feeding
on artificial feed only was associated with severe intestine
impairment. Liver pathology was not seen in any group.
KEYWORDS
artificial diet, coregonid, larviculture, live feed, weaning
Received: 4 April 2020 Revised: 12 December 2020 Accepted: 3 March 2021
DOI: 10.1111/jwas.12785
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
© 2021 The Authors. Journal of the World Aquaculture Society published by Wiley Periodicals LLC on behalf of World Aquaculture
Society.
J World Aquac Soc. 2021;52:829–842. wileyonlinelibrary.com/journal/jwas 829
1|INTRODUCTION
The maraena whitefish Coregonus maraena (Bloch 1779) is a commercially and ecologically valuable species showing promise
for inland freshwater aquaculture in eastern, central (Mukhachev & Gunin, 1999), and northern Europe, particularly Finland
(Jobling et al., 2010), Germany (Bochert, Horn, & Luft, 2017), and Norway (Siikavuopio, Knudsen, Amundsen, Sæther, &
James, 2011). Several decades ago, predation by the great cormorant Phalacrocorax carbo (L.) led to dramatic decline in
maraena whitefish populations in Europe (Suter, 1997). Depletion has been exacerbated by overfishing (Jackson et al., 2001),
hybridization (Luczy
nski, Falkowski, Vuorinen, & Jankun, 1992), habitat eutrophication (Thomas & Eckmann, 2007), degrada-
tion of natural spawning sites (Winfield, Fletecher, & James, 2004), pollution, and environmental changes (Walther
et al., 2002). At present, re-establishment of natural whitefish populations must be supported by culture in intensive rec-
irculating aquaculture systems (RASs) (Matousek et al., 2017; Matousek, Stejskal, Prokesova, & Kouril, 2017). The RAS is an
important model for aquaculture worldwide, given its cost-effectiveness and low environmental impact, along with allowing
control of water quality and manipulation of characteristics of the final product (d'Orbcastel, Person-Le Ruyet, Le Bayon, &
Blancheton, 2009). Successful whitefish production in RAS requires identification of optimal larviculture conditions and proto-
cols, including water physicochemical parameters, stocking density, nutrition, and feeding regime (Lahnsteiner & Kletzl, 2015).
Feeding strategies can influence a range of physiological and production parameters (Geng et al., 2019; Lall, Lewis-
McCrea, & Tibbetts, 2018; Orihuela et al., 2018). Farmed fish may display considerable species-specificity in feeding pat-
terns. It is standard practice to start fish larvae on live feed (LF) before weaning to a commercial formulated diet. Brine
shrimp Artemia salina (L.) nauplii comprise approximately 40% of the LF used in aquaculture and are particularly suitable
for hatchery operations, as they can be stored for long periods and are readily available when needed (Lavens &
Sorgeloos, 2000). Feeding on LF is essential to many fish species, including coregonids such as lake whitefish Coregonus
clupeaformis (Mitchill, 1818) (Harris, 1992). Alternatively, commercial dry feed can be used for the first exogenous feeding
of coregonids (Enz, Schäffer, & Müller, 2001; Leithner & Wanzenbock, 2015), usually with nutritional supplementation.
For maraena whitefish, this is generally propionic acid (Lahnsteiner & Kletzl, 2015). Larval feeding on nematodes (Hundt
et al., 2015), rotifers, or a combination of rotifers and Artemia (Bochert et al., 2017) has also been tested in this species.
Diet is closely tied to fish development and has a major influence on welfare and health, a crucial factor in profit-
able aquaculture. The intestine and liver are directly related to digestion and nutrient utilization (de Alcântara
et al., 2019; Yanes-Roca et al., 2018). A fatty liver indicates wasted calories, as there is little benefit to supplying an
energy-yielding nutrient that is deposited and stored unused in adipose tissue (Hansen et al., 2008). On the other
hand, it has been reported that small hepatocytes may indicate reduced lipid and glycogen storage ability. Changes
in hepatocyte histomorphology, such as a reduced number and size of lipid vacuoles, could reflect an inappropriate
feeding regime (Ostaszewska, Korwin-Kossakowski, & Wolnicki, 2006).
Research into effects of diet and feeding approaches on coregonids is critical to their productive culture. The goal of
the present study was to identify feeding strategies optimal for survival, growth, intestine development, and liver status
of maraena whitefish larvae to support intensive culture for commercial exploitation and conservation efforts.
2|MATERIALS AND METHODS
2.1 |Eggs and larvae
Maraena whitefish broodstock were obtained from lagoons in Szczecin in the River Odra, north-western Poland.
Gametes of 35 female and 35 male naturally spawning (no hormone stimulation) fish were stripped manually by com-
mercial fishermen in December 2016 shortly after capture and transported to local hatcheries for fertilization and
incubation. Eggs (100 mg) were fertilized with 0.5 mL milt mixed with 50 mL hatchery water and incubated at the
ambient water temperature of the river (2–3C) with initial water inflow 3 L/min, oxygen saturation to 90%, and pH
near 7.0. In February 2017, the eggs were taken to the Department of Lake and River Fisheries (Olsztyn, Poland)
830 STEJSKAL ET AL.
where they were distributed among five 8-L Zug jars (Sebesta, Kucharczyk, Nowosad, Sikora, & Stejskal, 2018;
Sebesta, Stejskal, Matoušek, & Lundova, K., 2018) (n=150,000 eggs/jar) in a recirculating system and incubated at
3.0–3.5C with water inflow 3 L/min, oxygen saturation to 90%, and pH near 7.0. In total, 750,000 eggs were incu-
bated. After 60 days, eggs were transferred to a second set of 8-L Zug jars and incubated at 8–9C to accelerate
development and hatching. After 5 days, temperature was increased to 10C for mass hatching. Hatching success
was estimated at 90%, and 675,000 larvae were available for the experiment. Hatched larvae swam into a 1 m
3
tank underlain with 0.2 mm mesh. Larvae at 2 days post hatching (dph) were transferred to tanks in the RAS.
2.2 |Experimental system and rearing conditions
Seven groups of larvae in three repetitions were transferred to the experimental system consisting of 21 two-L
tanks, 96 154 200 mm. Two-hundred-fifty larvae (initial body weight, 7.4 ± 0.1 mg, mean ± standard error of
mean (SEM); initial total length, 13.0 ± 0.1 mm) were placed in each tank. A total of 5,250 larvae were used in the
experiment lasting 30 days.
Oxygen level, water temperature, and pH were checked daily at 8.00 and 16.00. The pH range was monitored
using an OXYGUARD H04PP Handy pH meter (OXYGUARD International, Denmark). The initial temperature with-
out supplemental heat was 10C. Temperature was elevated to 15C by 24 hr (0.2C/hr), 19C at 48 hr (0.2C/hr),
and maintained at 19C by an HC-1000A cooler (HAILEA, China). Oxygenation was maintained using two Syncra
5.0 pumps (5,000 L/hr) (SICCE, Italy). Temperature and oxygenation were monitored using probes connected to a
central electronic software program, Pacific Insatech A/S (OXYGUARD, Denmark). Ammonia, nitrate, and nitrite con-
centrations were checked twice weekly using LCK 304, LCK 339, and LCK 341 kits (HACH, Germany) with a
DR5000 spectrophotometer (HACH, Germany). Disinfection used a 30 W UV MCT sterilizer (Transformatoren
GmbH, Germany). Sodium chloride was added at 1 g/L weekly to maintain a 16:1 chloride: nitrogen ratio to prevent
nitrite toxicity. A constant inflow of 0.4 L/min was ensured. Dead larvae were removed and counted during daily
cleaning. Over the course of the 30 day trial, basic physico-chemical parameters were temperature =19.1 ± 0.0C,
pH =8.7 ± 0.0, O
2
saturation =85.8 ± 0.9%, O
2
concentration =7.9 ± 0.1 mg/L, NH
4
+
=0.1 ± 0.0 mg/L,
NO
2
=0.8 ± 0.1 mg/L, and NO
3
=21.2 ± 5.4 mg/L.
2.3 |Feeding
Larvae were fed manually during the light phase (12 hr:12 light:dark) beginning at 2 dph. The artificial feed
(AF) group were fed PERLA LARVA PROACTIVE 4.0 (particle size 0.1 and 0.2 mm) (SKRETTING, Nutreco, Nether-
lands) to excess. The LF group were fed fresh Artemia metanauplii (Ocean HE >230,000, NPG Nutrition, Belgium)
(20–24 h, 0.4–0.5 mm) at 10 mL homogenous suspension/tank at approximately 3-hr intervals (08.30, 11.00, 14.00,
and 16.30). AF was provided manually every 10 min during 4-hr-long feeding periods (08.30–09.30, 11.00–12.00,
14.00–15.00, and 16.30–17.30). This feeding practise was based on the character of the diet, as Artemia metanauplii,
with its swimming ability, color, and enzyme secretions acting as visual and chemical stimuli, extend feeding activity.
As AF has limited attraction, it is advised to present it more frequently. Feeding level was fixed at 500–700 Artemia
sp. metanauplii/fish/day. The daily ration was based on a previous experiment (unpublished data) and was in slight
excess, as some uneaten metanauplii were observed in tanks at the end of the day. The feeding level was adapted
according to fish body weight and loss of larvae during the experiment. The nutritional composition of the commer-
cial diet and Artemia is provided in Table 1.
The experimental feeding strategies (groups) were as follows (Figure 1):
AF—artificial feed throughout the 30 days;
LF—live feed throughout the 30 days;
STEJSKAL ET AL.831
FW5—first weaning from LF to artificial diet after 5 days;
FW10—first weaning from LF to artificial diet after 10 days;
FW15—first weaning from LF to artificial diet after 15 days;
FW20—first weaning from LF to artificial diet after 20 days;
FW25—first weaning from LF to artificial diet after 25 days.
2.4 |Sampling and measuring
Ten larvae from each tank (30 from each experimental group) were randomly taken for measurements of total length
(TL, ± 0.01 mm) and body weight (W, ± 0.1 mg) on days 0, 5, 10, 15, 20, 25, and 30 of rearing, as described by
Łaczy
nska et al. (2016) and Nowosad et al. (2013). Larvae were anesthetized (Propiscin—0.4 mL/L; IRS, Poland),
weighed on a digital microbalance (ABJ 220-4 M KERN, Germany) and measured manually from images taken with a
Leica MZ16 A stereomicroscope and a digital camera with 5 Mp resolution for Leica DFW420 image analysis. The
anesthetized larvae were lain singly on a rectangular net of known weight which was placed on paper to absorb
water. The dried fish and net were placed on a balance with accuracy to 0.1 mg, weighed, and the weight of net was
subtracted to obtain the weight of the fish. Fish sampled for measurements and histological analysis were humanely
killed using anesthetic overdose. To avoid underestimation of final survival rate, these fish were not included in the
calculation of final cumulative survival.
TABLE 1 Nutritional composition of
Skretting feed and Artemia
(manufacturer's data) used for intensive
culture of maraena whitefish larvae
(Coregonus maraena)
Skretting
Particle size Mm 0.1–0.2
Crude proteins g/kg 620
Crude lipids g/kg 110
Crude ash g/kg 90
Crude cellulose g/kg 11
Vit A IU/kg 672
Vit D3 IU/kg 671
Na g/kg 8
Ca g/kg 22
P g/kg 17
MnSO
4
H
2
O mg/kg 69.3
FeSO
4
H
2
O mg/kg 182.4
ZnSO
4
H
2
O mg/kg 369.8
CuSO
4
5H
2
O mg/kg 29.5
KI mg/kg 3.9
Artemia
Artemia size NPG HE >230,000
Crude proteins g/kg 540
Crude lipids g/kg 110
Crude ash g/kg 50
Moisture g/kg 80
832 STEJSKAL ET AL.
Experimentation was carried out in accordance the European Communities Council Directive of November
24, 1986 (86/609/EEC).
The survival rate (SR), size heterogeneity (SH), larval yield (LY), and condition factor (K) were assessed as follows:
SR %ðÞ¼100 Nf=Ni
ðÞ
in which N
i
and N
f
=initial and final number of larvae, respectively.
LY g=groupðÞ¼
Ni
100
SR
W
with SR and W=% surviving and mean W(g) in larva groups, respectively.
SH %ðÞ¼100 SD=WðÞ
in which SH =size heterogeneity; SD =mean standard deviation of body weight of 10 randomly selected lar-
vae/tank; W=mean body weight (mg) of 10 larvae/tank.
K¼100, 000 W=TL3
in which W=mean body weight (g) of 10 larvae/tank; TL =mean total length (mm) of 10 larvae/tank.
2.5 |Histology
Five larvae from each group were sampled for histology on days 0, 5, 10, 15, 20, 25, and 30. Whole larvae were fixed
in Bouin's fluid for 24–48 hr depending on size. The fixed samples were washed in an ethanol series (75, 80,
90, 95%), acetone, xylene, and liquid paraffin at 54C. The obtained material was embedded in paraffin blocks, cut
into 6 μm sections on a rotating microtome (Leica RM 2155), and sections were placed onto protein-coated slides.
The slides were stained with Mayer's hematoxylin and eosin (Baginski, 1965). Subsequently, the stained preparations
were sealed in Histokitt mounting medium (Glaswarenfabrik Karl Hecht GmbH & Co KG, Germany). After drying, the
preparations were examined microscopically (Axio Scope A1, Zeiss, Germany) with AxioVs40 v. 4.8. 2.0 software
(Carl Zeiss MicroImaging GmbH, Germany).
FIGURE 1 Feeding regimen of live and artificial feed for maraena whitefish Coregonus maraena larvae in a 30-day
trial
STEJSKAL ET AL.833
Five larvae from each group were photographed, and intestine diameter (ID), villi length (VL), villi thickness (VT),
hepatocyte nucleus diameter, and hepatocyte diameter (HD) were measured. The measurements were compared
among groups on days 5, 10, 15, and 20. At the completion of trial, the presence of intestine and liver pathology was
assessed and compared using criteria of McFadzen, Coombs, and Halliday (1997) to categorize liver condition. Each
specimen was assigned a grade (1–3), with a healthy specimen scoring 1 to degraded liver scoring 3 (Table 2). Intes-
tine degradation was evaluated, and each fish was assigned a grade (-, +,++,+++), from healthy to severe degrada-
tion (Table 3).
2.6 |Statistical analysis
The data are presented as mean ± SEM. Statistical analyses were conducted using STATISTICA 12.0 (StatSoft, Praha,
Czech Republic). The effects of feeding strategy on W, TL, SR, LY, SH, K, ID, VL, VT, ND, HD, and IIS were analyzed
by one-way ANOVA with feeding as fixed variable. The level of significance used for all tests was α=.05
(Zar, 1999). Prior to ANOVA, survival percentages were arcsin-transformed. All data were tested for homogeneity of
variance using the Cochran, Hartley, and Bartlett test, and for normality with the Shapiro–Wilk normality test.
Tukey's test was used for identifying significant differences among groups. All applicable international, national, and
institutional guidelines for the care and use of animals were followed by the authors.
3|RESULTS
3.1 |Growth performance, survival, size heterogeneity, condition factor, yield
At the conclusion of the trial, the highest values of W(171.4 ± 8.9 mg), TL (32.2 ± 0.3 mm), LY (23.1 ± 1.24 g/tank),
SH (28.4 ± 2.0%), and K(0.51 ± 0.01) were observed in the FW15 group (Figure 2, Table 4). The W(p=.00083), TL
(p=.00052), and LY (p=.00075) differed significantly among some groups, while no significant differences were
observed in SH (p=.317), K (p< .146), and SR (p< .658) (Table 4). Significantly greater W(p< .05) was observed in
FW15 compared to LF, AF, FW5, and FW10 and in FW20 compared to the AF and FW5 groups. Similarly, signifi-
cantly greater TL (p< .01) was observed in FW15 and FW20 in comparison with the LF, AF, FW5, and FW10 groups.
The LF group showed the greatest growth/body weight at the first 20 days of rearing, and AF showed poorest
TABLE 2 Classification of liver and intestine degradation in maraena whitefish Coregonus maraena larvae
Tissue
Grade
1. Healthy 2. Intermediate 3. Degraded
Liver nuclei Nuclei lightly granular, small
and indistinct
Nuclei with abundant dark
granules; nucleoli
Nuclei small dark and pyknotic
Liver
hepatocyte
cytoplasm
Structured: Varied texture,
scattered granules with eosin
positive patches
Homogenous, granular, slight
variability in staining
property
Hyaline, lacking texture, dark
small and often separated
from the cell boundary
Intestine
mucosa
Enterocytes intact, villi with
deep, longitudinal folds,
cytoplasm homogenous, no
vacuolation, microvilli intact
Separation of enterocytes in
basal region, coarse dark
cytoplasm, frequent areas of
microvilli degeneration
Enterocytes small dark and
separated, extensive
intercellular cells may be
present, microvilli often
indistinct
Note: Adapted from McFadzen et al. (1997).
834 STEJSKAL ET AL.
TABLE 3 Classification of degradation and histomorphometry of intestine of maraena whitefish Coregonus maraena larvae at the end of a 30-day trial
Lesion Groups
LF AF FW5 FW10 FW15 FW20 FW25
Hyperplasia of mucosa +++++++++
Villus oedema +++ ++ + + + -
Exfoliation of intestine epithelium ++++++++-
Intestine diameter (μm) 629.3 ± 18.30 690.1 ± 23.24 717.6 ± 32.42 744.9 ± 58.31 686.7 ± 82.21 646.7 ± 11.92 672.0 ± 22.46
Length of villi (μm) 148.4 ± 1.83
ab
133.9 ± 4.10
a
151.4 ± 5.21
ab
136.2 ± 5.53
a
163.5 ± 9.48
ab
152.9 ± 9.71
ab
176.6 ± 9.03
b
Width of villi (μm) 54.5 ± 2.42 52.4 ± 3.01 58.2 ± 1.79 56.5 ± 3.04 54.5 ± 1.12 51.1 ± 3.39 53.1 ± 0.74
Intestine injury score 0.18 ± 0.05
a
2.03 ± 0.41
b
0.49 ± 0.05
a
0.22 ± 0.05
a
0.18 ± 0.04
a
0.17 ± 0.03
a
0.06 ± 0.02
a
Note: Histomorphometry parameters indicate mean ± SEM (n=3). Different letters indicate significant differences (p< .05). Degradation score: hyperplasia of mucosa, villus oedema, and
exfoliation of intestine epithelium ranges from - (none) to +++ (severe). Intestine injury score was calculated using information presented in Table 2. First weaning (FW) from live diet to
a commercial diet at 5 days (FW5), 10 days (FW10), 15 days (FW15), 20 days (FW20), and 25 days (FW25).
Abbreviations: -, none; +, mild; ++, moderate; +++, severe.
STEJSKAL ET AL.835
results over the course of the trial. The Wand TL increments in 5-day periods are shown in Figure 2. Significantly
higher LY (p< .01) was obtained in FW15 compared to LF, AF, FW5, and FW10, and in FW20 compared to AF.
3.2 |Histology
Significantly lower ID was observed in AF compared to LF (p=.0026) and FW5 (p=.0063) on Day 10, as well as in
AF compared to LF (p=.022), FW5 (p=.0065), and FW10 (p=.0012) on Day 15. Significantly longer villi were
observed in LF compared to AF (p=.00018), FW5 (p=.0030), and FW10 (p=.0035) and in FW5 (p=.0013) and
FW10 (p=.00025) compared to AF on Day 15. Significantly greater villi width was observed in LF compared to AF
(p=.019) and FW15 (p=.019) on Day 20 (Figure 3). At the conclusion of the 30-day trial, the LF, FW5, FW10, and
FW15 groups exhibited no serious intestine degradation. The AF group was the only treatment to receive a (+++)
grade on any aspect of intestinal degradation scoring (Table 3 and Figure 4).
Significantly greater hepatocyte nucleus diameter was observed in AF compared to FW5 (p=.040) on Day 10
and in LF compared to FW10 (p=.0066) on Day 20. Significantly greater HD was observed in LF compared to AF
on Day 5 (p=.0018), and in AF compared to LF (p=.00022), FW5 (p=.00019), FW10 (p=.0085), and FW15
(p=.0091) on Day 20 (Figure 3). Over the 30-day trial, liver of fish from all groups was normal (Grade 1).
4|DISCUSSION
The present study reported a feeding strategy in which LF (Artemia) was applied for 15 days with subsequent abrupt
weaning to dry feed as favorable to maximize growth and survival rate in Coregonus maraena larvae. Larva survival
FIGURE 2 Mean body weight (mg) and mean total length (mm) of maraena whitefish Coregonus maraena (Bloch
1779) at 5-day intervals during a 30-day feeding strategy trial. First weaning (FW) from live diet to a commercial diet
at 5 (FW5), 10 (FW10), 15 (FW15), 20 (FW20), and 25 days (FW25). LF and AF indicate groups with live and
artificial feed, respectively. Different letters indicate significant differences (p< .05). Bars represent means, and
whiskers indicate standard error of mean (SEM) of three replicates (n=3)
836 STEJSKAL ET AL.
TABLE 4 Effects of feeding strategy on growth and survival of maraena whitefish Coregonus maraena larvae in a 30-day growth trial
Group LF AF FW5 FW10 FW15 FW20 FW25 SS df F MS p
SH (%) 22.5 ± 1.0 22.7 ± 3.1 23.9 ± 2.5 18.4 ± 1.5 28.4 ± 2.0 23.0 ± 1.2 25.7 ± 5.0 168 6 28 1 .32
K0.49 ± 0.01 0.47 ± 0.02 0.47 ± 0.01 0.49 ± 0.01 0.51 ± 0.01 0.48 ± 0.01 0.50 ± 0.01 0 6 0 2 .13
SR (%) 90.5 ± 0.3 85.8 ± 1.1 90.0 ± 2.1 90.2 ± 0.4 90.8 ± 2.0 89.7 ± 1.0 88.2 ± 2.9 42 6 7 0 .66
LY 17.3 ± 0.73
ab
15.2 ± 0.62
a
16.9 ± 1.19
ab
17.2 ± 0.85
ab
23.1 ± 1.24
c
20.7 ± 0.32
bc
18.7 ± 1.27
abc
127 6 21 7 0
Note: Data are means ± SEM. Identical letters indicate no significant differences (p> .05) among groups. First weaning (FW) from live diet to a commercial diet at 5 days (FW5), 10 days
(FW10), 15 days (FW15), 20 days (FW20), and 25 days (FW25).
Abbreviations: df, degrees of freedom; F, distribution fitting; factor parameter: FS, feeding strategy; K, condition factor; LY, larva yield, MS, mean square; SH, final size heterogeneity; SR,
survival rate; SS, sum of square.
STEJSKAL ET AL.837
and growth are affected by starter feed, which must satisfy nutritional needs immediately after depletion of the yolk
sac (Puvanendran & Brown, 1999), and feed composition and feeding strategy are of critical importance (Lee, 2003).
The timing of weaning is considered to be the most important factor in successful larva feeding in peled Coregonus peled
(Gmelin) (Stejskal et al., 2017), pikeperch Sander lucioperca (L.) (Hamza, Mhetli, & Kestemont, 2007), totoaba Totoaba
macdonaldi (Gilbert) (Mata-Sotres, Lazo, & Baron-Sevilla, 2015), burbot Lota lota (L.) (Pali
nska- _
Zarska et al., 2014), golden
pompano Trachinotus ovatus (L.) (Ma et al., 2015), fine flounder, Paralichthys adspersus (Orihuela et al., 2018), Japanese
flounder, Paralichthys olivaceus (Geng et al., 2019), and butter catfish Ompok bimaculatus (Bloch) (Pradhan, Jena, Mitra,
Sood, & Gisbert, 2014). The majority of these reports also described a positive effect of LF for initial feeding, and exclu-
sive use of starter diets in early stages of rearing is often suggested to have negative effects on later development
(Bochert et al., 2017). This is supported by Leithner and Wanzenbock (2015) who observed dramatically reduced sur-
vival rate at 30–40 dph when feeding dried feed only in different strains of Coregonus lavaretus.Resultswerecom-
promised by unidentified disease in some groups near the end of their experiment. A similar dramatic increase in
mortality from 30 to 40 days of rearing was observed by Esmaeilzadeh-Leithner and Wanzenböck (2018). Importance
of using LF during early phases of larval rearing is also highlighted by Bochert et al. (2017).
We found no significant differences in SR, SH, and Kamong the feeding regimes. The SR of larvae fed the com-
mercial diet was lower than in the other groups, but did not reach significance. This was also observed by
Mahmoudzadeh, Ahmadi, and Shamsaei (2009), who reported that larvae fed dry feed showed comparable SR to
those fed a live and a live/artificial mixed diet during the first 4 weeks. Bochert and Luft (2019) found superior sur-
vival in C. maraena larvae fed exclusively with dry feed compared to those receiving Artemia and a mixture of live
and dry feed from the initiation of exogenous feeding, irrespective culture temperature (6–20C). A similar feeding
technique with Artemia only for the first phase of rearing, followed by a cofeeding 50% Artemia and 50% dry feed
and a final transition to dry feed only, is used in Atlantic whitefish Coregonus huntsmanui as described in a rearing
handbook (Whitelaw et al., 2015).
The survival rate of the most successful group in the present study (FW15) was higher than other reported
results (Beltran & Champigneulle, 1992; Bochert & Luft, 2019; Leithner & Wanzenbock, 2015) for a comparable
length trial. Ostaszewska et al. (2018) reported considerably lower survival at the end of a 35-day experiment
(82.9%) for a feeding strategy similar to FW15 in the present study. Our higher survival rate (91.8%) could imply an
effect of the commercial feed used. Survival rate in the present study was also considerably higher than reported for
C. peled (Stejskal et al., 2017, Matousek et al., 2020), as a related species.
At the end of our trial, significantly higher larva TL, W, and LY (p< .05) were observed in FW15 and FW20 com-
pared to other treatments. The LF group showed highest length and body weight values during the first 20 days of
the trial, and AF produced inferior results throughout the trial. Our results are similar to those of Bochert
FIGURE 3 Intestine diameter (μm), vili width (μm), vili length (μm), mean hepatocyte diameter (μm), and mean
nucleus diameter (μm) in maraena whitefish Coregonus maraena (Bloch 1779) at 5-day intervals during the 30-day
feeding trial. First weaning (FW) from live diet to a commercial diet at 5 (FW5), 10 (FW10), 15 (FW15), 20 (FW20).
LF and AF indicate groups with live and artificial feed, respectively. Different letters indicate significant differences
(p< .05). Bars represent means and whiskers indicate SEM (n=3)
838 STEJSKAL ET AL.
et al. (2017) at 30 days, who reported enhanced growth in maraena whitefish larvae fed live Artemia nauplii at first
feeding. Fish fed Artemia 6–16 dph and AF 17–42 dph displayed the highest TL and Wfrom Day 7 to Day 42. With
respect to growth, our results are superior to those obtained by Bochert et al. (2017). Growth of fish reared at low
water temperatures (Leithner & Wanzenbock, 2015) has been reported lower compared to findings of the present
study. Hundt et al. (2015) confirmed the highest growth at 17 dph in European whitefish larvae fed Artemia com-
pared to a dry diet or live nematodes. Stejskal et al. (2017) observed the lowest Wwith AF in all weekly increments
from 7–35 dph, with LF producing the highest Wvalues, except at 28 and 35 dph. Fish weaned at 20 and 25 dph
tended to have lower final body weight compared to those weaned at 15 days. A similar phenomenon was observed
by Ostaszewska et al. (2018).
A study of the characid black tetra Gymnocorymbus ternetzi reported successful larva weaning prior to gastric dif-
ferentiation, which results in reduced dependence on Artemia during early stages of rearing (Lipscomb, Yanong,
Ramee, & DiMaggio, 2020). Interaction of intestine and liver function is assumed to be a key factor for growth and
welfare of farmed fish. Histological examination revealed the most severe intestine degradation (Grade 3; IIS =2.03)
in the AF group, corresponding to the lowest ID, VL, and VT (Table 3). The AF group also produced the lowest
growth, survival, and larval yield (Table 4).
The ID, as well as VL and VT, displayed a trend to higher values with LF and later weaning time, in conjunction
with higher growth and survival in these groups. This may be attributed to the digestive enzymes obtained from
LF. However, it remains to be clarified whether the digestibility of dry diets is comparable to that of live diets.
It has been reported that production of specific pancreatic digestive enzymes occurs at different times after the
onset of first feeding and that their activity increases rapidly. Appearance of gastric enzymes is linked to develop-
ment of the stomach toward the end of the larval period and, hence, occurs later as reported for pigfish Orthopristis
chrysoptera (Thompson, Faulk, & Fuiman, 2019).
In our investigation, no group presented evidence of liver pathology (Table 2), and all showed a level of fat deposit
within the normal range. Ostaszewska et al. (2018) also did not find significant pathologies using similar feeding tech-
niques, but a different diet (OtohimB1, Red Mariculture, USA). Escaffre and Bergot (1986), in a study of rainbow trout
Oncorhynchus mykiss (Walbaum), reported that the diameter of hepatocyte nuclei reflects the nutritional status of the
fish. Segner, Rösch, Schmidt, and Jürgen von Pocppinghausen (1988) stated that European whitefish Coregonus lararetus
(L.) larvae fed on zooplankton exhibited the largest nuclei, with those of larvae reared on dry diets being significantly
smaller. This was confirmed by Ostaszewska et al. (2018). In our study, the hepatocyte nucleus diameter was similar
among tested groups, and no significant differences (p> .05) were found in larvae fed LF compared to AF. LF may stim-
ulate liver metabolic activity, in particular protein metabolism, which enhances growth of maraena whitefish larvae.
FIGURE 4 Intestine of maraena whitefish Coregonus maraena (Bloch 1779) larvae after 30-day feeding trial. (a) LF
(live feed), (b) AF (artificial feed), (c) FW5 (first weaning from live feed to a commercial dry diet at Day 5). (a) healthy
(intestine injury score =0.18), (b) moderate damage (intestine injury score =2.03), and (c) slight damaged (intestine
injury score =0.49)
STEJSKAL ET AL.839
We used a feeding regime in which we manually provided dry food at 28 feedings per day during the first
30 days of rearing. This is labor intensive and may be excessive. Further research should be aimed at establishing
optimal feeding frequency during European whitefish larval stage, as such frequent offering of dry feed may bring
unsustainable labor costs.
Present study investigated a single artificial diet in combination with Artemia. However, it was demonstrated
that different formulations of artificial diets can provide significantly different results during larval rearing as demon-
strated for Gulf killifish Fundulus grandis (Patterson et al., 2016). Therefore, next research should be focused on com-
parison of performance available commercial diets in early rearing protocols for C. maraena.
5|CONCLUSIONS
The reduction or elimination of Artemia from the early feeding protocol for maraena whitefish would be economically
advantageous. However, this 30-day investigation shows initial weaning from LF to an artificial diet after 15 days to
be the optimal strategy for beneficial effects on growth, body weight, and yield. Efficacy of other tested feeding
strategies can be ranked FW20 > FW25 > FW10 > LF > FW5 > AF. LF and appropriate time of weaning to artificial
diet is beneficial for intestine development, while a diet consisting of only AF is associated with severe intestine
impairment. Live/dry feeding strategies are not associated with liver pathology.
ACKNOWLEDGMENTS
The study was financially supported by the Ministry of Agriculture of the Czech Republic, project NAZV project
(QK1810296).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ORCID
Vlastimil Stejskal https://orcid.org/0000-0002-7892-695X
Tatyana Gebauer https://orcid.org/0000-0002-4943-1923
Joanna Nowosad https://orcid.org/0000-0001-9491-0141
Dariusz Kucharczyk https://orcid.org/0000-0002-0889-0656
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How to cite this article: Stejskal V, Gebauer T, Sebesta R, et al. Effect of feeding strategy on survival, growth,
intestine development, and liver status of maraena whitefish Coregonus maraena larvae. J World Aquac Soc.
2021;52:829–842. https://doi.org/10.1111/jwas.12785
842 STEJSKAL ET AL.
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