Aquaculture is intensifying in almost all regions of the world in order to supply the increasing demands for fish and shrimp.

1. March | April 2014
Poultry hydrolysates enhance stress
resistance and stress tolerance in Pacific
white shrimp
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3. A
quaculture is intensifying in
almost all regions of the world
in order to supply the increasing
demands for fish and shrimp.
In Thailand the average stocking den-
sity has quadrupled from approximately 40
shrimps/m2 to almost 160 animals/m2.
However, this intensification is often com-
bined to sub-optimal conditions for fish and
shrimps. Problems associated with higher
disease susceptibility have to be faced.
Some of these stress conditions may
be compensated by aquaculture production
practice and/or by an increase use of pharma-
ceuticals and antibiotics which in turn affect
food safety.
A ban of antibiotic growth promoters in
many importing countries has forced the indus-
try to search for environmentally friendly alter-
natives. And a natural solution to combat stress
conditions is the use of hydrolyzed proteins.
Fish protein hydrolysates (FH) are consid-
ered as ‘biological active ingredients’ due to
their beneficial properties to be antioxidative
or antimicrobial (Klompong et al. 2007).
The use of FH in diets has proven to
improve growth and performance in fish
for example, Atlantic salmon (Berge and
Storebakken, 1996),
catfish (Herault et
al. 2012) and tiger
prawns P. monodon
(Anggawati et al.
1990) by enhancing
stress resistance and
immunity. Anggawati
et al. (1990) found that
three percent FH was
enough to enhance
shrimp growth.
The immune
boosting effect of FH
is reported due to it´s
content of peptides,
free amino acids and nucleotides. Although
these peptides can be found in FH, they are
not restricted to marine proteins, only.
Poultry protein derivatives, especially
hydrolyzed poultry proteins, are (as FH) rich
in bioactive peptides, which are able to com-
pensate stress conditions.
The benefits of poultry peptides
The article describes the beneficial effects
of poultry peptides in respect of enhancing
stress resistance/tolerance and immunity in
Pacific white shrimps (Litopenaeus vannamei)
under controlled conditions.
Under controlled laboratory conditions
hydrolyzed poultry peptides (enzymatically
hydrolyzed liver (eL), enzymatically hydro-
lyzed meat & bone-mush (eMB)) have been
tested to see the effect on growth perform-
ance in shrimp. The properties of those
peptides are given in Figure 1, indicating the
small molecular size distribution.
Poultry hydrolysates enhance stress resistance
and stress tolerance in Pacific white shrimp
by Orapint Jintasataporn Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand and Franz-Peter
Rebafka, GePro Gefluegel-Protein Vertriebsgesellschaft mbH & Co. KG, Germany
table 1: Composition of the experimental diets
raw material Control el eMB
Fishmeal, tuna 30 7 7
Soybean 9 34 34
Shrimp meal 15 4 4
Squid meal 5 1 1
Wheat flour 31.4 29.2 28.85
Hydrolyzed poultry
liver
0 5 0
Hydrolyzed poultry
meat & bone
0 0 5
Soy protein isolate 0 6 6
tuna fish oil 3 3 3
Soya oil 2 2.75 3.1
Squid liver oil 1 1 1
Met + lys 0 0.45 0.45
Mono-cal 1 4 4
Binder 1.7 1.7 1.7
Vitamin-mineral
premix
0.9 0.9 0.9
14 | InternAtIonAl AquAFeed | March-April 2014
FEATURE

4. The trial was conducted in June 2012 at
Kasetsart University, Bangkok, Thailand, by the
Department of Aquaculture, Faculty of Fisheries,
in a complete randomised design (CRD) with
three treatments and four replicates.
Shrimps were fed with isonitrogenous
diets (see Table 1), including the control (50
percent marine protein) and the test diets
with either five percent hydrolyzed poultry
liver or with five percent hydrolyzed poultry
meat & bone-mush.
Pacific white shrimps (Litopenaeus van-
namei) of 3.1+-0.1g were stocked at a density
of 70 animals/m2 in a 240 litre glass container
(aquarium) in brackish water of 12ppt. During
an eight week period, feed was given three
times per day to match 2-2.5 percent body
weight. Two hours after feeding, the uncon-
sumed feed was collected to determine total
feed consumption.
Growth performance was evaluated every
two weeks by collecting data on average daily
gain, feed consumption, feed conversion ratio
and survival rate.
An acute salinity and chemical stress test
with a high or low level of salinity and toxic
concentration of nitrite (NO2-) and ammonia
(NH3+) was conducted at the end of four-
week feeding period.
Under salinity stress 30 shrimps per treat-
ment were transferred from a tank with 12ppt
water salinity to tanks of 20ppt for one week
March-April 2014 | InternAtIonAl AquAFeed | 15
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5. and then moved to with 0ppt or 40ppt saline
water in separate trial for stress test.
In the chemical stress test, 30 shrimps
per treatment were transferred to a tank
with 20ppt salinity for one week and a toxic
concentration of ammonia of 50+- 2ppm
(pH7-8) and – in a separate trial – of nitrite of
20+-2ppm, respectively.
Nitrite and ammonia concentrations in the
water were controlled daily to maintain the
toxic concentrations. Shrimps were fed once
a day. Mortality rates were recorded daily
during a 10-day test phase.
Typical immunological parameters, such
as hemolymp protein, total hemocyte cell
count, phenol oxidase activity and percentage
of oxyhemocyanin (live cell hemocyte) were
measured. Mortality were recorded daily dur-
ing a 10-day test phase.
After feeding shrimp with the experimental
feed for four weeks, 30 shrimp from each
treatment were collected to challenge with
White Spot Syndrome Virus (WSSV) and
– in a separate trial – with Vibrio harveyi by
subcutaneous injection. Mortality rate was
determined daily for a 10-day test phase.
The study was conducted in completely
randomised design (CRD). All data were ana-
lysed by one-way ANOVA. The significance of
differences between means was tested using
Duncan´s Multiple Range Test at a 95 percent
level of confidence (p<0.05).
Shrimp did better
Growth performance of shrimp fed with
enzymatically hydrolyzed liver (eL) and meat
& bone mush (eMB) tended to be better
than fed with the control diet (p=0.0787); the
best growth performance was achieved by
feeding shrimp with hydrolyzed poultry liver
(see Table 2).
Feed utilisation in term of total feed
consumption, feed conversion and survival
rate were not significantly different from the
control feed containing 30 percent fish meal,
15 percent shrimp and five percent squid meal
(see Table 1).
Shrimp fed hydrolyzed poultry meat &
bone showed the best hemolymp protein
and total hemocyte cell count (p<0.05)
(see Table 3 and Figures 2a/b).
Environmental factors
Environmental factors, particularly salin-
ity, nitrite (NO2-), nitrate (NO3-),
ammonia (NH3+) and diseases (WSSV,
Vibrio) affect the immunity of L. van-
namei shrimps. When shrimps are main-
tained under unsuitable environment
over a longer period, or come under
stress, the immune system is depressed
and shrimps become more susceptible
to disease.
The addition of hydrolysed poultry
protein can promote growth perform-
ance, enhance immune responses and
decreased mortality in shrimp stressed
by high and low salinity, poor chemi-
cal water quality and diseases such as
White Spot Syndrome Virus (WSSV)
and Vibrio harveyi.
Due to the high content of low
molecular weight compounds, includ-
ing nucleotides, free amino acids and
bioactive peptides, hydrolyzed poultry
derivatives are improving aspects of
feed transformation, anti-oxidation and
immunity. As a result, growth rate and
health status is enhanced.
16 | InternAtIonAl AquAFeed | March-April 2014
FEATURE

6. The activity of hemocyte cell in terms of
phenol oxidase activity and percentage of
oxyhemo-cyanin (live cell hemocyte), was
highest (P<0.05) in shrimps fed by marine
protein (fish-, squid-, shrimp-meal) (see
Table 3).
Under both high (40ppt) and low salini-
ty (0ppt) stress, the best immune response
was shown by shrimps fed hydrolysed
poultry meat and bone (see Figures 3 and
4). In low salinity the mortality (for 96
to 240 hours) was highest
(p<0.05) with shrimps fed
on fishmeal (the control
diet) (see Figure 3).
Under high salinity mor-
tality was not significant dif-
ferent among diets used (see Figure 4).
Shrimps under both high nitrite (20ppm)
or ammonia (50ppm) stress test conditions
showed the best immune response after
being fed with hydrolysed poultry liver
and hydrolyzed meat & bone, respectively
(see Figures 5 and 6). The mortality rate
after nitrite stress test (up to 240 hours)
was highest (p<0.05) in the control group
(fed with marine protein); a significant
lower (10 times lower) mortality rate was
observed in shrimps fed on hydrolysed
poultry protein (see Figure 5).
Ammonia stress test (up to 240 hours)
was in the same range (p>0.05). All shrimp
die after 96 hours (see Figure 6).
Infection with Vibrio harveyi decreased
the total homocyte count while shrimps
fed on a diet with hydrolyzed poultry prod-
ucts were able to maintain total hemocyte
count.
Interestingly, the mortality rate after
Vibrio challenge (up to 240 hours) was
highest in shrimps fed on marine protein
(100 percent mortality) and lowest in
shrimps fed with hydrolyzed poultry
liver (60 percent mortality) (see Figure
7).
table 2: Growth performance and feed utilization of white
shrimp fed with hydrolyzed poultry liver (el) and meat & bone
(eMB), respectively
Control 5% el 5% eMB P-value
Production after 8
wks (g/aq)
230.1b
±
13.40
270.8a
± 20.64
267.4a
± 13.20
0.0089
average daily
weight gain at 8
wks (g/ind/d)
0.08a
± 0.01
0.10a ±
0.01
0.091 ±
0.01
0.0787
total feed intake at
8 wks (g/ind/aq
189.7a
±
20.28
222.0a
± 15.60
215.1a
± 20.14
0.1072
Daily feed intake
at 8 wks (g/ind/d)
0.11a
± 0.01
0.12a ±
0.01
0.12a ±
0.01
0.5412
Feed conversion
ratio at 8 wks
1.35a
± 0.10
1.25a ±
0.06
1.30a ±
0.07
0.4025
Survival rate at 8
wks (%)
87.14a
± 6.80
90.71a
± 5.89
93.57a
± 3.60
0.4659
table 3: Immune status of white shrimp fed hydrolyzed poultry proteins
(el=hydrolyzed liver; eMB=hydrolyzed meat & bone) at normal condition
Parameter Control el eMB p-value
total hemolymph
protien (g/dl)
4.50b ±
0.20
6.88a ±
0.62
6.61a ±
0.25
0.0001
Phenol oxidase
activity (unit/min/
mg protein)
169.2a ±
4.12
109.2b ±
10.66
118.8b ±
2.92
0.0001
oxyhemocyanin
(%)
22.3a ± 2.5
15.25b ±
0.74
21.96a ±
4.47
0.0586
total hemocyte
count (x106 cell/
ml
4.63b ±
0.41
4.91b ±
0.15
6.05a ±
0.23
0.0006
March-April 2014 | InternAtIonAl AquAFeed | 17
FEATURE

7. The immune responses of shrimp
after challenged with white spot syn-
drome virus (WSSV) showed that the
group of shrimp fed hydrolysed poultry
products had better immune response
than control. The mortality rate was 100
percent after 72 hours in all treatments
(see Figure 8).
An important sources of
protein and energy
According to the result from this study, it can
be concluded that growth of shrimps, immune
responses and mortality rate induced by chemical
stress and disease challenge are related to the
usage and uptake of hydrolysed proteins and
peptides.
Generally, rendered poultry by-products
are important sources of protein and energy.
The enzymatic hydrolysis is widely used
to improve and enhance nutritional and func-
tional properties of feedstuff. During the
enzymatical hydrolyses poultry protein is con-
verted into free amino acids, short peptides
and small molecular proteins.
Based on their good functional properties
and nutritive value, these ingredients are able
to replace or complete fishmeal-based diets
in fish and shrimp feed. (Aguila et al., 2007;
Huong et al., 2012).
Several studies have described the anti-
oxidant activity of protein hydrolysates from
chicken (Wu et al., 2005; Rosa et al., 2008),
tuna liver (Je et al., 2009), sardinelle by-prod-
ucts (Bougatef et al., 2010), backbone of Baltic
cod (Zelechowska et al., 2010) and marine
skin gelatins (Alemán et al., 2011).
Furthermore, free amino acid and small
molecular weight compounds released during
the hydrolysis might also act as feed attract-
ants for shrimps, increasing feed intake, weight
gain and enhance shrimp immunity (Hardy,
1991).
When shrimp are exposed to high ammo-
nia and nitrite concentration in water, the
immune response is depressed and mortality
is increased.
Hence, robust and healthy shrimp show
a higher immunity resistance by expressing
typical immune parameters, such as hemo-
lymp protein, total hemocyte cell count,
phenol oxidase activity and percentage of
oxyhemocyanin (live cell hemocyte) than
weaker ones.
Althought L. vannamei can adapt to a wide
range of salinity, shrimps are more susceptible
to ammonia toxicity. Shrimps under low salin-
ity spend more energy to compensate their
additional energy demand for regulation of
the osmotic pressure (osmoregulation)(Liu
Chun-Hung et al., 2004; Li et al., 2007).
The results are in accordance with previ-
ous studies by Kvale et al. (2002) with pre-
digested protein in Atlantic halibut and Cahu
et al. (1999) describing effects of hydrolysed
protein in sea bass, revealing that survival in
fish can be improved by supplementing (fish)
protein hydrolysates in the diet.
In conclusion, poultry hydrolysates are
able to enhance stress resistance/tolerance in
Pacific white shrimp (Litopenaeus vannamei)
under salinity change, chemical stress and
disease challenge.
More inforMation:
GePro Gefluegel-Protein
Vertriebsgesellschaft mbH & Co. KG
Im Moore 1
49356 Diepholz
Germany
Website: www.ge-pro.de
Email: franz-peter.rebafka@ge-pro.de
18 | InternAtIonAl AquAFeed | March-April 2014
FEATURE

8. March-April 2014 | InternAtIonAl AquAFeed | 19
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The Role of prebiotics in
Pangasius production
Poultry hydrolysates enhance stress
resistance & stress tolerance
– in Pacific white shrimp
Volume 17 Issue 2 2014 - m ARCH | APRIl
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