Global farmed crustacean production has been increasing at much faster rate than major farmed species. It is expected to reach 6.8 million metric tons (MMT) in 2015, of which, 69 percent or 4.7 MMT will be from the marine shrimps (Figures 1A, 1B). In the last 15 years, the production increased six-fold driven more by intensification rather than expansion of the area cultivated.

1. G
lobal farmed crustacean pro-
duction has been increasing at
much faster rate than major
farmed species. It is expected
to reach 6.8 million metric tons (MMT) in
2015, of which, 69 percent or 4.7 MMT will
be from the marine shrimps (Figures 1A,
1B). In the last 15 years, the production
increased six-fold driven more by intensi-
fication rather than expansion of the area
cultivated.
The development of specific pathogen
free (SPF) broodstock of Pacific white shrimp
has been the major contributing factor to this
intensification, and the accompanied spectacu-
lar growth. This has resulted in a much higher
demand for compounded feed. The current
demand for feed is estimated to be 6.8 MMT
or 90 percent of the production. This is about
four times higher than 1.75 MMT or 77 per-
cent of the production in 2000 (Tacon and
Metian, 2015).
However, the over-
intensification and improp-
er management, the lack
of proper knowledge on
nutritional requirements at
various stages and over or
under formulation, and the
increasing use of poorly
digestible and poorly char-
acterised protein sources
while replacing fishmeal,
usually cause unwanted
stress and poor stress
responses in the shrimps.
As a result, occasional dis-
ease outbreaks have been
very common creating
considerable economical
losses in many countries.
The addition of dietary
protease is considered to be an important
tool for better nutrient utilisation. It is hypoth-
esised that a better nutrient profile and utilisa-
tion can compensate poor immune or stress
responses. Specially, when high quality ingre-
dients are partially or completely replaced by
more economical protein sources, which are
often of poor quality.
The study
The study, consisting of five treatments,
was conducted at the Guangdong Ocean
University of China. The objective of the
study was to assess the changes in immune
response to low fishmeal diets supplemented
with a commercially available dietary protease.
Two diets were prepared: one with 20
percent fishmeal (positive control) and the
other with 10 percent fishmeal (negative con-
trol). The fishmeal was replaced by increas-
ing peanut meal (16 percent) and soybean
meal (28 percent) contents compared to 11
percent and 16 percent, respectively from
the positive control diet for equal amount of
crude protein. Another three diets were pre-
pared by adding graded level (125, 150 and
175 ppm) of a commercial protease complex
(Jefo Nutrition Inc., Canada) to the mash of
the negative control diet.
The activities of acid (ACP) and alkaline
(AKP) phosphatases, superoxide dismutase
(SOD) and polyphenol oxidase (PO) were
analysed in both serum and hepatopan-
creas of 15 shrimps per dietary treatment.
The malon-di-aldehyde (MDA) content was
analysed only in serum. Regression analysis
was performed to assess the effects of
the intake of dietary protease on these
responses.
No differences in enzymatic activities and
MDA contents were observed between the
shrimps fed the 20 percent fishmeal diets and
the 10 percent fishmeal diets supplemented
with 175 ppm protease. The results of the
regression analysis with protease intake (µg/
shrimp) at 0, 125, 150, 175 ppm of the 10
by Hoongli Song, Guangdong Ocean University, Zhanjiang, China, Yao Liu, Nutritech Solutions Inc., Shanghai,
China, Xiao-hui Dong, Guangdong Ocean University, Zhanjiang, China and M. A. Kabir Chowdhury, Jefo Nutrition
Inc., Saint-Hyacinthe, Quebec, Canada
Dietary protease improves immune responses in
Pacific White Shrimp, Litopenaeus vannamei
IMMUNE RESPONSE
Table 1. Regression of the protease intake per shrimp with various immune parameters and 96-h cumulative mortality of
shrimps fed graded level of protease
Min Max Adj R2 MSE t-value
Serum Acid phosphatase - ACP (KAU/ml) 2.6 5.9 72% 0.37 6.28***
Alkaline phosphatase - AKP (KAU/ml) 2.6 6.6 70% 0.55 5.9***
Superoxide dismutase - SOD (U/ml) 345.9 411.1 75% 73.43 6.8***
Phenol oxidase - PO (U/ml) 515.7 958.2 60% 8230.00 4.9***
Malon Di-Aldehyde - MDA (nmol/ml) 9.1 15.8 60% 2.40 -4.8***
Min Max Adj R2 MSE t-value
Hepato-pancreas Acid phosphatase - ACP (KAU/ml) 55.9 132.9 46% 234.00 3.7***
Alkaline phosphatase - AKP (KAU/ml) 73.7 170.6 28% 556.00 2.6*
Superoxide dismutase - SOD (U/ml) 4.1 7.3 75% 0.30 6.8***
Phenol oxidase - PO (U/ml) 657.4 1057.4 52% 8787.00 4.1***
Cumulative mortality (%) 25% 57% 61% 0.00 -4.9***
KAU - King-Armstrong Unit, amount of enzyme that will liberate 1 ml of phenol in 15 min; SOD U - inhibits the rate
of increase in absorbance at 550 nm by 50%; PO U –the change in absorbance per min at 490 nm. For t-values: ***
significant at 0.001; **significant at 0.01, *significant at 0.05.
32 | INTERNATIONAL AQUAFEED | May-June 2015
FEATURE

2. percent fishmeal diets and their consequences
are discussed below.
Better gut health
Phosphatases are lysosomal enzymes play-
ing protective roles during the initial stages
of wound healing processes in animals. They
are also deemed to be the indicators of the
functionality of brush-border membranes.
In this study, ACP and AKP level (King-
Armstrong Unit - KAU/ml) in both serum
and hepatopancreas increased linearly with
the increasing intake of the protease (Table
1). However, the increase of AKP in hepato-
pancreas was less pronounced than those in
serum.
Better immunity
The production of superoxide dismutase
(SOD) increases naturally in response to pho-
to-oxidative stresses. A similar phenomenon
was also observed in animals being starved or
under limited nutrient supply.
Enzymes like catalase and SOD are natural
anti-oxidative enzymes specific for scavenging
superoxide radicals. The higher the SOD
activity, the more superoxide radicals need
to be reacted.
In this study, SOD activity in both serum
and hepatopancreas (Figure 2) of shrimps fed
diets supplemented with 175-ppm protease
was significantly higher than those in shrimps
fed 10 percent fish meal diets. However, we
observed much higher SOD activity in serum
than those observed in the hepatopancreas
of shrimps.
In animals, the phenol oxidase (PO) activity
usually increases in response to the pathogen
attacks to activate the “pro-phenol-oxidase”
(ProPO) system. The activated POs generate
highly cytotoxic quinones that help to inacti-
vate the viral pathogens.
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Figure 1. Total farmed crustacean and corresponding feed production (A)
and marine farmed crustacean and corresponding feed production (B)
Figure 2. The superoxide dismutase (SOD) activity in serum and
hepatopancreas of shrimps fed graded level of a protease
May-June 2015 | INTERNATIONAL AQUAFEED | 33
FEATURE

3. A trend similar to the SOD activity was
observed in the PO activity in the serums of
shrimps (Figure 3). However, unlike the SOD,
where activity in serum was much higher than
those found in hepatopancreas, no such dif-
ference in the PO activity between the two
was observed.
Reduced oxidation of lipids
Malon-di-aldehyde (MDA) is defined as
the marker for tissue damage. High level of
MDA in serum indicates high level of lipid
peroxidation in tissues.
In this study, the MDA level was signifi-
cantly higher in the serum of shrimps fed 10
percent fishmeal diets. The level decreased
significantly with increasing dietary protease
intake and was the lowest in shrimps fed diets
supplemented with 175-ppm protease. The
reduction was even much lower than those
fed the 20 percent fishmeal diets (Figure 4).
Disease resistance
A challenge test with shrimps fed the test
diets was performed against pathogen V. para-
haemolyticus. A significant relationship with
protease intake and reduction in cumulative
mortality was observed.
The reduction in mortality appeared to be
closely related to the reduction in the MDA
level in shrimps (Adj R2 – 0.55) (Figure 5).
Increasing MDA level in serum corresponded
well to the increasing 96-h cumulative mortal-
ity in this study.
Supplementation with digestive proteases
in animal diets has been shown to improve
growth, feed conversion and protein utilisa-
tion.
This study showed a significant improve-
ment in immune response parameters in
shrimps fed low fish meal diets supplemented
with a protease complex compared to those
fed the same diets.
These findings also showed why growth
performance such as weight gain, feed con-
version and protein efficiency are normally
better when fed protease supplemented
diets.
Results from this study are an early evi-
dence of improved disease resistance when a
protease is supplemented to the diets. Further
investigations are required to confirm these
findings.
Citations: Tacon and Metian, 2015,
Rev. Fish. Sci. Aquac. 23(1): 1-10.
Figure 5. Regression of cumulative mortality by MDA in serum
Figure 3. The phenol oxidase (PO) activity in serum and
hepatopancreas of shrimps fed graded level of a protease
Figure 4. The malon-di-aldehyde (MDA)
activity in serum of shrimps fed graded level
of a protease.
34 | INTERNATIONAL AQUAFEED | May-June 2015
FEATURE