FEATURE

IMMUNE RESPONSE
Dietary protease improves immune responses in
Pacific White Shrimp, Litopenaeus vannamei

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

lobal 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.

protease is considered to be an important

tool for better nutrient utilisation. It is hypothesised that a better nutrient profile and utilisation can compensate poor immune or stress
responses. Specially, when high quality ingredients 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 control). The fishmeal was replaced by increasing 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 prepared 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 hepatopancreas 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 (g/
shrimp) at 0, 125, 150, 175 ppm of the 10

The development of specific pathogen

free (SPF) broodstock of Pacific white shrimp
has been the major contributing factor to this
intensification, and the accompanied spectacular 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 percent of the production in 2000 (Tacon and
Metian, 2015).
However, the overintensification and improp- Table 1. Regression of the protease intake per shrimp with various immune parameters and 96-h cumulative mortality of
er management, the lack shrimps fed graded level of protease
of proper knowledge on

Min
Max
Adj R2
MSE
t-value
nutritional requirements at Serum
Acid phosphatase - ACP (KAU/ml)
2.6
5.9
72%
0.37
6.28***
various stages and over or
Alkaline phosphatase - AKP (KAU/ml)
2.6
6.6
70%
0.55
5.9***
under formulation, and the
Superoxide dismutase - SOD (U/ml)
345.9
411.1
75%
73.43
6.8***
increasing use of poorly
Phenol oxidase - PO (U/ml)
515.7
958.2
60%
8230.00
4.9***
digestible and poorly characterised protein sources
Malon Di-Aldehyde - MDA (nmol/ml)
9.1
15.8
60%
2.40
-4.8***
while replacing fishmeal,

Min
Max
Adj R2
MSE
t-value
usually cause unwanted
Hepato-pancreas
Acid phosphatase - ACP (KAU/ml)
55.9
132.9
46%
234.00
3.7***
stress and poor stress
Alkaline phosphatase - AKP (KAU/ml)
73.7
170.6
28%
556.00
2.6*
responses in the shrimps.
Superoxide dismutase - SOD (U/ml)
4.1
7.3
75%
0.30
6.8***
As a result, occasional disease outbreaks have been
Phenol oxidase - PO (U/ml)
657.4 1057.4
52%
8787.00
4.1***
very common creating Cumulative mortality (%) 25%
57%
61%
0.00
-4.9***
considerable economical
KAU - King-Armstrong Unit, amount of enzyme that will liberate 1 ml of phenol in 15 min; SOD U - inhibits the rate
losses in many countries.
of increase in absorbance at 550 nm by 50%; PO U the change in absorbance per min at 490 nm. For t-values: ***
The addition of dietary significant at 0.001; **significant at 0.01, *significant at 0.05.
32 | INTERNATIONAL AQUAFEED | May-June 2015

FEATURE
percent fishmeal diets and their consequences
are discussed below.

Better gut health

Phosphatases are lysosomal enzymes playing 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 (KingArmstrong 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 hepatopancreas was less pronounced than those in
serum.

Figure 1. Total farmed crustacean and corresponding feed production (A)

and marine farmed crustacean and corresponding feed production (B)

Better immunity

Figure 2. The superoxide dismutase (SOD) activity in serum and

hepatopancreas of shrimps fed graded level of a protease

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 inactivate the viral pathogens.

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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

MYCO

The production of superoxide dismutase

(SOD) increases naturally in response to photo-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

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May-June 2015 | INTERNATIONAL AQUAFEED | 33

FEATURE

Figure 3. The phenol oxidase (PO) activity in serum and

hepatopancreas of shrimps fed graded level of a protease

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 difference in the PO activity between the two
was observed.

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).

Reduced oxidation of lipids

Disease resistance

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-

A challenge test with shrimps fed the test

diets was performed against pathogen V. parahaemolyticus. A significant relationship with
protease intake and reduction in cumulative
mortality was observed.

Figure 5. Regression of cumulative mortality by MDA in serum

34 | INTERNATIONAL AQUAFEED | May-June 2015

Figure 4. The malon-di-aldehyde (MDA)

activity in serum of shrimps fed graded level
of a protease.

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 mortality in this study.
Supplementation with digestive proteases
in animal diets has been shown to improve
growth, feed conversion and protein utilisation.
This study showed a significant improvement 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 conversion and protein efficiency are normally
better when fed protease supplemented
diets.
Results from this study are an early evidence 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.