Global 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 studyThe 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

percent fishmeal diets and their consequences are discussed below.

Better gut healthPhosphatases 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 immunityThe 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.

Naturally ahead

MYCOFIX

MY

COTOXIN RISK

M

A N A G E M

EN

TMycofix®

Proven protection. Mycotoxins decrease performance and interfere with the health status of your animals.

Mycofix® is the solution for mycotoxin risk management.

mycofix.biomin.net

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

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 lipidsMalon-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 resistanceA 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