Wednesday, July 15, 2015

15/07/2015: 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

First published in International Aquafeed, May-June 2015

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.


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 over-intensification and improper 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 characterised protein sources while replacing fishmeal, usually cause unwanted stress and poor stress responses in the shrimps. As a result, occasional disease 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 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 (µg/shrimp) at 0, 125, 150, 175 ppm of the 10 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 (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 hepatopancreas was less pronounced than those in serum.

Better immunity
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 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 inactivate the viral pathogens.

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.


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 significantly 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. parahaemolyticus. 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 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.

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The Aquaculturists
This blog is maintained by The Aquaculturists staff and is supported by the
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