March | April 2014

Poultry hydrolysates enhance stress resistance and stress tolerance in Pacific

white shrimp

The International magazine for the aquaculture feed industry

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INCORPORAT ING F I SH FARM ING TECHNOLOGY

48 | InternAtIonAl AquAFeed | March-April 2014

Aquaculture 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 peptidesThe 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 shrimpby 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

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March-April 2014 | InternAtIonAl AquAFeed | 15

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

14 | InternAtIonAl AquAFeed | March-April 2014 March-April 2014 | InternAtIonAl AquAFeed | 15

FEATURE

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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 betterGrowth 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.

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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 ±

0.7421.96a ±

4.47 0.0586

total hemocyte count (x106 cell/ml

4.63b ± 0.41

4.91b ± 0.15

6.05a ± 0.23 0.0006

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FEATURE

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. KGIm Moore 149356 Diepholz Germany Website: www.ge-pro.deEmail: franz-peter.rebafka@ge-pro.de

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The Role of prebiotics in Pangasius production

Poultry hydrolysates enhance stress resistance & stress tolerance

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