FEED SUPPLEMENT AND ITS USE FOR AQUATIC ANIMALS

The present invention relates to feed supplements comprising vitamin E, vitamin C, beta- Glucan and nucleotides and the use of such supplements for improving performance and health of aquatic animals including fish and shrimp, especially for cold water fish as for ex ample salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish.

More particular, this invention relates to the use of the at least four substances as defined above for the improvement of the body length, feed conversion ratio, growth rate and/or daily weight gain in fish and shrimp, for reducing mortality by regulating the micro flora of the gut of the animal and/or by protecting the animal against infections caused by pathogen ic microorganisms.

The present invention also relates to the use of the at lest four substances as defined above for enhancing immunity of aquatic animals including fish and shrimp, especially for cold wa ter fish as for example salmon, bream, bass and for warm water fish as for example carp, ti lapia, catfish.

Furthermore, the present invention relates to a novel fish feed composition comprising as active ingredients vitamin C, vitamin E, beta-Glucan and nucleotides derived from natural sources.

The term feed or feed composition means any compound, preparation, mixture, or composi- tion suitable for, or intended for intake by an animal.

One important factor in aquaculture is the turnover rate. Turnover rate is determined by how fast the fish grow to a harvestable size. As an example, it takes from 12 to 18 months to raise Atlantic salmon from smolt (the physiological stage when the Atlantic salmon can first be transferred from fresh water to sea water) to harvestable size. A fast turnover has several positive results. First, it helps cash flow. Second, it improves risk management. Es pecially, a high mortality rate is a substantial risk for fish farmers.

It is generally known that mortality rate increases by an unbalanced microflora and/or by in- fections caused by pathogenic microbes. Fish diseases are common, and the likelihood of an outbreak is higher over a long growing period. There is also a risk that fish will escape due to accidents, e.g. when shifting nets, or due to bad weather causing wrecked fish pens.

For other farm animals it is well known to use antibiotics and vaccines to prevent the devel opment of diseases. In aquaculture, antibiotics are not so much used - at least in cold water aquaculture - due to the fact that disease spread very quickly, diseased fish do not eat much and also due to the negative impact on the environment of the wasted medicated feed. Vac cines are widely used when available but they are not developed for all diseases. As an alternative to synthetic drugs, the use of plant extracts and essential oils in animal feed is described in the literature. For example, patent W02004/091307 describes the use of polyphenols, and other natural actives in feed to increase survival rate of Artemia after hatching. W02004/091307 is however silent with regard to the selection of compounds to be used. Moreover, the application of polyphenols to reduce mortality is in the disclosed case above only useful at time of hatching.

It therefore remains a need in aquaculture to prevent the development of diseases, thereby reducing mortality by any prophylactic means including antimicrobial activity at the gut level.

The inventors of the present application surprisingly found that substances as defined above have a great potential for use in shrimps and fish feed, e.g. for improving the body length, feed conversion ratio, growth rate and/or daily weight gain and in particular for reducing mortality and for enhancing immunity. Further, the inventors surprisingly found that the substances, which are hereinafter also referred to as compounds, have also antimicrobial ac tivity resulting in a reduced mortality. The unique selection of active compounds of the pre sent invention allows for the first time controlling a number of fish and shrimp diseases caused by a number of different pathogens.

Therefore, in a first particular embodiment, the invention relates to methods for using the combination of vitamin E, vitamin C, beta-Glucan and nucleotides in shrimps and fish feed for improving the body length, feed conversion ratio, growth rate and/or daily weight gain and/or for reducing mortality by preventing diseases caused by pathogenic microorganisms and/or for enhancing immunity. For example, it has been shown that selected compounds of the invention exhibit excellent effects inhibiting the growth of marine strains, such as Vibrio parahaemolyticus, vibrio harveyi, vibrio anguillarum, Yersinia ruckeri or Vibrio haemolyt- icus which causes Acute Hepatopancreatic Necrosis Disease (AHPND) found especially in shrimps and causing high mortality.

It is therefore the object of the present invention to arrive at a new feed supplement that would improve health and performance in aquatic animals like shrimps and fish, especially during stress conditions and when high performance where demanded.

It has now been found surprisingly that feed additive compositions as hereinafter defined improves immune status, growth and feed conversion of shrimps.

The scope and special features of the invention are as defined by the attached claims.

The feed additive composition according to the present invention comprises

· vitamin C added for example as ROVIMIX ^® STAY-C ^® 35 (DSM Nutritional Prod ucts, Switzerland) which is at 35% vitamin C

• vitamin E added as ROVIMIX ^® E50 (DSM Nutritional Products, Switzerland) which is 50% vitamin E

• beta-glucan added as Leiber ^® beta-S plus (Leiber GmbH, Germany)

· nucleotide mixture derived from a natural source (for example ROVIMAX ^® NX plus from DSM Nutritional Products, Switzerland)

The inventors have been able to demonstrate that a mixture of these active ingredients used in synergy and in combination exhibits, in totally unexpected manner, the effects sought by the present invention of improving digestibility, growth and bone development and of boost ing the immune system of shrimps.

In a specific example it has been found that body length, growth rate, weight gain and feed conversion ratio can be improved in fish and shrimps by administering to the animals an ef fective amount of a feed supplement composition consisting vitamin C, vitamin E, beta- glucan and nucleotides.

As used throughout the specification and claims, the following definitions apply.

The term feed conversion ratio (FCR) is determined on the basis of a growth trial comprising a first treatment in which the composition according to the invention is added to the animal feed in a suitable concentration per kg feed, and a second treatment (control) with no addition of the composition to the animal feed.

As it is generally known, an improved FCR is lower than the control FCR. In particular embodiments, the FCR is improved (i.e. reduced) as compared to the control by at least 1.0% or 5%.

The term“mortality” as used herein refers to the ratio of life animals at the end of the growth phase versus the number of animals originally included into the pond. It may be determined on the basis of a fish challenge trial comprising two groups of fish challenged by a particular fish pathogen with the aim to provoke a mortality of 40 to 80% of the animals in the untreated group. However, in the challenge group fed with a suitable concentration per Kg of feed of a mixture of at least two compounds according to the invention, the mortality is reduced compared to the untreated group by at least 5%, preferably at least, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or at least 50%.

The term“immunity” as used herein is characterized by total phenoloxidase activities (U/mg), serum bactericidal activities (Ua), lysozyme activities, expression of immune genes

(such as proPO, PEN-3 and FSZ) of laemocytes, and Haemocyte and granulocyte counts in the animals.“Enhancing immunity” means the increase of the activities of the enzymes, the increase of the genes expression, and or the increase of Haemocyte and/or granulocyte counts.

Fig. 1 shows immune gens expression of Shrimps with different DSM health premix on 4th and 8th week: (A) 4th week, (B) 8th week.

Fig. 2 shows the cumulative mortality rate of shrimp challenged with Vibrio.

Fig. 3 shows the cumulative mortality of whiteleg shrimp fed the three different DSM diets and then challenged with a pathogenic strain of Vibrio parahaemolyticus. The shrimp were monitored every three hours until the experiment was terminated 187 hours post-infection. In particular, the inventors of the present application surprisingly found that the compounds according to the invention and mixtures thereof are effective against a number of pathogenic microorganisms of cold and warm water fish. Compositions according to the invention were shown to exhibit inhibitory effect against Vibrio anguillarum, a shrimp pathogen causing vibriosis. Compositions according to the invention were shown to exhibit inhibitory effect against Aeromonas salmonicida which is the pathogen causing a disease known as furunculosis. Compositions according to the invention were shown to exhibit inhibitory effect against Edwardsellia tarda causing systemic infection in fish.

Compositions according to the invention were shown to exhibit inhibitory effect against Lactococcus garvieae which is the etiological agent of Latococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over l6°C in summer months.

Compositions according to the invention exhibit excellent inhibitory effects on the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids.

Compositions according to the invention are shown to exhibit inhibitory effect against:

(i) Vibrio salmonicida, which is a psychrophilic bacterium that is the causative agent of cold-water vibriosis in Atlantic salmon.

(ii) Aeromonas hydrophila, causing ulcers and hemorrhagic septicaemia. This patho gen is very resistant to conventional simple antimicrobials like chlorine.

(iii) Photobacterium damselae, formerly Pasteurella piscicida : a pathogen causing high losses in the culture industry of economically important marine fishes such as seriola and red grouper in Japan and striped bass and white perch in the United States.

(iv) Streptococcus iniae, which is highly pathogenic in marine fish and is highly lethal: outbreaks may be associated with 30-50 % mortality.

Other aquatic pathogens such as

(i) Piscirickettsia salmonis, the causative agent of piscirickettsiosis or salmonid rick ettsial septicaemia (SRS),

(ii) Vibrio viscosus, recently renamed Moritella viscosa, etiologically responsible for the disease referred to as“winter ulcer”,

(iii) Ich (parasite), one of the most prevalent protozoan parasites of fish,

(iv) Vibrio harveyi, responsible for luminous vibriosis, a disease that affects commer cially-farmed prawns

(v) Vibrio parahaemolyticus, Yersinia ruckeri or Vibrio haemolyticus which causes AHPND in shrimps will also be inhibited by the compound mixture described in the present invention.

The vitamins E and C are commercially available or can easily be prepared by a skilled per son using processes and methods well-known in the prior art. For example vitamin E is available under the Trademark ROVIMIX ^® E50 (DSM Nutritional Products, Switzerland), vitamin C under the Trademark ROVIMIX ^® STAY-C ^® (DSM Nutritional Products, Swit zerland).

Beta-glucan may be obtained from any source, and a composition thereof may be prepared using convenient technology selenium. A preferred commercially available product is Leiber ^® beta-S plus (Leiber GmbH, Germany), a product comprising beta-l,3-l,6 glucan. A mixture of nucleotides according to the invention is commercially available (for example under the Trademark ROVIMAX ^® NX, supplied by DSM Nutritional Products, Kaiseraugst, Switzerland) or can easily be prepared from a yeast source by a skilled person using pro- cesses and methods well-known in the prior art.

By the expression“natural” is in this context understood a substance which consists of compounds occurring in nature and obtained from natural products or through synthesis. In a first aspect, this invention relates to the use of composition consisting vitamin C, vita min E, beta-glucan and nucleotides for improving body length, feed intake, weight gain, feed conversion ratio, growth rate and/or bone development in fish and shrimps and for boosting the immune system of the animals.

This aspect encompasses also a method of feeding of an animal with a feed supplement composition comprising as main ingredients composition consisting vitamin C, vitamin E, beta-glucan and nucleotides.

Vitamin C, vitamin E, beta-glucan and nucleotides are suitably administered together with the feed. The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. The term feed as used herein comprises both solid and liquid feed as well as drinking fluids such as drinking water.

Particularly, the combination of ingredients according to the invention can be added as a formulated feed supplement composition directly to the regular animal feed or to a premix containing other vitamins, minerals, amino acids and trace elements which is added to regu lar animal feed and thorough mixing to achieve even distribution therein.

In a second aspect, a feed supplement composition is provided which comprises vitamin C, vitamin E, beta-glucan and nucleotides.

A preferred feed supplement composition comprises vitamin C, vitamin E, beta-glucan and nucleotides in amounts sufficient to reach the following concentrations in the final feed: from about 500 to 2000, preferably 1000 ppm vitamin C

- from about 100 to 800, preferably 200 or 400 ppm vitamin E

from about 500 to 2000, preferably 1000 ppm beta-glucan

from about 50 to 300, preferably 150 ppm nucleotides.

A more preferred feed supplement composition comprises:

- 1000 ppm vitamin C added as ROVIMIX® STAY-C® 35 which is at 35% vitamin C

400 ppm vitamin E added as ROVIMIX® E50 which is 50% vitamin E

1000 ppm beta-glucan added as Leiber ^® beta-S plus

150 ppm nucleotides added as ROVIMAX ^® NX.

A third aspect of the invention relates to a premix or regular animal feed which comprises a feed supplement composition according to the invention. The incorporation of the feed supplement composition as exemplified herein above to fish or shrimps feed is in practice carried out using a concentrate or a premix. A premix desig nates a preferably uniform mixture of one or more micro-ingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. A premix can be added to feed ingredients or to the drinking water as solids (for ex ample as water soluble powder) or liquids.

Further, optional, feed-additive ingredients are aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; and/or at least one enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4., phospholipase Al (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6). Examples of antimicrobial peptides (AMP’s) are CAP18, Leucocin A, Protegrin-l, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins.

Examples of polyunsaturated fatty acids are g, C ₂ o and C ₂₂ polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such as perborate, persul phate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

A premix can contain, for example, per ton of fish feed, 50 to 200g of a propylene glycol solution of the mixture of the active compounds, 20 to lOOOg of an emulsifying agent, 50 to 900g of cereals and by-products, 20 to lOOg of a proteinic support (milk powder, casein, etc.) and 50 to 300g of a mineral component (expanded silica, feed quality lime, bi-calcium phosphate, etc.).

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The invention is further explained in connection with the following example.

EXAMPLE

Example 1 MATERIALS AND METHODS

The experimental diets were composed according to Table 1. The experimental products were added to the diet at the concentrations described in Table 2 and homogenised thor- oughly. The weight of the experimental products was subtracted from the weight of Sepio- lite (inert).

In order to pelletize the resulting mixtures, a feed binder and water were added. The result ing paste was passed through a pelletizing machine. The temperature during the procedure did not exceed 80°C.

Treatment A, which was considered to be the control diet, was supplemented with ROVIMIX® STAY-C® 35 experimental product.

Table 1 : Basel composition of diets

Table 2: Dietary treatment codes and supplementation

EXPERIMENTAL ANIMALS

Penaeus vannamei were imported as postlarvae (PLs) from Shrimp Improvement Systems, Florida, USA. These shrimps were certified to be specific pathogen free (SPF) for the fol lowing pathogens: WSSV, YHV/GAV/LOV, TSV, IHHNV, BP, MBV, BMN, IMN, Mi- crosporidians, Haplosporidians and NHP bacteria. Upon arrival, shrimp PLs were transported to the facilities and reared in a water recircula tion system containing artificial seawater at a salinity of 20 g L ¹ . They were raised on an ar tificial diet (replacement for alive feed) and after weaned onto a pelleted feed. Both feeds were distributed automatically 6-10 times a day. Water temperature was kept constant at 27°C ± l°C by means of an automatic heating system. A complete biological/mechanical fil ter and regular water changes kept the total ammonia ( H3/NH ) at <0.05 mg L ¹ and ni trites (NCfy) at <0.8 mg L ¹ .

EXPERIMENTAL BACTERIUM

The Vibrio Parahaemolyticus strain TW01 was isolated from Acute Hepatopancreatic Ne crosis Disease (AHPND) infected shrimp ponds and characterised in laboratory. Stocks of all bacteria are permanently kept frozen at -70°C at the facilities. After thawing, the stocks will be aseptically and separately inoculated in culture medium and grown using standard conditions. The optical density of the resulting bacterial suspensions will be determined spectrophotometrically. These data will be used to determine the concentration of bacteria in the suspension in colony forming units per millilitre (CFU/ml).

EXPERIMENTAL SETUP

Groups of shrimp were fed with the experimental diets during 28 days. This procedure roughly assessed the effect of the experimental diets on the growth performance of shrimp. After this period, shrimp were randomly selected from each dietary treatment replicate and transferred to the disease challenge facility for challenging with Vibrio parahaemolyticus strain. The inoculation of Vibrio parahaemolyticus strain was done by immersion following a standard protocol. This evaluated if the experimental products could protect shrimp from AHPND-provoked mortality.

FEEDING TRIAL

Standard conditions:

• Plastic tanks with a capacity of 290L (feeding units) are filled with artificial seawater com posed by adding a commercial sea salt mixture to purified water at 20 g L ¹ .

• Each tank is equipped with an individual filtration system composed by a protein skimmer, a mechanical filter and a biological filter.

• The biological filter is supplied with a given volume of filtration media, which was previ ously colonized with nitrifying bacteria.

• No water is shared in between the tanks; each tank is a true experimental replicate.

• The total weight of the groups is taken at the beginning and every 7 days until the end of the trial. The shrimp’s mean body weight (MBW) is calculated at the beginning of the trial for each group.

• The daily feeding rate for each group is calculated based on shrimp’s MBW (standard feeding percentages per weight) and adjusted daily according to the expected shrimp growth and mortality. This is corrected weekly after weighing the groups.

· The feed distribution is done automatically 6 times a day.

• Water quality is maintained by the filtration system and regular water changes.

• Water temperature is kept at 27 ± l°C by means of an automatic room temperature con trol system.

• Room is illuminated 12 h per day. • The setup is maintained daily.

Specific conditions:

A total of 600 shrimp with a mean body weight (MBW) of 0.4 g were used to randomly compose 12 groups of 50 individuals. Each group was housed in one feeding unit.

Each experimental diet was randomly assigned to 3 tanks according to the setup described in Table 3. During the feeding trial period, the groups of shrimp received the respective di ets at the predetermined percentages of their initial MBW and expected daily growth. The groups were weighed at the start, 7, 14, 21 and 28 days.

The feeding period served a double purpose, to roughly evaluate the growth performance of the shrimp and to prepare them for disease challenge by allowing the products to induce their putative effect and an adaptation of the animal’s organism to the experimental prod- ucts. This eventually maximised the assumed protection against AHPND.

The growth performance was assessed by the following parameters:

• Weight gain (WG)

• Specific growth rate (SGR)

· Feed conversion ratio (FCR)

• Survival rate (SR)

DISEASE CHALLENGE

Standard conditions:

• The shrimp are housed individually in 10L glass tanks (infection units) filled with artificial seawater.

• The seawater is composed by adding a commercial salt mixture to purified water at 20 g L- ¹ .

· The tanks are equipped with an individual mechanical/biological filter.

• The filters are supplied with filtration media previously colonized with nitrifying bacteria.

• No water is shared between the tanks; each tank is a true experimental replicate.

• The dietary treatment replicates correspond to 3 blocks of 10 individual shrimp per treat ment (in total 30 shrimp individually housed). The 3 blocks are placed at a different loca- tion of the challenge setup. This is carried out in order to account for possible variations induced by the location (light and temperature) within the setup. For purposes of statisti cal analysis, all shrimp are taken as housed individually.

• Water quality is maintained by filtration, aeration and regular water changes.

• Water temperature is kept at 27°C ± l°C by means of a room temperature control system. · Room is illuminated 12 h a day.

• Each shrimp is labelled as small, medium or big due to normal size variation in these ani mals. This categorization is made visually. The weight correspondent to each category will be determined by individually weighing 50 random shrimp. This information is used to determine the amount of feed that each shrimp receives per day.

· Individual shrimp are fed manually twice daily.

• Shrimp are monitored twice daily for clinical signs of disease and mortality.

• The setup is maintained daily. Seven days before the main AHPND challenge, we performed a pre-challenge aiming at ad justing the Vibrio parahaemolyticus strain dose necessary to induce a final mortality of 50- 80% in the specific shrimp batch used in this trial. A dose of 4* 10 ⁵ CFU/ml was determined during the pre-challenge and subsequently used in the main AHPND challenge.

Three days before starting the disease challenge, shrimp were transferred from the feeding trial facility to the disease challenge facility and housed in the infection units (1 shrimp per tank) for acclimatization. Shrimp from each feeding group replicate were used to compose a certain amount of infection units, according to the setup in Table 3. At the time of inocula- tion, each tank was inoculated (in water) with the dose pre-determined for each trial, as above described. One hour after inoculation, each shrimp received the first portion of the respective experimental diet. This procedure was specially designed to maximise the interac tion of the experimental products with the disease and ensure that the experimental products (or its effect) were present in the shrimp organism at the highest level possible during the critical moments of infection.

The clinical outcome will be evaluated by the following parameters:

• Cumulative mortality by day 7

• Onset of mortality

· Cessation of mortality

• Median lethal time

The mortality outcome in each feed treatment group was statistically compared with the control treatment group using a custom-made statistical methodology. The dimensioning of this challenge test was set to detect significant differences (treatment groups versus control groups) for survival improvements equal or higher than 25%.

Table 3: Overview of the experimental setup used for AHPND challenge with previous feed adaption period.

RESULTS

FEEDING TRIAL

The feeding trial was executed according to the protocol described in the materials and methods section. No technical problems were encountered during the procedure. All the evaluated parameters were within the ranges normally observed in our feeding sys tem.

Statistical differences for the specific growth rate and feed conversion ratio parameter were found as seen in Table 4. For other growth performance parameters, no significant differ ence has been found.

Table 4: Overview of the results of the growth performance parameters recorded during the feeding trial. Values are presented as average ± standard deviation. Values sharing the same label are not significantly different from each other tp>0.05 ). Values that have no label in common are significantly different from each other (p<0 05). Significant differences were found between dietary treatments for SGR and FCR.

AHPND CHALLENGE

The disease challenge was executed according to the standard procedure described in the materials and methods section and no problems were encountered during its execution.

We should note that the Control group (positive control for disease) was based on the standard diet, but it was supplemented with an experimental product ROVIMIX® STAY- C® 35. With the control feed, the expected range of 50-80% mortality is normally reached. The current control feed behaved similarly. Additionally, the mortality levels of the Mock (negative control for disease) group was 0% (Table 5), which demonstrates the correct exe cution of the experiment.

We observed significant differences in final mortality between treatment C (RC-RE50- LBS+-RNX+) and other treatments (Table 5), namely 64,4% versus 90 to 93,3% mortality was reached.

Table 5: Overview of the results recorded during the challenge with Vibrio parahaemolvti- cus. Values are presented as average of 3 dietary treatment replicates ± standard deviation Values sharing the same label are not significantly different from each other (p>0 05). Val ues that have no label in common are significantly different from each other tp<0.05 ). Sig nificant differences were found between dietary treatments for final mortality.

Further, at 48hpi, the mortality level was 43%, 33%, 20% and 47% for A, B(RSC-RE50), C (RSC-RE50-LBS+-RNX+) and D (RSC-ALA) treatments. At 78hpi, the mortality re mained lowest in treatments B and C, namely 57% and 44%, whereas A and D reached al ready 70% and 87% mortality.

However, best performing group is RSC-RE50-LBS+-RNX+ (treatment C), with lowest mortality levels from the beginnning untill the cessation of the trial.

CONCLUSIONS

The conclusions that can be drawn from the trial are:

• The experimental products had shown a positive effect on the growth performance of shrimp. SGR and FCR was significantly different between some of the treatments.

• During the challenge trial, treatment C (RSC-RE50-LBS+-RNX+) performed very well with significantly lower (and delayed) mortality compared to all other treatments, namely 64,4% (C) versus 90-93% (A, B and D).

• The different experimental products in treatment C can exert their synergistic effect on the performance/robustness of the shrimp in the most optimal way.

Example 2

MATERIAL AND METHOD

Experimental animal

The feeding trial was performed at an indoor industrial shrimp farm in Shandong Yellow River Delta Ocean Sci-Tech Co., Ltd. (Dongying, Shandong, China). An eight-week growth trial was carried out from November 2017 to January 2018. After 20 days of nurse ry, Pacific white shrimps postlarvae 25 (PL25) were moved to grow-out tanks. Shrimps (PL 25, 0. l7g, initial weight)) were randomly distributed into nine concrete tanks (38m ² ) in greenhouse with 27000 shrimps per tank (stocking density: 710 shrimp/m ² ). Concrete tanks were equipped with water exchange system receiving a constant supply of filtrated seawater and uninterrupted oxygenation system.

Nine treatments consisting of commercial shrimp pelleted feed mixed with 0% (control), 0.5% and 0.8% of DSM Health Premix were used in the experiment with three tanks per treatment.

DSM Health Premix comprised:

2000 ppm vitamin C added as ROVIMIX® STAY-C® 35 which is at 35% vitamin C 800 ppm vitamin E added as ROVIMIX® E50 which is 50% vitamin E

- 2000 ppm beta-glucan as Leiber ^® beta-S plus

300 ppm nucleotides as ROVIMAX ^® NX.

Growth and Survival study

Shrimp from nine treatment groups were fed four times daily at the satiation rate according to standard feeding rate. Feeding rate was adjusted according to shrimp weight through the 60 days experimental period. Water temperature (27-30 °C), pH (7.8-8.3), salinity (35 ppt), dissolved oxygen (5.3-6.1 mg L ¹ ), ammonia-nitrogen (0.10-0.30 mg L ¹ ), and nitrite- nitrogen (0.03-0.10 mg L ¹ ) were determined daily throughout growth trial. In order to as sess the growth performance of shrimps in all the tank, approximately 30 shrimps from each replicates of group were sampled randomly on 4th and 8th week to measure individual weight. Mean final weight, and specific growth rates were determined.

Field trial

Each week, sixty shrimp were sampled using two 1.52 m cast nets (monofilament net, 1.22 m radius and 0.95 cm opening) to determine average weight. The field trial was terminated on January 26, 2018. Following harvest, tanks were drained, shrimp collected and, weighed and total production per tank were calculated.

Tmmune parameters study

1. Haemolymph collection

During the feeding period, 20 shrimps from each replicates of group were sampled random ly on 4th and 8th week for immune parameters and immune genes expression assays. Hae molymph was collected from the ventral sinus cavity of shrimp using 1 mL tuberculin sy- ringe (26 gauge) containing chilled (4°C) anticoagulant solution (10 mM EDTA-Na2, 450 mM NaCl, 10 mM KC1, 10 mM HEPES, pH 7.3) at a proportion of one part hemo lymph to three parts anticoagulant solution. Haemolymph was centrifuged at 3 000 rpm for 10 min at 4°C, supernatant was used for determination of enzyme activity, hemocyte pellet will be rinsed twice and used for determination of immune-related genes expression.

2. Total phenoloxidase (PO) activity

Total phenoloxidase activity was determined by using L-DOPA as substrate. Briefly, 100 mΐ of centrifuged haemolymph was mixed with 50 mΐ PBS solution and 50 mΐ of enzyme inducer trypsin (Hi Media, 1 mg ml ¹ ) and incubated for 15 min at 25°C in 96-microliter plates. In controls, trypsin and serum was replaced by PBS. After incubation, 100 m 1 of a L-DOPA solution (lOmg lml ¹ ) was added to that mixture and incubated for 10 min at 25°C. Then the sample was read at 490nm using a microplate reader.

3. Serum antibacterial activity

100 mΐ of vibrio harveyi suspension (1 x 105 CFU mL ¹ ) in PBS was added with equal vol ume of shrimp serum and incubated with shaking at 28°C for 1 h. Then, 50 pL of the sam ple was removed, serially diluted with sterile saline and a 50 pL aliquot of each dilution was spread on TCBS agar plates. Plates were incubated at 28°C for 24 h and bacterial colonies counted. Bacterial suspension added with PBS instead of serum was served as the control. Serum antibacterial activity was calculated as described. One unit of serum antibacterial ac tivity was defined as a difference of 0.01 between R (control) and R (treatment), where R represents the ratio of change in bacterial counts from the beginning and end of the reac tions and calculated as: where, A ₀ and A _t are the bacterial counts at the beginning and end of the reactions, respec tively.

4. Lysozyme activity

Lysozyme activity was measured by the modified method. In this turbidometric assay, 0.03% lyophilized Micrococcus lysodeikticus in PBS was used as substrate. Ten microlitres of haemolymph were added to 250 m 1 of bacterial suspension in duplicate wells of a micro- titre plate and the reduction in absorbance at 490 nm was determined after every minutes (1, 2, 3, 4 and 5) of incubation at 22°C using a microtiter plate reader. One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 per min.

Expression of immune-related genes

1. Total RNA isolation and reverse transcription

Total RNA was extracted from the different tissue (haemocytes, hepatopancreas and intes- tine) according to the Trizol protocols (Invitrogen). The sensitivity of gene expression was compared on 4th week and 8th week. RNA was quantified at 260 and 280 nm using NanoDrop-lOOO (Thermo Scientific). Only RNAs with absorbance ratios (A260:A280) range in 1.8-2.0 were used for further experiments. First strand cDNA was generated with 20ul reaction volume containing 2mg total RNA, l x RT buffer, lmM dNTP, 0.2 mM Oli- go(dTl5), 10U of RNase inhibitor and 50U Multi-Scribe Reverse Transcriptase (Applied Biosystems) and the reaction was conducted at 37°C for 2 h.

2. Quantitative real-time PCR analysis of gene expression

Gene expression was analyzed by quantitative real-time PCR (qPCR) using Bio-Rad CFX384 (Bio-Rad Laboratories, Richmond, CA) using SYBR Green Supermix kits. 3 im mune-related genes commonly found in shrimp were selected for the analysis of expression levels: prophenoloxidase (proPO), penaeidin-3 (PEN), lysozyme (LSZ).The qPCR condi tions were 95 °C for 3 min and 50 cycles of 95°C for 10 s and 58 °C for 30 s. Relative gene expression was calculated as 2_DDCT, where Ct is threshold cycle. The expression levels of the housekeeping gene beta-actin were not altered with any of call population (data not shown). Data was be analyzed by one way ANOVA with post hoc LSD to adjust P values for multiple comparisons. P < 0.05 was considered statistically significant.

Challenge with Vibrio parahaemolyticus In the experiment, the isolated bacterial pathogen Vibrio parahaemolyticus (VP1) was used as a test organism. The bacterial strain VP1 was subcultured and centrifuged at lOOOOg for 10 minutes at 4°C. The supernatant was discarded and the bacterial pellet was washed three times and resuspended in phosphate buffered saline (PBS) at pH 7.4. Based on preliminary experiments, in order to control mortality of shrimps >50% within 3 days, infection dosage of 10 ⁸ cfu/ml was determined. The Vibrio doses, in immersion-route groups, were produced by dilution of bacteria in sterile seawater to 10 ⁸ cfu/ml. All shrimps were monitored after Vibrio infection. The experiment last for 3 days. Shrimps were continued feeding with the corresponding diets twice daily after challenge. The survival rate was recorded and calculat- ed.

Statistical analysis

All statistical analyses were performed using SPSS program version 22.0 (SPSS, Chicago, IL, USA), an independent samples T test was conducted to compare the significant differ ences of immune parameters and gene expression between treatments. A probability (P) value of less than 0.05 was considered significant.

Results

Growth performance

Growth performance showed a tendency to increase in DSM health premix treatments as compared to 0% (control), especially in 0.8% DSM health premix (Table 6). At the end of the trial, average body length (BL) and final body weight (FBW), were significantly higher than that of the control group except of the group with 0.5% DSM health premix on body length (P < 0.05). The highest BL and FBW were recorded on treatment group fed 0.8% DSM health premix. The results of sampled randomly to measure average weight on 4th and 8th week shown the tendency that fed 0.8% DSM health premix were significantly higher than that of the control group (P < 0.05) (Table 7). In this study, with the increase of dietary DSM health premix, the growth performance of shrimp was significantly improved when DSM health premix level up to 0.8%, indicating that DSM health premix might pro mote the growth of shrimp at a suitable dose.

Table 6 The weight and body length with different DSM health premix of shrimps

Treatment body length (cm) weight (g)

control 8.220±0.102 ^a 6.520±0.299 ^a

DSM health premix 0.5% 8.747±0.456 ^ab 7.989±1.167 ^b

DSM health premix 0.8% 9.098±0.223 ^b 9.028±0.833 ^b

Table 7: The weight with different DSM health premix of shrimps on 4th week and 8th week

Weight(g)

Treatment

0(week) 4(weeks) 8(weeks)

control 0T7 1.37 L52

DSM health premix 0.5% 0.17 1.43 7.98

DSM health premix 0.8% 0.17 1.88 9.03 Field trial production

The groups fed DSM health premix weight gain was pronounced than the control group, and the highest growth rate was observed in the 0.8% DSM health premix treatment (Table 8). Shrimp reared in production tanks, fed 0.5% DSM health premix and 0.8% DSM health premix, were significantly different (P > 0.05) with regard to average yield throughout the 8 weeks farming period (Table 9). No significant differences (P > 0.05) were observed among the weight gains of shrimps fed the two different DSM health premix.

Table 8: The weight with different DSM health premix of shrimps in every week lth 2nd 3rd 4th 5 th 6th 7th 8th

Treatment Initial week week week week week week week week

(g) (g) (g) (g) (g) (g) (g) (g) (g) control 0.17 0.37 069 086 137 238 094 446 6.52

DSM health „ . _

0.37 0.74 0.98 1.43 2.78 3.71 5.12 7.98 premix 0.5%

DSM health ,

0.50 0.82 1.16 1.88 3.44 4.54 6.25 9.03

Table 9: Production of Shrimps with different DSM health premix

Treatment Average Yield(kg/ha)

Control 327±2.65 ^b

DSM health premix 0.5% 334.3±2.89 ^a

DSM health premix 0.8% 332.6±1.00 ^!l

Immunological parameters

The immunological parameters were measured on 4th and 8th week of the feeding trial. Shrimp fed 0.5% DSM health premix and 0.8% DSM health premix had a significantly higher immune response (p<0.05), including Total phenoloxidase (PO) activity, Serum anti bacterial activity and Lysozyme activity, compared with the control group (Table 10). Shrimp fed 0.5% DSM health premix had higher PO, Serum antibacterial and Lysozyme activity than the control group but less pronounced than the 0.8% DSM health premix. Some previous study have reported that PO, LSZ and antibacterial activity were essential immunological parameters in most living organisms. Thus, the results of this study proved that the 0.5% DSM health premix and 0.8% DSM health premix were effective im- munostimulatory in shrimp by immune modulation mechanisms. Thus, the results of this study proved that DSM health premix were effective immunopotentiator in shrimp.

Table 10: Dietary effect of DSM health premix on immunological parameters of Shrimp on 4th and 8th week immunological pa 4th week 8th week

rameters control 0.5% 0.8% control 0.5% 0.8%

Total phenoloxi- 4.13±1.32 6.93±1.81 7±1.11 6.92±1.8 7.44±0.3 7. 94±0.6 dase activi- ties(U/mg)

Seram bactericidal 0.07±0.01 0.202±0.0 0.135±0.0 0.12±0.01 0.202±0.0 0.22±0.01 activity(Ua) 3 3 15 3 3 5

Lysozyme activi- 0.763±0.1 5.085±0.2 3.559±0.3 1.073+0.2 5.818±0.1 5.724±0.1 ty(ug/ml) 6 5 2

Gene expression analysis by real-time PCR

The expression ratio of target immune genes (proPO, PEN-3 and LSZ) of the haemocytes of shrimp fed with 0.5% DSM health premix and 0.8% DSM health premix is shown in Fig. 1. Dietary inclusion of DSM health premix significantly enhanced the mRNA expressions of immune genes on 4th and 8th week (P < 0.05) when compared with DSM health premix and control diet groups at all the time points tested throughout the experimental period. The immune genes expression was no significantly different (P < 0.05) on the 4th week between fed 0.5% DSM health premix and 0.8% DSM health premix group. However, 8th week shrimps, showed significantly higher level of PEN and proPO when compared with that of 0.5% and 0.8% DSM health premix diet fed shrimps (P < 0.05). Present study reports that DSM health premix enhances regulation of the immune effector mechanisms in P. vannamei by increasing the expression of immune genes. A dose dependent increase in the expression of immune genes was observed in the DSM health premix diet fed groups.

Cumulative survival rate

After fed diet experiment with DSM health premix, shrimp were challenged with Vibrio parahaemolyticus (VP1) observed for the cumulative mortality rate. The unchallenged, negative control ( control 1 ) group survived throughout the challenge trials, which indicat ed that the shrimp used in the current challenge trial were in a good health and nutritional status throughout the trial. The cumulative mortality (C _M ) of challenged shrimps following feeding with different experimental diets was significantly lower (P < 0.05) than that of challenged control shrimps (Figure 2). The cumulative gross sign appearance in positive control (control 2) reached up to 96.67 % by day 3 of challenge. The typical gross sign of AHPND, like, shrinkage of hepatopancreas, reduction in lipid content and empty stomach, started appearing from the day of challenge. The shrimp supplemented 0.8% DSM health premix tended to show much lower cumulative mortality rate (49.17%) compared to con- trol after 72h challenges. The group fed 0.5%

DSM health premix showed lower cumulative mortality rate (65%) . Therefore, in the immersion challenge shrimp supplemented with 0.5% DSM health premix and 0.8% DSM health premix showed a slower mortality initially compared to control.

CONCLUSION

In conclusion, under standard conditions, the study results showed the positive effects of the dietary intake of DSM health premix on the growth performance and innate status of Pacific white shrimp P. vannamei. These data indicated that DSM health premix feed addi tives could bring up survivals rate after vibrio immersion challenge. Several trials present DSM health premix as an ideal immunopotentiator. Especially in critical phases such as dis ease, maybe they show their potential and decrease mortality rates. The immunostimulatory effect of DSM health premix was observed in the present study, the Total phenoloxidase (PO) activity, Serum antibacterial activity and Lysozyme activity were enhanced after feed ing with DSM health premix supplemented diet. The DSM health premix feed additive products can enhance the immunity of shrimp and upregulated the expression of immune genes. Real time analyses of cDNA from feeding DSM health premix shrimp for immnue re sponse-related genes like proPO, PEN-3 and LSZ brought out upregulation. The DSM health premix also can be used as a potential supplemented diet in shrimp culture system with the view of enhancing the immune status of shrimp.

Example 3

MATERIAL AND METHOD

Experimental animals

The whiteleg shrimp ( Penaeus vannamei ) used for the trial were acquired as post-larvae (PL stage 14) from a specific pathogen free (SPF) hatchery supplied through Charoen Pok- phand. On receiving the post-larvae, a subsample of 150 PL split into 10 x 10-15 samples were selected at random and fixed in 95% ethanol and then submitted for disease testing. The shrimp were screened for AHPND, EHP, IHHNV, IMNV, TSV, WSSV and YHV and were found to be negative for all diseases. The PL were then nursed in ca. 30°C, bio floe, 15 ppt water at a stocking density of ca. 9 PL L ¹ and fed ad libitum a commercial pelleted feed until they reached an average weight of ca 1.0 g.

Once the shrimp reached the target weight of 1.0 g, a further sample of 30 shrimp were se lected at random and retested for the seven key shrimp diseases to ensure they were free of infection prior to stocking the tanks for the growth component of the trial. All shrimp, test ed as several pools, were negative for each disease.

Experimental diets

Diets 1, 2 and 3 were prepared with or without the product Health Boost according to table 11 and the stability of these in water and the acceptability to shrimp were tested. The Health boost comprises:

- 2000 ppm vitamin C added as ROVIMIX® STAY-C® 35 which is at 35% vitamin C

800 ppm vitamin E added as ROVIMIX® E50 which is 50% vitamin E

- 2000 ppm beta-glucan as Leiber ^® beta-S plus

300 ppm nucleotides as ROVIMAX ^® NX.

Table 11 : Ingredients and content of diets 1 GP ). 2 (T2) and 3 (T3)

Allocation of shrimp to experimental tanks and shrimp weights

The shrimp were then graded and allocated in batches of 25 animals to each experimental tank. A total of 16 replicates per dietary group were requested but an extra tank per diet was added. In addition, a further five tanks were added to the design to appraise the accep- tibility of the DSM experimental diets to a commercial brand of shrimp feed; for the current trial CP Starbird was used.

The inclusion of the CP Starbird feed was not to directly test and compare the growth per formance of the shrimp fed this diet but was added to ensure the shrimp consumed the ex perimental diets.

The starting weights of the shrimp allocated to each tank (Table 12) were then subjected to an ANOVA and Tukey post-hoc tests to ensure that there were no significant differences between dietary groups. The F test used tested for the effect of treatment. This test is based on the linearly independent pairwise comparisons among the estimated marginal means. The shrimp in each tank were fed five times a day at 08.00 am, 11.00 am, 14.00 pm, 17.00 pm and 21.00 pm hours. The feed was placed in submerged feeding trays and the shrimp will be given two hours to feed on the diet after which the trays were removed. Uneaten feed pellets at each meal were counted and then stored at -80°C for subsequent dry matter analysis by the study monitor.

Growth rates and FCRs After the 21 -day period of feeding on the DSM diets, the individual weight of all shrimp in each tank were taken and the survival of the population determined. A summary of the key metrics are presented in Table 12 below.

Table 12: A summary of shrimp performance following the 21 -day feeding period on the

DSM experimental diets. The average final weight, total biomass, survival and growth met rics of each dietary group are presented. Abbreviations: ADG = average daily growth rate; AGR = average growth rate; FCR = food conversion ratio.

From the results presented, it can be seen that DSM Diet 2 differed significantly from the other diets in terms of the average weight of the shrimp at the end of the 21 -day feeding pe- riod, the total biomass was also higher, as was the survival of the shrimp (i.e. 92.9% com pared to 88.7% for Diet 3 the next best peforming diet in terms of survival).

Further statistical differences were seen in the average daily growth rate per animal between dietary groups, where a high growth rate for shrimp fed DSM Diet 2 and a low growth rate for those fed Diet 4 (CP Starbird) were responsible for the differences.

Vibrio parahaemolyticus challenge test

After the feed component of the trial, a Vibrio parahaemolyticus (“Vp”) challenge was conducted. Representative whiteleg shrimp from each diet were allocated to the 20 L chal- lenge tanks. Each tank was sub-divided into 10 equal sized sections by sterile future board dividers. 15 tanks were used for each diet, i.e. a total of 45 tanks were set-up. Mesh cov ered panels were inserted into each side wall to allow for the free movement of bacteria be tween compartments. The tanks were filled with 7.5 L pre-treated, dechlorinated, 15 ppt seawater and maintained at 29°C degrees within a temperature controlled room within the Fish Vet Group Challenge Facility. A single shrimp was placed in each section.

The inoculum for the challenge trial was prepared by inoculating the fpAHPND isolate FVG0001 into tryptone soya broth (TSB) supplemented with 2% NaCl and culturing for 12 h at 28°C with shaking (ca. 250 rpm). Bacterial cells were collected by centrifugation at 900 xg for 10 min at l0°C and then the bacterial pellet was re-suspended in sterile 15 ppt sea water. The number of colony- forming units (CFU) mL ¹ in the suspension was estimated by measuring the optical density at 600 nm (OD600), as an OD600 of 1.0 equated to ca. 1.0x 108 CFU mL ¹ . The suspension was adjusted to the desired OD600 with sterile 15 ppt seawater, and then CFU mL ¹ verified by diluting and plating suspensions across tryptone soya agar and incubating at 28°C until CFU could be enumerated. The quantity of bacteria required for each challenge was determined from virulence pre-tests performed typically <48 h earlier to the main challenge. Each virulence pre-test was conducted on shrimp from the same population intended for use in the trial and under the same conditions as the actual challenge. The pre-tests used a minimum of three bacterial concentrations and three individ ually-housed shrimp per dose to determine the CFU mL-l required to give a ca. 66% mor tality at 48 h post-infection.

From the virulence pre-test, 60 mL of Vp inoculum was added to each challenge tank. Then the shrimp were monitored for survival every 3 h up to 187 h by which point the mortalities had stabilised. At 24 h, a further 7.5 L of 15 ppt seawater was added to each tank and the shrimp were maintained on their respective diets. At 48 h and 72 h, 5 L of tank water was removed and replaced.

A total of 150 shrimp were used for the challenge and sub-divided into two clusters. Cluster A comprised 100 shrimp divided into 5 replicates of 10 shrimp per tank representing the challenge group, and then a further 5 replicates of 10 shrimp per tank representing the non- challenged group. Cluster B consisted of 50 shrimp which were used for the sampling of haemocytes on days 1, 3, 5 and 7 days’ post-challenge.

Haemocyte and granulocyte counts

To determine the total haemocyte count at each time point and each sample, the method de tailed in Sritunyalucksana et al. (2015) was followed where 0.1 ml of haemolymph was withdrawn from the ventral sinus of the first abdominal segment into a 1 ml syringe contain ing 0.1 ml of 10% formalin in 0.45 M NaCl and then transferred to a labelled Eppendorf tube. After 10 min, 20 mΐ of the haemocyte sample was mixed with 20 mΐ Rose Bengal solu tion (1.2% Rose Bengal in 50% ethanol) and allowed to sit at room temperature for 20 min. Thereafter a 10 micro litre sample was transferred to an improved Newbauer bright line haemocytometer (Boeco, Germany) and the number of haemocytes counted in 5 of the 25 squares (volume of 1 square = 0.2 x 0.2 x 0.1 mm ³ ). The total haemocyte count was deter mined using the following conversion formula (5 x count x 104 x a dilution factor of 4).

To determine the proportion of granulocytes in each sample, a second 10 microlitre sample was transferred to a clean microscope slide and a smear was prepared. After air drying, the smears were then counterstained with a haemotoxylin solution for 7-10 min, then rinsed in tap water for 10 min and then dehydrated in 95% ethanol (10 dips) and then in 100% etha nol (10 dips). The preparations were then dehydrated by immersion in xylene, 3 x 3 min and then coverslipped. The proportion of granulocytes was determined from a count of 200 haemocytes and then the total number was recorded by using this proportion and multiply ing it by the total number of haemocytes (i.e. count/200 x THC).

Statistical analysis of shrimp challenged with Vibrio parahaemolyticus

The survival of each shrimp group following experimental challenge were plotted (see Fig ure 3.

The analysis of the haemocytes and granulocytes is presented in Tables 13 and 14. It can be seen that the THC in Diet 1 (see Table 13) continued to rise throughout the 7-day post challenge evaluation period, while the number in Diet 2 appeared to stay constant until day 7 (by which point there were no further mortalities in the V. parahaemolyticus challenged shrimp) and then the number of THCs fell. Likewise for Diet 3, the number of THCs peaked on Day 3 and then dropped thereafter. It is worth noting that most AHPND related mortali- ties occurred within the first 60 hours post-challenge.

Table 13: The corrected total haemocyte counts (THC) from shrimp experimental chal lenged with a pathogenic isolate of Vibrio parahaemolyticus causing AHPND at different time points post-challenge.

Table 14: The total number of granulocytes in samples taken from whiteleg shrimp at differ ent time points following their experimental challenge with a pathogenic isolate of Vibrio parahaemolyticus causing AHPND. The counts were calculated using the percentage granu locyte count and the total haemocyte count

From Tables 14, the total number of granulocytes in Diet 2 fed shrimp appear to be at a higher level than those determined in Diets 1 and 3. It is not known if the values obtained on Day 1 for Diet 2 are anomalous but the number of granulocytes appear to be maintained from Day 0 until Day 7 and then drop. Diets 1 and 3 follow similar responses to one another with gradual increases in the number of granulocytes, but at levels lower than Diet 2. The results suggest that the number of granulocytes in Diet 2 fed shrimp were higher at the point of pathogen challenge which may have contributed to their lower rates of observed mortali ty.

CONCLUSION The current trial set out to assess the performance of three DSM formulated diets. Growth and survival was assessed over a 21 -day feeding period during which five feeds were given a day. The tanks of shrimp fed Diet 2 had significantly: 1) higher average tank biomass (>12% higher than the other two diets); 2) heavier average body weights (4.27 ± 0.31 g shrimp-l); higher rates of survival (92.9%); the highest average daily growth rate per tank (3.61 g d-l); highest average daily growth rate per animal (0.14 g d-l shrimp-l); and had the lowest FCR of 1.37 although this was not significantly different from the other two diets. The final FCRs were higher than the 1.2 expected by the study monitor and were 1.37-1.73 for the three DSM formulated diets (in a rank order of Diet 2, 3 and 1). When challenged with V. parahaemolyticus, the shrimp fed Diet 2 had statistically higher rates of survival (74% versus 34% for Diet 1 and 32% for Diet 3).