Aquafeed vol 9 issue 2 2017 by Aquafeed Media

VOL 9 ISSUE 2 June/July 2017

A D VA N C E S I N P R O C E S S I N G & F O R M U L AT I O N An Aquafeed.com publication

Interview with Dr. Rick Barrows Survey of nutrient levels in commercial shrimp feeds in India

Mycotoxins in aquafeeds and why processing does not prevent it Taurine leaching in fish feeds Nucleotides in Fish Nutrition The importance of pre-extrusion process design

Mixed feed nut meal for aquafeeds Algae as aquaculture feed ingredients Computer-assisted image analysis to monitor whiteleg shrimp

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AQUAFEED

Volume 9, Issue 2

A D V A N C E S I N P R O C E S S I N G & F O R M U L AT I O N

Contents •

Interview with Dr. Frederic T. (Rick) Barrows

•

Survey of nutrient levels in commercial shrimp feeds in India

•

How mycotoxins get into aquafeeds — and why processing does not prevent it

•

The importance of pre-extrusion process design

•

* Taurine leaching in fish feeds

•

The use of algae as aquaculture feed ingredients

•

Mixed feed nut meal — natural and sustainable plant based protein for aquaculture

•

Nucleotides in fish nutrition — the best strategy to enhance immunity and intestinal health

•

Use of computer assisted image-analysis to monitor health and nutritional status in whiteleg shrimp (Penaeus vannamei)

•

Calendar of Events

*Cover story

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4 Volume 9, Issue 2

A D V A N C E S I N P R O C E S S I N G & F O R M U L AT I O N

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Dr. Frederic T. (Rick) Barrows is one of a very few number of scientists with expertise in aquaculture nutrition, feed production and fish physiology. He has devoted his long career with the U.S. Department of Agriculture, working at the Bozeman Fish Technology Center, Montana, to the evaluation of novel ingredients as alternatives to fishmeal and oil. He retired a year ago and formed Aquatic Feed Technologies, LLC., with the aim of assisting in the development of a safe, sustainable, seafood supply, working with companies with feed ingredient development and evaluation, regulatory approval and alternative feed development. This includes feeds ranging from larval, weaning or starter feeds through grow out feeds and brood feeds Rick has over 130 peer-reviewed publications through out his 28 years of innovative feed research and has received numerous awards for his work.

Interview with Dr. Frederic T. (Rick) Barrows AQUAFEED.COM You have gained a reputation over many years for your work on new protein and oil sources as potential alternatives to fishmeal/oil. Spirulina does not seem like a “new” ingredient in aquaculture. Would you talk about your work with this microalgae and why you believe it to hold such potential? FTB Yes, you are correct spirulina has been evaluated as a fish feed ingredient

for decades. There does seem to be an interaction of spirulina and other ingredients for select marine species. When spirulina was added to a fishmeal based diet for white sea bass or yellowtail there was no beneficial effect. When spirulina was supplemented to a fishmeal free diet, however, there was a large increase in both growth and survival. This suggests that spirulina is supplying a nutrient that is present in fishmeal but not in the other ingredients.

The one “new” aspect of spirulina might be that the price seems to be coming down to a more reasonable level as more is produced overseas. There is another source of spirulina product that could be beneficial for aquafeeds. Spirulina contains phycocyanin and this is now being extracted from the meal to produce a blue pigment for foods and it is approved in the U.S. and Europe. The residual

6 biomass should be more affordable and may contain the essential nutrients that the unextracted spirulina contains, but this needs to be demonstrated.

AQUAFEED.COM What are some of the other promising ingredients you have investigated and how do they hold up in terms of their nutritional value? FTB There is a lot of interest and investment going into single cell proteins (i.e. bacterial proteins) and insects (i.e. black soldier fly and meal worms) and production scale plants are being built around the world. The products themselves are very nutritious; the cost per ton and the scalability are primary questions now, but we will soon see. I think we will be seeing the development of regional or local ingredient sources. One example is what AAFCO calls mixed nut meal. This is nuts that cannot be certified due to size, color or broken pieces and are used for animal feed. This material is processed to produce a meal of approximately 62% protein and 10% lipid. Most times the meal is comprised of pistachios and almonds and this meal increases feed intake when included at 10% or greater in the diet. Most barley farmers want to produce malting grade barley, but barley that is too high in protein gets discounted and is sold as feed grade. The feed grade barley can then be processed to produce ethanol and a barley protein meal. This process is designed to protect the protein quality of the meal and it has been demonstrated to have high protein, dry matter and energy digestibility. Both barley protein concentrate and mixed nut meal are in the pilot scale phase, but

the ingredients have been fed commercially for several years now.

AQUAFEED.COM Are changes in animal health and/or behavior or quality of the finished product major issues with use of alternative ingredients youâ&#x20AC;&#x2122;ve looked at?

FTB These are also important considerations when evaluating new ingredients. If a new ingredient has a negative effect on these criteria it would be dropped immediately until further modified. There are certain prebiotic effects of some ingredients, and there are several fermented or microbially enhanced products that show great potential especially for the shrimp industry. One of these fermented products was tested on a farm scale with 8 different farms and a significant increase in survival was observed from approximately 40% in the control groups to approximately 80% in the test groups. The shrimp fed the fermented product were also more uniform in size. Many consumers and fish producers are concerned with the effect of alternative ingredients on the flavor of the final product. It is fairly clear that changing protein sources does not change the flavor of the fillet, but changing lipid sources from fish oil to plant oils does change the flavor. Depending on the consumer group this can be a positive or a negative effect but it seems a blend of oil sources is often most effective from a product quality and cost effectiveness perspective.

AQUAFEED.COM Have you looked into the physical properties and processing parameters of any of these ingredients?

FTB Yes, as you know this is of vital importance to feed manufactures to know that the ingredient will not have an adverse effect on pellet quality. This is difficult to determine for every type of diet due to interactions among ingredients, but a good time to evaluate the effect of the ingredient on processing and pellet quality is when producing diets for a digestibility study. In these studies there is a reference diet which is fed to three tanks of fish and typically 9 test diets which are each fed to three tanks of fish. The test diets contain 70% of the reference diet and 30% of the test ingredient. The 30% inclusion rate is greater than I would recommend in a practical type diet so if there is no effect on processing or pellet quality at this level I would expect none at lower levels. This is also a good time to evaluate the effect of an ingredient on the palatability of the diet by measuring feed intake. A good example of the effect of ingredient on pellet quality was with diets containing 30% Schizochytrium algae. This is the algae that contains high levels of lipids and high levels of DHA, the fatty acid known for its heart and brain healthy attributes. We processed these product obtained from 4 different sources and found a large difference among the products. Since this algae can contain over 50% lipid some of the products felt very greasy and some were â&#x20AC;&#x153;dryâ&#x20AC;? to the touch. One of the greasy products, however, processed well and produces a good quality pellet. Three of the products produced good quality floating pellets, but the fourth product we could not get the pellets to float no matter how much energy we put into the process. This inclusion level is way over the suggested level but demonstrates

7 that even among related ingredients you can have an effect on pellet quality. We still don’t know if the difference was due to strain of algae or how the algae was processed.

AQUAFEED.COM Will changes in major feed components require a change in process technology – new equipment or refinements to existing machines - or just the establishment of new parameters for existing processes? How will the feed industry need to adapt? FTB I think that the pace for change in processing is being set by the equipment manufacturers. As equipment refinements are developed it will allow for new type of ingredients to be used. For example, the addition of “wet” ingredients to an extruder system could decrease production costs for the ingredient since drying an ingredient can be costly only to rehydrate during feed production. Of course this raises questions on product stability and could require production of the ingredient in close proximity to the feed mill. This is an exciting combination of innovative ingredients, manufacturing process, and nutrition. Progress in this area will be interesting to watch in the future.

AQUAFEED.COM What are the research and/or commercial application gaps that need to be met in order to expand industry use of promising alternative ingredients? Is this important, and if so why? FTB The available nutrient content is of course a primary concern for new ingredients, but it is often the price or the potential scalability of an ingredient that is most often limiting. Increasing the

scale of production from the laboratory scale (hundred of kilograms) to pilot scale (hundreds of tons) can be challenging, but these challenges are often on the technical side. The development of new Ingredients often get stuck moving from pilot scale to production scale. This is because of the cost of building a production scale plant is usually large and the uncertainty of market acceptance. A practical consideration often limits the flexibility of feed mills to adopt new ingredients even if the nutritional and other characteristics have been clearly evaluated. This is the number of ingredient bins available whether it is 6 or 12 or 20 there are always ingredients already in them. So a feed mill would have to replace a known ingredient with the new ingredient. I have often recommended that ingredient providers work the cost of new ingredient bins into the business plan to at least get started.

AQUAFEED.COM Finally, what is the one thing you'd like to see happen in aquaculture before you really retire? FTB Well, there are actually a two related things I would like to see happen for aquaculture feeds and for human health. We do have many sources of protein ingredients, but there are no products that contain the essential fatty acids, and probably other nutrients, that is contained in fish oil. Fish oil is a very nutritious product but there just isn’t enough available to meet the needs of the growing aquaculture industry and the growing human population even at current harvest rates from the oceans. I would like to see an affordable lipid product that contains an equal level of essential nutrients to fish oil developed and commercialized. This would allow aquaculture to continue to grow and provide nutritious products and improve the health of the humans that consume those products. To reach that goal and beyond I think there should be a system put in place to

“It is fairly clear that changing protein sources does not change the flavor of the fillet, but changing lipid sources from fish oil to plant oils does change the flavor. Depending on the consumer group this can be a positive or a negative effect but it seems a blend of oil sources is often most

effective from a product quality and cost effectiveness perspective.” speed innovation and commercialization and to better communicate success’s around the globe. There are high quality research and development projects going on everywhere, but sometimes it seems that as researchers we are reinventing the wheel again. International travel is expensive, but with modern technology and some organization the rate of development and adoption of new ingredients could be increased. Also many new ingredient providers often do not know how to determine the nutritional and economic value of their products and a system that allowed them to do this would assist the industry greatly. AFΩ See page 33: “Mixed feed nut meal – natural and sustainable plant based protein for aquaculture” for more on this promising feed ingredient.

Page 21

Survey of nutrient levels in commercial shrimp feeds in India By Alexander van Halteren and Peter Coutteau, PhD., Nutriad International NV, Dendermonde, Belgium

Since the introduction of white leg shrimp in 2009, shrimp culture has boomed in India to over 485,000 Mt in 2016. During the past

years, Indian shrimp culture has spread from one main culture belt, Andhra Pradesh, into two more areas, Orissa and Gujarat. Culture conditions vary in salinity, water source (borehole/seawater), temperature and length of the winter period in the different culture areas.

The number of shrimp feed producers has increased dramatically in recent years. Initially, fish and chicken feed producers were the first to see shrimp feed as a potential for diversification. They were followed by large farmers

setting up their own feed-mill operations. Currently large seafood processors are installing new feed-mills. It is expected that this trend will further fragment the feed market, challenging the existing key feed producers. Globally there was a decline of fishmeal production in 2016 and also Indian domestic fishmeal production was affected, causing shortage of fish meal and fish oil during the summer crop. Contrary to earlier days when the bulk of the marine ingredients such as fishmeal, squid meal, and krill meal were imported, today shrimp feeds are formulated for more than 95 % with locally produced raw materials. Thanks to the implemen-

tation of modern production technology, most domestic producers of fishmeal/oil in India meet international quality standards nowadays. Although this allows feedmills to avoid the complex importation and stocking of marine ingredients, the prices of these local raw materials have increased to international levels. The selection of raw materials will influence the overall nutrient profile of the feed. Reducing marine ingredients will directly affect the level and availability of the essential lipids such as n-3 HUFA and cholesterol, unless the formulator compensates this by adding specialty ingredients providing these nutrients.

10 Compared to other (aquatic) species, formulating shrimp feed is more based on experience then on exact science. The optimal nutrient profile of a shrimp feed will depend on many factors, including the culture density, environmental conditions (temperature, salinity, oxygen, …), productivity of the pond water, the stability of the feed, feeding method and frequency, … These factors often are different depending on the season, region, farm or even pond which makes the selection of the ideal feed rather a complex and often unstable decision for the farmer. As a result, research in optimizing feed formulations under practical conditions continues to be a major objective for feed producers. Also, we can expect a wide variety in nutritional specifications among commercial shrimp feeds as composition may depend on the target market. Increasing cost and fluctuating availability of raw materials in combination with an increasingly competitive market is demanding a creative mind from the shrimp feed formulator. The nutritional strategy is key to maintain or gain market share. Aside from that, diseases like white spot, vibriosis and white gut/feces are an emerging risk during the production cycle of shrimp in India and require a good nutritional support to the animal. The present study investigated the different nutritional strategies in commercial shrimp feeds during 2016, when the number of shrimp feed suppliers increased sharply. Feed samples of 8 major brands were collected in the market and analysed for proximate composition as well as a number of essential nutrients (amino acids, phospholipids, cholesterol, n-3 highly unsaturated fatty acids).

Sample collection and analysis Since 2013, Nutriad has surveyed the composition of commercial shrimp feeds in India. Feeds have been analyzed on a range of nutritional parameters. For the present study, we restricted the samples to one pellet size, i.e. 3P, which constitutes the main consumed volume of commercial shrimp feed in India. Pellet 3P typically has the following specifications : crude protein (35 – 36 %), crude fat (4.5 – 6 %), crude ash (< 13 %) and crude fiber (2 – 5 %). The selected feeds were collected from the market during the second quarter of 2016 from farmers and feed distributors. All feed samples were produced during Q2 of 2016 and stored under typical lab conditions, before sending for analysis. Three different samples of each type of feed was pooled into one representative sample. Therefore, the results of this survey are representative for the feed specifications during a specific window of time during the culture cycle of 2016. Crude protein was analyzed following the Kjeldahl method (Commission Directive 93/28/EEC.OJ No L179.22.7.93). Crude fat has been determined with acid

… the results of this survey are representative for the feed specifications during a specific window of time during the culture cycle of 2016. hydrolysis following the Soxhlet method (AOAC 996.06). Fatty acid composition was determined with the gas chromatographic method following fat extraction (AOAC 996.06; expressed as g/kg). Cholesterol was determined by direct saponification using the gas chromatographic method (AOAC 994.10; expressed as g/kg). Phospholipids were analysed with 31P-NMR spectroscopy using the internal standard method (SAAMET002-03, expressed as % as is). The analyses of amino acids and nitrogen were performed by Evonik Degussa GmbH (official European method of amino acid analysis in feed, COMMISSION DIRECTIVE 98/64/EC of 3 September 1998; official method code 994.12 of the AOAC International 2000, and expressed % as is).

11 Crude protein and fat Crude protein levels varied between 33.9 % and 40.7 %, with an average of 36.4 % ± 2.3 % of the analyzed samples (Fig. 1). 5 feeds out of 8 were found to have a higher protein content than the minimum specification on the label. On average, the feeds were 4% above the labelled feed specifications of 35 % crude protein, but in one case the analysed crude protein level was 20% higher than the level specified in the label. The crude fat specifications vary between different feed producers between 4.5 – 6 % with the majority having the fat specification above 5 %. All the analyzed feeds have crude fat contents higher than 5 %, with an average of 6.18 % ± 0.54 %. This is approximately 25 % above the specification. There was no clear relation between crude protein and crude fat content. This confirms the lack of consensus among shrimp nutritionists on the importance of the protein/fat ratio in the diet. The analysis of crude fat and protein does not reveal the origin or quality of the fats and proteins used.

Figure 1. Crude protein versus crude fat of 8 commercial shrimp feeds.

Amino acid profile The amino acid profile gives a first idea on the quality of the proteins used as well as the nutrient density. This is far from a complete picture of protein quality which would include an assessment of the ingredients regarding processing parameters and protein digestibility. Amino acid nutrition in shrimp is complex due to the interaction from the leaching of amino acids prior to ingestion, lack of digestibility values, and the role of specific amino acids in feed attractiveness. The current knowledge on the requirements of amino acid

Figure 2. Amino acid profile for 8 commercial shrimp feeds (represented by different line colors).

requirements in white shrimp is still very limited (NRC 2011). In the absence of scientific studies determining absolute requirements for essential amino acids (EAA), the composition of the whole

body could give guidelines on the desired profile of essential amino acids in the diet (ideal protein concept). In this study the average (± standard deviation for 8 samples) lysine content was 2.11% ±

12 0.17% and methionine 0.63% ± 0.09% (Fig. 2). Arginine an amino acid that could play a role it the attractivity of the feed has an average of 2.41% ± 0.12%.

Phospholipids, n-3 HUFA, cholesterol The n-3 HUFA content is an indicator for the quantity of marine fats used in the feed, either derived from fish oil or from marine protein meals containing fat. Marine shrimp do not have the ability to biosynthesize n-3 HUFA and the dietary requirements of Litopenaeus vannamei were found to be at least 1 % (Kanazawa et al. , 1979; Kontara et al., 1995; Shiau, 1998). Five out of the 8 feeds had n-3 HUFA values around 5 g/kg, whereas crude fat levels varied between 5.8 and 7.1 g/kg (avg 6.18 % ± 0.54; Fig. 3). One feed exhibited more than 7g/kg n-3 HUFA and two feeds were below 3 g/kg n -3 HUFA. Cholesterol requirement studies show a wide range in cholesterol requirements from 0.5 to 5 g/kg for L. vannamei (Chen, 1993, Duerr and Walsh, 1996; Gong et al., 2000). Duerr and Walsh (1996) showed that dietary cholesterol levels below 1 g/kg limit growth in L. vannamei. Morris et al. (2011) reported the cholesterol requirement for L. vannamei grow-out to be somewhere between 0.76 and 1.1 g/kg, however, a regression analysis predicted the cholesterol requirement for maximum growth to be 1.5 g/kg. Gong et al. (2000) estimated that the cholesterol requirement for L. vannamei was 3.5 g/kg in the absence of supplemented phospholipids. At 1.5% and 3% phospholipids, dietary cholesterol requirements reduced to 1.4 and 1.3 g/kg, respectively. The cholesterol levels

Figure 3. Dietary level of n-3 HUFA versus crude fat for 8 commercial shrimp feeds.

Figure 4. Dietary level of cholesterol versus crude fat for 8 commercial shrimp feeds.

found in the analysed feeds were all well below 1 g/kg (Fig. 4). The average cholesterol level was 0.64 g/kg ± 0.16, with half of the feeds around 0.7-0.8 g/kg and one feed sample as low as 0.34 g/kg. Dietary phospholipid is required for optimal growth of penaeid shrimp including L. vannamei (Glencross et al. 1998; Paibulkichakul et al. 1998; Thongrod and Boonyaratpalin 1998).

Addition of 1.5% of phosphatidyl choline (PC) from either a 95% pure soybean source, 94% pure chicken egg source, or deoiled soybean lecithin (23% PC) increased growth of L. vannamei relative to a PC-deficient diet (Coutteau et al. 1996). Recommended levels of dietary phospholipids from soybean sources range from 1.25% to 6.5%, depending on shrimp species, developmental stage, as well as purity of the lecithin (Coutteau et

13 al., 1997). The average levels of phospholipids in the current study were 1.74 % ± 0.43, with the lowest level being 1.13% and the highest 2.5% (Fig. 5). Comparing different studies on phospholipid requirements can be troubled by the differences in analytical methods used. The present study used NMR spectroscopy to quantify total phospholipid content in the shrimp feeds. This method is more accurate than traditional methods based on HPLC or iatroscan as the NMR quantification is independent from fatty acid composition and phospholipid profile of the phospholipids. The level of the essential fat nutrients, cholesterol, n-3 HUFA and phospholipids, were not correlated at all with the total level of dietary fat (Fig. 3, 4, 5 : nonsignificant correlations). This indicates that the dietary fat originates from blending fats from marine as well as vegetable origin.

Trends revealed from comparing surveys 2014 and 2016 Comparing the present survey for 2016 with a similar survey in 2014 (based on 4 commercial feeds), we see some significant trends. Overall the average crude protein increased from 35.8% to

Figure 5. Dietary level of phospholipids versus crude fat for 8 commercial shrimp feeds.

36.4% in 2016. Average crude fat levels are similar in both surveys, ie around 6.2%. n-3 HUFA and cholesterol levels have dropped with 16% and 24%, respectively, , whereas the average inclusion of phospholipids increased with 38 % between 2014 and 2016. The changes in the lipid profiles reflect possibly the effects of the trend to replace fish meal and fish oil, particularly in 2016 when the domestic supply of these raw materials was insufficient. Average levels indicate that overall reduced levels of cholesterol and n-3 HUFA, likely due to increased replacement of marine by vegetable ingredients, were compensated with increased levels of phospholipids and crude protein.

Table 1. Variation in fat composition in commercial shrimp feeds between 2014 versus 2016 (data represent average and stdev).

Crude fat (%) Crude protein (%) n-3 HUFA (g/kg) Cholesterol (g/kg) Phospholipids (%)

2014 Average ± s.d. (n=4) 6.28 ± 0.86 35.82 ± 0.20 5.23 ± 2.48 0.84 ± 0.16 1.26 ± 0.29

2016 Average ± s.d. (n=8) 6.18 ± 0.54 36.44 ± 2.25 4.42 ± 1.74 0.64 ± 0.16 1.74 ± 0.43

Reducing the level of essential lipids like cholesterol, phospholipids and n‐3 HUFA significantly affected growth, feed conversion and protein efficiency in white shrimp in a controlled feeding trial in clear water (van Halteren et al., 2016). The above trends in the feed industry promote the application of digestibility enhancing additives which improve the absorption efficiency of the increasingly limited levels of cholesterol and n-3 HUFA. Digestive enhancers like bile salts and phospholipids are natural emulsifiers capable of enhancing the digestive capacity for lipids in the digestive system of shrimp by improving the lipid emulsification and micelle formation, resulting in a faster absorption of lipids in the hepatopancreas. Furthermore, bile salts constitute an alternative source for the steroid ring which shrimp cannot synthesize, which is at the basis of their requirement for dietary cholesterol. Adding bile salts to the diet lower in essential lipids restored the performance of the shrimp to the same level as the control diet with elevated levels of essential lipids (van Halteren et al., 2016). By improving the utilization

14 efficiency of dietary lipids, shrimp formulations can be made more costeffective by reducing the formulated values for phospholipids, cholesterol and n-3 highly unsaturated fatty acids (HUFAs) without affecting the performance (Coutteau et al., 2011).

crude fat, 2.11 ± 0.17% lysine, 0.63 ± 0.09% methionine, 2.41 ± 0.12% arginine, 4.42 g/kg ± 1.74 g/kg n-3 HUFA, 0.64 ± 0.16 g/kg cholesterol, 1.74 ± 0.43% phospholipids. Furthermore, the survey showed an overall trend in the industry between 2014 and 2016 to offer feeds with lower levels of cholesterol and n-3 HUFA, whereas crude protein

Conclusions The present study collected samples from 8 commercial shrimp feed brands (pellet size P3) in India during 2016 for the analysis of selected nutrients. The shrimp feed samples exhibited a wide range of levels for the nutrients analysed. The shrimp feeds contained on average (± s.d. for the eight samples) 36.4 ± 2.3% crude protein, 6.28 ± 0.86 %

More information Alexander van Halteren Business development manager Aquaculture Nutrition, Nutriad International, Belgium. E: a.vanhalteren@nutriad.com Peter Coutteau, PhD Business Unit Director Aquaculture, Nutriad International, Belgium. E: p.coutteau@nutriad.com

and phospholipid levels were increased over the same period, likely due to the increasing replacement of marine ingredients by vegetable raw materials.

AFΩ

How mycotoxins get into aquafeeds and why processing does not prevent it Rui Gonรงalves, Aquaculture Scientist, Biomin

The topic of mycotoxin has started to draw increasing attention from the aqua industry in recent years. There is, however, nothing new about fungal contamination. Fungi have always held biological, ecological and economic importance when it comes to plants, aquafeeds and aquaculture. The role fungi play ranges from positive to neutral to negative, depending on the species and circumstance. The Good, The Bad and The Friend The importance of fungi to the ecosystem is immeasurable. Fungi are present in all terrestrial habitats from Antarctica to the hot deserts of Namibia, and in most aquatic environments. Being opportunistic heterotrophs, they have specialized in penetrating solid substrates (rock, bark, dead branches, bare soil or grains), nutritionally exploiting almost any food substrate. Fungi are essential to recycle nutrients. Most of the known taxa are specialized in decomposing complex plant and animal debris.

The Good Several species of fungi, namely from genus Penicillium, play an important role in industry. For example penicillin, produced by P. chrysogenum (formerly P. notatum), discovered by Alexander Fleming in 1929, was probably the most important discovery of the last century and changed the course of medicine.

Fusarium verticillioides (major producer of FUM) colonies morphology on petri dish.

16 and utilize substrates efficiently by growing over their surfaces and penetrating into their matrices. Fungal metabolic processes originate an apparently endless diversity of organic compounds which are not obviously required for normal growth and metabolism: these are called secondary metabolites. Not all secondary metabolites are mycotoxins. Simplistically, we could split them in 3 broad groups, being 1) toxic to bacteria (antibiotics), 2) toxic to plants (phytotoxins) and 3) toxic to animals (mycotoxins).

Aspergillus-flavus (major producer of AF) colonies morphology on petri dish.

Fungi also play a central role in the food industry (cheese and various meat products) and are becoming increasingly important in the biotechnology field, especially in the production of enzymes (e.g. gluconic, citric, and tartaric acids, several pectinases, lipase, amylases, cellulases, and proteases).

The friend Fungi are vital to agriculture and forestry through their global involvement in mycorrhizae (fungus roots). They also established mutualistic symbioses with a wide range of organisms like cyanobacteria (blue-green â&#x20AC;&#x2DC;algaeâ&#x20AC;&#x2122;) and chlorophycota (green algae) in lichens.

The bad Minor fungi taxa can have a parasitic behavior, and in certain cases can be

pathogenic. The ability to penetrate almost any surface can be used to invade host organisms. Fungi attack almost all known taxa of plants and animals, including shrimp (e.g. Fusarium sp in penaeids) and fish (e.g. Saprolegniasis). Focusing on fungi as plant pathogens, they attack all parts and all stages of crop plants (from root hairs to apical buds, grains or fruits). The fungal infections may be restricted to small leaf spots, or may be systemicâ&#x20AC;&#x201D;killing their host very quickly or remain invisible until it is time to appropriate crucial energy resources, such as those concentrated by the host in anthers, bulbs or seeds.

Toxic secondary metabolites Taking into account the available nutrient content, plant stuffs are desirable targets for fungus. Fungi successfully colonize all plant parts, at all stages of crop/storage

Mycotoxin contamination in aquatic species is often associated with poor growth and low feed efficiency. The lack of obvious pathological signs make it difficult to identify the source of the problem.

Mycotoxin producing fungi Aflatoxins (AF), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FUM) and ergot alkaloids are within the most common mycotoxins found in agriculture commodities and responsible for millions of dollars annually in losses worldwide. These toxins are produced by just a few species from the common genera Aspergillus, Penicillium, Fusarium, and Claviceps. All Aspergillus and Penicillium species either are commensals, growing in crops without obvious signs of pathogenicity, or invade crops after harvest and produce toxins during drying and storage. The most important Aspergillus species, occurring in warmer climates, are A. flavus and A. parasiticus, which produce aflatoxins in maize, groundnuts, tree nuts, and, less

17 frequently, other commodities. Penicillium verrucosum also produces ochratoxin A, but occurs only in cool temperate climates, where it infects small grains. In contrast, the important Fusarium and Claviceps species infect crops before harvest. F. verticillioides is ubiquitous in maize, with an endophytic nature, and produces fumonisins, which are generally more prevalent when crops are under drought stress or suffer excessive insect damage. It has recently been shown that Aspergillus niger also produces fumonisins, and several commodities may be affected. F. graminearum, which is the major producer of deoxynivalenol and zearalenone, is pathogenic on maize, wheat, and barley, and produces these toxins whenever it infects these grains before harvest.

How mycotoxins reach aquafeeds Despite efforts to control fungal contamination both on the field and in storage, extensive mycotoxin contamination has been reported in both plants and finished feeds. The type and prevalence of mycotoxin contamination will depend on the type of substrate (plant meal type; finished feed characteristics) as well as geographical area, seasonal and local weather conditions during critical plant growing stages or storage. Factors contributing to the presence or production of mycotoxins include: environmental (temperature, humidity) and ecological conditions (insect attacks, physical plant damage and general stress)â&#x20AC;&#x201D;though these are often times beyond human control.

Incredibly durable, even in processing The mycotoxins commonly occurring in plant stuffs are not destroyed during most processing operations. On the contrary, processing affects mycotoxins distribution and concentrates mycotoxins into fractions that are commonly used as animal feed (plant by-products; e.g. corn gluten meal, DDGS, etc). The fate of mycotoxins during cereal processing, such as sorting, cleaning, milling and thermal processes has been studied by several authors. However, their level in feedstuffs is variable and affected by several factors such as the type of mycotoxins, the level and extent of fungal contamination, and the complexity of the cereal processing technology.

The mycotoxins commonly occurring in plant stuffs are not destroyed during most processing operations.

Penicillium-verrucosum (major producer of OTA) colonies morphology on petri dish.

On the contrary, processing

Mitigation methods

affects mycotoxins

Not all molds produce mycotoxins and even the ones that have that capacity may be present without producing any toxin. Thus, the confirmation of mold contamination is not the same thing as the demonstration of mycotoxin contamination. As a result, the use of mold inhibitors does not guarantee that feed is free of mycotoxins, as they are also produced in crops and not destroyed during processing. It is recommended that aquafeed and aquaculture producers regularly monitor raw commodity feed ingredients and finished feeds for mycotoxin contamination—either through on-site rapid testing or through an external laboratory that may be equipped with more powerful detection equipment. In cases where feed quality has been compromised by mycotoxins, the use of a mycotoxin deactivator is advised.

distribution and concentrates mycotoxins into fractions that are commonly used as

animal feed (plant by-products; e.g. corn gluten meal, DDGS, etc).

More information

Rui Gonçalves Aquaculture Scientist, Biomin E: rui.goncalves@biomin.net

AFΩ

The importance of pre-extrusion process design By Hennie Pieterse

Extrusion is the heart-beat of an aquatic feed processing plant. Still it largely depends on a well designed pre-extrusion and

post-extrusion process to ensure the desired nutritional and physical outcome of the extruded final product is achieved. Knowledge and understanding of the raw

materials, both dry and wet (or fresh) to be used in formulations are of utmost importance in designing and specifying a process that will optimize extrusion performance. Pre-extrusion

process design plays a key role in producing extruded aquatic feeds that are feed safe and environmentally friendly.

Some critical pre-extrusion processing steps It is difficult to accentuate only a few of the processing steps that make up a complete pre-extrusion process. The next topics cover some of the more critical ones:

Dry ingredient intake, cleaning and storage. The range of ingredients we use to manufacture modern-day aquatic feed are becoming increasingly more expensive. We should therefore take care as to how we acquire, accept, handle and store these ingredients on site. The following design criteria should be taken into consideration:

20 1. Intake sampling and quality control 2. Flexibility in intake & storage design allows for utilizing a wide range of raw ingredients 3. Good aspiration and dust removal from the intake pits. 4. Cleaning of the raw ingredients and removing foreign plants and other materials including metals, sand and stones 5. Safe storage of raw ingredients in bulk storage silos, flat storage and dosing silos

Dry ingredient batching Accurately combining ingredients together to create a proper formulation to achieve species health, species performance and production costs. All ingredients whether dry, liquid or fresh go through an accurate batching process ensuring the final extruded product

meets the species nutritional and lifecycle requirements. Process control and communicating formulation details between least cost formulation software and the field devices controlling the process play a key role in a successful feed mill design.

Size reduction 1. Size reduction is the first processing step where the physical and potentially nutritional characteristics of ingredients are changed. Physical ingredient size is reduced to an average particle size. Heat damage to the nutritional value of amino acids and some micro ingredients may take place when the size reduction system is not well designed or specified. The traditional size reduction rule during extrusion is to have no particle larger than one third of the die openings in the extruder die plate. This is easily

achievable using a single grinding step when producing larger extruded pellet sizes. It becomes a challenge when producing smaller pellets for shrimp and micro feed for hatcheries and ornamental fish. Traditional hammer mill designs were in most cases inadequate for fine grinding in a single step. It was and still is common to use a course grinding hammer mill followed by a fine grinding hammer mill to achieve the required fine grinding specifications. For ultra-fine grinding, to achieve an average particle size below 300 micron, a course grinding hammer mill is used in conjunction with a pulverizer. This is a common setup for producing shrimp feed and micro feed for hatcheries or ornamental fish. A range of high efficiency hammer mills are now on the market, capable of producing narrow particle size

Figure 1. This flow shows the steps we have to take before extrusion. The numbers show: 1. First Stage grinding, 2. Fine grinding , 3. Vitamin & mineral additions, 4. Fresh additions.

21 distribution curves with average particle size down to the 300-350 micron mark. The design of these hammer mills allow for shorter grinding chamber retention times and therefore lower grinding temperatures. Coupled with a variable frequency drive and special features in terms of breaker plate design and sieve types, these hammer mills can mill a large range of cereals and other aquatic feed ingredients at lower cost compared to traditional systems. Figure 2 shows typical application range for size reduction technology in relation to average ingredient size and feed type requirements. In addition an attempt in design should be made to mill only the ingredients in the formulation that requires size

reduction. Funneling unnecessary ingredients through the size reduction system increases operational costs through wear and could also increase capital cost through a higher capacity requirement.

More critical pre-extrusion processes Other pre-extrusion processing steps of critical importance are: 1. Mixing: a range of single shaft ribbon mixers, single and twin shaft paddle mixers are on the market. The ultimate goal is to deliver a homogeneous mix to the extruder surge bin, representing the specific formulation to be extruded. A

well-designed mixing environment makes provision for additional liquids and dry ingredients as well. 2. Vitamins & minerals addition: it is common for large feed mills to have dedicated micro ingredient dosing sections. Such capability provides more control and could limit costs when compared to pre-packed vitamins and minerals. 3. Fresh ingredient intake & preparation: the demand to include fresh meat, fish and other wet by-products into the formulations of aquatic species is increasing. Great care should be taken to ensure ingredient streams do not cause cross-contamination hazards. Keeping dry, fresh and final product separated. Fresh products could be pre-processed

Figure 2. Size reduction technology should be specified with final product requirements and ingredient characteristics in mind.

22 into slurries, hydrolysates, concentrates and other forms prior to inclusion into the feedstock stream.

cross contamination and segregation of mixed formulations

Critical process design aspects

Ultimately the pre-extrusion process should supply meal to the extruder that will allow the extrusion operation to be consistent, constant and easily controllable. And all of this in a cost efficient and reliable way.

A range of operations make up the preextrusion process. The discussion in this article covered some aspects of the more important operations. Ultimately the process has to be engineered into a three dimensional space that will meet operational requirements and other feed milling aspects that are frequently overlooked: 1. Sound insulation 2. Dust and odor control 3. A safe environment for workers 4. Layout design and transitional equipment sections that will minimize

5. Feed plant zoning in line with functional and feed-safety requirements

AFâ&#x201E;Ś

More information Hennie Pieterse is a graduated agricultural engineer and has 25 years experience serving the extrusion industry. He represents Ottevanger Milling Engineers in Australia and New Zealand through hp dezign Pty Ltd. He is also full-time on-board Ottevanger Milling Engineers as part of the Aquatic Feed Technology team . E: h.pieterse@ottevanger.com

Taurine leaching in fish feeds By Guillaume Salze1, T. Gibson Gaylord2, D. Allen Davis1 1

School of Fisheries Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849

US Fish and Wildlife Service, Bozeman Fish Technology Center, Bozeman, MT 59715

Leaching of water soluble compounds is an issue for fish and shrimp feeds often discussed among nutritionists. Indeed, such compounds include critical nutrients and supplements such as vitamins and free amino acids.

Yet, this issue has received limited attention in terms of dedicated, quantitative studies. Taurine has been the subject of an intensive research effort, and

Photo: Guillaume Salze, Ph.D

is now widely recognized as an essential nutrient in many fish species, such as European

seabass, California yellowtail, or Japanese flounder. Traditionally supplied through animal meals such as fishmeal, taurine must be supplemented in formulations containing high levels of taurinepoor ingredients such as plantbased protein sources. As of January 2017 taurine is listed as an approved feed additive for fish feeds in the United States; thus it is only appropriate to look at this important nutrient and its retention in fish feeds.

Descriptions of symptoms of taurine deficiencies in fish have mostly been limited to depressed growth, reductions in immune response and occurrence of green liver syndrome in juveniles of some species, and development retardation in larvae. In mammals taurine is attributed many functions spanning from bile-salt formation, osmoregulation, and inhibition of oxidative stress (Huxtable, 1992). In yellowtail taurine deficiency plays a role in osmoregulation (Takagi et al., 2006), and taurine-deficient red seabream exhibit a decrease in taurine conjugated bile-salts (Matsunari et al., 2008). In Florida pompano, taurine deficiency caused significant changes in several biomarkers of liver function, including a decrease in alkaline phospha-

tase activity, accumulation of glycogen and lipid with marked vacuolation, and reduced mitochondrial function in the liver. Taken together, these findings indicate severe abnormalities in liver metabolism in fish fed taurine-deficient diets, and provide an explanation for the reduced growth, feed efficiency and liver issues commonly observed with taurine deficiency (Salze et al., 2016). Taurine is a polar molecule, and since it is a beta-amino acid and cannot form a peptide bond to form a protein, it is almost always found in free form. Taurine is therefore prone to leaching, and it is important to quantify leaching rates from practical feeds. Indeed, there are a number of factors that will affect

24 the rates of nutrient loss due to leaching such as time of exposure, pellet size, and extrusion technology. The time of water exposure can vary dramatically with slow -eating species, as well as when feeding response is reduced with medicated feeds or during winter months. Both time in water and pelleting technology will affect the physical integrity of the pellet at the time of ingestion. Pellet size will change with fish species and across fish life stages. Quantitative knowledge of factors driving leaching rates is critical to adapt formulations accordingly and ensure that taurine intake in susceptible species are sufficient to meet the dietary requirement and avoid the consequences of a deficiency as described above. Therefore, the goal of this study was to quantify the effects of major drivers of taurine leaching and evaluate possible interactions between duration of pellet immersion, size of the pellet, along with the effects of diet manufacturing process.

Experimental design To shed some light on factors influencing leaching, a series of four diets varying in size and extrusion temperature (cold- or hot-extruded) were produced and then immersed in water for 1, 5, 20, and 40min. Table 1 summarizes the general experimental setup. Diets were manufactured using commercial methods starting with ingredient weighing of the control grind mixture. Ingredients minus fish oil were mixed in a paddle mixer in a 100-kg batch followed by grinding to a particle size of <200 µm using an air-swept pulverizer. The 2mm and 4mm pellets were manufactured with a twin-screw

Table 1: Experimental setup

Pellet size

Extrusion temperature

Time points

2.0 mm

Cold

1, 5, 20, 40 min

2.0 mm

Hot

1, 5, 20, 40 min

4.0 mm

Cold

1, 5, 20, 40 min

4.0 mm

Hot

1, 5, 20, 40 min

Table 2: Formulation of the diet mash

Ingredient Menhaden fishmeal Corn protein concentrate

Inclusion (% as-is) 12.00 10.00

Diet composition (% dry matter) Crude protein Crude lipid

45.07 ± 0.36 14.61 ± 0.78

Soybean meal Poultry by-product meal

10.00 10.00

Taurine

1.17 ± 0.03

Wheat gluten meal Blood meal, spray dried Wheat flour, durum

4.00 2.50 30.03

Menhaden fish oil Other (premixes, supplements, etc.) Taurine Yttrium oxide

10.01 10.36 1.00 0.10

extruder at the U.S. Fish and Wildlife Service, Fish Technology Center, Bozeman, Montana. The processing parameters were set to either cooking extrude or cold process the pellets. Cooking extrusion is defined when temperatures above 110oC are used to gelatinized the starch, and cold is defined as when the starch is not gelatinized and ingredients are primarily pressed into a form. Hot-extruded diets were cooked using conventional settings on the extruder. Diet mash was exposed to an average of 110oC for approximately 14 seconds in five barrel sections and the last section was maintained at 62oC. Pressure at the die head was approximately 50 bar, and screw speed was maintained at 423rpm.

The 4.0mm floating pellets were manufactured through a 3mm die while the 2 mm floating pellets were manufactured through a 1.5 mm die. The diets were dried in a pulse bed drier until moisture readings were below 6%. Drier conditions were maintained with incoming air temperature at approximately 107°C with an upper limit outflow air temperature of approximately 88°C. The diets were then cooled at ambient air temperatures for final moisture levels of <10%. The remaining fish oil was applied using a vacuum infusion coater after the pellets were cooled. The cold-extruded diets had all the oil mixed in the mash prior to pelleting. No additional heat was added and barrel tempering units were set at 15oC, which

Photo: Ryan Hagerty/USFWS

resulted in an average barrel temperature mid-way through the production of only 23.2oC. Solids feed rate was half of the cooking extruded pellets which resulted in a longer retention time in the barrels (28 seconds), but only 13 bar of pressure at the end plate. The 2mm and

4mm pellets were manufactured with 2 and 4 mm dies, respectively. The resulting extrudate did not expand and resulted in sinking pellets. Drier conditions and diet storage were as described for the cooking extruded pellets. These temperatures and pressures are typically observed in what is described as a cold extrusion process.

transformation prior to statistical analysis. The model was run using Program R (version 3.3.2, www.rproject.org) and predicative equations developed to determine estimates of taurine leaching.

The leaching trials were conducted following methods described by Watson et al. (2015) with modifications. At 1, 5, 20, and 40min after immersion, two flasks were removed from the water bath and the pellets were dewatered as quickly as possible, dried, and frozen. All leached pellets were analyzed for amino acids and yttrium oxide, in duplicates.

As expected, immersion duration, pellet size, and extrusion temperature all significantly affected leaching of taurine: higher losses were seen with longer immersion time, smaller pellets, and lower extrusion temperature. Regardless of the conditions, taurine leached out of the pellet in a logarithmic fashion: quickly during the first few minutes after immersion, after which the leaching rate slowed down progressively (Figure 1A). Therefore this relationship can be plotted on log-scaled x-axis to observe a straight line, facilitating interpretation (Figure 1B). Taurine losses after 40min immersion ranged from 44.5% to 93.6% across diets and conditions.

Taurine leaching was expressed as the percentage lost relative to the initial diet content. Effects of duration of immersion, pellet size, and extrusion temperature were evaluated in a linear model along with interactions. The duration of immersion was linearized by log-

Results and interpretation

Figure 1: Typical leaching response observed with increasing time spent in water (A) and on logarithmic scale (B). The logarithmic trajectory indicates fast leaching rates immediately after immersion, and slower rates thereafter.

26 There were significant interactions between the factors we tested, meaning that data must be interpreted according

to these four pairs of factors. Figure 2 illustrates the relationship between pellet size and immersion duration.

Figure 2: Interaction between immersion duration and pellet size. The 2mm pellets leached 35% faster than the 4mm pellets.

Regardless of extrusion temperature, the 2mm pellets leached faster than the 4mm pellets. While differences in taurine loss are barely noticeable after 1min in water, the slope of the regression lines separates the two groups by 5min of immersion. The ratio of the slopes indicates that the 2mm pellets leached about 35% faster than the 4mm pellets. This is directly attributable to the increased area of contact between the pellet and surrounding water in a smaller pellet: the larger the surface area, the more and faster the leaching. The cold-pelleted diets had a higher leaching rate than the hot-extruded pellets (31% overall), which is most likely explained by a lower physical integrity of the cold-extruded pellets vs. hotextruded pellets. Indeed, the hotextrusion process increased carbohydrate gelatinization, which traps soluble compounds in the matrix more effectively than a cold-extrusion where carbohydrates gelatinization is limited. However, there is an interaction between pellet

Figure 3: Interaction between pellet size and extrusion temperature. The cold-extruded 2mm pellets leached 54% faster than the 4mm pellets, whereas this difference was reduced to 20% in hot-extruded pellets.

27 References Huxtable, R.J., 1992. Physiological actions of taurine. Physiological Reviews 72 (1), 101-163.

Photo: Ryan Hagerty/USFWS

Matsunari, H., Yamamoto, T., Kim, S.-K., Goto, T., Takeuchi, T., 2008. Optimum dietary taurine level in casein-based diet for juvenile red sea bream Pagrus major. Fisheries Science 74 (2), 347-353.

size and extrusion technology (Figure 3), where that difference was only noticeable in a 2mm pellet, whereas leaching rates were similar in a 4mm cold- or hotextruded pellet. As a result, a 2mm coldextruded pellet leached 54% faster than its 4mm counterpart, whereas hotextrusion reduced this difference to 20% of the 4mm pellet.

Conclusion Beyond confirming the importance of pellet size, extrusion technology and immersion duration, these results quantify these effects. More importantly, they reveal and quantified interaction effects between these factors. This information can be used toward adjusting the inclusion of crystalline taurine in dietary formulations. For example, Salze et al. (2014) estimated the taurine requirement of Florida pompano at 0.54% of the diet, using cold-pressed pellets. Assuming that the pellets were immersed for about 15 seconds before being ingested by the fish (a conservative estimate allowed by an aquarium-based research system),

leaching can be estimated at approximately 5%, i.e. the fish would have eaten a pellet containing 0.51% taurine. If formulation were to be transferred from a 2mm cold-extruded to a 4mm hotextruded for commercial purposes where immersion time are expected to be up to 5min (i.e. leaching rates of about 25%), then such pellets should be formulated with 0.64% taurine to account for leaching and achieve the same ingested dose. Accounting for leaching rates as illustrated allows nutritionists to reduce the risks of either under-formulating (leading to deficiencies and reduced performances) or overformulating (reduced economic performances as well as potential water quality issues from sub-optimal nutrient utilization).

Salze, G.P., Davis, A.D., Rhodes, M.A., 2014. Quantitative requirement of dietary taurine in Florida pompano Trachinotus calorinus. Aquaculture America 2014, Seattle, WA, Feb 11th.pp. Salze, G.P., Spangler, E., Cobine, P.A., Rhodes, M., Davis, D.A., 2016. Investigation of biomarkers of early taurine deficiency in Florida pompano Trachinotus carolinus. Aquaculture 451, 254-265. Takagi, S., Murata, H., Goto, T., Hayashi, M., Hatate, H., Endo, M., Yamashita, H., Ukawa, M., 2006. Hemolytic suppression roles of taurine in yellowtail Seriola quinqueradiata fed non-fishmeal diet based on soybean protein. Fisheries Science 72 (3), 546-555. Watson, A.M., Barrows, F.T., Place, A.R., 2015. Leaching of taurine from commercial type aquaculture feeds. Aquaculture Research 46 (6), 1510-1517.

More information Guillaume Salze, Ph.D., Research Associate III, School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA. E: gzs0010@auburn.edu

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The use of

ALGAE as aquaculture feed ingredients High-quality microalgae (Reed Mariculture Shellfish Diet 1800®)

By Eric Henry, Ph.D., Research Scientist, Reed Mariculture Inc.

Interest in using algae as fish feed ingredients In recent years there has been great interest in the potential of algae as ingredients in aquaculture feeds. Even a cursory Internet search of the topic will find numerous web sites describing studies of the nutritional value of algae, or touting the potential of algae as a feed ingredient, or even announcing the launch of new aquaculture feeds made with algae as ingredients, although technical information about the products may be conspicuously absent. All this attention is largely driven by the need to find replacements for fish meal and fish oils, as awareness grows of the unsustainability of the practice of feeding wildcapture fish to support a rapidly growing farmed fish industry. Demand for aquaculture feeds that provide the high

nutritional value found in fish meal and oils is also spurred by the expansion of aquaculture of high-value fish such as Sea Bass, Sea Bream, Red Drum, Seriola, Grouper, etc. These carnivores require high-quality protein and omega-3 fatty acids in their diets, nutrients that are difficult or impossible to supply from conventional plant-based feeds.

Why Algae? Algae, including both macroalgae (“seaweeds”) and microalgae (e.g. phytoplankton), are the base of the aquatic food chains that generate the nutritional resources that fish are adapted to consume. Certain algae are already recognized as premium aquaculture feeds, for both direct feeding and to produce zooplankton (e.g. rotifers,

copepods, Artemia) for fish and shrimp larviculture, and for bivalve larviculture. So it is not surprising that many algae are nutritionally superior to the land plants used in formulated aquaculture feeds.

Which Algae? It is often not understood that the term “algae” commonly refers to what is really an arbitrary grouping of organisms that encompass a bewildering variety of forms, and an even more bewildering biochemical diversity. Algae may vary in the properties of their cell walls, which can impede digestion or extraction of nutritional components, presence of toxins or anti-nutritional factors, as well as desirable nutritional components. It is therefore impossible to make meaningful generalizations about the nutritional

30 value of this extremely diverse group of organisms, so it is always necessary to consider the particular qualities of specific algae.

Nutritional value of Algae Protein Fish meal is so widely used in feeds largely thanks to its substantial content of high-quality proteins, containing all the essential amino acids. A critical shortcoming of the crop plant proteins commonly used in fish feeds is that they are deficient in certain amino acids such as lysine, methionine, threonine, and tryptophan (Li et al. 2009). By contrast, analyses of the amino acid content of numerous macro- and micro-algae have found that they generally contain all the essential amino acids (Brown et al. 1997,

Dawczynski et al. 2007, Ortiz et al. 2006, Rosell & Srivastava 1985, Wong & Peter 2000). Lipids Certain fish oil lipids, called “PUFAs” (polyunsaturated omega-3 and omega-6 fatty acids), have become highly prized for their contribution to good cardiovascular health in humans. But it is not always appreciated that these “fish oil” fatty acids are in fact originated by algae at the base of the aquatic food chain. These desirable algal fatty acids are passed up the food chain to fish, and they are indeed essential nutrients for many fish. Algae have been recognized as an obvious alternative source of these “fish oil” fatty acids for use in fish feeds (Miller et al. 2008), especially eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA),

and arachidonic acid (ARA). There is a substantial literature devoted to analysis of the PUFA content of microalgae, particularly those used in aquaculture, because they have long been recognized as the best source of these nutrients that are essential for production of the nutritious zooplankton necessary for the first feeding of larval fish (Holt 2011), as well as filter-feeding shellfish.

Tests of Algae in formulated fish feeds Various species of macroalgae and microalgae have been incorporated into fish feed formulations to assess their nutritional value, and many have been shown to be beneficial for fish such as Tilapia (Tartiel et al. 2008), Korean Rockfish (Bai et al. 2001), Sea Bream (Yone et al. 1986, Mustafa & Nakagawa

31 1995), European Sea Bass (Valente et al. 2006), Striped Mullet (Wassef et al. 2001), Gilthead Sea Bream (Wassef et al. 2005), Atlantic Cod (Walker et al. 2009, 2010), and Salmon (Norambuena et a. 2015). But unfortunately, it is often impossible to determine the particular nutritional factors responsible for these beneficial effects, either because no attempt was made to do so, or due to the poor design of the studies. In recent years there has been great interest in the potential of algae as a biofuel feedstock, and it has often been proposed that the protein portion remaining after lipid extraction could be a useful input for animal feeds (e.g. Chen et al. 2010). However, the algae chosen for biofuel production may not be optimal for use as a feed input, and the economic pressure for the lowest-cost methods of fuel production can result in protein residues with contamination that makes them unfit for use as feed (e.g. Hussein et al. 2012).

Choosing the right Algae Just as it would be senseless to arbitrarily substitute one conventional crop plant for another (e.g. potatoes for soybeans) when formulating a feed, the particular attributes of each alga must be carefully considered. In addition to the protein/ amino acid profile, lipid/PUFA/sterol profile, and pigment content, there are important additional considerations. The type and quantity of extracellular polysaccharides, which are very abundant in certain algae, can interfere with nutrient absorption, or conversely be useful binding agents in forming feed pellets. The thick cell walls of microalgae such as Chlorella can prevent absorption

of the nutritional value of the cell contents. Inhibitory compounds such as the phenolics produced by some kelps, and brominated compounds produced by red algae, can render an alga with an excellent nutritional analysis unsuitable for use in a feed. Depending on growth and processing conditions, algae can contain high concentrations of trace elements that may be detrimental.

Economic constraints Despite the high nutritional value provided by some algae, their adoption as ingredients for aquaculture feeds remains constrained by the high cost of production and processing. Recent economic analyses (Beal et al. 2015, Maisashvili et al. 2015, Voort et al. 2015) provide useful insights toward understanding how technological progress and market forces will determine whether algae will soon become significant inputs into the aquaculture feed market. Such factors as the current moderation in fishmeal prices (Byrne 2017) will continue to have a strong influence on how rapidly such changes in feed formulation are adopted by the aquaculture industry.

Just as it would be senseless to arbitrarily substitute one conventional crop plant for another (e.g. potatoes for soybeans) when formulating a feed, the particular attributes of each alga must be carefully considered.

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More information

Eric Henry, Ph.D., Research Scientist, Reed Mariculture Inc. E: techsupport@reedmariculture.com References available by request.

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Mixed feed nut meal – natural and sustainable plant based protein for aquaculture By John Bowman, General Manager, Adaptive Bio-Resources

Aquaculture feed formulations have undergone significant change in recent years as researchers sought plant based alternatives to supplement fish meal and fish oil. The staff at Adaptive Bio-Resources, LLC has worked with esteemed aquaculture nutrition researchers for over 10 years to identify viable alternative protein sources, and recently collaborated with Dr. Frederic Barrows and his team at the USDA ARS Fish Technology Center in Bozeman, Montana, in researching several potential sources of plant based proteins. The key criteria used to determine the commercial potential of a newly developed feed ingredient include: •

Nutrient profile

•

Fish performance (growth and health)

•

Availability

•

Economics (1)

Most of the plant based feeds had significant challenges to overcome, including vastly different nutrient and anti-nutrient profiles from fish meal, high cost, limited availability, poor performance, and/or lack of sustainability. Dietary inclusion levels were often limited due to the presence of antinutrients or an imbalanced nutrient profile that resulted in reduced feed performance (growth and health). The collaborative research project culminated with the identification, testing, development of an exciting new source of protein, Mixed Feed Nut Meal, an all natural material that has a nutrient profile similar to fishmeal with no discernible anti-nutrients. The study found Mixed Feed Nut Meal produced comparable feed performance (growth and health), at virtually the same cost as fishmeal. The following summarizes the technical results of the research and testing against the key criteria. The Nutrient Profile of nut meal typically includes 50-55% protein, 8-11% lipids, and little fiber or ash. The specific nutrient profiles for materials tested in

the study are shown in Table 1. Amino acid profiles showed similarities in the ingredients, making nut meal a strong candidate for further testing. Several different forms of almond and pistachio nut material were tested in a series of trials to discern the feed performance versus alternative diets. Other nut meals such as walnut were evaluated but did not perform as well and were discarded from consideration. The Performance of the diets was tested over the life stages of rainbow trout from fry to juvenile. The first trial was a six week fry screening study, with nut meals, spirulina, Algae, and mussel meal tested against the baseline fishmeal diet. The positive performance results from this initial trial suggested that Nut Meal (pistachio and/or almond) is palatable, digestible and does not contain detrimental anti-nutrients (Table 2). The trout fed a diet containing the nut meals had survival and growth that was similar to trout fed a diet with 55% fish meal. Both pistachio and almond meals had strong apparent digestibility coefficients for crude protein, lipid, and amino acids.

34 Table 1. Nutrient compositions of nut meals, soy protein concentrate and whey for macro-nutrients and minerals.

g/100g Dry weight

g/100g Dry Weight

Test Ingredient

Lipid

Protein

Energy

Almond Meal, first cut, Adaptive Bio-Resources

92.4

8.5

53.4

4986

1.09

0.00

0.18

6.23

Almond Meal, second cut, Adaptive Bio-Resources

93.0

10.8

51.2

5053

1.09

0.01

0.18

6.28

Blanched Almonds, Adaptive Bio-Resources

93.6

9.1

61.5

5032

1.77

1.42

0.29

5.71

Almond meal, Adaptive Bio-Resources

93.7

8.6

54.4

4986

1.53

1.43

0.28

7.69

Pistachio meal, Adaptive Bio-Resources

94.2

10.7

58.8

5267

1.90

1.67

0.49

8.89

Whey Protein Isolate

94.1

1.8

79.7

5645

0.67

0.61

1.09

0.75

Soy Protein Concentrate, Profine VF, Solae

94.6

1.5

69.9

4803

1.43

0.55

2.98

Fish meal, Menhaden, Special Selectâ&#x201E;˘, Omega Prot.

94.3

8.2

69.0

4705

5.08

1.31

0.81

10.68

Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014) Table 2. The effect of diet on growth and survival of first feeding rainbow trout

3 weeks Ingredient

6 weeks

g/fish

Gain, %

Survival

g/fish

Gain, %

Survival

Fish meal control

2.7

394

100

7.6

1285

99.3

Almond 1st cut

2.6

365

7.7

1297

96.7

Almond 2nd cut

2.4

331

7.1

1195

98.0

Almond blanched

2.3

324

100

6.8

1145

96.0

Almond meal

2.5

360

100

7.8

1331

97.7

Pistachio meal

2.6

372

100

7.4

1244

99.3

Spirulina

1.9

244

99.7

5.8

948

97.7

Algae strain 1

1.9

239

4.9

795

94.3

Algae strain 2

1.8

220

100

4.1

658

96.3

Mussel meal

2.9

427

4.1

636

39.0

The second trial compared digestibility, and it confirmed that Nut Meal had high levels of digestible protein, energy and amino acids consistent with, and in some cases exceeding digestibility of the reference diet (Table 3, Table 4). The third trial was a juvenile growth study in

which trout with an average initial weight of 20 g/f were fed one of ten experimental diets for 12 weeks. This trial confirmed the results of the previous fry and digestibility trials (Table 5), with comparable to superior weight gain and survival rates. Trout gained between

600% and 900% of their initial weight. There were two series of diets, one where protein from each of the nut meals replaced fishmeal at a 50% or 100% rate, and the second where the protein from soy protein concentrate (SPC) was replaced with the nut meals

35 Table 3. Apparent digestibility co-efficient of nut meals, soy protein concentrate and whey for amino acids.

Test Ingredient

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Tyr

Val

Sum AA

Almond meal, Adaptive Bio-Resources

Pistachio meal, Adaptive Bio-Resources

Whey Protein Isolate

100

Soy Protein Concentrate, Profine VF, Solae

100

Fish meal, Menhaden, Special Selectâ&#x201E;˘, Omega Prot.

Reference diet #3

Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014) Table 4. Apparent Digestibility Co-efficient of nut meals, soy protein concentrate and whey for macro-nutrients and minerals.

Dry Matter

Fat

Crude Protein

Energy

Almond Meal, first cut, Adaptive Bio-Resources

100

Almond Meal, second cut, Adaptive Bio-Resources

Blanched Almond meal, Adaptive Bio-Resources

Almond meal, Adaptive Bio-Resources

Pistachio meal, Adaptive Bio-Resources

Whey Protein Isolate

100

Soy Protein Concentrate, Profine VF, Solae

Fish meal, Menhaden, Special Selectâ&#x201E;˘, Omega Prot.

Reference diet #3

Test Ingredient

(Table 6). Both pistachio and almond meals could serve as a complete alternative to fish meal and Soy Protein Concentrate with similar or improved performance. These trials confirmed that almond and pistachio meals have good levels of available nutrients, support a high level of fish performance, are highly palatable, digestible, and support high levels of growth whether replacing fish meal or soy protein concentrate.

The trials confirmed the first two criteria, nutrient profile and performance, but the last two criteria must also pass the test of commercial viability, adequate availability and cost.

Availability: The U.S. tree nut industry is based in California with almond and pistachio production estimated at 2.9 billion pounds (1.45 million tons) in 2016 (USDA, California Pistachio Growers). Most of this high value crop is consumed

directly by humans, with only a small portion of broken, chipped, or off color material available for feeds and oil production. ABR estimates that with conservative 30% diet inclusion rates, about 2.5% of raw nut material has the potential to provide for the diet needs of current U.S. trout and west coast salmon aquaculture. Check criteria #3. Economics/ Cost: ABR has developed proprietary production methods to safely

36 Table 5. Effect of replacing fish meal or soy protein concentrate with almond or pistachio meals at two levels on growth of juvenile rainbow trout.

Diet

Gain/fish, grams

Gain, % of initial

Survival, %

Feed Intake, % bw/day

FCR

HSI

Fish Meal Control

131.2 b

753 ab

93.3

0.92

0.78

1.33 ab

Fish, Almond, mid

104.5 d

614 d

100.0

0.95

0.79

1.10 cd

Fish, Almond, high

124.5 bc

711 bc

98.3

0.88

0.74

1.04 cd

Fish, Pistachio, mid

131.0 b

752 ab

98.3

0.91

0.76

1.16 cd

F-Pistachio, high

138.7 ab

790 ab

90.0

0.92

0.77

1.00 d

Soy Protein Control

111.8 cd

648 cd

96.7

0.99

0.83

1.39 a

SPC, Almond, mid

126.8 bc

728 bc

90.0

0.97

0.82

1.33 ab

SPC, Almond, high

111.0 cd

650 cd

95.0

0.97

0.81

1.16 cd

SPC, Pistachio, mid

123.1 bc

718 ab

90.0

0.97

0.82

1.09 cd

SPC, Pistachio, high

149.3 a

831 a

88.3

0.95

0.8

1.17 bc

Menhaden Special Select, Omega Proteins Corp, 610 g/kg crude protein b Adaptive Bio-Resources, 490 g/kg protein c Adaptive Bio-Resources, 546 g/kg protein d IDF Inc., 832 g/kg protein e Solae, Pro-Fine VF, 693 g/kg crude protein f Cargill, Empyreal 75, 756 g/kg crude protein g Manildra Milling, 120 g/kg protein h Omega Proteins Inc., Virginia Prime menhaden oil iARS 702; contributed, per kg diet; vitamin A 9650 IU; vitamin D 6600 IU; vitamin E 132 IU; vitamin K3 1.1 gm: thiamin mononitrate 9.1 mg; riboflavin 9.6 mg; pyridoxine hydrochloride 13.7 mg; pantothenate DL-calcium 46.5 mg; cyancobalamin 0.03 mg; nicotinic acid 21.8 mg; biotin 0.34 mg; folic acid 2.5 mg; inostitol 600 mg. j Contributed in mg/kg of diet; manganese 13; iodine 5; copper 9; zinc 40. k Stay-C, 35%, DSM Nutritional Products l Carophyll Pink 10, DSM Nutritional Products Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014) Table 6. Effect of replacing fish meal or soy protein concentrate with almond or pistachio meals at two levels in the diet of juvenile rainbow trout on growth performance; factorial analysis.

Diet

Gain/fish, grams

Gain, % of initial

Survival,%

Feed Intake, % bw/day

Fishmeal control

131.2

753

93.3

0.92

Fish, Almond, mid

104.5

614

100.0

0.95

Fish, Almond, high

124.5

711

98.3

0.88

Fish, Pistachio, mid

131

752

98.3

0.91

F-Pistachio, high

138.7

790

90.0

0.92

Soy Protein Control

111.8

648

96.7

0.99

SPC, Almond, mid

126.8

728

90.0

0.97

SPC, Almond, high

111

650

95.0

0.97

SPC, Pistachio, mid

123.1

718

90.0

0.97

SPC, Pistachio, high

149.3

831

88.3

0.95

37 modify the nutritional profile of nut material to meet the specifications for aquaculture feeds without degrading the basic proteins. This results in the conversion of a low value raw material into a high value product with very little waste. ABR began producing commercial volumes of Mixed Feed Nut Meal at its central California facility in 2014, with ample room for expansion. Our customers have shown strong interest in building a sustainable aquaculture industry that produces the highest quality farmed fish. Some might expect there to be an economic penalty for early adopters of plant based feeds, but that is clearly not the case. Mixed Feed Nut Meal is priced at virtually the same level as fishmeal. In addition there is another substantial benefit as the taste, texture, and overall quality of the sustainably farmed fish has opened up premium

market opportunities. Check criteria #4. We at ABR are committed to maintain and consistently supply high quality commercial ingredients needed to support this growing premium market at competitive costs. A truly sustainable, viable, and high quality aquaculture industry is not a far off dream dependent on new technology. The all natural alternative is here today for those ready to embrace the opportunity.

More information

References: (1) Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co -products from the nut industry, algae and an invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014) AFâ&#x201E;Ś

John Bowman, General Manager, Adaptive Bio-Resources E: abrjbowman@gmail.com

Nucleotides in Fish Nutrition: The best strategy to enhance immunity and intestinal health By Oriol RoigĂŠ (Bioiberica), Barcelona, Spain

Nucleotides and aquaculture: a review The aquaculture feed industry, as well as other animal feed industries, is constantly screening the market and the latest research looking for new ingredients that can give an added value to their formulations in terms of quality and functionality, having a direct impact on the production parameters and performance of the animals. In such a growing industry that is aquaculture, where fish is being cultured more and more intensively and production of new species is being researched, the addition of new functional ingredients to the diet plays a very important role and will become even more important in the future.

One of these new ingredients are nucleotides. Nucleotides are low-molecular-weight intracellular compounds that play key roles in literally every biochemical and cellular process of all living beings. This is because nucleotides are the building key parts of DNA, the molecule of life that contains all the information about everything that happens and defines the living being. Dietary nucleotides have been researched and studied in animal and fish nutrition for some years, although in a small extent. They are formed by three different units: a pentose (sugar), a nitrogenous base and a phosphate group. The pentose is either a ribose or a deoxyribose (forming RNA and DNA respectively), the nitrogenous base can be a purine or a pyrimidine depending on the molecular structure, and the phosphate group can contain from one to three phosphates. Nucleotides can be acquired by the animals from two different sources: an endogenous route through de novo synthesis, and

an exogenous route through diet. Because nucleotides can be synthetized by living organisms, they are not considered essential nutrients. However, in specific life periods with stress, diseases, fast growth or limited nutritional support, nucleotide de novo synthesis can be limited and might not cover the needs of the animal. This is because endogenous production of nucleotides is a highly energy-demanding process for the body, and during these periods energy can be limited. For this reason, nucleotides are considered semiessential nutrients, because their supplementation through the diet is very important during some specific and crucial stages of the development of the animals, such is early stages of life, vaccination, disease...

Nucleotides, as building blocks of DNA, are involved mainly in processes where cell replication is important and necessary. Cell replication is related to global growth and tissue development and reparation. For this reason, nucleotides are very useful in early stages of life, where the animals are growing quickly and tissues need to replicate fast. More specifically, some key systems benefit directly from this property of nucleotides: the immune system and the digestive system. Both systems are of huge importance for animal production in aquaculture: the immune system protects the body from diseases and is responsible for survival and healthy development, while the digestive system is responsible of the correct development and health of the intestines, absorption of nutrients and overall growth and production. Thus, the more developed these systems are, the better their survival, performance and productivity will be.

39 Dietary nucleotides in fish and immunity There are many reasons why dietary nucleotides are necessary in aquaculture. Economic losses of fish producers caused by pathological problems are becoming more and more common in the industry. These diseases have multiple causes: unbalanced nutritional conditions, wrong production management (superpopulation) or poor water quality. All this factors affect the fish by causing Figure 1. Weight gain was higher in the nucleotide group compared to the control group. Data stress, which has a direct impact on the was collected every 15 days. immune system of the animal lowering fed with control diet, (3) a group with placebo vaccination fed their resistance to infections and diseases and limiting their with an experimental diet containing 300ppm of nucleotides feed intake. This, of course, means a decrease on survival and (Nucleoforce Salmondis, Bioiberica), (4) and a group with SRS production. vaccination fed with an experimental diet containing 300ppm A trial carried out by Bioiberica, supplementing nucleotides of nucleotides (Nucleoforce Salmondis, Bioiberica). Salmons (Nucleoforce Fish, Bioiberica) to Tilapia, showed a positive had an average weight of 10g at the start of the trial, were impact on performance and health of the animals. A supplevaccinated at 21d, challenged at 71d and results were mentation of 500 ppm in the feed during 135 days was enough collected to 103d. As nucleotides are immunomodulators, the to increase survival and significantly improve productivity idea was to see whether nucleotides had any effect as vaccine parameters such as body weight and FCR (Figure 1 & Table 1). enhancers, considering that vaccines require the activation and development of the immune system. Results are shown in This trial proved that nucleotides are very helpful as they are Figure 2. immunomodulators: they get the immune system ready sooner and better, as they help immune cells to replicate and grow faster. At the end, it is translated into higher survival, body weight and improved FCR, which means better production in a more economical way.

Surprisingly, it was found that the group of salmons fed with experimental diet containing nucleotides but with placebo vaccination (3) improved their survival rate even more than the group fed with a control diet but with SRS vaccination (2). This is a clear indicator of how nucleotides are useful in the In another trial in Chile, nucleotides were tested against early maturation of the immune system, which allowed the Pisciricketsia salmonis infection, one of the main pathogens salmons to be strong enough to overcome the infection even affecting aquaculture of salmons. The experiment was better than the SRS vaccinated salmons. However, the group designed with four experimental groups of salmons: (1) a that showed the best results was the one combining SRS group with placebo vaccination fed with a control diet, (2) a vaccination and a diet supplemented with nucleotides (4). This group with SRS (Salmon Rickettsial Septicaemia) vaccination combination showed the best reduction on Table 1. Productive parameters were significantly higher in the nucleotide group. Survival rate mortality. Furthermore, when productivity also increased when compared to the control group. parameters were analysed before the challenge (at day 70), it was observed that Treatment Inital weight (g) Final Weight (g) FCR Survival (%) the groups supplemented with nucleotides showed a higher body weight than the Control 1.86 130.14 1.582 85 salmons fed with the control diet (Figure Nucleoforce 1.43 182.5 1.264 91 3). FISH

Figure 2. Accumulated mortality of the different experimental groups with different combinations of diet and vaccination.

P<0,05

Figure 3. At day 70 (before challenge) salmons with control diets and experimental diets (with nucleotides) were compared. Salmons that had been taking nucleotides showed a significantly higher body weight.

Dietary nucleotides and intestinal health: compensating the negative impact of vegetable proteins. It is a growing trend to include proteins from vegetable origin

Figure 4. Comparison of the body weight of fish with control diets and experimental diets (supplemented with nucleotides).

in fish diets (among other species), and the inclusion rate of these proteins is also increasing in feed formulations. It is known, however, that plant-based proteins have a negative impact on the intestinal health of fish, as sources like soy contain antinutritional factors that damage the intestine causing inflammation and limiting its correct development, which has a direct relation with a decrease in productivity. Considering this, it is logic to think that supplementation with nucleotides could be a good solution to compensate the negative impact of vegetable proteins: Nucleotides help replication of tissues and thus tissue repairing; intestinal mucosa is damaged when using vegetable proteins, and has a limited capacity of de novo synthesis of nucleotides. In Bioiberica, several trials have been done to prove this thesis. In a first trial in Portugal, supplementation with 1000 ppm of nucleotides (Nucleoforce Fish, Bioiberica) was tested in juvenile meagre with initial average weight of 37,2g over a 60day period. Nucleotide group and control group had diets with high vegetable protein content (75%) (Figure 4) Results showed a significant improvement on body weight after 60 days in the nucleotide group and a significant increase in leucine aminopeptidase activity (data not shown), suggesting improved enterocyte status and increased digestion capacity. Histological sections also showed a better developed intestine (Figure 5).

41 over a 134-day period. Both nucleotide and control group received 100% plantbased fishmeal-free diets. Results are shown in Figure 6 and Figure 7.

Figure 5. Histological sections of the intestine at 60 days. Figure 5A is from fish with supplementation of nucleotides, 5B is from fish with control diets.

Fish supplemented with 250 ppm of nucleotides performed significantly better than fish fed with control diet. After 134 days, there were significant improvements in body weight, growth rate and FCR when compared to the control group.

Both trials suggest that nucleotides are an excellent strategy to significantly reduce the bad impact of vegetable proteins in fish feed. In other words, supplementing fish diets with nucleotides is very useful to increase the vegetable protein content and reducing fishmeal, as they reduce significantly the negative impacts of these proteins.

Conclusions

Figure 6. Differences in weight gain of gilt-head sea bream fed with a control diet and with a diet supplemented with nucleotides.

Nucleotides are semi-essential nutrients that have key roles in the body of fish and animals. Nucleotides are very useful in some specific stages of development, especially in those where cell and tissue replication is very important: early stages of life, infection and diseases, vaccinations, stress… Nucleotides help the immune system to develop faster, making it ready to any challenge. Furthermore, they contribute to the healthy development of intestines, improving productivity and overall performance.

AFΩ

More information Figure 7. Differences in Individual Growth Rate (IGR) and Feed Conversion Rate (FCR) of gilt-head sea bream fed with a control diet and with a diet supplemented with nucleotides.

In a second trial in Valenica (Spain), supplementation with 250 ppm of nucleotides (Nucleoforce Fish, Bioiberica) was tested in juvenile gilt-head sea bream with initial average weight of 11g

Oriol Roigé Product Manager Animal Nutrition Bioiberica Barcelona, Spain. E: oroige@bioiberica.com References and bibliography are available by request.

Use of computer-assisted image analysis to monitor health and nutrition status in whiteleg shrimp (Penaeus vannamei) By Fabio Cervellione, Charles McGurk, Skretting ARC and Wim Van den Broeck, Ghent University

The aquaculture of penaeid shrimp has grown from its experimental beginnings three decades ago into a major industry. Almost from the start, diseases and adverse environmental conditions were recognized as threats to the shrimp industry, causing serious economic losses. Feeding strategies form an important part of modern farm management. Feed manufacturers invest in R&D to develop and offer new feeds and feeding solutions aimed at helping shrimp farming to grow, and to support against emerging health issues and adverse environmental conditions. Current methods to assess health and nutritional status in shrimp mainly rely on clinical examination followed by laboratory investigations and evaluation of growth performance. One of the key indicators of health in shrimp is the perigastric organ (formerly known as the hepatopancreas), making it one of the most useful to examine. The perigastric organ is the site of synthesis and secretion of digestive

enzymes, digestion and nutrients absorption, reserve storage and detoxification. The perigastric organ is bilobed and composed of many blindly ending tubules, which wrap over the dorsal and lateral sides of the posterior part of the stomach and the anterior part of the midgut. It is the principal organ of the digestive tract and vulnerable to pathophysiological changes.

Feeding regime affects the perigastric organ. Under starvation and refeeding conditions, beneficial lipid droplets are rapidly depleted and haemocytes (comparable to the white blood cells in mammals) are recruited to mitigate degenerative processes and inflammation occurring in the organ. Haemocyte infiltration in the intertubular spaces of the perigastric organ is also observed in instances of tissue damage (traumatic or due to infection), toxicity and environmental stress. In current practice, histological analysis of the perigastric organ is based on qualitative features interpreted by

pathologists. Pathologistsâ&#x20AC;&#x2122; quantification is generally time-consuming and poorly objective, with significant discrepancies in scoring results reported between observers. This has motivated the development of computer-assisted image analysis (CAIA) methods for producing unbiased, objective, reproducible, and reliable data. The basic principle of automated image analysis for histology is the use of a series of mathematical algorithms to process images, enabling the segmentation of picture elements into regions of interest based on their colour, texture and/or context. CAIA is currently used by Skretting Aquaculture Research Centre (ARC) to investigate a number of organs (gills, gut and skin) across various fish species.

The success of these methods in fish recently led to the development of a semi-quantitative histological method to monitor health and the nutritional status in whiteleg shrimp using CAIA on microscopic sections of the perigastric organ. After the optimization of fixation

44 methods (both for paraffin and frozen sections) and staining protocols for the perigastric organ, customized software was used to develop and validate protocols for the quantification of following morphological parameters of the organ: areas of haemocyte infiltration (inflammation), F-cells (production of digestive enzymes), B-cell vacuoles (site of intracellular digestion), lipid droplets (reserve energy stored in the R-cells) and their ratios to total tissue area and total lumen area. Figure 1 shows the development of one protocol for CAIA. The effect of feed deprivation and refeeding was studied using the developed method. Shrimp of 2 ± 1 g were divided into three groups (40 shrimp each), receiving three treatments (one treatment per group) for 15 days: FE (fed), ST (starved), and REF (starved for 5 d and then re-fed for 10 d). Shrimp were sampled at day 0, 5, 10, and 15 and selected for the C-intermolt stage. Software was used to quantify the following morphological parameters, indicating different parameters: inflammatory index (HIA:TTA), nutritional index (LDA:TTA), and other parameters indicating the enzymatic and digestive

Figure 1. Development of computer assisted image analysis protocol for the detection of haemocytes and B-cell vacuoles in the hepatopancreas (HP) of whiteleg shrimp (Penaeus vannamei). Paraffin section (5 µm) of the HP stained with immunohistochemistry using monoclonal antibodies WSH8 counterstained with Mayer´s haematoxylin (a). Protocol workflow (b,c,d): pre-processing (b), classification (c), post-processing (d). In d: total tissue area (pink), lumen area (grey), B-cell vacuoles (light blue), haemocytes (blue). lu = lumen; tb = hepatopancreatic tubule; vB= B-cell vacuole; black arrows = haemocytes. Scale bar = 100 µm

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45 activity of the organ. Starvation led to significant morphological changes in the HP. Significant changes were measured for the inflammatory index (Figure 2) and nutritional index (Figure 3) during starvation (increase of inflammation associated with decreased of lipid reserves) and starvation followed by refeeding (decrease of inflammation associated with increase of lipid reserves). Refeeding did not result in a complete recovery of the perigastric organ. The main findings of this three-year PhD study can be considered as a starting base to implement image analysis in the shrimp histology labs for diagnosis purposes and to start collecting reference values of the developed morphological parameters to be used for screening health and nutritional status in crustaceans. The development of specific indices (inflammatory and nutritional) will be used in the future to collect reference values for monitoring health, assess the impact of different diets and feeding regimes, facilitate early diagnosis of diseases and study the pathophysiology of the perigastric organ.

Fig 2. Effect of starvation and refeeding on the inflammatory index of the perigastric organ in whiteleg shrimp (Penaeus vannamei). Note: HIA:TTA = ratio of Haemocytic Infiltration Area to Total Tissue Area (as %). FE = fed; ST = starved; REF = refed; R = start of refeeding after 5 days of starvation.

The PhD has been funded by Skretting ARC and the Research Council of Norway.

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46 References: Cervellione, F., McGurk, C., Silva, P., Owen, MAG & Van den Broeck, W. (2016) Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp, Penaeus vannamei (Boone). Journal of Fish Diseases, doi:10.1111/jfd.12531. Cervellione, F., McGurk, C., Berger Eriksen, T. & Van den Broeck, W. (2016) Use of computer-assisted image analysis for semi-quantitative histology of the hepatopancreas in whiteleg shrimp Penaeus vannamei (Boone). Journal of Fish Diseases, doi:10.1111/jfd.12599. Cervellione, F., McGurk, C. & Van den Broeck, W. (2017) Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer-assisted image analysis. Journal of Fish Diseases, doi:10.1111/jfd.12599. Cervellione, F., McGurk, C. & Van den Broeck, W. (2017) “Perigastric organ”: a replacement name for the “hepatopancreas” of Decapoda. Journal of Crustacean Biology, doi: 10.1093/ jcbiol/rux020.

Fig 3. Effect of starvation and refeeding on the nutritional index of the perigastric organ in whiteleg shrimp (Penaeus vannamei). Note: LDA:TTA = ratio of Lipid Droplet Area to Total Tissue Area (as %). FE = fed; ST = starved; REF = refed; R = start of refeeding after 5 days of starvation.

AFΩ

More information Fabio Cervellione, DVM, MSc, PhD, Research Pathologist, Skretting ARC, Norway E: fabio.cervellione@skretting.com Charles McGurk, DVM, MSc, PhD, Fish & Shrimp Health Manager, Skretting ARC, Norway E: charles.mcgurk@skretting.com Wim Van den Broeck, DVM, MSc, PhD, Professor of Histology and Cell Biology, Ghent University, Belgium E: wim.vandenbroeck@ugent.be

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