Aquafeed Magazine Vol 15 Issue 2 2023

Vol 15 Issue 2 April 2023

AQUAFEED Advances in processing & formulation An Aquafeed.com publication

NOVEL CANOLA PROTEIN CONCENTRATE Feed additives Mycotoxins Plant extracts and botanicals Published by: Aquafeed Media, S.L.U. www.aquafeed.com info@aquafeed.com

IF YOU THINK ALL LYSOPHOSPHOLIPIDS ARE THE SAME...

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AQUAFEED

VOL 15 ISSUE 2 2023

Contents

MYCOTOXINS RISK AND MANAGEMENT 35 How mycotoxins impact through feed and feed ingredients and their consequences on fish and shrimp health.

HIGH-PROTEIN CORN FERMENTATION PRODUCTS 14 Increased protein and reduced fiber content has allowed its use in carnivorous fish species.

HARNESSING PLANT SYNERGIES 49

REPLACING VITAMINS E AND C 59

How incorporating microencapsulated botanicals into aquafeeds improves performance.

Selected polyphenols have the potential to partially replace vitamins E and C in animal feed.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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AQUAFEED CONTACT US Editorial: editor@aquafeed.com Editor/Publisher: Lucía Barreiro Consulting Editor: Suzi Dominy Technical Editors: Peter Hutchinson, Albert Tacon, Ph.D Assistant Editor: Marissa Yanaga Conferences and webinars: info@aquafeed.com Advertising enquiries/request media pack: sales@aquafeed.com Accounts & all other enquiries: info@aquafeed.com

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VOL 15 ISSUE 2 2023

Contents 6

Interview with Dustin Schulz

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News Review

11 A novel canola protein concentrate for aquaculture *Cover story

14 Use of high protein corn fermentation products in the diet of rainbow trout

22 Fermented plant proteins in fish feeds: Playing an especially powerful role in improving animal health

25 Precision nutrition and corn-fermented protein: A valuable concept

29 A 60% sustainable vegetable protein for aquaculture derived from the corn renewable fuels industry

33 Algal oil gives control of omega-3 levels in farmed salmon 35 Mycotoxins in aquaculture: Risk and management

We are grateful to the following companies for sponsoring this issue of the magazine.

Their support allows us to make our publications available without charge.

Adisseo...................................................................................... 2 Mixscience................................................................................ 5 Wenger........................................................................................ 8 Extru-Tech..................................................................................10 The Andersons..........................................................................21 POET ..........................................................................................28 Phodé........................................................................................32 Phytosynthese...........................................................................34 Trouw..........................................................................................41 Oxiris ..........................................................................................44 Vetagro.....................................................................................52

42 Designing antioxidants: The effect of molecular structure 45 Health program based on functional feed additives to fortify gut integrity and protect against parasitic infestations in aquaculture

49 Harnessing plant synergies to enhance performances, health and sustainability in fish and shrimp

53 Benefits of supplementing herbs and plants as feed additives and complementary feeds for sustainable aquaculture

59 B eneficial replacement of vitamins E and C in feeds for tilapia 62 Calendar of events

Nuproxa.....................................................................................57 Victam.......................................................................................63 WAS ..........................................................................................64

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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Dustin Schulz is VP of Operation at Optimal Aquafeed

INTERVIEW with Dustin Schulz AQ: What has been your journey in aquafeeds? How did you get to where you are today?

DS: One of my first childhood memories was trying to make fish food with my brother. I didn't know how things were made, so I thought you put similar items together, mixed them with water, and then waited until they became something new. I saw my goldfish flakes were thin and colorful, so we collected all our crayon shavings; I knew it smelled like dog food, so we went and got some dog food; I knew fish in the quarry liked crickets, so we went and caught some of those. Then we mixed them with water and checked the concoction every morning to see if we had made fish food.

Thankfully, I've learned a lot since then.

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I eventually went to grad school at South Dakota State University, where I worked in nutrition research.

During this time, a medical doctor and a biochemistry Ph.D., a fish nutritionist Ph.D., my brother, and I laid the foundation of Optimal Fish Food. In the evenings and on weekends, I would hand-weigh ingredients, run a small extruder, ship out finished fish feeds, and talk to customers on the phone. The appreciation and support throughout the pond and lake community were encouraging and helped develop the customer service we continue to focus on today at Optimal. As each year passed, we learned new things about running a business, built new relationships, and continued to see the company grow.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

INTERVIEW A few years after the start of Optimal, we started Optimal Aquafeed, a joint venture with Green Plains Inc. focused on aquaculture feeds. As we develop Optimal Aquafeed and continue to grow Optimal Fish Food, it is with great pride that we have kept strong relationships with our customers throughout the years and have continued to advance our knowledge for producing highquality fish feeds.

AQ: How did the company benefit from the joint venture with Green Plains?

DS: Partnering with a much larger company always has obvious benefits, like resources, experience, and reach.

Optimal Fish Food was, and remains, devoted to the pond and lake management marketplace. When word of our feed performance got out, we were contacted by aquaculture producers, but their volume needs far exceeded our capacity. Optimal Aquafeed is a joint venture that allows us to take what we love about feed and customer service and bring that to the larger aquaculture market. With Green Plains' commitment to producing high-quality proteins at a commercial scale and Optimal's commitment to creating high-quality feeds, this joint venture offers a unique opportunity for an ingredient company and a feed company to work together to make quality feeds with quality ingredients.

It's a rare opportunity for us to play a pivotal role in guiding the research to develop ingredients that we, and our customers, want in our diets.

AQ: Where is the company today in terms of size, markets and species served?

DS: We are a small feed company servicing the pond and lake industry as well as creating specialized formulations, feeds, and ingredients for the aquaculture industry. We aim to see the overall marketplace of fish feed grow and continue to raise the bar in ingredients, feed quality, fish health, and feed conversion. We see an opportunity to work alongside other feed manufacturers to provide the products and services necessary to continue developing and growing aquaculture in America. We will never compete with the scale of the larger established companies in regards to grow-out diets, but we see a lot of opportunities with specialized feeds and ingredients that will benefit any aquaculture producer, especially in the areas of starter feeds, finishing feeds, or unique challenges a specialized diet could provide the solution for.

We see a lot of opportunities with specialized feeds and ingredients that will benefit any aquaculture producer, especially in the areas of starter feeds, finishing feeds, or unique challenges a specialized diet could provide the solution for.

AQ: Optimal Aquafeed uses a different approach than traditional aquafeeds in terms of diet formulation.

Would you let us know a bit more?

DS: At Optimal Fish Food, all our formulations are driven around making the best diets for growth, health, and longevity. For pond and lake management, the goal is to grow trophy fish. And trophy fish are generally old fish in comparison to aquacultured fish. We achieve this by creating diets utilizing high-quality ingredients and putting these formulations through multiple trials to ensure the best results.

At Optimal Aquafeed, we use this same approach; combined with our small size, we can access and utilize many modern novel ingredients that aren't being produced at a commercial scale to focus species-specific diets designed to not only grow large, healthy fish but achieve market size growth as fast as possible.

AQ: Optimal Aquafeed has an innovation center in Omaha, Nebraska, USA that performs aquafeed processing trials. What does this center add to the company’s capacities?

DS: Our innovation center in Omaha is an example of how we benefit from our joint venture with Green Plains. The facility does a lot under one roof. We have a small feed mill complete with a pulverizer and vacuum coater to produce very specialized feeds. We also have an aquaculture lab where we can do all the classic trials to evaluate the palatability, digestibility, and long-term growth implications of new ingredients or formulations. The facility also has a small twinscrew extruder lab for making smaller batches of trial feeds. Green Plains also has a centralized analytical lab and a pilot-scale biorefinery lab there. It is an incredible

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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INTERVIEW space that allows people from the many aspects of ingredient production to take a multidisciplinary approach to solve problems, create new technology, and provide ongoing evaluations.

AQ: Being an innovation-driven company, what are the current trials in your pipeline?

DS: We always have many things being worked on at any given time. We are currently very excited about our work tackling the off-flavor problems associated with RAS. There are a lot of demands on a RAS setup, and like many others, we are working on trying to make our feeds perform better and developing solutions for whatever unexpected challenges our customers might face. Along with our multiple RAS trials in the works, another big area of focus has been on starter feeds. We’ve seen the importance of starter feeds in growing trophy fish, and are excited about how those same approaches impact what we are seeing within our aquaculture research, development, and trials.

AQ: Looking ahead, what are the company projections in terms of product developments and production capacity?

DS: Nowadays, when I walk into the feed mill and look at the array of available ingredients, it reminds me of searching for ingredients around the house when I was little. But, unlike back then, we now have a great team of enthusiastic, knowledgeable, and skilled colleagues that are always looking for the next process, the next ingredient, the next formulation, or the next idea to challenge how we do things at Optimal. We endeavor to provide solutions to help grow and strengthen the aquaculture industry. Our goal has always been to grow as fast as we can and still offer high-quality feeds and customer service we stand behind. We don’t know what the future has in store for us, but we are growing far faster than we had ever thought when we started. It has been a strange last couple of years, but we believe there is a bright future for aquaculture, and we intend on being there to support it.

Wenger.com

The Future Awaits Built on partnership and innovation, Wenger is providing more opportunities for client success.

For almost a century, Wenger has delivered extrusionbased innovations to our partners. We’ve worked alongside you to develop new processing solutions and better products, providing our industry-leading expertise and ongoing support every step of the way.

We don’t plan on stopping any time soon.

Wenger’s global food processing family is growing, and we look forward to the exciting opportunities that lie ahead. We will continue to deliver even more innovations and technologies to benefit companies that share our vision of tomorrow.

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

NEWS

NEWS REVIEW Highlights of recent news from Aquafeed.com

Skretting India opens stateof-the-art shrimp and fish feed facility in Surat

Maharashtra Feeds opens fish feed mill in India

Skretting opened a EUR 18.5-million state-of-theart production facility for shrimp and fish feed in Mangrol, Surat. The facility has three production lines with a production capacity of 50,000 metric tonnes per year. It can produce both extruded/ floating and pelleted/sinking feed as per the requirement of the species and customers.

DSM presents fillet color measurement tool

The company recently inaugurated a new state-of-theart floating fish feed plant in Lucknow, Uttar Pradesh, India. With a production capacity of 35,000 metric tons per year, the company aims to serve the North, Central, East and parts of Western India. “Our advanced and latest grinding, mixing and extrusion technology with a 10 TPH twin screw extruder ensures the production of premium hatchery feeds as well as grow-out floating fish feeds with sizes ranging from 0.6mm to 4mm,” Garvit Jain, vice president at Maharashtra Feeds, told Aquafeed.com in an interview.

De Heus opens its first feed mill in Ivory Coast The company opened a new greenfield animal feed factory with an initial capacity of 120,000 MT. It will manufacture feeds for fish, poultry and cattle for farmers in Ivory Coast and surrounding export destinations.

The company unveiled a new technology, Digital SalmoFan™, to accurately measure fillet color throughout the entire value chain. The Digital SalmoFan™ is a portable and user-friendly device measuring the color of salmon fillets according to a numeric scoring system.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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NEWS BioMar partners with Benson Hill to boost sustainability of aquafeed The companies signed a strategic collaboration to advance BioMar’s responsible sourcing program by leveraging Benson Hill soy and further assessing its sustainability impact on high-performance aquafeed formulations. Benson Hill advances sustainability goals throughout the ingredient development process, from monitoring regenerative and deforestation-free practices on the farm to producing

Whisky byproducts turn into omega-3 product Scottish biotech company MiAlgae launched its omega3 product NaturAlgae to market thanks to the recent expansion of its commercial production site in Scotland.

The company recycles the co-products from the Scotch whisky industry as a feedstock to grow omega-3-rich microalgae which can then be used in aquafeed recipes.

ingredients that reduce water and energy-intensive protein-concentrating steps in processing.

AB Vista unveils natural resin acids product

Progres® is a patented natural resin acids product that supports gut integrity, helping producers mitigate the impact of different stress factors and improve animal productivity and resilience.

The product was originally developed by Finnish biosciences company Hankkija and acquired by AB Vista as part of its strategy to ramp up its gut health portfolio.

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

NEW PLANT-BASED INGREDIENTS

A novel canola protein concentrate for aquaculture Andre Dumas, AD Aquaculture Nutrition Services Inc., William Riley, WWR & Associates, David Dzisiak, Botaneco Inc., John Brennan, JBW Nutrition Consulting Inc.

Continued expansion of aquaculture production is driving the demand for new sources of sustainable, nutritious, and cost-effective protein. Canola (oilseed rape) is the second largest oilseed grown globally after soybean, with crop production mainly located in Canada, northern Europe, and Australia. Over 50 MM mt is produced annually, containing over 10 MM mt of high-quality protein.

This makes canola highly available on a global scale, and it provides one of the most balanced amino acid compositions among plant-based protein sources.

Historically, inclusion rates of canola protein in salmonid diets were limited due to the presence of antinutritional factors that reduced feed intake and growth.

Botaneco has developed a novel aqueous process for canola that allows for the high concentration of protein (75%) and the removal of the anti-nutritional factors.

Alofin™ canola protein concentrate is manufactured using new production technologies, different from traditional oilseed processing, delivering a clean and highly palatable protein ingredient.

A series of nutritional studies were conducted to estimate nutrient digestibility in fresh and saltwater and assess the long-term efficacy of Botaneco’s Alofin canola protein concentrate (Table 1) in the diets of Atlantic salmon. These studies were conducted at the Center for Aquaculture Technologies, Prince Edward Island, Canada.

Nutrient digestibility with pre-smolt salmon Atlantic salmon (mean initial body weight = 57.3±6.7g) were hand-fed four digestibility diets containing different ratios of a Reference diet (51.2% crude protein; 19.1% crude lipid; dry matter basis) to Botaneco’s Alofin canola protein concentrate: Diet A served as the Reference diet and contained no Alofin; Diet A was

diluted with 10, 20 and 30% Alofin to create test diets B (Alofin 10), C (Alofin 20) and D (Alofin 30), respectively.

Each diet was randomly allocated to three 100-L digestibility tanks in recirculating aquaculture system (RAS) in freshwater, and feces were collected daily by

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS Table 1. Analyzed nutrient composition of typical Alofin canola protein concentrate.

Nutrient

Units (as-fed)

Proximate composition (%)

Dry matter

Crude protein

Crude lipid

Crude fiber

Ash

95.6 75.8 4.4 <0.2 6.2

Essential amino acids (%)

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Trp

Val

5.0 2.2 3.1 5.7 4.9 1.6 3.2 3.3 1.2 3.8

Minerals (% or mg/kg)

Phosphorus

Sodium

Calcium

Magnesium

Potassium

Iron

Manganese

Zinc

0.70 % 1.27 % 0.14 % 0.17 % 0.65 % 41.9 mg/kg 36.1 mg/kg 58.9 mg/kg

the settling method over 34 days. Titanium dioxide (0.5% inclusion) served as the digestibility indicator.

Alofin contains highly digestible nutrients, which reveal the promising value of this alternative protein source for the salmon feed market. The mean (±standard deviation) apparent digestibility coefficient (ADC, % dry matter) for crude protein was 91.6 (5.5)%. The mean ADCs of essential amino acids (EAA) varied between 84.7 (8.2)% for leucine and 97.5 (7.8)% for tryptophan.

The mean ADCs for lysine and methionine were 90.9 (6.3) and 93.9 (5.3)%, respectively. Nutrient digestibility was not affected by Alofin inclusion level (P≥0.05).

Nutrient digestibility with post-smolt salmon Post-smolt Atlantic salmon (227.0±4.1g) were handfed four digestibility diets containing Reference diet to Botaneco’s canola protein concentrate ratios identical to those of the pre-smolt digestibility trial. Each diet was randomly allocated to four 750-L tanks in recirculating aquaculture system (RAS) in saltwater (25 ppt), and feces were collected weekly using the manual stripping method over 55 days. Titanium dioxide (0.5% inclusion) served as the digestibility indicator.

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Like with pre-smolt salmon, nutrients of Alofin were highly digestible in saltwater. The mean (±standard deviation) apparent digestibility coefficient (ADCs, % dry matter) for crude protein was 95.6 (2.9)%. The mean ADCs of essential amino acids (EAA) varied between 93.8 (2.3)% for threonine and 98.0 (2.2)% for arginine.

The mean ADCs for lysine and methionine were 95.7 (2.1) and 97.1 (2.0)%, respectively. Again, nutrient digestibility was not affected by Alofin inclusion level (P≥0.05).

Long-term efficacy study with post-smolt salmon Graded inclusion levels (0, 10, 15, and 20%) of Alofin were assessed in post-smolt Atlantic salmon (228.0 ± 4.9 g) over a six-month growth study. Eight experimental diets containing 0 (Diets A, E), 10 (Diets B, F), 15 (Diets C, G), and 20% (Diets D, H) Alofin were randomly allocated to 24 750-liter tanks at 33 fish per tank. Diets A through D were formulated to contain processed animal proteins (PAPs), whereas diets E through H included no PAPs. Feed intake and growth (thermal-unit growth coefficient, TGC) were not significantly affected by Alofin inclusion (Table 2). The best feed conversion ratio (FCR) was observed at 10% Alofin. Results from histological examination of the distal intestine and blood plasma biochemistry indicated no adverse effect of Alofin. To conclude, Alofin stood as a safe and nutritious protein alternative up to at least 20% inclusion in salmon diets.

Study with shrimp Continued growth in the world shrimp production creates another high-value market opportunity for Alofin. In anticipation, Botaneco conducted a study to determine the efficacy of Alofin as a replacement for fishmeal (FM) in Pacific whiteleg shrimp diets. The study assessed growth performance, nutrient digestibility, and shrimp immunity at several levels of Alofin inclusion.

Alofin (78.9% crude protein) replaced 65% FM at 0, 5, 10, and 20% dietary inclusion (0, 5.7%, 11.5%, 20% FM replacement), with the latter treatment completely replacing FM as well as 7% dietary SMB. The eight-week trial employed juvenile whiteleg shrimp (~2.6 gm) in 10-15 ppt saline water, with 25 shrimp/aquarium and 6 repetitions/treatment. The shrimp were fed three times per day at 3-4% body weight. By the end of the trial, the weight gain and

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS Table 2. Effect of canola protein concentrate (Alofin) inclusion on feed intake (FI), feed conversion (FCR), and thermal-unit growth coefficient (TGC) of post-smolt Atlantic salmon. Values are means ± standard errors.

ALOFIN (%)

FI (g fish-1)

FCR

TGC [g1/3 (oC d)-1]

0 10 15 20

724.0 ± 34.5 755.2 ± 20.2 739.2 ± 21.7 736.6 ± 12.8

1.05 ± 0.00ab 1.04 ± 0.01b 1.08 ± 0.00a 1.07 ±0.01a

0.152 ± 0.005 0.158 ± 0.003 0.151 ± 0.003 0.152 ± 0.002

P-value

0.8286

0.0017

0.3875

Figure 1. Growth trajectories of post-smolt Atlantic salmon fed graded inclusion levels of canola protein concentrate (0, 10, 15 and 20%) in diets with (A through D; left) and without (E through H; right) processed animal proteins.

specific growth rate of shrimp fed 5-20% Alofin exceeded that of shrimp fed no Alofin. Feed conversion was improved at these same inclusion levels through 6 weeks (but unchanged at 8 weeks). The digestibility coefficients for protein (86%), lipid (96%), phosphorus (96%), and energy (79%) were high. Shrimp immunity was improved with the addition of Alofin under normal (non-challenged) conditions and after a challenge with Vibrio parahaemolyticus. Increased phenoloxidase activity, a reduced Vibrio count in the gut, and improved post-challenge survivability were evident in the Alofintreated shrimp.

These results support the use of Alofin in the diets of whiteleg shrimp as a replacement for FM at an inclusion rate of 5-20%. Alofin had high protein and lipid digestibility, and it improved growth performance, immunity, and disease resistance against Vibrio parahemolyticus at an inclusion rate of 10-20%.

Conclusions Canola production in Europe, Canada, and Australia provides a large-scale, existing source of feedstock for Alofin production. Taking advantage of existing production and upscaling the meal into high-quality protein concentrates provides for a sustainable

production system that adds value to farmers, shortens supply chains, supports circular economies, and helps reduce the carbon footprint of aquaculture feeds.

The scale of canola available and Botaneco’s process technologies would allow for pricing under fishmeal.

These feeding trial results strongly support the potential of canola protein as a macro ingredient in salmon and shrimp diets. Alofin delivers a nutritious, scalable, and sustainable protein feed ingredient for global aquaculture.

Acknowledgements Botaneco would like to recognize Protein Industries Canada and the Global Innovation Cluster for their project support.

More information: David Dzisiak Chief Operating Officer Botaneco E: ddzisiak@botaneco.com

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS

The use of high-protein corn fermentation products in the diet of rainbow trout Scott Tilton, Julia Green, John Hagios, The Andersons, Inc.

Global farmed trout production continues to grow, with rainbow trout (Oncorhynchus mykiss) leading the way. It is estimated that world production of rainbow trout was 959.6 thousand tons in 2020 (FAO, 2022), making it the second-largest farmed salmonid species.

Rainbow trout is a carnivorous fish species that requires high levels of both protein and lipid, with commercial diets commonly containing 40-45% protein and 14-25% lipid depending on the stage of production and performance objectives. Historically, these diets have relied heavily on fishmeal as their primary protein

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source; however, recent research has focused on reducing or eliminating fishmeal from feed formulations to make trout diets more sustainable.

Recently, the U.S. ethanol biorefinery industry has started to evolve, creating novel protein products ranging in protein contents from 40% to 60% on an as-fed basis. These products are corn protein-based, and most contain significant amounts of protein from the spent yeast (Saccharomyces cerevisiae) used in the fermentation process. To date, there are at least four different technology suppliers and six different high

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS protein feed products commercially available from the United States. Currently, these products are marketed in North America under the AAFCO feed ingredient definition 27.5 Distillers Dried Grains (AAFCO, 2023), however, this ingredient definition covers products with a crude protein range of 24% to 60%. As such, the Distillers Grains Technology Council developed a group of industry trade terms to attempt to describe potential products more accurately. These terms are Distillers Dried Grains (DDG) for products from 24-35% crude protein, High Protein Distillers Dried Grains (HPDDG) for products from 36-48% protein, and Fermented Corn Protein for products higher than 48% crude protein.

Growth, in volume of these high protein products, has been gradual, starting at one biorefinery in 2010, to over twenty facilities either in production or currently under construction. Two of these new high protein biorefinery co-products are the ANDVantageTM 40Y and ANDVantageTM 50Y protein products, 40% and 50% crude protein products produced by The Andersons, Inc. These products are produced using ICM, Inc.’s patented processing technologies.

Briefly, fiber is removed prior to fermentation.

The remaining low-fiber material is then fermented and distilled to separate alcohol from protein and corn oil fractions. Material is processed using ICM’s Thin Stillage Solids Separation SystemTM to make the high protein products. Further processing utilizing ICM’s Feed Optimization TechnologyTM is required to reach 50% crude protein. Differences in processing by utilizing the Feed Optimization Technology result in increased protein and yeast content of the final protein product.

With the increased protein and reduced fiber content of these new ethanol biorefinery feed products, the potential for use in carnivorous fish species like trout and salmon is possible. To further evaluate the potential use of ANDVantage 40Y and ANDVantage 50Y in rainbow trout production, two experiments were conducted by the United States Fish and Wildlife Services’ Bozeman Fish Technology Center. The first of these experiments was to determine amino acid, phosphorus, and energy digestibility of both the 40% and 50% protein products, while the second was to evaluate the growth performance of rainbow trout fed varying levels of either product.

It's all about digestibility ANDVantage 40Y and ANDVantage 50Y were provided to the Bozeman Fish Technology Center (BFTC) by The Andersons, Inc. for analysis and subsequent digestibility determination. Additionally, Special Select Menhaden Fishmeal was used as reference material for digestibility. Compositional analysis on a dry weight basis of the tested ingredients are provided in Table 1. Digestibility was determined using the methods of Cho et al. (1982), Bureau et al. (1999) and Forster (1999). This involves formulation of a basal diet that meets or exceeds all known nutritional requirements of rainbow trout. Test diets were then blended using a 70:30 ratio of reference diet to test ingredient, and digestibility was determined by difference. All diets contained yttrium oxide as an inert marker for use in calculating digestibility.

Digestibility was determined using feces collected by manual stripping while fish were under mild anesthesia.

Three replicates were used per diet, with 23 fish per replicate tank. Trout averaged 346g at the start of digestibility determination and were fed test diets for 7 days before feces collection. Feces were collected 18 hours after feeding. After collection, feces were freezedried, ground, and stored at -20°C until analyzed.

Overall digestibility of both the ANDVantage 40Y and ANDVantage 50Y products were quite good, with crude protein digestibility values of 81.7% and 83.0%, respectively (Table 2), and did not differ from fishmeal. Crude fat digestibility was also high with 98.1% and 98.3% for ANDVantage 40Y and ANDVantage 50Y. Amino acid digestibility values ranged between 69.3% and 90.0% for amino acids reported. Digestibility values for lysine, threonine and tryptophan were lower than those for fishmeal but were well within the range of common plant-based proteins used in aquaculture diets.

Phosphorus digestibility of the ANDVantage 40Y was quite high and is an artifact of the use of a heatlabile phytase enzyme in the fermentation process.

At the time of this research, the plant responsible for the production of the ANDVantage 50Y product was not utilizing phytase as a processing aid and had phosphorus digestibility values similar to fishmeal.

Steps are being taken to implement phytase use in the production of ANDVantage 50Y as this allows for reduced total phosphorus inclusion in aquaculture

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS Table 1. Analyzed composition of test ingredients. Reported as percent on a dry matter basis unless otherwise noted.

Ingredient

ANDVantageTM 40Y

ANDVantageTM 50Y

Menhaden Special Select Fishmeal 70.8

Crude Protein (%)

44.2

54.8

Crude Lipid (%)

10

10.1

9.5

Dry Matter (%)

93.2

93.6

93.1

Energy (kcal/kg)

5406

5608

4666

Alanine

3.32

4.22

4.3

Arginine

1.86

2.4

4.22

Aspartic Acid

2.97

3.76

6.35

Cysteine

0.88

1.16

0.66

Glutamine

7.93

10.23

9.31

Amino Acids (%)

Glycine

1.61

2.05

4.91

Histidine

1.18

1.54

1.73

Isoleucine

1.89

2.42

2.9

Leucine

5.8

7.21

4.85

Lysine

1.38

1.84

5.54

Methionine

0.99

1.43

1.98

Phenylalanine

2.53

3.02

2.75

Proline

3.75

4.65

3.1

Serine

2.03

2.57

2.63

Taurine

0.12

0.12

0.56

Threonine

1.67

2.11

2.92

Tryptophan

0.4

0.54

0.87

Tyrosine

1.88

2.39

2.15

Valine

2.29

2.9

3.31

Phosphorus (%)

0.56

0.6

3.79

Minerals Copper (ppm)

6.62

7.69

3.16

Iron (ppm)

76

35

1291

Manganese (ppm)

6.81

14.84

58.4

Zinc (ppm)

45

182

117

diets while maintaining adequate levels of digestible phosphorus in the diet.

Growth performance and feed conversion After completion of the digestibility research, a growth study was designed to evaluate potential inclusion rates of either ANDVantage 40Y or ANDVantage 50Y in rainbow trout diets. ANDVantage 40Y or ANDVantage 50Y was added in replacement of a combination of menhaden fishmeal and poultry meal. ANDVantage 40Y and ANDVantage 50Y were included at 0%, 7.5%, 15%, 22.5%, or 30% of the diet (Table 3). Diets were

16

formulated to contain approximately 42% digestible protein and 18% digestible lipid, with 3.82%, 1.30%, 2.14% and 0.5% lysine, methionine, threonine, and taurine respectively, and were formulated to contain 0.6% available phosphorus. Diets were fed to juvenile rainbow trout, average initial weight of 38.0±0.7g, for 12 weeks. Fish were stocked at 20 per tank, with three tanks per dietary treatment.

Fish were fed twice daily during weekdays, and once daily on weekends to apparent satiation. At the end of 12 weeks, final weights were taken and feed intake and feed conversion ratios were calculated.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS Table 2. Apparent digestibility coefficients for ANDVantage protein products in rainbow trout.

Ingredient

ANDVantageTM 40Y

ANDVantageTM 50Y

Menhaden Special Select Fishmeal

Protein (%)

81.7

83.0

83.3

Fat (%)

98.1

98.3

104.9

Dry Matter (%)

59.8

59.9

83.3

Energy (%)

70.7

72.5

104.2

Amino Acids (%) Alanine

87.5

86.8

87.8

Arginine

87.4b

89.7a

90.4a

Glutamine

89.3

88.1

91.5

Glycine

72.3

76.0

77.3

Histidine

83.0b

85.4b

90.1a

Isoleucine

81.4b

82.9b

92.1a

Leucine

a

90.3

b

88.5

93.4a

Lysine

69.3c

b

76.4

91.5a

Methionine

90.0

89.7

90.7

Phenylalanine

87.5

87.0

90.2

Threonine

74.3b

77.2ab

89.5a

Tryptophan

79.2b

86.5ab

92.6a

Valine

b

82.4

b

84.3

90.4a

86.3a

40.3b

35.5b

Minerals Phosphorus (%) a,b,c

Treatments within row with different superscripts differ P < 0.05.

In addition, three fish per tank were harvested for compositional analysis, and an additional three fish per tank were euthanized for determination of condition factor, hepatosomatic index, viscerosomatic index and muscle ratio.

Growth performance Inclusion of up to 15% of either ANDVantage product in the diet resulted in no differences in final fish weights, FCR, or feed intake as a percent of body weight compared to the control diet (Table 4).

Increasing inclusion rates to 22.5 or 30% for either of the ANDVantage products resulted in poorer growth performance. This may be due to either an amino acid deficiency or imbalance as increases in feed intake are common with these nutritional challenges.

An interaction between ingredient and inclusion level was observed for both feed intake as a percent of body weight, and feed efficiency. For these two variables, values were equal to the control for the 7.5%

and 15% inclusion levels but got worse at the higher inclusions, but at a slower rate for the ANDVantage 50Y treatments. Protein retention efficiency was slightly poorer for the 7.5% and 15% diets containing either of the ANDVantage protein sources compared to the control diet due to slightly higher crude protein contents of the basal diets for these treatments to meet similar digestible protein and amino acid levels.

Energy retention efficiency was not different between the control diet and either of the ANDVantage diets at 7.5% or 15% inclusion. Increases in either product above 15% of the diet resulted in reductions in efficiency with the poorest values observed for the 30% inclusion rate treatments.

An interesting observation was made for phosphorus digestibility of the diet as overall an increase in phosphorus digestibility of the growth diets was observed with an increasing inclusion rate of the ANDVantage proteins, which is in line with the initial digestibility data showing improved digestibility of both

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

17

PLANT-BASED INGREDIENTS Table 3. Diet formulations for growth performance evlauation of graded levels of ANDVantage 40Y or ANDVantage 50Y in diets for rainbow trout.

ANDVantage 40Y

ANDVantage 50Y

Ingredient (%)

0% Control

7.5%

15.0%

22.5%

30.0%

7.5%

15.0%

22.5%

30.0%

ANDVantage 40Y

0

7.5

15

22.5

30

0

0

0

0

ANDVantage 50Y

0

0

0

0

0

7.5

15

22.5

30

Menhaden Fishmeal

15.25

12.94

10.65

8.36

6.06

12.34

9.46

6.58

3.69

Poultry Meal

11.72

9.95

8.19

6.43

4.66

9.49

7.28

5.06

2.84

Soybean Meal

15

15

15

15

15

15

15

15

15

Corn Protein Conc.

5

5

5

5

5

5

5

5

5

Blood Meal

5

5

5

5

5

5

5

5

5

Feather Meal

5

5

5

5

5

5

5

5

5

Menhaden Fish Oil

12.33

12.22

12.11

11.99

11.88

12.32

12.31

12.29

12.28

Wheat Gluten Meal

0

0.48

0.97

1.5

1.94

0.4

0.8

1.2

1.6

Wheat Flour

20.84

16.78

12.61

8.43

4.34

17.49

14.05

10.7

7.28

Lecithin

1

1

1

1

1

1

1

1

1

Vitamin Premix

1

1

1

1

1

1

1

1

1

Stay-C 35

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

Choline Chloride 50%

1

1

1

1

1

1

1

1

1

Salt

0.28

0.28

0.28

0.28

0.28

0.28

0.28

0.28

0.28

Magnesium Oxide

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

Potassium Chloride

0.56

0.56

0.56

0.56

0.56

0.56

0.56

0.56

0.56

Moocalcium Phosphate

2.15

2.2

2.3

2.4

2.5

2.5

2.9

3.2

3.6

DL-Methionine

0.54

0.54

0.55

0.55

0.55

0.53

0.52

0.52

0.51

Lysine HCl

1.68

1.86

2.05

2.23

2.42

1.89

2.1

2.32

2.53

Threonine

0.46

0.5

0.54

0.58

0.62

0.51

0.55

0.6

0.64

Taurine

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Trace Mineral Premix

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Astaxanthin

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

Calcium Propionate

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.15

0.15

Yttrium oxide

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

Formulated Nutrient Content Crude Protein (%)

45.7

46.1

46.5

47

47.4

46.3

46.6

47.1

47.6

Digestible Protein (%)

41.7

41.9

42.1

42.3

42.4

41.9

42.1

42.3

42.5

Digestible Lipid (%)

18

18

18

18

18

18

18

18

18

Digestible Energy (%)

4606

4571

4534

4499

4461

4559

4512

4467

4419

Total Phosphorus (%)

1.35

1.29

1.25

1.2

1.16

1.33

1.33

1.3

1.3

Digestible Phosphorus (%)

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

sources compared to fishmeal. However, phosphorus digestibility of the ANDVantage 40Y diets was much higher than those of ANDVantage 50Y diets. On review of ethanol plant production practices, the basis for the improved phosphorus digestibility observed for ANDVantage 40Y is due to the use of low levels of

18

phytase enzyme in the fermentation process resulting in reductions in phytate content of the resulting feedstuff.

From a feed manufacturing perspective, diets containing the ANDVantage products had improved pellet durability compared to the control diet. Durability values increased with increasing inclusion rate of either

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS Table 4. Growth performance of rainbow trout fed diets with increasing levels of ANDVantage TM 40Y or ANDVantageTM 50Y corn protein products

ANDVantage 40Y

ANDVantage 50Y

ANOVA1

0.0%

7.5%

15.0%

22.5%

30.0%

7.5%

15.0%

22.5%

30.0%

SEp2

I

L

IxL

Survival (%)

93

100

98

100

100

100

100

100

95

2.36

0.74

0.22

0.86

Final Fish Weight (g)

368A

367A

357A

297�

212C

376A

359A

293B

235C

21.38

0.29

0.0001

0.35

Weight Gain (G)

A

330

A

328

A

319

B

258

C

174

A

338

321

A

B

255

C

197

21.64

0.16

0.0001

0.38

Feed Intake (% BW/d)

1.87cy

1.88c

1.90c

2.14b

3.08a

1.88y

1.89y

1.90y

2.36z

0.1

0.0001

0.0001

0.0001

Feed:Gain

0.79cx

0.80c

0.81c

1.04b

1.73a

0.79x

0.81x

0.92y

1.33z

0.08

0.0001

0.0001

0.0001

Protein Retention Efficiency

48.7az

42.4b

41.8b

33.3c

19.9d

42.6y

43.5y

37.9x

26.6w

2.06

0.0001

0.0001

0.0005

Energy Retention Efficiency

53.0A

49.7A

54.1A

37.7B

19.7C

52.6A

50.5A

41.1B

24.4C

7.38

0.34

0.0001

0.44

Phosphorus Digestiiblity

31.3cy

45.3b

48.3b

63.4a

62.0a

34.5xy

36.5xyz

49.7z

45.4yz

1.7

0.0001

0.0001

0.02

Pellet Durability

80.0

89.5

91.9

95.3

95.6

83.8

88.3

94.1

97.6

1.32

0.0001

0.0001

0.0001

3

dv

c

b

a

a

w

x

y

z

Ingredient (I), Inclusion Level (L) and interaction terms. When no ingredient by inclusion interactive effects are observed, main effect of level is differentiated using capital superscripts A, B, C. When interactive effects were detected, the superscripts a, b, c, d were used to differentiate treatment differences for ANDVantage 40Ytreatments, and v, w, x, y, z were used to signify differences for the ANDVantage 50Y treatments 2 Pooled standard error 3 Pellet durability index as measured with a Holmen NHP100 manual pellet durability tester (TEKPRO Ltd., Norfolk, UK)

1

Table 5. Effect of graded inclusions of ANDVantage 40Y or ANDVantage 50Y on various condition factors, indices and proximate composition

ANDVantage 40Y

ANDVantage 50Y

ANOVA1

0.0%

7.5%

15.0%

22.5%

30.0%

7.5%

15.0%

22.5%

30.0%

SEp2

I

L

IxL

Condition Indices 1.45A

1.41A

1.44A

1.34AB

1.29B

1.44A

1.38A

1.42AB

1.32B

0.1

0.48

0.002

0.43

4

Hepatosomatic Index

AB

1.28

B

1.16

B

1.17

B

1.17

A

1.38

B

1.15

B

1.08

B

1.20

A

1.33

0.15

0.52

0.0002

0.84

Viscerosomatic Index5

10.3B

10.3B

10.4B

12.1A

14.0A

10.2B

9.9B

12.2A

12.5A

1.72

0.27

0.0001

0.56

Muscle Ratio

az

60.2

a

58.9

a

57.8

b

54.8

b

51.7

z

59.2

z

59.9

y

52.6

55.2

2.47

0.19

0.0001

0.01

Dry Matter (%)

32.4A

32.6A

32.2A

30.8B

27.8C

32.6A

32.7A

30.5B

28.1C

1.18

0.74

0.0001

0.87

Crude protein (%)

17.4

17.6

17.7

17.8

17.5

17.5

17.9

17.4

17.5

0.57

0.53

0.34

0.62

Crude lipid (%)

A

12.9

A

13.0

A

13.4

11.6

8.5

13.1

A

A

12.5

11.0

C

8.9

1.4

0.51

0.0001

0.66

Ash (%)

1.8

1.8

1.6

1.9

1.7

1.7

1.6

1.5

1.7

0.35

0.22

0.48

0.52

Energy (kcal/g)

2.27A

2.03AB

2.27A

2.10AB

1.83B

2.13AB

2.13A

2.03AB

1.80B

0.31

0.67

0.002

0.81

Condition Factor3

6

y

Proximate Composition

B

C

B

Ingredient (I), Inclusion Level (L) and interaction terms. When no ingredient by inclusion interactive effects are observed, main effect of level is differentiated using capital superscripts A, B, C. When interactive effects were detected, the superscripts a, b, c, d were used to differentiate treatment differences for ANDVantage 40Y treatments, and v, w, x, y, z were used to signify differences for the ANDVantage 50Y treatments 2 Pooled standard error 1

ANDVantage product, with some interaction between ingredient and inclusion rate being reported. Feed mill personnel also expressed that it was difficult to keep bulk densities below 450g/l when using more than 15% ANDVantage 40Y or 22.5% ANDVantage 50Y. This makes it difficult to produce floating feeds but likely contributes to the increased product durabilities.

Condition indices Condition factor was reduced for fish fed the 30% ANDVantage diets compared to all diets except the 22.5% ANDVantage treatments. No differences in hepatosomatic index (HSI) were observed when comparing the ANDVantage-containing diets to the control diet. However, the 30% ANDVantage diets

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

19

PLANT-BASED INGREDIENTS

had increased HSI values compared to the other ANDVantage inclusion levels. Viscerosomatic index and muscle ratio both showed no difference between the control diet and the 7.5% and 15% ANDVantage 40Y and ANDVantage 50Y treatments but values were poorer for the 22.5% and 30% treatments.

Whole body composition No differences in whole body dry matter and crude protein content were observed between the control diet and either the 7.5% or 15% inclusion diet for either of the ANDVantage products. The 22.5% and 30% treatments were significantly lower in whole body dry matter and lipid content compared to the 0%, 7.5% and 15% diets, with the 30% treatments being lower than the 22.5% diets as well. No differences were observed for crude protein or ash content of fish in this experiment. Results for whole body energy density found the lowest values for the 30% ANDVantage-fed fish but were only significantly lower than the control and 15% ANDVantage treatments.

An excellent alternative protein for aquaculture feeds Both ANDVantage 40Y and ANDVantage 50Y, combinations of fermented corn protein and spent

20

yeast from the production of ethanol in the USA, were shown to be suitable for use in rainbow trout diets at up to 15% of the diet and could possibly be used at higher inclusion rates in diets that did not contain other corn protein products as our diets also contained 5% corn protein concentrate. These products are high in crude protein, and due to the spent yeast from the fermentation process, contain improved amino acid profiles compared to traditional corn protein ingredients like corn gluten meal.

Overall, digestibility of the ANDVantage products was quite good, with protein digestibility values similar to fishmeal in this experiment. Dry matter, crude fat and gross energy digestibilities were numerically improved compared to the work of Davies et al. (2021). However, Davies' research utilized a fermented corn protein product produced using alternative technology resulting in lower lipid content of the final product.

In this study, the corn protein products were replacing a combination of fishmeal and poultry meal, with fishmeal inclusions ranging from 15.25-3.69%, unlike previous research where fishmeal was held constant at approximately 21% of the diet (Davies et al., 2021; Overland et al., 2013). As such, it is difficult to determine whether inclusion levels of ANDVantage 40Y or ANDVantage 50Y could have increased to as high as 24% as observed by Davies et al. (2021) without adversely affecting growth performance.

In summary, ANDVantage 40Y and ANDVantage 50Y corn protein products can be effectively utilized in diets for rainbow trout while significantly reducing feed costs by replacing much more expensive proteins in the diet.

Inclusion rates of up to 15% of the diet are proven to produce results similar to the control diet while reducing reliance on fishmeal and improving sustainability of rainbow trout production.

More information: Scott Tilton Food Animal Nutritionist & Technical Advisor The Andersons, Inc. E: stilton@andersonsinc.com

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

GIVE YOUR FISH EVERY ANDVANTAGE

™

HIGH-PROTEIN FEED INGREDIENTS FOR AQUACULTURE The Andersons is proud to offer ANDVantage™ 40Y and ANDVantage™ 50Y. Highly digestible plant-based feed ingredients rich in protein and spent distillers’ yeast.

ANDVANTAGE™ 40Y

ANDVANTAGE™ 50Y

40% protein product containing corn protein and spent yeast from the ethanol fermentation process

50% protein product produced using ICM’s APP technology to produce a high protein product containing 22-25% yeast.

• • •

•

•

High fat content 16-18% yeast 45% more lysine than corn gluten meal Free-flowing meal

• • •

80% more lysine than corn gluten meal 50% more methionine than soybean meal Highly digestible Plant based protein with yeast

STRONG RELATIONSHIPS, STRONG PARTNERSHIPS. WWW.ANDERSONSGRAIN.COM

PLANT-BASED INGREDIENTS

Fermented plant proteins in fish feeds: Playing an especially powerful role in improving animal health Brandon White, Luke Fredrickson, Sergio F. Nates, Houdek Direct effects of special ingredients such as fermented plant proteins can provide significant amounts of biologically active factors that can increase gut microbiota, reduce intestinal inflammation, and boost metabolic processes for improved animal health.

Changes in production technology, marketing, and feed ingredients are key structural transformations necessary for the aquaculture sector to grow.

Supplementing aquaculture feeds with functional ingredients represents the largest opportunity for improvement and optimization in aquaculture.

Physical characteristics of such ingredients include high viscosity, which can improve fecal material stability, low dust, and small particle size, all of which can improve water quality. Some functional ingredients, such as fermented co-products, also offer significant amounts of short-chain peptides and free amino acids that confer excellent attractability and palatability properties. The presence of biologically active factors can increase gut microbiota, reduce intestinal inflammation, and boost metabolic processes leading to improved animal health.

Results from numerous feeding trials have indicated that fermentation may improve the acceptance and utilization of soy protein products in feeds for carnivorous fish. This article presents a summary of several studies in which we evaluated the inclusion of a commercial fermented plant protein (FPP), ME-PRO®, as a protein source and fishmeal replacement in diets for several commercially important fish species.

22

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS Fish feeding trials Studies have shown that the use of ME-PRO in practical diets is a promising solution for the production of sustainable aquaculture feeds. Besides having over 70 percent crude protein content and highly available phosphorus content, ME-PRO can be manufactured from non-GM soybeans, which also makes it a perfect match for the European aqua industry. The protein is processed at a state-of-the-art plant using either GMO or non-GMO soybean meal and a natural occurring, non-toxigenic, fungi, Aureobasidium pullulans.

Trial results have demonstrated that ME-PRO can sustain fish health, high-performance growth, and feed efficiency with inclusions of up to 20% of the total amount of ingredients in fish diets. Feeding trials using rainbow trout, European seabass, and Atlantic salmon have shown that fish fed ME-PRO based feeds consistently utilized feed more efficiently than the fish fed control feeds.

Rainbow trout feeding trials Performance and palatability of ME-PRO have been evaluated in rainbow trout (Oncorhynchus mykiss), in multiple studies. In a six-week growth trial, three commercially available salmonid feeds (>42% crude protein) and three diets containing ME-PRO were randomly assigned to five replicate tanks in a 30-tank recirculating aquaculture system (RAS). Commercial diet 1 exhibited the best growth performance during the six-week period. The diets containing 15% ME-PRO

Table 1. Feed conversion ratio (FCR) and specific growth rate (SGR) of RBT at final weigh point in trial (n=5). Superscripts of different letters within the same column denote significance.

Treatment

FCR

SGR

Commercial 1

1.14 ± 0.15

a

1.66 ± 0.12 c

Commercial 2

1.61 ± 0.06 b

1.14 ± 0.14 a

Commercial 3

1.18 ± 0.14 a

1.36 ± 0.12 ab

36% ME-PRO

1.22 ± 0.07 a

1.24 ± 0.13 ab

Non-GMO ME-PRO

1.15 ± 0.04

a

1.46 ± 0.15 bc

GMO ME-PRO

1.18 ± 0.15 a

1.41 ± 0.12 bc

(both GMO and non-GMO) outperformed the remaining commercial feeds, as well as the diet containing 36% ME-PRO. The inclusion of ME-PRO in rainbow trout diets showed similar or improved feed conversion ratios and specific growth rates compared to the three commercial control diets (Table 1). The results of this study highlight the importance of the inclusion of ME-PRO within future salmonid diets and how this protein ingredient can help change the aquaculture industry.

European seabass feeding trials Few studies have been conducted on the replacement of fishmeal with different types of fermented soy proteins in formulated feeds for European seabass (Dicentrarchus labrax). The results from our studies have shown that replacing FM with 5-8% ME-PRO didn’t affect feed intake as shown by similar or lower FCR’s to the control (Fig. 1). In addition, replacing fishmeal with ME-PRO yielded better fish weight

Figure 1. Specific growth rate (SGR) and feed conversion ratio (FCR) of juvenile European seabass fed diets containing different levels of ME-PRO.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

23

PLANT-BASED INGREDIENTS Table 2. Hematological and innate immune parameters measured in fish sera (mean± SEM; n=21). Asterisks represent significative differences between fish fed the control diet and fish fed ME-PRO at different dietary doses (simple contrast analysis & Tukey’s t-test for hemoglobin concentration or Dunnett’s t-test for alkaline phosphatase activity).

Diet

Osmotic Hemoglobin Nitricoxide fragility

Ceruloplasmin activity

Trypsin Alkaline Lysozyme inhibition phosphatase

E. coli inhibition

V. harveyi inhibition

FM

83.39 ± 2.78

15.41 ± 0.66

74.16 ± 6.10

37.31 ± 6.45

94.025 ± 1.13

142.61 ± 6.45

379.78 ± 18.02 59.34 ± 1.31

58.24 ± 4.98

MP5

76.37 ± 4.19

19.06 ± 0.74*

71.20 ± 5.76

50.28 ± 9.49

95.97 ± 0.43

148.55 ± 4.70

345.37 ± 12.78 63.80 ± 1.96

60.16 ± 4.97

MP8

87.00 ± 2.93

16.47 ± 0.87

78.13 ± 6.87

37.21 ± 5.15

95.80 ± 0.35 175.79 ± 11.84* 386.77 ± 13.98 62.60 ± 1.84

63.35 ± 4.63

MP12

80.32 ± 2.80

18.07 ± 0.55*

78.07 ± 4.46

51.59 ± 8.87

94.79 ± 0.93

59.73 ± 4.37

144.85 ± 6.53

377.02 ± 17.08 63.04 ± 1.47

the eighth day. Digestibility coefficients for protein were the highest in FPP (93) followed by FM (92), SPC (91), and SBM (89). The results of the digestibility study showed that ME-PRO, an ingredient with higher crude protein content and higher protein digestibility, provides more bioavailable protein to a diet than fishmeal or other soy protein ingredients.

Another feed trial was designed in response to the digestibility results. Juvenile Atlantic salmon were offered one of two diets, control or 8% ME-PRO (MP8).

The growth performance was not significantly different between the two diets. The control diet had slightly better growth rates but ME-PRO had a better FCR during the 10-week trial (Fig. 2).

Figure 2. Specific growth rate (SGR) and feed conversion ratio (FCR) of Atlantic salmon fed diets.

and reduced total feed cost per kilogram of gain.

Likewise, the inclusion of ME-PRO slightly improved the immune and hematological status of juvenile European seabass (Table 2). Similar to previous studies conducted in other aquatic species, dietary concentrations of ME-PRO up to 5% improved the immunology status of the fish, which may be able to resist better environmental or infectious stress.

Atlantic salmon feeding trials Measuring the apparent digestibility of nutrients is an important tool for evaluating feed ingredient quality. In vivo digestibility values for fishmeal (FM), soybean meal (SBM), ME-PRO (FPP), and soy protein concentrate (SPC) were determined in a trial using Atlantic salmon (Salmo salar). Four hundred adult salmon (2 kg average weight) were used for this study. A reference diet was fed to all tanks for seven days. Experimental diets containing a 30% blend of experimental ingredient (FM, SBM, FPP, SPC) and chromic oxide (Cr2O3) were offered to start on

24

Conclusion The studies performed to date have shown that ME-PRO is a valuable feed ingredient that can provide benefits to many species in the aquaculture industry.

The improvements in feed conversion, digestibility, and animal health are highlighted by just a few of the research trials we have conducted on fish. Future work by our team will investigate the performance of different novel ingredient combinations in aquaculture feeds to not only improve animal health and growth performance but also improve their environment.

References available upon request

More information: Sergio F. Nates Executive Vice President Houdek E: sergio@prairieaquatech.com

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS

Precision nutrition and corn-fermented protein: A valuable concept Dr. Kevin Herrick, POET

The concept of Precision Nutrition represents a relatively new approach in animal nutrition, especially when it relates to the use of distillers co-products. Historically, the industry has often viewed distillers' co-products as a highly variable ingredient that provides value for species like beef and dairy (greater inclusion and lesser value). However, the development of fiber separation technologies to create products like corn-fermented protein provide a tremendous opportunity to support formulation concepts like Precision Nutrition.

What does Precision Nutrition mean? Although the definition of Precision Nutrition could differ between individuals, generally speaking, it defines the concept of matching an animal’s nutrient requirements with the nutrients supplied in the diet or formulation.

In order for this concept to work, nutritionists need to select ingredients that 1) provide a consistent source of nutrients and availability; 2) have a nutrient profile that closely matches the nutrient requirements of the animal; and 3) provide these characteristics at a reasonable price (Fig. 1). An ingredient with

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

25

PLANT-BASED INGREDIENTS ingredients. This ensures that the total diet will contain sufficient nutrients in the event a certain batch of an ingredient contains less/more of a certain nutrient.

Although corn-fermented protein represents a relatively novel ingredient, nutritionists have a significant amount of available research, which should improve confidence in the nutrient profile when formulating diets.

As a final note, the availability of an ingredient also relates to consistency. Fortunately, the bioethanol industry continues to invest in corn-fermented protein technology, which will make the ingredients readily available.

Figure 1. Venn diagram depicting the concept of Precision Nutrition

consistency and an ideal nutrient profile will work well in the formulation but may not improve profitability for the producer. Similarly, an inconsistent ingredient with a great nutrient profile and low cost will probably not support optimal performance. Finally, a consistent ingredient with a reasonable cost but a poor nutrient profile for the targeted species may not provide much benefit.

Consistency and corn-fermented protein Although bioethanol and corn co-product production use a biological process that can have variability, ingredients like corn-fermented protein remain very consistent. Bioethanol producers recognize the importance of consistency and utilize quality control practices to ensure a very consistent product. These practices include frequent testing and monitoring of several different characteristics of the co-product streams throughout the production process.

Additionally, the production process for corn-fermented protein includes opportunities to adjust production parameters that affect product quality. As a result, if a nutrient like protein starts to change, operators can change parameters in the production process to move the protein content back to typical concentrations.

We should also highlight that consistent ingredients provide nutritionists with more confidence when formulating diets. Nutritionists often over-formulate certain nutrients in the diet when using inconsistent

26

Nutrient content and corn-fermented protein The elevated protein in corn-fermented protein makes it an ideal ingredient for those species of aquaculture that have greater protein requirements. Additionally, the amino acid profile matches well with the requirements.

Corn protein contains less lysine than other protein sources, and as a result, we often consider corn as lysine deficient. Fortunately, yeast biomass produced during bioethanol fermentation (as much as 25% of the ingredient) gets included with the corn co-products.

This yeast biomass has an excellent amino acid profile, and as a result, ingredients like corn-fermented protein contain more lysine than many other protein alternatives. Additionally, research has shown the amino acids from microbial biomass have excellent digestibility and could possibly have some functional characteristics.

Figure 2 highlights this relationship. The Essential Amino Acid Index (EAAI) compares the digestible amino acid content of an ingredient and the nutrient requirement of an animal in order to create a ranking.

Values of 1 or higher indicate the ingredient matches up very well, while lesser values indicate a poorer match.

Ingredients like corn-fermented protein, soybean meal, and fishmeal look very similar and complement the general aquaculture nutrient requirements very well.

This relationship could change depending on the species and nutrient requirements. However, the EAAI provides a quick and easy method to screen novel ingredients.

Value of corn-fermented protein As previously mentioned, the concept of Precision Nutrition will not work if it does not improve profitability. This becomes slightly more complicated because we now start talking about not only the cost

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS

Figure 2. Essential Amino Acid Index for ingredients used in aquaculture formulations

of the ingredients but also the nutrient content of the ingredient and how the animal performs when fed the ingredient. Ideally, formulation using best-cost strategies (as opposed to least-cost strategies) will identify the most profitable alternatives. This typically involves a greater time commitment and only provides an analysis unique to certain situations.

We can perform a quick evaluation by using software that calculates the economic value of selected nutrients in the ingredient and then compare it with the market price of that ingredient. Ingredients with more nutritional value than cost represent a “good

buy.” Meanwhile, ingredients with less nutritional value than cost represent a “poor buy.” Figure 3 uses this type of comparison to calculate a relative value between distillers' grains, soybean meal, and cornfermented protein. This analysis used current ingredient prices and typical nutrient profiles based on nutrients typically considered in aquaculture formulations. This small comparison shows that the value of the nutrients in corn-fermented protein exceed the cost of the ingredient. It also highlights the advantages in value compared with other plant proteins like traditional distillers grains and soybean meal.

Figure 3. Predicted minus actual value of selected ingredients used for aquaculture

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS Precision nutrition and sustainability The topic of sustainability continues to become more important to both consumers and farmers/producers.

Although the intent of Precision Nutrition does not directly address sustainability, the outcome of Precision Nutrition plays a significant role. Most individuals would probably agree that minimizing waste represents a key component of any discussion related to sustainability.

Matching the nutrient content of the diet with the nutrient requirement of the animal minimizes waste and potential negative contributions to the environment.

Additionally, a well-balanced formulation should promote better efficiency of production and minimize waste even more.

Although this discussion on Precision Nutrition could include additional topics, the previously highlighted talking points provide the key components of Precision Nutrition related to aquaculture formulations. Taking into consideration the consistency, nutritional value,

and cost of not only ingredients like corn-fermented protein but all potential ingredients form the basis for the formulation strategy. Nutritionists also need to embrace this concept. The era of “over-formulating” animal diets to make sure the animals receive enough nutrients has probably ended. In the end, incorporating the Precision Nutrition concept can improve animal production, improve producer profitability, and also help sustainability efforts.

More information: Kevin Herrick Technical Services Director POET E: Kevin.Herrick@poet.com +1 (605) 965-2224

CHOOSE THE SUPERIOR PROTEIN INGREDIENT.

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WHERE PERFORMANCE MEETS SUSTAINABILITY. POET’s next-generation protein product is a world-leading, plant-based ingredient sourced from the highest-quality American corn. NexPro® is a 50% corn-fermented protein ingredient derived from the dry-mill bioethanol production process. Its high protein content and small particle size make it an ideal protein ingredient for a variety of markets including pet food, aquaculture, poultry, swine and dairy.

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS

A 60% sustainable vegetable protein for aquaculture derived from the corn renewable fuels industry Peter Williams, Louis Rens, Green Plains

Sustainability (meeting energy and carbon emissions reduction targets) and demographics (e.g. the age of those in the labor market) have been identified by the International Feed Industry Federation as key global issues facing the industry. This is why indexes of sustainability, such as Green House Gas (GHG) and Land Use Change (LUC), are likely to be two new parameters that will become components of feed specification sheets in addition to the nutritional data that has been the norm. Major aquafeed manufacturers (e.g. Aller Aqua and Cargill) have already started to include data referencing the sustainability

characteristics of their feed. Feed is one of the major components in the determination of the sustainability characteristics of any livestock production enterprise and decisions on how a feed is formulated are already being driven by the sustainability characteristics of individual feed ingredients.

Marine sources remain important livestock feed ingredients in many parts of the world, especially in aquaculture. Glencross (2022) reported that although there was increasing pressure on fish stocks, sustainably harvested fishmeal will remain a significant component of aquaculture feed formulation for the foreseeable

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS Corn is the major crop grown in the USA but it has traditionally been considered 1. Canola Meal an energy crop with limited relevance New agronomy 2. Pulses (Peas, Beans, Lupins) to aquaculture. It has been grown for 3. Cottonseed/Sunflower the energy that it can produce in the Fermentation Technology 4. Single Cell Protein form of starch and oil and it has taken many years of development in breeding Co-products 5. Brewers Spent Grains and agronomic practices to achieve the 6. Processed Animal Proteins current levels of production. Specific Discarded materials 7. Insect Protein regional varieties, genetic modification to protect the crop and control weeds and future. It is important, therefore, that alternative cultivation practices such as no-till and cover crops all sources of protein that are used with fishmeal combine to optimize the production of corn. The value complement and are not detrimental to the value of elite corn varieties can be seen from the range of of fishmeal. In a recent article covering trends in the Green House Gas (GHG) (kgCO2e/ton dry matter from development of animal feed, four of the seven major 4400 in SE USA to 3700 in the corn-belt). Therefore, trends related to the increasing demand and use of turning corn into a multi-purpose crop that can produce both energy and protein and specifically an application protein in feed were outlined. in aquaculture is a highly worthy pursuit.

Alternative protein for feed Sourcing an alternative protein for feed formulation, Producing a commercially viable feed product and particularly for aquaculture, is a challenging One overriding aspect of the production of raw exercise. Crops and vegetables are the primary sources materials that impacts the whole of feed production is of protein, and soybean meal, for many years, has scale. Feed for aquaculture is no exception. Commercial been the protein of choice. However, recently there feed production, for the major livestock species and has been a movement to reduce the reliance on soy aquaculture, is driven by large-scale production – up products with more sustainable and more benign to 500,000 metric tons per annum of a specifically sources of protein. Processed animal proteins are formulated feed is a norm for commercial feed being reintroduced into feed formulations and work production. A batch of the formulated feed of up to 100 is ongoing to produce a range of single-cell proteins, tons is made, then mixed in 10-12 tons mixer batches.

Storage space for feed ingredients is mainly in feed bins insect proteins and microbial proteins (Table 1).

Attention is being paid to producing alternative and large-scale integrators are limited to 3-4 feed bins crops high in protein. However, the challenges to on site which limit the number of individual ingredients the diversification of large acre production of a which can be stored. Therefore, the availability of feed different new crop are considerable. Elite varieties ingredients must merit bin space.

Resilience in the supply chain, Redundancy in the of crops are bred specifically to achieve maximum production in different geographic locations, with supply chain and Reliability in product consistency (the different environmental conditions and under the 3 R’s) relate to the fact that if for any reason there is impact of day length. It takes several years to breed a need to change a feed formulation, this can be an an elite variety that achieves maximum production in expensive and time-consuming exercise. Resilience in a specific location. Logistic factors such as appropriate the supply chain relates to the fact that feed producers machinery and not least, the intrinsic knowledge will not work with a single source of supply in case of that has been accumulated by farmers and built a plant stoppage. It is essential to have one or more into the local agronomy, all serve to favor the status sites of manufacture of the product for there to be quo. Planting alternative agronomy is challenging an alternative source of supply. For the same reason, which is why diversifying the existing agronomy has a there needs to be redundancy in volume production to account for increased demand. With the resilience in major advantage.

Table 1. Alternative protein sources

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS the supply chain, there is an additional need for product consistency across different suppliers.

Corn Fermented Protein (CFP) from the dry grind ethanol industry Plant-based co-product, secondary products generated during a manufacturing process, have a unique position in that they do not demand additional acreage and do not compete with human food consumption.

The original co-product from the dry-grind ethanol process, distillers dried grains with solubles (DDGS), while being a valuable feed product, was never designed for any specific nutritional purpose.

Corn fermented protein, a plant-based protein produced from the mechanical separation of whole stillage in the dry grind bioethanol process with Fluid quip Technologies’ Maximized Stillage Co-Products™ system, is a new, specifically designed high-protein product (up to 60% crude protein) that has been extensively and very successfully tested as a protein supplement for aquafeed. Producing a new protein product from maize turns the major crop produced in the USA into a multi-purpose protein plus energy source. It benefits from the extensive research that has gone into the agronomy of corn, making it possible to use every part of the corn plant for productive purposes.

The MSC™ process separates the protein from the whole stillage by mechanical centrifugation and

alternate washing steps. The dry grind ethanol process starts with a 50-55h fermentation of ground corn. The fact that during fermentation there is a significant net generation of protein from the yeast that is grown in the fermenter to facilitate fermentation is not well appreciated. In addition to recovering corn protein, the MSC™ process also recovers a high proportion of spent fermentation yeast. The 50% protein product is therefore a combination of the protein fraction of corn with approximately 24% of the dry matter consisting of spent yeast components. Furthermore, plants producing CFP are able to operate without the use of antibiotics to control the fermentation process and are able to be compliant with No Antibiotics Ever (NAE). Annually, over 600,000 tons of CFP is produced in the USA from ten dry grind ethanol plants with high consistency in the nutritional composition and bioavailability of the products.

Based on standardized digestibility measured in caecetomized cockerels (Parsons et al., 2023) over a period of ten years and from different plants, the coefficient of variation of available digestible protein is <2.0% from samples of CFP. The digestibility of protein in a variety of different aquaculture species is >87%. This new product is also produced in a highly competitive, more sustainable manner with lower GHG emission values compared with a range of different protein products (Fig. 2). Using CFP in ration formulation as a partial replacement for soybean meal has been

Figure 2. GHG values for a selection of feed materials used in aquaculture feed. Corn Fermented Protein from Sphera; the rest from the GFLI 2.0 database October 2022 edition.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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PLANT-BASED INGREDIENTS reported to reduce the GHG value of feed formulation (Burton et al., 2021). In addition, components of fermentation plus spent yeast components have been demonstrated to improve gut health in salmon. Cornfermented protein is a commercially viable alternative protein that provides increased flexibility in ration formulation for a wide range of aquaculture species.

As outlined, the complimentary benefits of spent yeast and fermented carbohydrates provide CFP with functional benefits. Furthermore, the production of CFP conforms to the 3Rs – it is a new protein product that formulators of diets for aquaculture can use with confidence and, at the same time, lower the GHG index of their formulated feed products.

References Burton E, Scholey D, Alkhtib A, Williams P. Use of an Ethanol Bio-Refinery Product as a Soy Bean Alternative in Diets for Fast-Growing Meat Production Species: A Circular Economy Approach. Sustainability. 2021; 13(19):11019. https://doi.org/10.3390/su131911019

Glencross B. 2022 Marine Ingredients: Setting the benchmark for sustainable and responsible sourcing.

Towards Sustainable Aquafeed VICTAM Conference 30/09/2022 Life cycle inventory (LCI) database for the agriculture and feed sector from the Global Feed LCA Institute (GFLI) https://globalfeedlca.org/gfli-database/ Parsons BW, Utterback PL, Parsons CM, Emmert JL. Standardized amino acid digestibility and true metabolizable energy for several increased protein ethanol co-products produced using back-end fractionation systems. Poultry Science, Volume 102, Issue 2, 2023, 102329.

More information: Peter Williams Senior Nutritionist Green Plains Inc. E: peter.williams@gpreinc.com

SUPPORT OF NATURAL DEFENSES www.phode.com / phode@phode.com Follow us

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

PLANT-BASED INGREDIENTS

Algal oil gives control of omega-3 levels in farmed salmon Ian Carr, Veramaris Inclusion of microalgal oil in salmon feed improved omega-3 profiles, reduced the marine footprint and preserved pigmentation and sensory properties.

The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential for human health. They are known to reduce the risk of cardiovascular disease and are important contributors to a healthy and nutritious diet. Their primary sources are oily fish, including Atlantic salmon, that are rich in both EPA and DHA. Omega-3 fats are also equally important for fish health. However, they are not produced in sufficient quantities by the fish themselves and need to be supplemented in aquaculture feeds.

The Atlantic salmon industry must continue to grow to meet the increasing global demand for healthy seafood.

However, the overuse of marine resources needed to

produce fish oil (FO), the main lipid source in salmon aquafeeds, has become a bottleneck.

Vegetable oils are used as alternative lipid sources, but these are rich in omega-6 and deficient in omega-3 fatty acids. Because of this, EPA and DHA levels in salmon fillets have decreased by more than 50% in recent decades, which is detrimental for both farmed salmon and humans alike. Vegetable oils in feed also unbalance the optimal omega-3 to omega-6 ratio which is essential for fish and human health.

Microalgal oil (AO) from Veramaris is a viable alternative for addition to fish feed owing to its richness in EPA and DHA, ease of integration into fish feed

Algal Oil Gives Control of Omega-3 Levels in Farmed Salmon DHA

Experiments conducted on post-smolt salmon

Atlantic salmon are rich in the omega-3 fatty acids (FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), essential for human health

EPA

Experiment 1

145 g

>500 g

Experiment 2

413 g

>550 g

Experiment 3

1.3 kg

>3 kg

Limited availability of fish oil (FO) is a bottleneck for industry growth, resulting in a >50% reduction in EPA and DHA in farmed salmon Is microalgal oil (AO) in salmon feed a viable solution to ensure sustainable growth of the industry?

Testing alternative sources to achieve same levels of EPA and DHA in diets

..from different sources and levels of EPA and DHA

To understand how AO inclusion affects performance, muscle FA profile, and sensory properties of fish fillet

Including AO in aquafeed

EPA + DHA

improves omega-3 FA profiles in Atlantic salmon and can meet

AO was a reliable source of omega-3 fats during the entire salmon life cycle

AO inclusion improved omega-3 profiles in line with recommended dietary intakes

Fish growth and health was equal between treatments, irrespective of EPA and DHA source

AO inclusion contributes to a reduced marine footprint for the aquafeeds

Pigmentation and taste were preserved in salmon fillets

the global demand for healthy seafood in a sustainable manner

ALGAL OIL GIVES CONTROL OF LONG-CHAIN OMEGA-3 LEVELS IN FULL-CYCLE PRODUCTION OF ATLANTIC SALMON, WITHOUT DETRIMENT TO ZOOTECHNICAL PERFORMANCE AND SENSORY CHARACTERISTICS SANTIGOSA et al. (2023) | DOI:10.1111/JWAS.12947

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

33

PLANT-BASED INGREDIENTS formulations, and environmental sustainability, resulting in a much lower marine footprint for salmon farming.

the replacement of FO with AO is not likely to affect consumer preference.

The demand for fish products rich in EPA and DHA continues to grow. This study provides useful insights for the efficient and healthy production of Atlantic salmon to meet the rising market demand, without harming the environment.

Effects on full life-cycle Replacement of FO with AO has been documented in rainbow trout, gilthead seabream, and Atlantic salmon, but until now comprehensive studies covering the full life-cycle were lacking. To address this gap, a series of experiments were conducted to assess the impact of the partial and entire replacement of FO with AO from Veramaris in all life stages of the Atlantic salmon.

When tested for growth, muscle fatty acid profile, and muscle quality, the experimental fish were found to be healthier and more nutritious. They also showed greater than 96% EPA and DHA digestibility in response to the altered diet.

The inclusion of AO instead of FO led to a huge reduction in the marine footprint of the farmed salmon.

Additionally, higher EPA and DHA levels mean that consumers can attain recommended intake levels by consuming just one portion of salmon per week, consistent with advice from national health authorities.

Moreover, the pigmentation and sensory characteristics of raw fish fillets were maintained intact suggesting that

Reference Algal oil gives control of long-chain omega-3 levels in the full-cycle production of Atlantic salmon, without detriment to zootechnical performance and sensory characteristics. Ester Santigosa, Rolf Erik Olsen, Angelico Madaro, Viviane Verlhac Trichet, Ian Carr. DOI: 10.1111/ jwas.12947

More information: Ian Carr Business Development Director Veramaris E: ian.carr@veramaris.com

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Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

FEED SAFETY

Mycotoxins in aquaculture: Risk and management Kai-J. Kühlmann, Trouw Nutrition Asia-Pacific, Saravanan Subramanian, Swamy Haladi, Selko Feed Additives

Aquaculture feeds and the raw materials they contain are very susceptible to fungal/mold infestation. Major plant-based feed raw materials around the world include maize, wheat, broken rice, millets, soybean meal, sunflower meal, rapeseed meal, rice bran, wheat bran and DDGS. In recent times, raw materials conventionally used in feed have tended to be in short supply and costs have risen. This situation has led to increased use of different agricultural grain and oilseed byproducts at different inclusion levels. Such byproducts unfortunately have proven to contain higher concentrations of mycotoxins compared to their parent grains or oilseeds since mycotoxins tend

to concentrate on the outer portion of grains or seeds. In tropical and sub-tropical climatic conditions, raw materials in storage are susceptible to mold and mycotoxin contamination due to the ideal air temperature range of 25-30°C, and water activity values above 0.7 with a concomitant higher relative humidity of 88-95%. These conditions, coupled with the greater inclusion of plant-based raw materials and their byproducts in aquaculture feed in recent years, have increased the risk of mycotoxicosis.

The following overview offers guidance on mycotoxin impacts through feed and feed ingredients, and their consequences on fish and shrimp health.

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FEED SAFETY Mycotoxicosis Mycotoxicosis is an impaired health condition observed in animals upon the ingestion of different mycotoxins present in their feed. The severity of mycotoxicosis depends on the type and concentration of mycotoxin intake, duration of exposure and life stage of the animal. Mycotoxins, a diverse group of toxic secondary metabolites of fungi, are a worldwide challenge in husbandry and aquaculture. The increased global trade of raw materials as well as climate change and changing agricultural practices make managing mycotoxin risk even more complex.

Although animals can be exposed to more than 600 different mycotoxins, most research to date has focused on six groups of mycotoxins: aflatoxins (AF), deoxynivalenol (DON), fumonisins (FB) ochratoxins (OTA), T-2 toxin and zearalenone (ZEN). Recent research in fish shows greater risk and potential negative effects of these 6 mycotoxins at much lower concentrations in feed as compared to the current regulatory limits (Table 1). In addition, the occurrence

and health effects of emerging mycotoxins such as fusaric acid, moniliformin, and enniatins are also getting more attention, but studies on the impact of these emerging mycotoxins on aquaculture species are very limited. Taking into consideration the presence of the “Big 6”, emerging and masked mycotoxins and their interactions, Selko, the feed additive brand of Nutreco, has developed practical guidance values for mycotoxins and these can be shared upon request.

In farmed aquatic species, mycotoxicosis can adversely affect feed intake, growth, reproductive capacity, feed conversion and other performance parameters.

It can also cause liver impairment and even increase mortality, resulting in serious economic losses (Table 2).

Mycotoxin occurrences and levels in feed raw materials Trouw Nutrition’s Mycotoxin Monitor used the average mycotoxin content of the “Big 6” (AF, DON, FB, OTA, T2HT2, ZEN) in 2018 as a reference for seven feed raw materials commonly used in Asian aquaculture (maize,

Table 1. The critical concentration of mycotoxins in fish feed has the risk of affecting health and performance of at least 5% (CC5) of the fish population (Pietsch, 2020).

Mycotoxins

AF-B1

Mean Critical Concentration in fish feed (CC5) μg/kg

EU regulatory limits and guidance values (μg/kg)

4.3

10

ZEN

2.3

2,000

DON

114.8

5,000

OTA

1.3

250

FB1

505.7

10,000

T2

21.9

500

Table 2. General mycotoxin symptoms and negative impacts in fish and shrimp

Mean Critical Concentration

EU regulatory limits and

AF

Poor performance and feed conversion, immune suppression, reduced gut integrity, pale gills, spiking mortality

DON

Feed refusal, poor performance and feed conversion, immune suppression, reduced gut integrity, spiking mortality

FB

Liver and kidney necrosis, lesions, high mortality, poor performance and feed conversion

OTA

Liver necrosis, posterior kidney lesions, high mortality, poor performance and feed conversion

T-2 Toxin

Feed refusal, poor performance and feed conversion, immune suppression, reduced gut integrity, spiking mortality

ZEN

36

Disruption of reproductive system, poor growth performance

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

FEED SAFETY

Figure 1a. Mycotoxin trend between 2019 to 2022 vs. average value of 2018 (ppb) for selected feed raw materials relevant in aquafeeds.

AF – Aflatoxin, FB – Fumonisin, DON – Deoxynivalenol, OTA – Ochratoxin, T2HT2-toxin, ZEN – Zearalenone

maize DDGS, rice, rice products, soybean meal, wheat and wheat bran) to determine a respective mycotoxin contamination trend of the used feed raw materials between 2019 to 2022. Considering 67,454 mycotoxin analyses from 52 countries, the obtained result was correlated to the resulting average mycotoxin content of these feed raw materials from 2019-2022 to obtain a respective mycotoxin contamination trend (Fig. 1a, 1b). Results show a strong increase of DON for rice by

617% compared to the average mycotoxin content of 2018 (67 ppb) (Fig. 1a). Likewise, the presence of AF, FB and ZEN in rice increased by 175%, 99% and 189%, respectively, while for maize, soybean meal and wheat, FB, T2HT2 and ZEN contents decreased slightly (Fig. 1a, 1b).

Impact of mycotoxins on aquaculture species Given the current trend of replacing animal protein

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FEED SAFETY

Figure 1b. Mycotoxin trend between 2019 to 2022 vs. average value of 2018 (ppb) for selected feed raw materials relevant in aquafeeds.

AF – Aflatoxin, FB – Fumonisin, DON – Deoxynivalenol, OTA – Ochratoxin, T2HT2-toxin, ZEN – Zearalenone

feed sources with plant-based proteins, containing mycotoxins is of growing concern in aquafeed production. In aquaculture, several cold and warm water species are intensely farmed. Considering their poikilotherm metabolism, mycotoxin sensitivity results in complex age- and species-dependent effects.

Table 3 summarizes the effects of mycotoxincontaminated aquafeeds on various tropical aquaculture species and references respective

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literature. AF is known to result in changes in body composition and oxidative stress Marijani et al. (2021).

Increased AF susceptibility caused by increased plantbased feed raw materials in Nile tilapia often results in impaired liver function, lower growth rate, loss of body weight, internal organ dysfunction and mortality Khaled et al. (2014). Channel catfish appear to be more resistant among aquaculture species when exposed to AF, while Nile tilapia are less tolerant.

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FEED SAFETY Table 3. Effects of mycotoxins in various aquaculture species. Channel catfish is more tolerant to mycotoxins compared to tilapia or shrimp.

Species

Channel catfish

Nile tilapia

Black tiger prawn Whiteleg shrimp

Fungi

Mycotoxin Content (ppb)

Effects

Reference

AF > 10,000 Aspergillus sp.

OTA > 20,000

Reduced growth, poor FCR Can detoxify AF Reduced growth performance

Lovell (1990) Manning (2010) Manning et al. (2003)

Fusarium sp.

DON FB T2

> 17,500 > 20,000 > 600

Reduced growth performance Reduced growth, increased liver Reduced growth performance

Manning (2005) Lumlertchada et al. (1995) Manning et al. (2003)

Aspergillus sp.

AF AF FB

> 2,500 > 1,800 > 40,000

Reduced growth, poor FCR and low haematocrit values

FB

> 70,000

Smaller fingerlings are more affected

AF > 20 Aspergillus sp.

OTA > 200

Reduced immune response, growth, increased FCR Reduced growth performance

Aspergillus sp.

Histological changes in hepatopancreas

Fusarium sp.

AF DON T2

> 60 > 200 > 100

The species-specific sensitivity to DON is related to its metabolism as reflected in substantial toxicokinetic studies in terrestrial species. In aquaculture species, however, studies are limited. Considering the significant DON-contamination increase in rice from 2019 to 2022 (Fig. 1a) and rice incorporation into aquafeeds by about 25%, fish or shrimp can experience significant DON toxicity in the absence of proper mycotoxin monitoring and risk mitigation practices. DON can impact aquaculture species’ feed intake with concomitant reduced growth and feed efficiency, pathological lesions in liver and kidney, and altered oxidative stress leading to poor immunity (Pietsch, 2020).

The differences in the sensitivity to DON-containing aquafeeds are not only observed between species but also more prominently between warm and cold-water species. Warm-water fish are more tolerant to DONcontaminated feeds compared to cold-water species, while channel catfish appear to be more resistant to DON than, e.g., salmonids (Hooft & Bureau, 2021).

A recent study predicts a reduction in feed intake (3.5%) and growth (3.7%) of rainbow trout fed on feed contaminated with 136 ppb DON (Koletsi et al. 2021).

OTA contaminations are attributed to embryotoxicity including severe deformities, reduced growth and hatching rates, culminating in increased embryo mortality and these effects are related to increased

Reduced growth performance

Manning (2010) Nguyen et al. (2002) Tuan et al. (2003) Bautista et al. (1994) Bintvihok et al. (2003) Boonyaratpalin et al. (2001) Supamattaya et al. (2006) Bintvihok et al. (2003) Trigo-Stockli et al. (2000) Supamattaya et al. (2006)

oxidative stress. Weight gain reduction, poorer feed conversion rate, lower survival, and hematocrit levels were observed in channel catfish fed with OTAcontaminated diets (Marijani et al., 2021). ZEN in fish has been shown to be immuno-toxic, genotoxic, hepatotoxic, and cytotoxic causing increased kidney damage (Pietsch, 2020).

In general, mycotoxins have various effects on fish and shrimp resulting in feed refusal and reduced growth performance (Table 3). The gastrointestinal tract (GIT) is a system constantly in touch with feedborne mycotoxins right from the oral cavity all the way to the large intestine. As a result, the GIT is particularly affected by mycotoxins. Generally, the intestinal barrier in the GIT functions as a filter against harmful mycotoxins. However, some mycotoxins have been found to exert detrimental effects on the GIT. For example, mycotoxins can alter normal intestinal functions such as barrier function and nutrient absorption (Fig. 2). Some mycotoxins also affect the morphology of the intestine. As the feed gets digested at different levels of the GIT, mycotoxins get released into the intestinal lumen and subsequently absorbed into the blood circulation. From blood circulation, mycotoxins reach the liver and various other organs resulting in toxicity. To prevent toxicity, sound gut health is critical.

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FEED SAFETY

Figure 2. Effect of mycotoxin exposure on gut structure and function (image created with BioRender).

Impact of mycotoxins on immunity The most underrated negative effects of mycotoxins in animals are on their immune system. The immune system is broadly classified into innate (macrophages/ dendritic cells), cell-mediated (DTH, cytotoxic T cells) and antibody-mediated (B cells, T helper cells, antibodies). Mycotoxins are known to affect all these arms of the immune system and subsequently make animals more susceptible to various bacterial, viral, and protozoan infections.

Mycotoxin management As shown above, all the “Big 6” affect the gut integrity, immunity, and growth performance of aquaculture species. Most mycotoxins are heat stable, and therefore, complete thermal destruction and elimination are not possible during the pelleting and extrusion process of fish feed production.

Mycotoxin-adsorbing agents are developed and targeted to encompass a high adsorption capacity and mycotoxin affinity in an acidic and alkaline digestive environment with low or no adsorption of essential nutrients (e.g., vitamins and minerals).

Such agents need to be heat stable and capable of

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withstanding the pelleting or extrusion temperatures during feed mash gelatinization. Although inorganic adsorbents such as bentonites can effectively bind aflatoxins, T-2, ergot toxins and bacterial (endo) toxins in the intestine, they only have moderate binding to OTA, and ZEN, while DON and FB are not easy to bind. Merely relying on a mycotoxin binding strategy, hence, may not help in protecting the aquaculture-farmed species, thereby urging the need for ingredients capable of maintaining immunity and gut health.

TOXO®-XL is Selko's broad-spectrum mycotoxin mitigation solution that can support aquatic species' health and performance beyond binding. Aquafeed is often contaminated with multiple mycotoxins and producers are looking for a broad-spectrum solution to safeguard their business. To understand the effects of multiple mycotoxin contamination in aquaculture species in more detail, further studies are needed to bring more insights into the combined effects of multiple mycotoxins on mycotoxin absorption, distribution, and elimination. Special attention is needed considering the poikilotherm metabolism of cold and warm water species.

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

FEED SAFETY Concluding remarks The latest research on mycotoxins in aquaculture has shown that both gut health and immunity are significantly compromised by multiple mycotoxins.

Research over the last few decades has also shown that mycotoxin binding alone is not sufficient to manage multiple mycotoxin risks in animals. Implementing an effective mycotoxin risk management strategy combining a mycotoxin binding concept along with means of supporting gut health and immunity is of paramount importance in not only enhancing animal health and performance but also improving the bottom line of business operations.

References available on request.

For more information about Selko feed additives or feed safety solutions, please e-mail Selko.marcom@trouwnutrition.com

More information: Kai-J. Kühlmann Feed Mill Solutions Manager Trouw Nutrition Asia-Pacific

Saravanan Subramanian Technical Commercial Manager Aquafeed Additives Selko Feed Additives

Swamy Haladi Commercial Technical Manager Mycotoxin Risk Management Selko Feed Additives

Your partner in customised aquafeed additive solutions

Enhance feed quality

Optimise nutrition

Maintain fish & shrimp health

Increase performance

Learn more, visit www.selko.com

Aquafeed: Advances in Processing & Formulation Vol 15 Issue 2 2023

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FEED ADDITIVES

Designing antioxidants: The effect of molecular structure Margarita Altable, Oxiris Chemicals

Phenolic antioxidants are able to break down the oxidative degradation processes which affect different kinds of products and materials. These degradative processes result in the reduction of the lifespan of these products, as well as of economic, material and energy losses. For all these reasons, manufacturers of antioxidants have adapted their products to customers’ needs, which have emerged from new regulations, and social and technological trends.

There are different types of antioxidants depending on how they act and protect materials against oxidation and whose mechanism of action is deeply related to their molecular structure. For instance, phenolic antioxidants deactivate free radicals by transferring their labile hydrogen atoms to them, thus transforming free radicals into non-reactive substances. The antioxidant, after losing its hydrogen, transforms into a radical (phenoxy radical) which is less reactive and does not participate in the oxidative degradation process. This is why it is said that the antioxidant is sacrificed because it is exposing itself to oxidation in order to prevent the oxidation of the components of the product.

Sterically hindered phenolic antioxidants are part of this phenolic family. These compounds are characterized by having voluminous functional groups in the ring, which provide them with interesting properties.

The molecular structure is paramount in dictating the effectiveness of hindered phenolic antioxidants in

their role as antioxidants. Chemists carefully consider a range of critical parameters, including the arrangement of atoms, the nature of their interactions, and the distribution of electronic density within the molecule, when designing these compounds.

The molecular structure Chemical modification of phenols has made it possible to add or modulate their properties through specific structural changes.

For example, BHT, one of the most popular hindered phenolic antioxidants, is synthetically obtained. It can be described as a phenol with two tertbutyl groups on either side of the hydroxyl group, and a methyl group in front of them. These groups play different roles in the molecule. One is the stabilization of the phenoxyl radical formed after the transfer of the hydrogen atom to the free radical, which enhances its antioxidant activity.

Another is the blocking of the reactive position of the ring, impeding secondary reactions.

In addition, these groups give BHT its apolar character, which is responsible for its low solubility in water, and its high solubility in oils and hydrocarbons. Thus, BHT is used to protect oils and fats, and other substances present in them.

Nutrition is one of the sectors where oils and fats are used extensively, so preventing their degradation is important to maintain the quality of many products.

Antioxidants are added to food or packaging as

Figure 1. Aromatic ring stabilizing phenoxyl radical

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FEED ADDITIVES technological additives to preserve them against oxidation. Antioxidants are also used during some industrial processes such as frying or rendering, which require high temperatures. Temperature, oxygen, metal ions and light are the main factors that promote the oxidation process.

The molecular structure is also related to other properties such as melting point, volatility, and optical properties. For instance, BHT is white because it does not have chromophore groups. It is therefore widely used to protect products where white color is a requirement, such as cosmetics or plastics. Moreover, BHT is practically odorless, so its addition to final products does not alter the user’s final perception.

Solid or liquid antioxidants?

Whatever the antioxidant and the type of application, the main requirement must be to ensure contact between the antioxidant and the substance to be protected. For this purpose, the nature of the antioxidant, its format and the carrier used to add it to the product are important.

Solid antioxidants Solid antioxidants are very useful when solid products, such as vitamin premixes, need to be protected.

Because the amount of antioxidants required is usually very small, antioxidants can be mixed with inert solids, such as silica or calcium carbonate, which facilitates dosage.

In all these cases, particle size is an important parameter. The evidence shows the smaller the particle size, the better the protection. However, other aspects, such as cost and others associated with solids handling, must be considered.

BHT is a molecule that can be obtained by different industrial chemical processes. Depending on the purification process, BHT can be an amorphous powder, a crystalline solid or flakes, and each of them has its own advantages.

Liquid formats BHT is one of the fewer examples of antioxidants with a low boiling point, 70°C. Thus, is relatively easy to melt and add as a liquid without adding any other solvent.

However, antioxidants are often dissolved in liquid carriers, such as vegetable oils, propylene glycols, water

Figure 2. Left: crystalline BHT. Right: flakes BHT

Figure 3. Left: blend of BHT in sunflower.

Right: suspension of BHT in water

or acids. These carriers facilitate addition, dosage and mixing with the product.

Vegetable oils are possibly the most widely used carrier of BHT in nutrition applications, due both to their safety for humans and animals and to the solubility of BHT in oils.

In liquid blends, the maximum concentration of BHT depends mainly on two parameters: the type of oils and the solubility of BHT in them; the temperature changes during production, transport, and storage which may lead to precipitation of BHT. However, it is possible to prepare stable mixtures using solvents in which BHT is not soluble such as water. In these cases, it is necessary to stabilize the antioxidant with some substances to prevent precipitation.

Some of these substances are: • Wetting agents that coat the solid particles and promote their interaction with the liquid phase.

• Surfactants that prevent the agglomeration and sedimentation of particles keeping the particles separated.

• Thickeners that increase the viscosity of the mixture by creating a three-dimensional net which keeps the dispersive phase stable.

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FEED ADDITIVES

Figure 4. Different types of physical instability of a suspension

• Preservatives that prevent the growth of microorganisms in the medium.

Suspensions, like other dispersions, tend to break down over time, so it is important to ensure microbiological, chemical and physical stability for as long as possible.

Some of the strategies to increase the stability of a suspension are: • Reduce particle size by micronization.

• Ensure the separation of particles with the best selection and proportion of surfactants.

• Increase the viscosity to prevent the particles get together.

• Avoid sudden temperature and pH changes which can break the interactions formed.

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In conclusion, the selection of the best antioxidant, and the best way to use, is an important decision that requires technical expertise. A seemingly simple change in the format of the antioxidant or the carrier used can greatly improve the results in protecting products against oxidation.

More information: Margarita Altable Sánchez PhD in organic Chemistry R&D Department, Oxiris Chemicals E: margarita.altable@ oxirischemicals.com

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FEED ADDITIVES

Health program based on functional feed additives to fortify gut integrity and protect against parasitic infestations in aquaculture Panos G. Kalatzis, Maria Mercè Isern-Subich, Waldo G. Nuez-Ortín, Adisseo

production, the expected outcome can be leveraged by putting in action preventive health programs that can be implemented daily through the administration of functional feed additives.

Why prevention Increasing food demand requires sustainable and efficient farming strategies in order to meet the nutritional requirements of the steadily increasing world population. Aquaculture has been an essential component of animal protein production and is further expected to play a critical role in the future. During the Global Seafood Alliance (GOAL) Conference, industry stakeholders agreed that despite turbulent market conditions and high costs, long-term growth was projected for aquaculture. Production intensification, which has led to a steep growth of the aquaculture industry over the past decades, has also led to the emergence of several diseases in culture fish and shellfish. Aquatic diseases are a constant threat that hampers productivity and leads to significant financial losses for the sector. Although corrective actions are necessary in order to mitigate fatal losses upon disease outbreaks, prevention is certainly the most sustainable, environmentally friendly and efficient strategy.

Since a good level of hygiene and plain standard operating protocols are in place to guarantee

Gut integrity and health, key in dealing with gut parasites The contribution of a robust gut to fish health is fundamental and complementary to its unequivocal role in nutrient absorption and growth performance.

Gut microbiota, gut epithelium and immune arm interact synergistically to maintain gut integrity and fish health. Aquaculture conditions entail multiple factors, such as high fish density, environmental stressors and nutritional ingredients, that may disrupt this delicate balance and leave room for parasites and pathogenic microorganisms to infiltrate.

Numerous research projects and publications have focused on the composition, diversity and functionality of gut microbiome, highlighting not only its important role in fish health but also the high level of complexity and the multifaceted approach that is required to address that. Opportunistic pathogens usually take advantage of external stressors, for instance, temperature changes, and colonize the fish tissues to complete their reproduction cycle. Disrupted gut integrity is typically demonstrated by an inflammatory response, while the presence of intraepithelial immune cells such as macrophages is increased in the area.

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FEED ADDITIVES

Figure 1. (A) Transepothelial resistance of anterior intestine with and without SANACORE GM supplementation (0.5%). Tissue resistance in the intestine of healthy seabream juveniles of 100g is expected to be >150 Ω cm 2, therefore a threshold has been set with dotted line. (B) A statistically significant difference of 30% higher tissue resistance is reported under SANACORE GM supplementation (0.5%). Statistical significance is stated by *.

An infamous gut pathogen in Mediterranean aquaculture, the myxosporean parasite Entreomyxum leei, can be a serious threat for a sensitive, unhealthy gut in species such as gilthead seabream (Sparus aurata), sharpsnout seabream (Diplodus puntazzo) and red seabream (Pagrus major). Enteritis, which is caused

by E. leei, may in some cases lead to a chronic condition of anorexia and weight loss but in other cases also to mass direct mortality (Henry et al., 2020; Palenzuela et al., 2020). Microsporidian infections have also been observed since the early 2000s in the Mediterranean with Enterospora nucleophila causing emaciative syndrome to mainly, yet not only, the early stages of gilthead seabream, eventually leading to severe weight loss and eventually mortality (Palenzuela et al., 2014).

SANACORE® GM by Adisseo is a broad-spectrum, health-promoting additive of natural origin. The positive impact of SANACORE® GM on gut integrity and health promotion has been both extensively documented in the literature and reported by EU aquaculture projects such as ParaFishControl. SANACORE® GM has been particularly effective against E. leei infections by limiting the abundance and extension of the parasite in various doses in gilthead and sharpsnout seabream (Henry et al., 2020; Palenzuela et al., 2020).

Compromised gut health and histopathological damages increase vastly the chances for secondary infections to be developed by the simultaneous infiltration of opportunistic bacteria. Chronic inflammation of the gut along with gut disruption is very common nowadays in cultured fish, whereas the

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increasing inclusion of plant-based ingredients in the diets does not help change the situation. Recently in another EU project, the contribution of SANACORE® GM was tested in a zero fishmeal diet leading to no undesired effects or impairments of gut microbiota and gene expression during a challenge against E. leei (Piazzon et al., 2022).

SANACORE® GM has been shown to support gut health by assessing transepithelial resistance (TER) as an indicator of gut integrity. In gilthead seabream fed 10% FM feeds, the additive increased TER on average by 30% (Fig. 1). In this line, a very recent peer-review publication corroborates the enhanced development of intestinal villi as a key mechanism of SANACORE® GM to optimize nutrient absorption and promote growth performance in gilthead seabream (Abdel-Tawwab et al., 2022).

The economic losses accompanied by hampered productivity are very substantial for the industry and the inclusion of functional feed additives in the diet is a preventive strategy that can protect fish and support gut health. SANACORE® GM is a robust solution to support gut integrity and reduce the severity of gut parasite infections.

Optimizing the external line of defense is key in the fight against ectoparasites and secondary infections Parasites that colonize the external surfaces, gills and skin of fish are naturally present in marine environments. Parasitism exists in nature but is not common in wild fish populations because the chances

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FEED ADDITIVES known to cause severe issues of anemia, compromising growth while opening doors to secondary opportunistic infections (Mladineo et al., 2021; Sitjà-Bobadilla et al., 2010). Over the past years, S. chrysophrii has been a constant concern for the Mediterranean aquaculture industry, due to culture conditions and environmental parameters.

APEX® BRANCHIA is a health-supporting functional feed additive composed of a synergistic blend of natural ingredients with antiparasitic, antimicrobial and immunostimulatory properties. Using guppy fish as a model, APEX® BRANCHIA has shown consistent and statistically signif icant efficacy against infection by Figure 2. Infection rates in guppies (Poecilia reticulata, n=18 per treatment) infected with Gyrodactylus turnbulli. Infection rates and mean parasite abundance in two individual groups the monogenean parasite of adult guppies (A) Trial 1, (B) Trial 2.

Gyrodactylus turnbulli. The additive significantly reduced of parasites meeting available hosts are scarce. the parasitic burden in adult-infected guppies by However, high stocking densities dramatically increase 43-47% (Fig. 2). the chances of parasites encountering their hosts and Besides antiparasitic action, APEX® BRANCHIA then horizontal transmission progresses quite fast. delivers antimicrobial activity to skin mucus. This The most notoriously known ectoparasite in the is a key benefit of the additive since opportunistic aquaculture industry is the sea louse Lepeoptheirus bacteria are a constant threat to parasitized fish, salmonis, which parasitizes the outer surface of particularly in cage production under high stocking Atlantic salmon, (Salmo salar). The salmon industry densities. The innate immunity components of has been struggling with sea lice for many years epidermal mucus can actively mitigate the threat investing also heavily in research grants, however, no posed by the favorable growth of pathogens in efficient solution has yet been developed. Mechanical aquaculture conditions. Skin mucus of gilthead treatments to delouse fish have been greatly applied seabream supplemented with the additive was able and that often leads to stressful conditions which to significantly delay the growth of Pseudomonas eventually facilitate the intrusion of opportunistic anguilliseptica (Fig. 3). When the antimicrobial pathogens such as Moritella viscosa and Tenacibaculum properties of the mucus extracted from gilthead spp.; which has been significantly increasing over the seabream skin were assessed, it was shown that the external defense line of the fish which were fed with past few years (Walde et al., 2022).

In Mediterranean aquaculture, Sparicotyle chrysophrii, APEX® BRANCHIA supplemented diet were able to which parasitizes the gills of gilthead seabream, is compromise the growth of pathogenic bacteria.

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Figure 3. Antimicrobial activity of skin mucus following APEX® BRANCHIA supplementation (0.6%) for four weeks. (A) Growth curve of P. anguilliseptica in regular medium (black), mucus from fish fed with the additive (white) and mucus from fish fed without the additive (blue). B) Percentage of P. anguilliseptica growth inhibition in mucus (blue) compared to growth in regular medium (white). *Significant differences (p<0.05) between control and additive.

Conclusions Increasing demands for fish production inevitably lead to the intensification of aquaculture. Maintaining good levels of fish health is a prerequisite for optimal performance but also for a profitable and sustainable aquaculture industry. Therefore, a reliable health program is recommended to be in place and the role of functional fish diets is becoming even more crucial. SANACORE® GM and APEX® BRANCHIA are two efficient preventive solutions that will boost gut integrity, support fish health and provide systemic support for the cultured organism so that it can

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successfully cope with the challenges that will face in the aquaculture environment.

References available on request.

More information: Panos G. Kalatzis Regional Manager Aquaculture Europe Adisseo E: panos.kalatzis@adisseo.com

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PLANT EXTRACTS & BOTANICALS

Harnessing plant synergies to enhance performances, health and sustainability in fish and shrimp Fabrizio Caruso, Vetagro Spa Incorporating microencapsulated botanicals into aquafeed

Complementing the power of aquafeeds Global aquaculture production, based on feed provision, accounted for 61 million tons in 2017 while industry estimates expect a further significant increase, to the point where the sector would require more than 73 million tons of feed by 2025 to meet global demands (FAO, 2019; Boyd et al., 2020). In order to achieve this blue evolution, it is the duty of fish

nutritionists worldwide not only to evolve their choice of ingredients but also to reach sustainability by exploiting the full potential of functional additives and micronutrients. With this in mind, Vetagro S.p.A, an Italian progressive science-based company specializing in microencapsulated feed additives, has recently entered the aquafeed sector to transfer the knowledge developed in the terrestrial livestock

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PLANT EXTRACTS & BOTANICALS

Microencapsulated essential oils and organic acids - fine granulometry

industry additives over the last 40 years. Committed to assisting aquaculture farmers worldwide, the company offers a scientifically proven cost-effective solution based on organic acids (OAs) and natureidentical compounds (NICs) capable of increasing fish and shrimp performance while boosting immunity through the use of patented microencapsulation and slow-release technology.

Exploiting nutrition potential through natural synergies After decades of first-hand experience with the results of slow-release additives in ruminants, swine and poultry nutrition, Vetagro started investigating the effects of supplementing botanicals such as OAs and NICs in aquaculture. Although these compounds are well-known for their potential to improve performance, intestinal health, immune status, and as antimicrobials, knowledge and research on them are still recent when compared to other bioactive molecules in aquafeed.

Organic carboxylic Acids (OAs), for example, are proven to be linked to lowering pH of feed and digestion, therefore, inhibiting the growth of acid-intolerant bacteria and enhancing feed hygiene; increasing in digestive enzymes, resulting in increased nutrient digestibility and feed utilization; and modulating microbiota and animal health.

On the other hand, NICs, synthetic but chemically identical molecules to pure botanical extracts, are used in agriculture as antimicrobials, antioxidants, and palatability enhancers. The NICs tested were thymol, carvacrol, vanillin, eugenol, cinnamaldehyde, geraniol, alpha-pinene, eucalyptol, menthol, linalool, and limonene. After carefully evaluating

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their in vitro activity against Vibrio harveyi and Vibrio anguillarum Vetagro experts selected four botanicals for further trials, proving not only the positive effects but also a strong synergistic relationship (Rossi et al., 2021). Once the formula with the bioactive blend of four different molecules was established the research carried on concerning the best way to deliver them to the digestive tract of fish and shrimp.

This was accomplished thanks to the native technology of microencapsulation. The microencapsulation process distributes the active compounds into fine and stable beads under 500um of a vegetable matrix. This technology protects the molecules before, during and after the inclusion in aquafeeds from external agents thus allowing higher stability together with easy and safe use. From a biological standpoint, this capsule makes the delivery of the product slow and constant all along the digestive tract, enhances palatability and grants efficacy at low dosages. This combination now holds the commercial name of Aviplus® Aqua.

This product was tested in vivo thanks to partnerships with research centers and universities all over the world and its combination is protected by numerous international patents. Continuous in vivo validation of Aviplus® Aqua began in 2017 in Italy, China, and Spain, and traveled through Mexico, Portugal, and the United States, testing health and growth performance on some of the most important fresh and marine aquaculture species, such as European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), hybrid catfish (Ictalurus punctatus x Ictalurus furcatus), rainbow trout (Oncorhynchus mykiss) and whiteleg shrimp (Penaeus vannamei).

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PLANT EXTRACTS & BOTANICALS

Statistical analysis of growth parameters: body weight and specific growth rate in S. aurata 24 hours and 15 days post infection with V. harveyi

Aviplus®Aqua boosts growth and immunity against vibriosis In Spain, experts conducted a trial on the growth of S. aurata. During the 56-day experiment, seabreams were fed three diets: one control and two enriched with 500ppm and 1000ppm Aviplus® Aqua. Six weeks into the trial, fish were then challenged with an intraperitoneal injection of V. harveyi to induce vibriosis.

Two weeks after, gilthead seabreams were sampled, and indicators were analyzed to evaluate the effect of the microencapsulated blend. To evaluate growth, body weight (BW) and specific growth rate (SGR) were sampled along the trial: i) at the end of the performance trial or day 42 and ii) 15-days post-infection or day 56.

The data was analyzed using one-way ANOVA, and results supported Aviplus® Aqua's positive effect on growth performance before, during and after vibriosis.

Encouraged by these findings, Esteban et al. (2019) conducted a second trial in which they demonstrated

not only what had previously been seen with growth performance (weight gain and FCR) but also integrated an immune status analysis of humoral and cellular immune parameters, such as immunoglobulin M (IgM), phagocytosis, and respiratory burst activities. The trial took place on a similar set-up with gilthead seabream from a commercial facility in Spain. Results presented: i) generally higher values and a significant increase for IgM with Aviplus®Aqua at 1000ppm; ii) significantly higher phagocytic capacity after 14 days; and iii) significantly higher respiratory burst activities after 14 days; supporting the hypothesis of a primary immunoboosting effect followed by a positive effect on the general health status of seabreams, appreciable on long term use of the product.

The way ahead The data presented underlines that Aviplus®Aqua boosts the immunity of aquatic animals against vibriosis

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PLANT EXTRACTS & BOTANICALS and increases growth rate and body weight, thanks to the harnessed power of pure natural compounds and organic acids. In light of these positive results, our research and development of new products for the Vetagro Aqua Collection will continue. The R&D department will coordinate several field trials to improve understanding of the role of OAs and NICs in aquatic animal nutrition and health, with the goal of tailoring the effects to the specific farmer's needs in the near future.

More information: Fabrizio Caruso Aquaculture Product Specialist Vetagro Spa E: fabrizio.caruso@vetagro.com

References FAO (2019). Fishery and aquaculture statistics. In Global aquaculture production 1950– 2017 (FishstatJ). Rome, Italy FAO Fisheries and Aquaculture Department. Retrieved from www.fao.org/fishery/statistics/ software/fishstatj/en Boyd, C. E., D'Abramo, L. R., Glencross, B., Huyben, D. L., Juarez, L., Lockwood, G. A., Valenti, W. C. (2020). Achieving sustainable aquaculture: Historical and current perspectives and future needs and challenges.

Journal of the World Aquaculture Society, 51(3), 578–633.

Rossi B, Esteban MA, GarcíaBeltran JM, Giovagnoni G, Cuesta A, Piva A, Grilli E. Antimicrobial Power of Organic Acids and Nature-Identical Compounds against Two Vibrio spp.: An In Vitro Study. Microorganisms. 2021 Apr 29;9(5):966. doi:10.3390/ microorganisms9050966. PMID: 33947155; PMCID: PMC8146449.

Esteban MA, Rossi B, GarcíaBeltran JM, Cuesta A, Piva A, and Grilli E. 2019. Dose-effect response of a blend of organic acid and nature-identical compounds on gilthead seabream (Sparus aurata L.), Book of Abstracts, Aquaculture Europe 2019.

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PLANT EXTRACTS & BOTANICALS

Benefits of supplementing herbs and plants as feed additives and complementary feeds for sustainable aquaculture Javier Gonzalez F., Nuproxa Switzerland Ltd.

Herbs and medicinal plants have been used for centuries in ancestral medicine to counteract human illness. Among these is Ayurveda, a traditional system of Indian medicine that originated over 5,000 years ago.

Ayurveda is based on the use of herbs and plants to treat different anomalies in humans and animals. The benefits are associated with their phytoconstituents which provide several health benefits. In the last decade, herbs, plants and their extracts have been used as feed additives and complementary feed to improve performance and welfare and replace synthetic additives, nutrients and drugs.

In fish and shrimp feeds, herbs and phytogenic feed additives (PFA) improve performance, immune function, gut health, disease resistance and stress control, and provide antimicrobial and liver protective properties (Prabu et al., 2018; Vaseeharan et al., 2011; Xie et al., 2018). Regarding gut health, PFA can

enhance digestive function and prevent the growth of harmful bacteria.

In addition, herbs are currently used for growthpromoting, as antimicrobial agents, immunostimulants and nutrient sources in commercial feeds for fish and shellfish species (Citarasu, 2010; AftabUddin et al., 2017) as well as improved feed conversion efficiency (FCR), enhanced antioxidant and anti-inflammatory activity.

Plants, including Aloe barbadensis (Aloe Vera, AV), Andrographis paniculata (Green Chiretta), Annona squamosa (Sugar Apple), Azadirachta indica (Neem Tree), Citrus aurantifolia (Lime), Coriandrum sativum (Coriander), Ocimum sanctum (Holy Basil), Ollium cepa (Onion) and Psidium guajava (Guava), have been used as natural immunostimulants for preventing several fish and shellfish diseases (Nya and Austin, 2009; Pedge and Ahirrao, 2012; AftabUddin et al., 2017; Kaur et al., 2020; AftabUddin et al., 2021; Syed et al., 2021).

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PLANT EXTRACTS & BOTANICALS This article will focus on the use of whole herbs, parts of them and/or their extracts and their applications in aquaculture species nutrition and health.

Plants and herbs with health-enhancing properties Aloe barbadensis (Aloe vera, AV) is known because of its multiple healing properties, with more than 75 bioactive compounds (Radha and Laxmipriya, 2015; Sanchez et al., 2020; Hamza, 2022).

Fehrmann-Cartes et al. (2022) used AV extract as an additive to reduce gut inflammation in Atlantic salmon (Salmo salar) fed with a high soybean meal (SBM) diet (30%). Fish fed with AV extract (0,4 g/kg) significantly reduced enteritis associated with SBM. These findings were associated with a reduction in the number of goblet cells, lamina propria thickness and sub-epithelial mucosa size, with a significant decrease in proinflammatory cytokine IL-1β, similar to the observed in fish fed a high fishmeal diet.

Andrographis paniculata (Green chiretta, AP) is commonly known as the “King of Bitters” in Acanthaceae family. AP exhibits anti-microbial (Guo et al., 2014), anti-inflammatory (Li et al., 2017) and antioxidant (Sivakumar and Rajeshkumar, 2015) activities.

AP promotes the immune system of aquatic animals to resist several pathogens. Rattanachaikunsopon and Phumkhachorn (2009) found that diets supplemented with AP leaf aqueous extract increased the survival rate of Nile tilapia after a challenge with Streptococcus agalactiae.

A more recent study (Yin et al., 2023) evaluated different doses of AP extract (0, 0.25, 0.50 and 1%) on shrimp performance and health after a challenge with Vibrio alginolyticus. In the pre-challenge phase, AP significantly (p<0.05) improved shrimp performance, total hemocyte count (Fig. 1) and the percentage of phagocytic cells in hemocytes. In addition, AP increased the activity of superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and decreased the levels of malondialdehyde (MDA) in hepatopancreas.

In the post-challenge phase, AP improved shrimp survival (Fig. 2) and attenuated the generation of superoxide radicals in hepatopancreas (Fig. 3). The authors concluded that the supplementation of AP (0.25% and 0.5%) enhanced performance, promoted the non-specific immunity and the resistance to Vibrio in

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L. vannamei and attenuated the oxidative stress and apoptosis induced by Vibrio infection.

Figure 1. Total hemocytes count (THC) of L. vannamei fed a diet containing 0.25%, 0.5%, or 1% of AP for 14 and 28 days. Data are presented as mean ± SD. *p<0.05, **p<0.01 (Yin et al., 2023).

Figure 2. Cumulative survival rate of L. vannamei fed either a control diet or a diet containing 0.25%, 0.5%, or 1% of AP for 28 days, and then challenged with V. alginolyticus (Yin et al., 2023).

Antibiotics and synthetic drugs are the first alternatives for controlling pathogens in aquaculture, however, conditions like antibiotic residues, microbial resistance and environmental pollution are a concern (Harikrishnan et al., 2009; Citarasu, 2010). The use of herbs has attracted considerable attention due to their natural antimicrobial, immuno-potentiating and antioxidant properties, rapid biodegradability, low toxic side effects and ease of availability (Prasad & Variyur, 1993; Saha et al., 2017).

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PLANT EXTRACTS & BOTANICALS pinene group, myrcene and cymene which are mostly monoterpenes (Nagulakshmi et al., 2000). Thymol is known for its antibacterial and antifungal properties.

Extracts of TA are active against Pseudomonas, Escherichia coli, Klebsiella, and Staphylococcus aureus (Shankaracharya et al., 2000; Usha et al., 2012). Ali et al. (2017) demonstrated that adding 1% of TA extract in rainbow trout feeds significantly improved their performance and survival.

Figure 3. Effect of AP over the levels of superoxide radical in hepatopancreas. Results are shown as mean ± SD. NS means p>0.05, *means p<0.05, ** means p<0.01 (Yin et al., 2023).

Phytogenics with benefits on performance and immune response in aquaculture species include Astragalus polysaccharides (APS), extracted from Astragalus membranaceus (Mongolian Milkvetch) (Du et al., 2022), chlorogenic acid (CGA), a class of phenolic acid present in coffee grains and berberine (BBR), an alkaloid isolated from various barberry (Asia) and goldenseal species (America) (Salehi et al., 2019).

A dietary supplement with APS significantly increased the activities of phenoloxidase (PO), total SOD and lysozyme, a in shrimp hemolymph (Chang et al., 2017).

CGA supplemented at 200 and 400 mg/kg feed could enhance non-specific immunity, serum antioxidant capacity and growth performance of carp (Cyprinus carpio) (Wen, 2012). In addition, BBR significantly reduced oxidative stress and apoptosis and enhanced the immunity of blunt snout bream (Chen et al., 2016).

Ding et al. (2020) tested different combinations of APS, CGA and BRR in shrimp feeds and later evaluated immunological indicators and antibacterial activity using Vibrio harveyi. The results indicated that 0.5 g/kg of APS + 0.5 g/kg of CGA showed higher total hemocyte counts, phagocytic, antibacterial and bacteriolytic activities during 6 days of feeding.

Trachyspermum ammi (Thymol seeds, TA) belongs to the Apiaceae family. The major components of the plant extract are thymol, terpinene, phlandrene,

Ocimum sanctum (OS) belongs to the family Lamiaceae. The leaves of OS contain water-soluble phenolic compounds, such as eugenol, tannins, saponins, flavonoids, terpenoids, methyl eugenol and caryophyllene, some of which act as immunostimulants (Bairwa et al., 2012; Nahak & Sahu, 2014). In aquaculture, OS extract was studied in several fish, including Oreochromis mossambicus (Logambal et al., 2000), Epinephelus tauvina (Sivaram et al., 2004), Catla catla (Chitra & Krishnaveni, 2011), Cyprinus carpio (Pavaraj et al., 2011) and Labeo rohita (Das et al., 2013).

Moreover, OS increase the resistance to pathogens, such as Vibrio harveyi (Sivaram et al., 2004) and Aeromonas hydrophila (Logambal et al., 2000; Chitra & Krishnaveni, 2011; Pavaraj et al., 2011; Das et al., 2013).

A study demonstrated that tilapia fed with 200 mg/ kg feed of an OS extract performed significantly better than the control and had significantly lower mortality when challenged with Streoptococcus agalactiae (Panprommin et al., 2017).

Salvia officinalis (sage, SO) is a member of the Labiatae/ Lamiaceae plant family. The therapeutic property of SO is due to its distinctive phytochemical constituents, comprising alkaloids, phenolic substances (e.g., coumarins, flavonoids, tannins), glycosidic derivatives (e.g., saponins, flavonoid glycosides, cardiac glycosides), steroids, poly acetylenes, terpenes/terpenoids and waxes (Ghorbani & Esmaeilizadeh, 2017; HrebienFilisinska & Bartkowiak, 2022; Poulios et al., 2020).

These phytomolecules act as anti-inflammatory and antioxidant, in addition to a wide range of woundhealing effects (Li et al., 2019; Salomon et al., 2020).

Earlier studies were carried out to test the beneficial application of sage as a feed additive on gilthead seabream (Salomon et al., 2020), mirror carp (Abdel Rahman et al., 2022), beluga (Dadras et al., 2020) and

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PLANT EXTRACTS & BOTANICALS rainbow trout (Tafi et al., 2020). The results indicated the potential role of sage as a beneficial supplement for growth performance, immune response and health.

Recently, Hussein et al. (2023) evaluated different levels of SO (0, 2, 4, 6 and 8 g/kg) leaves in the diet of European sea bass. The regression analysis showed that the optimum quantity of sage at 3.6 – 4.1 g/kg diet is recommended based on the WG, SGR, phagocytic activity, FCR, lysozyme activity, blood indices (Fig. 4), non-specific immune response and healthier gut flora.

Jindal, 2019). An extract of EO was tested against the toxic effect of triarylmethane dye (MG) in carp (Cyprinus carpio) red blood cells (RBC) (Sinhan & Jindal (2019).

Through scanning electron microscopy, the authors evaluated the effects of MG and demonstrated that EO extract fed at 1000 mg/kg feed significantly prevented RBC abnormalities (Fig. 5).

Figure 5. Graph depicting abnormalities (%) in erythrocytes of malachite green (MG)‐exposed and Emblica officinalis‐supplemented Cyprinus carpio. a,b indicate significant change (p<0.05) compared to control and MG treatment respectively (Sinhan & Jindal, 2019).

Figure 4. Lysozyme and phagocytic activities in serum of European seabass fed experimental diets for 90 days. Values (means ± SE, n=3) with different superscripts are significantly different at P≤0.05 (Hussein et al., 2023).

Emblica officinalis (Indian gooseberry, EO) is a plant that possesses antioxidative, hepatoprotective and immunostimulant properties (Chaphalkar et al., 2017; Hazra et al., 2010; Tahir et al, 2016; Tan et al., 2018).

The polyphenols found in EO, especially tannins and flavonoids are key responsible elements for major bioactivities. EO exhibits a broad spectrum of pharmacological activities including antioxidant, immunomodulator, anti-inflammatory, wound healing and cyto-protective properties (Yadava et al., 2017).

Major phytoconstituents present in EO extract are gallic acid (640 mg/g) and ascorbic acid (198 mg/g) (Sinha &

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Withania somnifera (Ashwagandh, WS) is a fastgrowing Indian herb. Various studies have shown the immunomodulatory effects of withanolides which is the principal phytoconstituent of the plant (Chandrasekaran et al., 2017). Dietary administration of WS root powder was reported to stimulate immunity and disease resistance in rohu carp Labeo rohita (Sharma et al., 2010). Mukherjee et al. (2019) determined that the main phytoconstituents in WS are tannins, saponins, alkaloids, flavonoids and terpenoids.

Nile tilapia were fed diets containing different concentrations (0, 0.3, 0.5 and 0.7 g/kg feed) of an ethanolic extract of WS for 30 days (Mukherjee et al., 2019). Fish fed WS extract (0.7 g/kg) showed the highest immunological, hematological, biochemical and growth parameters. In a subsequent study, Mukherjee et al.

(2022) observed a significant reduction in mortality when Nile tilapia were fed with a WS extract and challenged with Aeromona hydrophila.

Another research evaluated the effects of an aqueous extract of WS against cadmium chloride (CC)–induced toxicity (1.775 mg/L) in Nile tilapia (El-Sabbagh et al., 2022). Fish exposed to CC and co-supplemented with the extract at high doses (3 ml/kg BW) showed significant

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PLANT EXTRACTS & BOTANICALS ameliorative effects in hemogram parameters, total protein, globulin, IgM and lysozyme. In addition, the fish showed higher activities of CAT and SOD, reduced MDA formation and improved survival. This study showed that WS extract was an effective tool to overcome CC–induced toxicity and to improve immune response against invading bacterial pathogens.

Asparagus racemosus (Asparagus, AC) and WS were tested by Trivedi et al. (2023) for their immunostimulatory effect in spotted snakehead (Channa punctatus). Fish were fed with different levels (1 to 3 g/kg feed) of a dry powder of the plant roots either alone or in combination. Although WS significantly improved hematological and immune indicators, the best response was obtained with the combination of both herbs at the high dose (1.5 g of WS + 1.5 g of AC/kg feed). The blood concentration of neutrophils, lymphocytes, monocytes and eosinophils and the activity of lysozyme in liver and muscle were all improved with the combination of both herbs.

apoptosis, angiogenesis and immunomodulation.

Amongst the bioactive compounds, limonoids such as nimbolide and azaridachtin are extensively studied.

Different parts of neem tree show remarkable antioxidant activity with the potential to scavenge free radicals and reduce ROS-mediated damage to cells.

Additionally, neem extracts minimize the release of proinflammatory cytokines such as TNF-α and IL-6 and have immunomodulatory functions, increasing the count of CD4+ and CD8+ T cells. These studies are indicating the pivotal roles of AI in the regulation of different biological pathways (Sarkar et al., 2021).

Recently, Abidin et al. (2022) conducted a study to evaluate different dietary levels of an AI leaf extract (0, 5, 7 and 10%) on the performance of rainbow trout during a 90-day period. The results indicated that the best SGR was achieved at 7.5% of the extract.

Another study performed by Thanigaivel et al. (2015) revealed that AI pant extract at a concentration of 150mg/l in vivo can be used as an alternative to antibiotics for treating Citrobacter freundii in tilapia.

Azadirachta indica (AI) is a large evergreen tree (Neem) that contains numerous phytochemicals with a range of pharmacological properties against certain fungal, bacterial and viral infections and boost antioxidant properties (Talpur & Ikhwanuddin, 2013; Adamu et al., 2018). Phytogenics obtained from AI have a role in enhancing immunity against certain fish diseases and suppressing specific pathogens (FAO, 2007; Binh, 2016).

Sarkar et al. (2021) performed a comprehensive review of the health benefits of AI. The bioactive compounds in AI have different regulatory effects in inflammation,

Mix of herbs and plants as nutrient supplements Different polyherbal formulations have also been used to substitute synthetic nutrients in aquaculture species.

A mix of herbs based on Emblica officinalis and Ocimum sanctum was tested as a potential replacement of synthetic vitamin C monophosphate (VCMP 35%) in Nile tilapia for an 8-week period (Williamson, 2022). The negative control treatment had no addition of ascorbic acid (AA), the positive control group contained 50 mg/ kg of AA supplied by a commercial source of synthetic VCMP and the test diets contained 25, 50 and 100 mg/kg

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PLANT EXTRACTS & BOTANICALS Table 1. Effect of a polyherbal based on Ocimum sanctum and Emblica officinalis on the replacement of synthetic VCMP in Nile tilapia performance (Williamson, 2022).

Treatment

1 2

Source of AA1

Concentration in the feed (mg/kg)

SGR

FCR2

Negative control

None

3.71

1.14a

Positive control

VCMP

140

3.83

1.10ab

T1

Herbal Mix

25

3.80

1.10ab

T2

Herbal Mix

50

3.82

1.08b

T3

Herbal Mix

100

3.86

1.08b

VCMP: vitamin C monophosphate 35% a,b p <0.05

Table 2. Effect of a polyherbal based on Andrographis paniculata, Azadirachta indica and Trachyspermum ammi in Nile tilapia performance (Bombardelli, 2022)

1 2

Treatment

Source of Choline

Concentration in the feed (mg/kg)

Weight gain (g/d)

FCR2

Positive control

Choline chloride

1401

4.66ab

0.77a

Negative control

None

0

4.65

0.78a

T1

Herbal Mix

200

ab

4.47

0.77a

T2

Herbal Mix

400

4.28b

0.75ab

T3

Herbal Mix

600

4.84

a

0.79a

T4

Herbal Mix

800

ab

4.62

0.71b

ab

As pure choline supplied by choline chloride a,b p <0.05

of the herbal mix. The results of the study demonstrated that based on performance, the herbal mix could be used as a substitute of VCMP in tilapia feeds (Table 1).

Another mix of herbs including Andrographis paniculata, Azadirachta indica and Trachyspermum ammi was tested at different doses (0, 200, 400, 600 and 800 mg/kg) to evaluate their effects over Nile tilapia performance and as a substitute of synthetic choline chloride (CCH) in a 4-month growing period (Bombardelli, 2022). The control group was supplemented with 140 g of choline supplied by CCH. The results showed no differences in weight gain (g/d) between the fish fed CCH and the polyherbal. The fish fed the highest dose of the polyherbal (800 mg/ kg feed) achieved a significantly better FCR compared to the control groups and the lower doses of the herbal mix (Table 2). In addition, the study showed that fish fed the polyherbal product presented lower levels of liver fat and blood triglycerides, an indication of better lipid metabolism. These results, together with several antioxidant indicators and blood biochemistry parameters lead the author to conclude that the polyherbal not only improved fish performance but also allowed to fully replace synthetic CCH in the diet of Nile tilapia.

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Conclusions Overall, the use of herbs, plants and their extracts as feed additives or complementary feed ingredients in fish nutrition has shown huge promise as a natural and effective way to improve fish growth, health, performance and to substitute synthetic nutrients. Their complex mode of action demands profound research to fully understand their mechanisms of action, efficacy and optimal dosage for different fish species and life stages. Nevertheless, it is well accepted that in the near future these natural solutions would become a reliable alternative to replace synthetic drugs and nutrients in aquaculture feeds. It is recommended that aquaculture zootechnicians, pathologists and nutritionists be more aware and knowledgeable about these natural solutions available to them.

More information: Javier Gonzalez Director of Research and Development, Aqua Division Nuproxa Switzerland Ltd. E: javier.gonzalez@nuproxa.ch

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PLANT EXTRACTS & BOTANICALS

Beneficial replacement of vitamins E and C in feeds for tilapia Karsten C. Kjeldsen, R2 Agro A/S University research with tilapia has shown that a natural polyphenol product can substitute up to 80% of added vitamins E and C in fish feed.

Some fruits, berries and herbs have a high content of polyphenols that possess interesting properties in animal feeds. Cabanin® CSD (Cabanin) has a high content of polyphenols in the form of selected elements from grapes, citrus, blackcurrants, and chestnut with a particularly strong antioxidant effect. These polyphenols can be even more effective antioxidants than vitamins E and C. In addition to the antioxidant properties, the polyphenols in Cabanin also have good anti-microbial and anti-inflammatory properties.

These selected polyphenols have the potential to partially replace vitamins E and C in animal feed.

Animals, including fish, are often fed vitamins E and C far beyond the minimum requirement to ensure a sufficient level of antioxidants to counteract oxidative stress. It is possible to replace that part of the vitamin E and C content in the feed above the minimum standard requirements and even improve the results.

Enhance growth performance A trial conducted at Nong Lam University (Dr. Vo Van Tuan) in Vietnam in 2022 aimed to investigate the effects of Cabanin as a vitamin E and C antioxidant replacement above the minimum dosage of 50ppm.

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PLANT EXTRACTS & BOTANICALS

Figure 1. Effect on the overall weight gain. Initial fish weight 9.8g. ab Means with different superscripts within the row differ significantly (P<0.05).

Figure 2. Effect on the overall average feed conversion ratio. ab Means with different superscripts within the row differ significantly (P<0.05)

A total of 600 Nile tilapia fingerlings with an IBW of 9.8g were divided into 4 groups with 3 replicates. The trial lasted 10 weeks. The groups were fed a basal extruded pre-grow-out tilapia diet assigned to one of the following treatments: Low Vit. E & C, 50ppm of vitamin E (all-rac-α-tocopheryl acetate) and vitamin C; High Vit. E, 250ppm of vitamin E and C; Cabanin – 50% replacer, 150ppm of vitamin E and C + 200ppm of Cabanin® CSD; and Cabanin – 100% replacer, 50ppm of vitamin E and C + 400ppm of Cabanin® CSD. Results showed a significantly improved weight gain and feed efficiency when Cabanin was included as a partial replacement of vitamins E and C (Fig. 1, 2).

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At end of the growth trial, a seven-day ammonia challenge trial was conducted for testing the survival rate of tilapia. From day 5 and onwards, a significant difference was recorded with an improved survival rate when Cabanin was included.

Less oxidative stress and improved stability in refrigerated flesh Results showed that Cabanin also improves the antioxidant status in the body compared to low and high dosages of vitamins E and C. It was tested by taking blood samples (Table 1) for the test of stressrelevant parameters.

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PLANT EXTRACTS & BOTANICALS Table 1. Oxidative relevant parameters measured in blood and meat of tilapia

Parameters

Low Vit. E

High Vit. E

Cabanin 50% replacer

Cabanin 100% replacer

SOD in blood (U/ml) Higher = Higher protection Capacity

0.36a

0.34a

0.45b

0.51b

TBARS in blood (nmol/ml) Low = Less oxidative stress

0.23a

0.22a

0.17b

0.17b

TBARS in meat (nmol/mg protein) at day 7 of refrigeration

0.37a

0.36ab

0.30b

0.27b

Means with different superscripts within the row differ significantly (P<0.05) SOD = Superoxid-dismutase, TBARS = thiobarbituric acid-reactive substances

ab

can be monitored by TBARS level. The relevance of TBARS as a useful marker of meat stability in meat processing and packaging was shown by Salami et al. (2016) and Marzoni et al.

(2014).

This study showed that the polyphenolic antioxidant product Cabanin was dispersed, retained, and remained functional in blood plasma and in refrigerated flesh and hereby having the potential to improve shelf life in refrigerated flesh.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide radicals (O2−) to molecular oxygen (O2) and hydrogen peroxide (H2O2), providing cellular defense against reactive oxygen species. Endogenous protection against oxidative stress is achieved by enzymes that catalytically remove free radicals and other reactive species. The antioxidant defense systems include antioxidants (natural or synthetic) and the antioxidant enzymes present in the biological system. Increasing the activity of SOD would subsequently enhance the clearance capacity of oxygenfree radicals in the fish. Together with the increased activity of SOD, TBARS concentration in the blood plasma is reduced.

Malondialdehyde is formed as an end-product of lipid peroxidation and, therefore, the extent of lipid ROS

Conclusions Trials indicate that all vitamin E and C content above 50ppm can be replaced in feed for tilapia.

Our general recommendation is a replacement with 2mg Cabanin per mg of pure vitamins E and C, including a significant safety margin.

The replacement of vitamins E and C with Cabanin in aquafeeds is not only beneficial for performance reasons but also in terms of price to replace a big part of vitamin E and C in fish feed.

More information: Karsten C. Kjeldsen Product Manager R2 Agro A/S E: kck@r2ago.com

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