Shrimp is now established as one of the most consumed seafood products and is of considerable popularity worldwide, with increasing expansion in many countries and regions. It is a significant sector of the lucrative aquaculture industry.

The global shrimp market was US$18.30 billion in 2020 and is projected to be attaining some US$23.4 billion by 2026 with expansion of production in many regions of the world. Shrimp are available in various species such as L. vannamei, P. monodon, and M. rosenbergii, the giant freshwater prawn.

These are all highly popular in the international market, and many countries are encouraging shrimp production by giving incentives to farmers and offering financial incentives. In order to meet growing demands in Europe and North America, major shrimp producing countries such India, China, Vietnam, Indonesia, Thailand, Mexico and Ecuador continue to require a large enough supply to satisfy their strong domestic and export markets.

The need for efficient commercial diets
Like all intensive aquaculture enterprises, the main constraint is the need for efficient commercial diets that can satisfy their stringent nutritional requirements (NRC 2011) and based on a sustainable platform of ingredients that supply key nutrients in a balanced formulation to allow maximum growth and optimum feed efficiency.

Shrimp diets may contain low levels of marine ingredients such as fishmeal and fishery by-products (squid meal, and fish hydrolysates) but largely comprise of plant ingredients such as soybean meal, corn gluten meal and grains as filler ingredients and energy rich starch sources such as wheat middling fractions and corn, placing added burdens to sustainability of available resources.

There is now much consideration as to the long-term consequences of an expanding global farmed shrimp industry and the sustainable use of soybean meals due to environmental pressures associated with their production in countries like Brazil and increasing deforestation as well as the need for water and nutrients.

This is the main rationale for the controversial fish in: fish out FIFO Ratio for various species as recently refined by Kok et al 2020. Consequently, the aquaculture industry is shifting attention to novel ingredients that can meet the aims and requirements with reduced carbon footprint, greater transparency of the feed supply chain and gaining consumer recognition for meeting the sustainability agenda.

There is considerable potential for grain-based protein concentrates derived from the classical fermentation industries for potable alcohol but more recently co-products originating from the industrial bio-refining sector for ethanol fuel generation from corn and wheat is gaining momentum. These have been advocated and tested for aquaculture and in formulated diets for carp and tilapia with much success.

Innovative biotechnology platform
A US based company with this intent is POET Nutrition, who are based in Sioux Falls, South Dakota. Their innovative biotechnology platform associated with biofuel generation from corn has now yielded advanced co-products namely a range of corn fermented proteins combined with yeast and of very high digestible amino acid profile and digestible energy that can be used for both finfish and shrimp. 

NexPro® corn fermented protein, results from post-fermentation mechanical separation of the DDG product utilising a patented technology called Maximized Stillage Co-Products. By fractionating the material post-fermentation, enabling the fermentation process to augment separation as well as weaken the cellular wall matrix of the fibrous fractions.

This also allows further concentration of inactive Saccharomyces cerevisae yeast utilised for the production of alcohol. The solution has a superior crude protein (~50 vs ~28%), lower crude fibre levels and improved nutritional composition compared to traditional DDGS. 

Poet Nutrition have conducted a series of independent trials with various species including Atlantic salmon (Salmo salar) and rainbow trout (Onchorhyncus mykiss), tilapia and shrimp with excellent results. The salmonid work was previously reported in IAF in 2020, 2021. 

This article will present trials with L.vannamei to evaluate the performance of NexPro® under classical experimental conditions. 
In order to assess its effectiveness, feeding trials undertaken with the principle aim of evaluating the efficacy of a corn fermented protein (CFP) in shrimp, and its viability as a high- quality protein source for use in aquafeed formulations. 

Our objectives being to characterise the growth performance and feed utilisation metrics for juvenile shrimp and determine 
the optimum practical inclusion level of CFP in typical production type diets for L. vannamei. Sequential growth trials were conducted to evaluate the use of corn fermented protein in practical Pacific white legged shrimp (L. vannamei) feed formulations. 

Experimental diets & growth trials 
Sequential growth trials were conducted at E. W. Shell Fisheries Research Station in Auburn, AL, USA. The CFP products were obtained from Flint Hills Resources (Wichita, KS, USA) (now POET Nutrition group) A preliminary trial (Trial 1) with 4 diets and a second trial (Trial II) with five experimental diets (Tables 1, 2) were formulated using CFP as a replacement for fishmeal in trial 1 and then a combination of fishmeal and soybean meal in trial II. 

Proximate composition of the diets was analysed at the University of Missouri Agricultural Experiment Station Chemical Laboratories (Columbia, MO, USA) to AOAC methods (2000) and presented for the respective diet formulations (Tables 1 & 2). 
The diets were prepared by blending the dry ingredients in a mixer (Hobart, Troy, OH, USA) for approximately 15 minutes. 

Fish oil was then incorporated, followed by the graded addition of boiling water to the mixture until obtaining an appropriate consistency for pelleting. Diets were then be passed through a 2.5-mm die in a meat grinder. The wet pellets were then placed into a forced air oven (< 50 °C) overnight in order to attain a moisture content of less than 10. Dry pellets were crumbled, packed in sealed bags, and stored in a freezer until required. 

In the first trial, juvenile shrimp (L. vannamei) (1.24 g initial weight) were stocked into replicate aquaria (four replicates allocated randomly per treatment) with 80 L volume each as a component of a recirculating system at a stocking density of 10 shrimp per tank. The indoor recirculating culture system consisted of culture tanks, sump-tank with biological filter, bead filter, circulation pump and supplemental aeration.

Each experimental diet was randomly assigned to four replicated tanks per treatment. Shrimp were group weighed at the start and end of the growth trial (five weeks). They were
fed the experimental diets four times daily with two feedings in the morning and two in the afternoon.

Feed inputs were fixed based on historical growth and feed intake, with the amount
 of feed consumed carefully monitored for calculations of Feed Conversion Ratio (FCR) and related metrics such as Thermal Growth Coefficient (TGC) - a parameter of growth incorporating the effects of temperature on metabolism. 

Testing a more specific range of inclusion 
This study used the same ingredients as in the first trial but tested a more specific narrower range of inclusion to best find the inclusion for optimised performance, so 6, 12, 18 & 24 percent, for example. The same experimental holding systems and rearing conditions were employed. Again, post larval shrimp were utilised with a smaller initial mean weight of 0.25 g and capacity for higher Thermal Growth Coefficient (TGC).

During the rearing period for both trials I & II, dissolved oxygen (DO), temperature, salinity, and pH was measured twice daily in one of the rearing tanks using a YSI 556 MPS meter (Yellow Spring Instrument Co, Yellow Springs, OH, USA). Water samples were taken in one of the tanks to determine total ammonia-nitrogen (TAN) on a weekly basis.

All data were statistically analysed using one-way analysis of variance to determine significant differences (P < 0.05). The multiple comparison test was used to determine significant differences among treatment means if a significant treatment effect was observed. 
A Dunnet T test was utilised to compare the basal with the other treatments. All statistical analyses were carried out using SAS (V9.4. SAS Institute, Cary, NC, USA). 

Results summary 
In the first trial, fishmeal was replaced on an iso-nitrogenous and iso-lipidic basis with up to 30 percent CFP. Results indicated that up to 20 and 30 percent were feasible levels included in the diet. The full growth performance and feed utilisation data is displayed in Table 3. 

In the second trial, 5 diets with a maximum inclusion of 24 percent was evaluated at incremental levels of 0, 6, 12, 18 & 24 percent. In this trial, the lowest level (6% CFP) actually improved growth above the control group.

Albeit, there were no significant differences between the highest of inclusion and the basal diet, but a level of 24 percent was deemed to be maximal under these conditions as validated by the Dunnet T test when compared to the highest inclusion level of CFP to the basal diet fed shrimp (Table 4). 

An 18 percent optimal inclusion for NexPro® at a much higherlevel of inclusion, some nutrient limitations including marginal essential amino acid levels, digestibility difference or the fact fishmeal is at very low levels
of the diet become apparent. However, economic advantage remains for the farmer due to savings in overall feed costs. 

Sustainability is a major international goal 
The use of alternative ingredients for replacing fishmeal and soybean meal is now a major international goal, which is aiming to offset the environmental and ethical consequences associated with using marine and terrestrial high protein concentrates like soybean meal.

There have been many strategies to use other ingredients in commercial shrimp diets and more recently insect meal, algae and various single cell proteins (SCP's) from microbial and yeasts such as Roy et al (2009). 

These have been tested in shrimp with good success. However, these exotic ingredients have proved expensive due to supply limitations and high cost of production. They may also have some constraints due to the presence of cell wall structural components and variable nutritional specifications. 

The alternatives have included with success co-
products from industrial processes including streams from both potable and the bioethanol industries and the latter attracting much interest for aquaculture diets. Although many studies exist to promote the consideration
of distillers dried grains and various forms of High Protein DDG and DDGS these commodities are of variable quality for aquaculture and are with wide ranges of protein content and energy value.

They also often have appreciable insoluble fibre content and therefore may not be fully digested within the fish gastrointestinal tract or for shrimp where the gut is short and transit time for digesta is quite rapid. The present constraint margin for high protein HP-DDGS inclusion may be partly attributed to the fibre (non-starch polysaccharides NSP's) in grain-based products, but also to the reduced levels of cholesterol and phospholipids when fishmeal is reduced. These are considered essential for shrimp growth and health and must be included in diets.

Also, there are issues concerning overall digestibility and availability of indispensable amino acids and these may become limiting in higher inclusions of HD-DDGS type products. There is also a potential palatability issue when fishmeal is reduced in formulations and shrimp are particularly sensitive
in respect of the gustatory and olfactory properties of the diet.

These workers studied the capacity of tuna by-product protein hydrolysates (TBPH) to improve the quality and digestibility of Pacific white shrimp (Litopenaeus vannamei) fed low fishmeal diets. Many of these factors can be corrected by the use of exogenous enzymes to aid digestion such as proteases, carbohydrases that better degrade protein and non-starch polysaccharides (NSP's). The use of phytase can greatly increases P availability in formulated diets in aquatic species and shrimp.

Indeed, more work is needed to supplement high grain protein diets with essential amino acids like methionine, lysine and threonine to raise the barrier to meet the 'ideal amino acid' concept known to be important in attaining maximum performance and comply with requirements for protein accretion in key tissues, organs such as muscle during intensive growth phases. 

Diet palatability and acceptance can be greatly improved by the addition of attractants in the diet such as clam and squid meal extracts/oils and supplements like glycine-betaine, dried fish solubles–high biogenic amines in the diet to initiate a robust feed response. 

Very effective within optimal margins 
Our results indicate that NexPro® is very effective within optimal margins when accommodated within diets for shrimp at the expense of fishmeal and soybean meal whilst maintaining the balanced protein and lipid levels for L. vannamei

It was found that we could include without compromise
on performance up to 18-20 percent and significantly reduce soybean and fishmeal components of the diet. Shrimp growth performance and feed utilisation parameters were within the expected normality for this species and only at 24 percent did we observe some reduction in growth compared to the control diet groups for both trials. 

However, the performance of this treatment was cost effective due to the reduced cost of feed and would return a profit margin for the farmer under a grow-out of 100 days to harvest compared to a high fishmeal-based formulation. 

Future work is being directed to optimise the use of the product and extend its inclusion level. Also, there is a need to evaluate potential functional properties on gut health integrity in shrimp under typical farmed conditions. Investigations will include an examination of its effects on disease resistance based on pathogen challenge tests, survival and also gut microbial balance and examination of histological characteristics of gut and hepato-pancreas s indictors of health status. 

The inclusion of NexPro® already yields cost benefits as the price of diet formulation is significantly reduced with 20 percent inclusion as measured by growth rate and economic FCR. 

This promises increased profit for the farmer whilst meeting the important sustainable agenda and contributing to the reduction
of marine and terrestrial proteins by filling the emerging 'protein gap' in global aquaculture production. 

About the authors

This article was very kindly contributed to this publication by Professor Simon J Davies, Editor of International Aquafeed magazine, Adjunct Professor, National University of Ireland, Galway, Ireland & Derek Balk and Melissa Jolly-Breithaupt, Poet Nutrition, Sioux Falls, USA.

References are available on request 

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