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September | October 2011

Feature title: Challenges associated with carrying out a meta-analysis of essential amino acid requirements of fish

The International magazine for the aquaculture feed industry

F: Amino acid

High cost of fishmeal, the volatility in the price of agricultural commodities and the stagnant price of

aquaculture products are constraining aquafeed manufacturers to pay very close attention to the cost-effective-ness of their feeds.

Nutritionists are required to formulate to lower or narrower essential nutrient specifications in order to minimise feed cost. At the same time, these feeds must sustain high growth, feed efficiency, health and product quality of the animals at the farm.

Nutritionists also are relying on an increasingly diverse portfolio of ‘economi-cal’ protein sources, with different amino acid profiles.

Thus, formulating cost-effective aqua-feeds requires increasingly precise infor-mation on essential amino acid (EAA) requirements of aquaculture species.

Dynamic field of researchAquaculture nutrition is a very dynamic

f i e l d of research. A very large number of stud-

ies have been conducted

on EAA n u t r i -tion of t e l eos t

fish and penaeid shrimp over the past 50 years and the body of knowledge on EAA requirements of aquaculture species is continuously expanding.

One of the problems resides in keeping up with progress and developing a whole-some understanding of the ‘state-of-the-art’.

The great diversity of methodological approaches used and animal species and ingredients studied as well as the multitude of opinions with regards to optimal levels and modes of expression of EAA require-ments limits the ability of manufacturers to meaningfully improve the cost-effectiveness of feeds and/or adapt formulations to an ever changing commodities market.

A number of scientific reviews and publications have attempted to summarise the body of knowledge on EAA nutrition and requirements of aquaculture species (Wilson, 1989; NRC, 1993; Cowey, 1994; Lall and Anderson, 2005; Bureau and Encarnação, 2006; Hernandez-Llamas, 2009; NRC, 2011), and some concluded that the state-of-the-art on EAA nutrition of aquaculture species is still quite shallow.

The choice of the mode of expression (percent dry diet, percent crude protein, g/kJ digestible energy, ideal protein, etc.) of EAA requirement is a matter of much debate, and reflects the conflicting assump-tions authors make when considering what affects the requirements or not (Bureau and Encarnação, 2006; Bureau, 2008).

Together with methodological issues (notably limita-tions of the e x p e r i m e n t a l design used), the variability in achieved growth

and feed efficiency, as well as differences in the mathematical and statistical approaches used to analyse data, these result in high variability in estimates of EAA requirements.

Understanding the reasons underpinning this great inconsistency is important for developing more reliable and practical esti-mates of EAA requirement of aquaculture species.

All these issues point toward a need for the systematic integration and analysis of information from the large number of studies that have been published so far on EAA requirements of aquaculture species. Statistical meta-analysis offers a mean to realize by integrating and standard-izing information and allowing meaningful comparisons.

The goal of this project was to carry out a meta-analysis of EAA requirement of fish through the construction of a dataset gathering all available data on EAA require-ments of teleost fish.

Challenges associated with carrying out a meta-analysisof essential amino acid requirements of fish

Figure 1: Diagram illustrating the screening of studies, from the total number found to the

number of suitable studies that constituted our working data set

number of stud-ies have been

conducted

Figure 2: representation of EAA across the dataset

by Guillaume Salze*, Margaret Quinton and Dominique P. Bureau, UG/OMNR Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada, N1G 2W1Email: gsalze@uoguelph.ca

28 | InternatIOnal AquAFeed | September-October 2011

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28 | InternatIOnal AquAFeed | September-October 2011

HeadquartersEvonik Degussa GmbHHealth & Nutritionfeed additives Rodenbacher Chaussee 4 63457 Hanau-Wolfgang, Germanyphone +49 6181 59-2256 fax +49 6181 59-6734

Europe & Middle East Africa +49 6181 59-6766Latin America +49 6181 59-6761North America +1 678 797-4300Asia North +86 10 85 27-6400Asia South +65 6890-6861

Amino AcidsChoosing the right nutrients for your Aquafeed

Our amino acids help to•  replace costly and scarce raw materials •  improve protein balance and production efficiency •  reduce environmental pollution

feed-additives@evonik.com | www.aminoacidsandmore.com

09-01-148 vND AZ Aquaculture A4 us.indd 1 15.06.2009 10:25:04 UhrIAF11.05.indd 29 02/09/2011 09:26

poor growth performance achieved during the trial.

The large number of species studied and the large number of EAA resulted in a very fragmented dataset (see Figures 2 and 3).

For example, the dataset only includes four studies on phenylalanine require-ments, which were all conducted on dif-ferent species. Great differences in the body weight of fish used (<1g to more than 600g) introduce some challenges for standardisation of data.

Almost half of the studies did not report information on carcass composition.

Protein gain (retention), an important response variable for EAA requirements, could only be computed for a limited number of studies.

Finally, only 16 percent of the studies in the working data set included some evalua-tion of protein digestibility.

Consideration of digestibility would greatly strengthen and refine our under-standing of EAA requirements in fish by taking account of some of the variability due to bioavailability of EAA in different ingredients.

In order to standardise the data onto a common ground, two different modes of expression (percentage of EAA of interest in the dry diet, amount of EAA per MJ of digestible energy) were computed and two different growth response variables (weight gain per kg of metabolic body weight and thermal-unit growth coefficient).

Therefore, for each study we obtained four pairs of variables, each of which was analysed using four mathematical models: the broken-line model (BLM), the quad-ratic model (QM), the broken-quadratic model (BQM) and the saturation kinetic

model (SKM) (see Figure 4) in each study to allow estimation of EAA requirements.

Preliminary results and perspective for future studies

Figure 5 presents the computed arginine requirement for rainbow trout using six studies for which models fitted correctly, depending on the three modes of expression.

It illustrates that even after selecting suitable studies and stand-ardising data, very large discrep-ancies in estimates between the studies remain. Variations around estimates based on ingested EAA remain high (coefficient of variation between 20 and 35 percent overall,

the accu-racy of the non-linear r e g r e s -s i o n s analyses.

Studies had to r e p o r t i n f o r m a -tion on w a t e r t e m -perature ,

experiment duration, diet

composition (dry-matter basis), initial and final individual body weight, and feed intake. Screening of the studies with these selection criteria yielded a final dataset comprised 109 studies, which covered all 10 EAA in 28 teleost species (see Figure 1).

A fragmented & diluted body of knowledge

It is striking that less than half of the 249 original studies could be considered for the working dataset.

This highlights the limited scope of many studies and/or scientific manuscripts and the uneven quality of the research effort. An important cause of rejection of studies was simply a lack of reported information, which precluded us from calculating the various variables.

Simple parameters, such as feed intake (feed served) and the dry matter content of the diets, were frequently not reported by authors. Other major motives for rejecting studies included the use of too few graded levels of nutrient studied or

The main objectives of this effort were identifying factors that may affect estimates of requirement, highlighting the shortcom-ings in the existing body of knowledge, and providing guidelines for future research.

Building a working datasetA comprehensive search of papers on

EAA nutrition of commercially relevant teleost species (for example, salmonidae, cychlidae, cyprinidae) published in peer-reviewed journals and other technical pub-lications was carried out.

This search yielded 286 papers of which 249 were original research studies focusing on EAA requirement of teleosts.

As expected, a great variety of objec-tives, experimental designs, and analytical methodologies were employed in these studies. Selection criteria were therefore applied to the original dataset to identify studies suitable for a meta-analysis.

Amongst selection criteria, studies had to have at least five or more experimental diets with graded levels of an EAA. This criterion was established in order to ensure

Figure 3: representation of species across the datase

(a)

(c)

(b) (95%)

(d)

Figure 4: Diagrams illustrating the typical shape of the four models: (a) broken-line model (BLM), (b) quadratic model (QM), (c) broken-quadratic model (BQM), and (d) saturation kinetic model (SKM). Dashed line indicates the determination of the requirement for each model

(95%)(95%)

30 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 31

F: Amino acid

IAF11.05.indd 30 02/09/2011 09:26

Nutrición Acuícola, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, Mexico, pp. 29-54.

Bureau, D.P., 2008. Toward a better definition of essential amino acid requirements of fish, International Aquafeed. Perendale Publishers Ltd, pp. 14-17.

Cowey, C.B., 1994. Amino acid requirements of fish: a critical appraisal of present values. Aquaculture 124, 1-11.

Lall, S.P., Anderson, S., 2005. Amino acid nutrition of salmonids: Dietary requirements and bioavailability. Cahiers Options Méditerranéennes 63, 73-90.

Hernandez-Llamas, A., 2009. Conventional and alternative dose-response models to estimate nutrient requirements of aquaculture species. Aquaculture 292, 207-213.

NRC, 1993. Nutrient Requirements of Fish. National Acad. Press, Washington, DC.

NRC, 2011. Nutrient requirements of fish and shrimp. The National Academies Press, Washington, D.C., 360 pp.

Wilson, R.P., 1989. Protein and amino acid requirements of fishes. Progress in fish nutrition. In: Shiau, S.-Y. (Ed.), Proceedings of the fish nutrition symposium, pp. 51-77.

Simple steps can be taken to improve the quality and relevance of future studies and allow us to develop more precise estimate of EAA require-ments. Experiments should not ideally use fewer than six experimental diets, and report suf-ficient information on diet composi-tion, growth per-

formances and husbandry information. The BLM has now been clearly dem-

onstrated to underestimates require-ments, and thus should be avoided. Instead, the QM or BQM offer a better balance between accuracy and practicality (that is, parsimony and ease of fit).

Most importantly, studies should be designed so results fit a clear ‘diminish-ing return’ pattern. The graded dietary levels of the test EAA should include clearly adequate levels so an obvious plateau can be observed.

Concomitantly, equally clearly deficient levels should also be included to ensure sufficient d i f f e r e n c e s between treat-ments receiving suboptimal levels.

ReferencesBureau, D.P., Encarnação, P., 2006. Adequately defining the amino acid requirements of fish: The case example of lysine. In: Cruz Suárez, L.E., Ricque Marie, D., Tapia Salazar, M., Nieto López, M.G., Villarreal Cavazos, D.A., Puello Cruz y Armando Garcia Ortega, A.C. (Eds.), Advances en Nutrición Acuícola VIII - VIII Simposium Internacional de

14 and 23 percent for arginine requirement of rainbow trout).

This shows that much remains to be done to reconcile the existing data and determine factors underpinning variability.

Nevertheless, several valuable points arose from the data standardisation.

The accuracy of the models was impact-ed by the shape of the curve they are meant to fit. Since all of these models are based on the law of diminishing returns, they attempt to fit curves shaped as those illustrated in Figure 4.

Therefore experiments should be designed to produce such result curves.

For example, data points of a given study will approach the horizontal line if there is little difference between treatments, which will cause the model to fail.

Our meta-analysis shows that if the final body weight of the slowest-growing group is higher than 70 percent of the fastest growing group, then the probability of obtaining a good fit decreases significantly. When using the SKM, this number is closer to 50 percent.

Additionally, if the curve only increases but does not plateau, then models can only guess where the curves plateaus or peaks. Since the require-ment is estimated by the EAA level where the curve plateaus or peaks, such an estimation should not be considered reliable.

Studies which simultaneously incorpo-rated very deficient and obviously adequate levels of EAA produced a clear ‘diminishing return’ pattern and consequently had vastly improved chances of estimating the require-ment accurately.

Conclusions and recommendations

In this study we highlighted important variations in requirement estimates despite an attempt to standardise our working dataset.

Figure 5: Estimated arginine requirements for rainbow trout according to the mode of expression

30 | InternatIOnal AquAFeed | September-October 2011 September-October 2011 | InternatIOnal AquAFeed | 31

F: Amino acid

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EDITOR’S DESK

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The new Astec Aquaculture Business & Science Centre located on Nor th East

England’s Northumberland coast-line, is a purpose-built facility, which provides a unique and sup-portive environment for all types of aquaculture related business and research activities.

It has a specific focus on helping fledgling enterprises to become established and grow, and as such, provides a unique combi-nation of ‘plug-in and go’ facilities and specialist business support services.

Astec has a very unique offering that appeals to marine scientists and industry alike – a year round supply of near tropical temper-ature, flow-through seawater, enabling a wide range of aquatic plants and animals to be culti-vated there.

Further to the recent Common Fisheries Policy Reform, pre-

sented by Mar ia Damanaki, Commissioner for Mar itime Affair s and Fisher ies, Astec welcomes the EU’s recognition of the importance of developing ecologically viable aquaculture as

part of its proposals to manage fish stocks.

Kevin Haddrick, chief executive of Astec says, “We have previ-

ously highlighted the need for a coordinated approach to support the development of this dynamic but fragmented industry, so we are par ticular ly suppor tive of the EU’s plans to establish a new Aquaculture Advisory Council to give advice and guidance on industry-related issues.”

The repor t also states that “a better framework for aqua-culture will increase production and supply of seafood in the EU, reduce dependence on imported fish and boost growth in coastal and rural areas.”

By 2014, Member States will draft national strategic plans to remove administrative barriers and uphold environmental, social and economic standards for the farmed-fish industry, adds the report.

“As the UK’s first aquaculture business incubator, with a focus on supporting start-up businesses

in the sector, we very much welcome the EU’s intention to remove administrative and legis-lative barriers to trade.”

Mr Haddrick adds, “Astec shares the views of Commissioner Damanaki that there is great potential within the aquaculture sector.

“We look forward to seeing how the framework develops and strongly suggest the EU grasp this opportunity to ensure aqua-culture plays an integral part in developing a more sustainable approach to fisheries manage-ment.”

More inforMation:Astec Lyne Sands, WoodhornNorthumberlandNE63 9NWUnited Kingdom Tel: +44 1670 852771Email: info@astecaquaculture.comWebsite: www.astecaquaculture.com

Aquaculture centre welcomes CFP reforms

Niels Otto Damholt joins Hamlet Protein in the posit ion of

sales director.

Based in Horsens, Denmark, Niels Otto Damholt will be responsible for Hamlet Protein´s sales and customer ser vice outside NAFTA and will be reporting to chief commercial officer of Hamlet Protein, Søren Bank.

Niels Otto Damholt, aged 47, joins Hamlet Protein from a position as regional director in Danisco Animal Nutrition, br inging 20 year s of back-

ground in in ternat iona l sales management in the animal nutrition sector to the company.

The strengthening of Hamlet Protein’s com-mercial organisation comes as a result of the significant increase in the demand for Ham le t P ro te i n ’s products during 2010 and 2011, and to support the continued

quest to deliver the compa-ny’s strategic goals in the high quality soya protein market.

More inforMation:Søren BankChief Commercial Officer Hamlet ProteinSaturnvej 51DK - 8700 HorsensDenmarkTel: +45 75 631020Fax: +45 75 631025Website: www.hamletprotein.com

Strong profile in the global feed business joins Hamlet Protein

Niels Otto Damholt

September-October 2011 | InternatIOnal AquAFeed | 3

Aqua News

2 | InternatIOnal AquAFeed | September-October 2011

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Challenges associated with carrying out a meta-analysis

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