SUSTAINABLE ORGANIC EGG PRODUCTION

THROUGH ALTERNATIVE FEEDING STRATEGIES

Sadia Afrose

PhD THESIS: SCIENCE AND TECHNOLOGY. 2015

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Preface

Europe has a scarcity of feed ingredients, especially protein sources for organic

poultry and synthetic amino acids are banned for organic production systems. This

thesis suggests new feeding strategies for laying hens that can facilitate transition of

100% organic feed ingredients in organic poultry production. This was done by

different forage supplementations, and new animal protein sources as mussels and

starfish. Therefore, the aim of this study was to investigate effects of these

supplementations on production performance, bird welfare, nutrient digestibility

and environmental impact. The thesis is a part of the requirements of obtaining a

PhD degree at the Department of Animal Science at the Faculty of Science and

Technology, Aarhus University, Denmark, where the experimental work related to

The PhD project was performed. The PhD project was funded by The Graduate

School of Science and Technology (GSST), Aarhus University and the Department of

Animal Science.

Foulum, August 2015

Sadia Afrose

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Acknowledgements

I would like to offer my sincere thanks to my main supervisor Sanna Steenfeldt for

her invaluable, competent and kind academic support during challenging times until

the last hour of submission, as well as for encouraging and inspiring discussions.

Thanks also to my supervisors family for making my stay homely and friendly. I

would also like to thank my co-supervisor Ricarda Greuel Engberg for invaluable and

motivating discussions of the microbiology and nutritive aspects of the project and

for motivated me in writing mauscripts.

Some analytic parts of my project were performed at the Food Science Department,

Faculty of Science and Technology, Aarhus University and I would like to thank

Marianne Hammershøj for providing me with facilities and inspiring discussions and

a special thanks to Jens Askov Jensen for helpful assistance in the analytical work of

egg quality.

I would also like to thank my colleagues at the Department of Animal Science,

Faculty of Science and Technology, Aarhus University for their skillful assistance in

the lab and at the experimental facilities. I also thank to Stina Greis Handberg for her

skillful assistance in fibre and gross energy analyses. A special thanks to Lars Bilde

Gilberg and Ole H.Olsen, for their invaluable and skillful technical assistance during

the experimental part of the PhD project. Also a deep thanks to Leif Rasmussen, Tina

Hald, Kirsten Lund Balterzenand Bjørk Bonnichsen for hen management. I would

like to thank Lotte Hansen for her effort on English proof reading.

Finally, my deepest gratitude and thanks to my family for bearing with me when it

seemed as the only thing I could talk about was organic egg-production; my lovely

son, Md. Rajin Hossain and especially my dear husband, Md. Sharoare Hossain, for

irreplaceable help, support, encouragement, comfort and love throughout the PhD

project.

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Contents Preface........................................................................................................................................ 1

Acknowledgements .................................................................................................................... 2

List of Scientific papers and Manuscripts ................................................................................. 5

Abbreviations ............................................................................................................................. 6

Summary .................................................................................................................................... 7

Dansk Resume ......................................................................................................................... 10

Chapter 1: Introduction ........................................................................................................... 14

Chapter 2: Background ............................................................................................................ 17

2.1 Current status, rules and regulations of organic poultry .......................................... 17

2.2 Challenges of 100% organic diet formulation ........................................................... 19

2.3 Feeding strategy for feeding organic poultry ............................................................20

2.4 Nutrients requirements for protein and amino acids ............................................... 22

2.4.1 Animal protein ................................................................................................... 23

2.4.2 Forage material .................................................................................................. 24

2.5 Benefits of forage materials as supplement to poultry diet ...................................... 25

2.6 Capacity of hens to digest fiber rich forage ............................................................... 27

2.7 Effect of dietary fibre on nutrient digestibility ......................................................... 28

2.8 Welfare in organic poultry ........................................................................................30

2.9 Environmental issues ................................................................................................ 31

Chapter 3: Aim and Hypothesis ............................................................................................... 32

Chapter 4: Methods and Materials .......................................................................................... 33

4.1 Experimental design ....................................................................................................... 33

4.2 Experimental Diets ........................................................................................................ 35

4.3 Housing of laying hens ................................................................................................... 38

4.4 Experimental Procedure ............................................................................................... 40

4.4.1. Data Recording ...................................................................................................... 40

4.4.2 Egg quality .............................................................................................................. 40

4.4.3 Chemical analyses................................................................................................... 40

4.4.4 Welfare recordings .................................................................................................. 41

4.4.5 Digesibility experiement .......................................................................................... 41

4.4.6 Gastrointestinal characteristics and intestinal micro flora ..................................... 41

Chapter 5: Results .................................................................................................................... 41

5.1 Manuscript 1 ................................................................................................................... 43

5.2 Manuscript 2 ..................................................................................................................86

5.3 Manuscript 3 ................................................................................................................. 121

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Chapter 6: General Discussion ............................................................................................... 151

6.1 Production performance ................................................................................................ 151

6.2 Feed and forage intake ................................................................................................. 153

6.3 Egg quality .................................................................................................................... 155

6.4 Environment ................................................................................................................ 156

6.5 Nutrient digestibility .................................................................................................... 158

6.6 Animal welfare .............................................................................................................. 161

6.7 Gastro intestinal characteristics and microbiology ....................................................... 161

Chapter 7: Conclusions .......................................................................................................... 162

Chapter 8: Perspectives ......................................................................................................... 164

Chapter 9: References ............................................................................................................ 166

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List of Scientific papers and Manuscripts

List of Scientific papers and Manuscripts included in the

thesis

S. AFROSE, M. HAMMERSHØJ, R. M. ENGBERG and S. STEENFELDT.Effect of organic diets providing silages and vegetables on production performance, nitrogen and phosphorous excretion and plumage quality of organic layers

S. AFROSE, R. M. ENGBERG and S.STEENFELDTEffect of feeding 100% organic

diets and forage supplements on apparent nutrient digestibility, metabolisable

energy and microbial activity in laying hens

Sadia Afrose, Marianne Hammershøj, Jan Værum Nørgaard, Ricarda Greuel Engberg and Sanna Steenfeldt Influence of blue mussel (Mytilus edulis) and starfish (Asterias rubens) meals on production performance, egg quality and digestibility of nutrients of laying hens

List of other published work not included in thesis

Afrose S, Engberg, RM, Hammershøj M and Steenfeldt S 2014. Effect of feeding 100% organic diets including silages and vegetables on production performance in laying hens.Poster session presented at XIVth European Poultry Conference 2014, Stavanger, Norway. Steenfeldt, S, Afrose S and Hammershøj M.2014 Muslinge og søstjernemel som proteinkilder til økologiske høner.In: Dansk Erhvervsfjerkrae. 43, p. 768-771.

Steenfeldt S, Afrose S, Hammershoj M and Horsted K 2013. How to safeguard

adequate nutriention in organic poultry production. In Proceedings of the 19th

European Symposium on Poultry Nutrition. p. 60-66.

Shreshtha, Aruna; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl; Afrose, Sadia; Engberg, Ricarda M. 2013. Influence of maize silage supplementation on selected intestinal bacteria and the course of a Ascaridia galli infection on organic layers.In Proceedings of the 19th European Symposium on Poultry Nutrition. 160-160.

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Abbreviations

AMEn Nitrogen corrected apparent metabolizable energy

ADC Apparent Digestibility co-efficient

DF Dietary fibre

DM Dry matter

EC European Commission

EU European Union

FCR Feed conversion ratio

GIT Gastrointestinal tract

IFOAM International Federation of Organic Agriculture Movements

NRC National Research Council

NSP Non starch polysacharides

SAS Statistical Analyses Softwear

SCFA Short chain fatty acids

SEM Standard error mean

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Summary

The EU legislations for organic poultry production aims to introduce diets based on

100% organic ingredients before 1. January 2018, and it is expected that organic

diets for laying hens use increasing amounts of locally grown ingredients ,where the

diet can supply the hens with nutrients for maintenance as well as for production

providing good animal welfare, positive ecological impact, ensuring healthy food

through a sustainable way. In this thesis it was hypothesized that the use of locally

available ingredients and forage material or animal protein other that fish meal can

support transition to 100% organic feed formulation for organic laying hens in a way

to reinforce sustainability of production systems as well as improving animal welfare.

Concurrently, this may benefit feed cost and the environment.

The main aim of this thesis was to optimize 100% organic diet for laying hens in a

sustainable way with less negative impact on the environment and animal welfare.

PAPER І: One control diet without access to forage and 8 organic layer diets with 8

different forage supplements were fed to 810 laying hens from 22-46 weeks of age in

the outdoor facilities. The nutrient composition of the silages and vegetables used as

forage material were used to formulate the experimental diets. The results showed

that varying amounts of nutrients were present in the different supplements. The dry

matter content was higher in silages compared to vegetables. Some silages such as

alfalfa-, hemp- and grass-herb silages and kale contained the highest amount of

protein ranged from 182-268 g/kg DM, whereas, maize- and maize cob silages were

high in starch being 259 g/kg DM and 407 g/kg DM, respectively, and a high level of

sugar was found in beetroot and carrot. Silages were higher in dietary fibre (NSP +

lignin) compared to vegetable, however a higher amount of soluble NSP in percent of

total NSP was found in vegetables. Except for beetroot, carrot, maize silage, maize

cob silages, the other silages and kale contained high amount of methionine. The

highest laying rate (90.1%) was found with the diet with maize silage as supplement.

The intake of the experimental diets decreased 6%- 11% when fed maize- and hemp

silage. The highest egg weight and egg mass was found in diets with maize silage and

grass-herb silage. Silages and vegetables supplementations had a positive effect on

plumage quality, whereas the effect on mortality was very different. In the study

nitrogen (N) and phosphorous (P) balance were calculated to get an indication on the

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environmental impact and with some dietary treatments, especially diets with maize-

, hemp-, maize cob-, hemp silages and kale had a positive effect on N and P retention.

To conclude, local forage materials contribute with some nutrients to the hens and

when included in the total diet formulation, improving the sustainability of organic

egg production system. Further, these diets and supplements had positive effect on

production performance and provide a good animal welfare.

PAPER ІІ: The objective of this experiment was to study the effect of forage

supplemented diets on apparent nutrient digestibility, nitrogen corrected apparent

metabolisable energy and N, calcium (Ca) and P retention as well as microbial

populations and activity in the digestive tract. Starch was digested to a high extent in

all the diets and there was no effect of diets with forage supplements compared to the

control without forage. The digestibility coefficient (DC) of fat was high in all diets

being in the range from 0.862 to 0.895, with the lowest value obtained with the

carrot supplemented diet (0.846). There was a significant effect on nitrogen

corrected apparent metabolisable energy (AMEn) between some of the treatments;

diet plus kale had the highest level of AMEn (14.19 MJ/kg DM), whereas the lowest

was found with diets plus beetroot (12.87 MJ/kg DM) and carrot (12.96 MJ/kg DM).

A significant difference was observed in N-retention among the diets; highest N-

retention was found in diets with kale, grass-herb silage, hemp silage and beetroot

compared to diets with maize-, alfalfa, maizecob silage and carrot. The DC of total

amino acids significantly changed when diets with forage supplements were fed to

the hens, and a more pronounced total amino acid digestion was found in the diet

with kale. However, unfortunately methionine and lysine were not properly digested

in diets with beetroot and maize cob silage. Numerically higher amounts of organic

acids were found in the intestine of hens fed maize silage as supplemented to the

diet. To conclude, diets with some of the forage supplements, especially grass-herb

silage, hemp silage and kale had the most positive influence on nutrient digestion,

AMEn, and N, P and Ca retention in laying hens.

PAPER ІІІ: A total of 300 Hyline white laying hens from 21 to 31 weeks of age were

fed diets supplemented with three different inclusion levels of mussel meal (4%, 8%

and 12 %) and two starfish meal (4% and 8%) in replacement of fish meal to observe

the effect on production performance, egg quality, apparent nutrient digestibility,

AMEn and N-retention from the diets. The results indicate that mussels and starfish

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contained 60% and 70 % protein on DM basis, respectively; and 20% DM ash was

found in starfish meal. The diets did not have negative effect on egg production

compared to the control diet with fishmeal, and a numerically higher egg production

was obtained with 4% starfish meal diet. A significant higher egg weight was found

with diets having 8 % and 12 % inclusion of mussel meal in the diets compared wih

4% mussel meal and 8% starfish meal diet. Feed conversion ratio for diet 8% (2.27)

and 12% (2.32) mussel meal was comparable to that of control diet (2.30). Mortality

rate, yolk weight, albumen dry matter, albumen pH, egg shell strength were not

affected by diets. Increasing the inclusion of mussel meal influenced yolk color,

whereas, starfish meal had no effect on yolk color compared to the control with

fishmeal. The DC of fat was significantly increased by mussel meal compared to the

control diet. N-retention was improved when increasing different levels of mussel

meal and starfish meal in the diets. The DC of amino acids as well as AMEn increased

with 8% and 12% mussel meal in the diets. The fishy taste was found with 12%

mussel meal in the diet in a consumer test. In conclusion, a moderate level of mussel

meal, up to 8% might be a high quality protein source for laying hen to replace fish

meal without altering egg production.

The overall conclusion of this thesis is that different locally available forages can

partly contribute with nutrient to formulate 100% organic layer diets and mussel

meal or starfish meal can be used instead of fish meal as a protein source in the

organic layer diet aiming at a more sustainable organc egg production. Forage and

foraging ensure good welfare for the hens as they can express their natural foraging

behavior reducing the risk for feather pecking. Finally, if a main part of the feed

ingredients is grown locally on farm and used for animal feed, it will maintain the

nutrient cycle, reduce the feed cost and facilitate sustainable egg production.

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Dansk Resume

EU lovgivningen for økologisk produktion af fjerkræ arbejder for, at overgangen til

foder, der er baseret på 100 % økologiske råvarer skal igangsættes 1. januar 2018.

Der er desuden en forventning om at foder til høner i den økologiske ægproduktion

vil være baseret på stigende mængder af lokalt dyrkede råvarer, hvilket skal sikre en

mere bæredygtig produktion. Det er imidlertid vigtigt, at foder der er baseret på 100

% økologiske råvarer, forsyner hønerne med de nødvendige næringsstoffer til

vedligehold og produktion af æg, sikre god dyrevelfærd, samt have en positiv effekt

på miljøet. I dette Ph.D. studie har der været fokus på at undersøge hypotesen, at en

øget anvendelse af lokalet, tilgængelige råvarer, samt en øget viden om forskellige

grovfodertypers ernæringsmæssige værdi vil støtte overgangen til 100% økologisk

foder. Ved at indregne den ernæringsmæssige værdi af grovfoder forventes en mere

optimal sammensætning af de økologiske foderblandinger, så udskillelsen af stoffer

som kvælstof og fosfor til miljøet reduceres. Introduktion af alternative, animalske

proteinkilder som muslingemel og søstjernemel som erstatning for fiskemel i foder

til økologiske æglæggende høner, vil yderligere styrke bæredygtigheden i den

økologiske produktion.

Formålet med Ph.D. studiet har været at skabe viden om forskellige grovfodertypers

ernæringssmæssige værdi for æglæggende høner i den økologiske produktion, hvor

hønernes samlede næringsstofforsyning fra fuldfoder og grovfoder optimeres

løbende. Optimal udnyttelse af tilgængelige foderressourcer er nødvendig for en

bæredygtig økologisk ægproduktion ved overgang til 100 % økologisk foder, og en

øget anvendelse af lokalt dyrkede råvarer, samt alternative, animalske proteinkilder

har været vigtige parametre med henblik på udviklingen af et mere optimalt foder for

den økologiske ægproduktion.

PAPER І: En kontrolbehandling uden tildeling af grovfoder, samt 8

forsøgsblandinger, hvor der blev tildelt forskellige typer af grovfoder blev fodret til

810 æglæggende høner gennem forsøgsperioden med en hønealder fra 22-46 uger.

Forsøget blev gennemført i mobile huse i de udendørs faciliteter på Aarhus

universitet, Foulum. Den ernæringsmæssige sammensætning af de forskellige

ensilager og grøntsager, der blev tildelt i de forskellige behandlinger indgik i

formuleringen af forsøgsfoderet. Majs-, lucerne-, græs-urt-, hamp- og

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kolbemajsensilage, samt rødbeder, gulerødder og grønkål indgik som grovfoder

supplement til de forskellige forsøgsblandinger, der var baseret primært på lokalt

dyrkede råvarer. Kemiske analyser viste, at indholdet af næringsstoffer varierede

betragteligt mellem de forskellige grovfodertyper. Indholdet af tørstof var højest i

ensilagerne. Nogle ensilager såsom lucerne-, hamp- og græs-urt ensilage, samt

grønkål indeholdt den højeste mængde protein varierende fra 182-268g/kg tørstof,

mens majs- og kolbemajs ensilage havde et højere indhold af stivelse, henholdsvis

259g/kg tørstof og 407g/kg tørstof, mens indholdet af sukker var højt i rødbeder og

gulerod. Ensilagerne var karakteriseret ved et højere indhold af kostfibre (NSP +

lignin) sammenlignet med grøntsager, mens en større mængde opløseligt NSP i

procent af det totale NSP indhold blev fundet i grøntsager. Bortset fra rødbede,

gulerod, majs- og kolbemajs ensilage, havde de andre ensilager samt grønkålen et

forholdsvist højt indhold af methionin. Den højeste ægproduktion (90,1%) blev

fundet med behandlinger, hvor majsensilage blev tildelt sammen med foderet.

Foderforbruget af de forskellige forsøgsblandinger faldt 6% - 11%, når majs- og hamp

ensilage blev tildelt. Den højeste æg vægt og æg masse blev fundet med de

foderblandinger, hvor majsensilage og græsurt ensilage indgik. Tildeling med

ensilager og grøntsager havde en positiv effekt på fjerdragtens kvalitet, mens effekten

på dødeligheden var meget forskellig. I undersøgelsen blev kvælstof (N) og fosfor (P)

balancen beregnet for at få en indikation af N og P udskillelsen og dermed effekt på

miljøet, og resultaterne viste, at især foder med majs-, hamp-, kolbemajsensilage og

grønkål havde en positiv effekt på N og P retentionen. Det kan konkluderes, at

økologisk foder, der er formuleret på basis af lokale råvarer, samt indholdet af

næringsstoffer i de anvendte grovfodertyper, bidrager med en mere optimal

næringsstofforsyning, samt en øget velfærd, hvilket forbedrer bæredygtigheden i den

økologiske ægproduktion.

PAPER ІІ: Formålet med dette forsøg var at undersøge effekten af foder, hvor

økologiske høner tildeles forskellige typer af grovfoder som et supplement, på den

tilsyneladende næringsstoffordøjelighed, kvælstof korrigeret omsættelig energi

(AMEn), kvælstof (N), calcium (Ca) og fosfor (P) retention, samt effekt på

mikrofloraens aktivitet i fordøjelseskanalen. Stivelsesfordøjeligheden var høj i alle

behandlinger. Fordøjelighedskoefficienten af fedt var ligeledes høj med et interval

mellem 0,862-0,895, hvor foder suppleret med gulerod var lavest (0,846). Der var en

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signifikant effekt på den tilsyneladende omsættelige energi (AMEn) mellem nogle af

de behandlinger, hvor foder med grønkål, som supplement havde det højeste niveau

af AMEn (14.19 MJ /kg tørstof), mens den laveste blev fundet med rødbeder (12,87

MJ/kg tørstof) og gulerod (12,96 MJ/kg tørstof). En signifikant forskel blev

observeret i N-retentionen blandt behandlingerne, hvor den højeste N-retention blev

fundet i foder med grønkål, græs-urt ensilage, hamp ensilage og rødbeder i forhold til

behandlingerne, hvor der blev tildelt majs-, lucerne-, kolbemajs ensilage og gulerod.

Fordøjeligheden af det totale aminosyre indhold blev ændret betydeligt, når der blev

fodret med grovfoder sammenholdt med kontrolbehandlingen, hvor den højeste

aminosyrefordøjelighed blev funder i behandlingen med grønkål. Den laveste

fordøjelighed af methionin og lysin blev funder i behandlinger med rødbeder og

kolbemajs ensilage. Indholdet af organiske syrer i tarmen var højest hos høner, der

fik foderet suppleret med majsensilage, men der blev ikke funder signifikante

forskelle mellem behandlingerne. Det kan konkluderes, at græs-urt ensilage, hamp

ensilage og grønkål havde den mest positiv indflydelse på

næringsstoffordøjeligheden, AMEn, samt N, P og Ca retentionen hos æglæggende

høner.

PAPER ІІІ: I alt 300 hvide Hyline høner i alderen 21-31 ugers blev tildelt

forsøgsfoder med tre forskellige niveauer af muslingemel (4%, 8% og 12%) og to

forskellige niveauer af søstjernemel (4% og 8% ) som erstatning for fiskemel for at

undersøge effekten på ægproduktionen,, æg kvalitet, næringsstoffordøjelighed,

AMEn og N-retention. Resultaterne viste, at muslinger og søstjerner indeholdt

henholdsvis 60% og 70% protein på tørstofbasis og der var 20% aske søstjerner. De

forskellige behandlinger med muslinge- og søstjernemel havde ikke nogen negativ

effekt på ægproduktion sammenlignet med kontrolgruppen, hvor foderet indeholdt

fiskemel, og en numerisk højere ægproduktion blev opnået med 4% søstjernemel i

foderet. En signifikant højere æg vægt blev fundet med 8% og 12% muslingemel i

foderet sammenlignet med 4% muslingemel og 8% søstjernemel. Foderudnyttelsen

for behandlingen med 8% (2,27) og 12% (2,32) muslingemel var sammenlignelig med

kontrolbehandlingen (2.30). Der var ingen effekt af behandlinger på dødelighed,

æggeblommens vægt, æggehvidetørstof, æggehvide pH og skalstyrke. Et øget indhold

af muslingmel påvirkede æggeblommens farve, mens søstjernemel ikke havde nogen

effekt på æggeblommens farve sammenlignet med kontrolfoderet med fiskemel.

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Fordøjeligheden af fedt blev signifikant forøget i foder med muslingemel

sammenholdt med kontrolbehandlingen. N-retention blev forbedret med stigende

mængder muslinge- og søstjernemel i foderet. Fordøjeligheden af aminosyrer samt

AMEn steg med 8% og 12% muslingemel i blandingerne. Der blev konstateret

fiskesmag i behandlingen med 12% muslingemel i foderet i en

forbrugerundersøgelse. Det kan konkluderes, at muslingemel med op til 8% i

æglæggerfoder kan betragtes som en proteinkilde af høj kvalitet til økologiske

æglæggende høner, og som kan erstatte fiskemel uden at ændre ægproduktionen.

Den overordnede konklusion af Ph.D. studiet er at forskellige, lokalt tilgængelige

grovfoder typer kan bidrage med næringsstoffer til at formulere 100% økologisk

foder til æglæggende høner og at muslinge- og søstjernemel kan bruges i stedet for

fiskemel som proteinkilde i økologisk foder for at sikre en mere bæredygtig

produktion af økologiske æg. Adgang til grovfoder og mulighed for fouragering kan

sikre en god velfærd for økologiske høner, da de kan udtrykke deres naturlige

fourageringsadfærd, hvilket reducerer risikoen for fjerpilning. Hvis hovedparten af

råvarer samtidig dyrkes lokalt og anvendes i foderet, vil det påvirke

næringsstofkredsløbet positivt, reducere omkostningerne på foder og sikre en mere

bæredygtig ægproduktion.

14

Chapter 1: Introduction

During the last 30 years, an increased concern considering poultry welfare has

developed in Europe and triggered the resurgence of interest in free-range egg

production, since these production systems differ from the conventional cage system

by having more focus on animal welfare (Magdelaine et al., 2010; Miao et al., 2005).

Denmark is one of the countries with a relatively high organic egg production

compared to other European countries, and Denmark has the highest market share

of organic eggs constituting 20.4% of the total production, and in 2013 20% of all

hens were producing under organic production conditions (Anonymous, 2014). This

increased production of organic eggs is a response to an increased consumer demand

for food that is expected to be fresh; wholesome and flavoursome; free of hormones,

antibiotics and harmful chemicals and produced in a way that is environmentally

sustainable and animal friendly (Blair, 2008). The International Federation of

Organic Agriculture Movements (IFOAM) has described guidelines for the general

principles and standards in organic agriculture (IFOAM, 2005), and the specific

standards and regulations for the actual organic production are given by the EU

legislation which is partly based on the guidelines from IFOAM. According to the

European Council (EC) regulations, the organic production system should aim to use

100% organically produced feed ingredients, and if grown on-farm, the production

will improve the nutrient cycle between animal and crop within the farm giving

benefit to the environment (EC Regulation 2092/91). However, if organic feed in

terms of quantity and quality is not obtainable, 5% of non-organic raw materials are

still allowed in organic poultry diets until 31 December 2017 (EU, 836/2014). The

transition to the 100% organic feeding to poultry has been an ongoing process that

has to be achieved by 2018. This decision gives rise to the challenge and concern

diets containing 100% organic ingredients may not supply sufficient levels of certain

essential amino acids, especially for young birds (Sundrum, 2005; Steenfeldt et al.,

2013).

The feeding of 100% organic diets is challenging because of insufficient amounts of

valuable organic feedstuffs available within the EU, especially with regards to protein

sources with a balanced content of essential amino acids. When comparing organic

and conventional poultry feed, the amino acid content is general lower in organic

15

feed (Velik, 2004), and the possible use of synthetic amino acids in conventional

poultry feed makes it less difficult to formulate a diet composition that meets the

nutritional requirements of poultry. The use of synthetic amino acids and protein

sources such as soya bean meal (due to use of chemical solvent) is not permitted in

organic feed. The fundamental basis for a balanced diet is that it can cover the

physiological requirements of animals.Feeding imbalanced diets might have a

negative effect on overall animal health and welfare. The deficiency of sulfur

containing amino acids (methionine and cysteine) in poultry diets can lead to feather

pecking and cannibalism (Tiller, 2001; Ambrosen and Petersen, 1997) which result in

poor production performance of the laying hens (Kjaer and Sorensen, 2002). Birds

require sufficient amounts of sulfur containing amino acids for the plumage

development, and a poor plumage quality cannot maintain body temperature which

subsequently can reduce the feed efficiency. The formulation of 100% organic diets

often includes excessive protein to supply sufficient levels of essential amino acids.

However, insufficient amino acids, or an imbalanced amino acid profile in the diet

increase the risk of nitrogen excretion to the environment (Scholtyssek et al., 1991;

Summer, 1993).

Considering all these aspects, balancing amino acids in organic diets that follow the

EU legislation is a very crucial step towards a sustainable organic egg production.

The major challenge of fulfilling the legislative requierement is that the quality and

quantity of organic protein sources within the EU is still low (Sundrum et al., 2005)

to meet the nutritional requirements of organic poultry. The feed for organic poultry

is based on cereals such as wheat, barley, maize and oat and protein sources such as

soya beans, sunflower cake, rapeseed, lupine, peas and faba beans. The average

protein content is approximately 23%, 30% and 40% DM in pea, faba beans and

lupine, respectively, but the content of methionine and cysteine is lower than in soya

beans (Pettorson, 2000; Sauvant et al. 2004). Lupin contain high levels of the non-

starch polysaccharides (NSP), which are almost twice as high as in other protein-rich

plants (Daveby and Åman, 1993; Bach-Knudsen, 1997), which might restrict the use

of lupin in poultry diets (Perez-Maldonando et al. 1999; Hammershøj and Steenfeldt,

2005). The most obvious feed ingredients like soya beans are not grown abundantly

in Europe due to the unfavorable climatic conditions (Steenfeldt et al., 2013).

However, it is indisputable that soya beans have an optimal amino acid profile for

16

poultry (Grieshop and Fahey, 2001, Sauvant et al., 2004). Therefore, a higher local

production of more climate robust soya bean cultivars in both Southern and

Northern Europe would be an important supplement to home-grown legumes with a

lower content of methionine. Many farmers can obtain feed ingredients in different

ways; for instance, some producers are rely largely on home-grown ingredients as

they grow cereals and some protein sources, sush as lupin and faba beans, on their

own land (Blair, 2008). The organic regulations also dictate that to some extent,

forage, fresh or dried fodder or silage must be used as supplements to the organic

feed for poultry on a daily basis (EC 1804/1999). It has been shown in previous

studies that despite the limited capacity to digest fibres, hens can utilise feed high in

fibre content (Hetland and Svihus, 2001; Engberg et al., 2004), and due to the

varying content of different nutrients, forage materials are capable of providing a

certain amounts of nutrients (Hammershoj and Steenfeldt, 2005; Steenfeldt et al.,

2007; Hammershøj and Steenfeldt, 2012).

Protein of animal origin is an important source of protein and essential amino acids

for organic poultry, where fish meal is a valuable source of methionine which at

present is used to quite an extent in organic poultry feed. However, fish is a limited

resource of fish and increasingcosts may reduce the probability of of the use of fish in

the future. Thus, the search for new potential protein sources rich in methionine is of

critical importance for the future survival of organic poultry production (Elwinger et

al., 2008; Wilson, 2009). A substantial amount of evidence suggest that mussels

have the potential as an alternative and as sustainable source of protein with an

amino acid profile comparable to that of fish meal (Jonsson and Elwinger, 2009;

Jonsson et al., 2011). Further, starfish, which have been a major problem for mussel

fishermen in Denmark, can be considered as a new animal protein source in organic

poultry diets, since starfish are also rich in protein and other essential nutrients

(Nørgaard et al., 2015).

Considering animal welfare and environmental impact, fulfilling all the necessary

nutrient requirements, especially the essential amino acids, is a very important step

for the organic poultry sector in Europe. To address the current challenges, it is

necessary to develope alternative feeding strategies that consider a better use of

organic ingredients based on locally available protein sources thus complying with

17

the organic principles. A sustainable supply of seasonal available vegetable as protein

rich forage materials and substituting fish meal with alternative animal protein

sources is a step in the direction towards a higher sustainability of organic

eggproduction.

Chapter 2: Background

2.1 Current status, rules and regulations of organic poultry

Egg production systems can broadly be classified in 4 different categories which is

the production in enriched cages, barn egg production, free-range as well as organic

egg production. The demand for organic egg has increased considerably in some

countries in Europe. In the EU, egg production in other systems than cages was 8%

in 1996, and increased to 21% in 2005, with increasing attention to bird health and

welfare, environmental impact as well as quality and safety of food (Magdelaine,

2007). Within Europe, France, Germany, Austria and Denmark are the leading

countries for keeping laying hens under organic condition, and in terms of volume,

Germany is the biggest player (Steenfeldt et al., 2013; Hammershøj, 2011). In

Denmark 20% of total numbers of laying hens used in the organic egg production in

2013 and 42.5% of layers farms (68 out of 160 farms) has produced 11 mio.kg eggs

in 2013, constituting 18% of the total egg production. According to the Danish

statistics, the market share has been increasing to 20.4% in 2013 (Anonymous,

2014). According to the study of Baltzer (2004), consumers in Denmark are willing

to pay more for free-range and organic eggs and consumers living in the urban areas

are more willing to pay the extra money than those living on the country side. This

attitude indicates that in future organic egg production will continue to increase in

Denmark.

18

Figure 1: Egg production in Europe in 2008 (Source: Anonymous ., 2013)

Figure 2. World Egg production (2013) (Source: Anonymous. 2013)

Table1.No of egg producers in Denmark (oct, 2013) (Anonymous, 2013 )

Production sites No of hens Shares(%)

Cage 39 1828,200 55.4

Barn 47 683.100 20.7

Free range 18 191.400 5.8

Organic 66 597.300 18.1

Total 170 3.3mill. 100

19

The basic standards of organic animal production were adopted by Council

Regulation (EC) No. 1804/1999(EC, 1999) which was a supplement to regulation

No.2092/91 on organic production of agricultural products. During the introduction

of the legislation, some further developments were identified and based on these new

Regulations EC No 834/2007 (EC,2007) and Commission Regulation No (EC)

889/2008 (EC,2008) were introduced by revision of Council Regulation N.2092/91

(EC,1991). The EU rules and regulations are partly derived from the standard of

International Federation of Organic Agriculture Movements (IFOAM), givingin the

European members states room for their own interpretation, wich allows them to

tighten the regulations according to their own demands (Hermansen et al., 2003).

Current regulations for organic poultry production systems permit 5% feed

ingredients of non-organic origin, primarily due to concerns that a 100% organic diet

would be unable to meet the demand for the essential amino acids, especially

methionine.

2.2 Challenges of 100% organic diet formulation

It is well accepted that achieving the goal of 100% organic diet within the stipulated

time is a burning issue for organic poultry production. Formulation of organic diets

is challenged by the situation that the availability of organic protein sources of high

quality, e.g. soya beans, within Europe is very scarce (Sundrum et al., 2005). Due to

the temperate climate condition in Northern Europe the production of protein

sources with a high quality with an ideal amino acid profile is still very limited (Van

Krimpen et al., 2013). In conventional production systems, requirement for

essential amino acids in poultry diets is covered by using synthetic amino acids,

however, the use of synthetic amino acids is not permitted in organic productions

(EU, 1804/1999) (Racheal, 2014). In Europe, the conventional poultry industry

relies on imported soya bean meal from South America (Aramyan et al., 2009), USA

(Euractiv, 2011) and China (van Krimper et al., 2011). Soya beans is one of the most

important protein sources in poultry diets (Van Krimpen et al., 2013), as it contains

about 40% protein and has a fine amino acid profile for poultry compared with

other legumes such as lupin and faba beans (Blair, 2008) However, it is difficult to

grow soya bean in Northern Europe due to the unfavorable climate conditions, and

Italy and Romania are the main producers of organic soya beans in Europe, where

the production is still low and not sufficient to cover the amounts needed for organic

20

feed in Europe. However, the results from several studies, including studies in

Denmark, indicate a potential for the growing of soya bean in Northern Europe

(Vollmann et al., 2000; Edlefsen et al., 2008; Palermo et al., 2012), but more

research in new robust cultivars and crop management is necessary to achieve a

higher and stable yield. A large amount of imported soya beans from overseas is

costly and has negative effect on the environment (van Krimpen et al., 2013).

Another protein source approved for organic poultry feed in Denmark is fish meal

which is used frequently and is an excellent source of protein with a high content of

essential amino acids. The quality of fish meal depends mainly on the quality of fish

used, the processing technique and oxidation processes (Seerley, 1991; Wiseman et

al., 1991). There are some limitations for use of fish meal in poultry diets such as

fishy smell in the eggs, gizzard erosion, poor processing and storage condition and

the risk of adulteration with cheap diluents (Blair, 2008). Overfishing of the seas

has triggered an intensive debate regarding the use of fish meal in feeds for animals,

and considering fish meal as a limited resource is reflected by increasing prices.

Therefore, the interest to use alternative animal protein sources such as mussel and

starfish has increased in recent years (Lindahl and Kollberg, 2009; McLaughlan et

al., 2014; Jan et al., 2015).

2.3 Feeding strategy for feeding organic poultry

Currently, organic poultry farmers can obtain the organic feed in different ways,

where some farmers purchase complete compound feeds from a feed manufacturer,

whereas others buy only a protein concentrate from a local feed manufacturer to mix

with cereals grown on-farm which can be more cost effective than purchased

compound feed (Blair, 2008). By legislations (EU, 2012) at least 20% of total feed

ingredients used in organic poultry diets must be produced on the farm or in the

same region and 70-80% can be purchased from certified organic farmers or feed

manufactures, however this target is yet to be achieved. The definition of region is

broad between the EU member states, where some countries extend the “region” to

be “Europe” (Madelaine et al., 2010).

Since laying hens and slow growing genotype of broilers can consume a considerable

amount of forage materials by developing the gizzard size (Horsted, 2006; Steenfeldt

21

et al., 2007 and Almeida et al., 2012), the supplementaion of high quality forage

materials or access to out-door range area with grass and herbs, can contribute

partially with energy and some nutrients the diets. The structure and color of the

feed are important characteristics that encourage or discourage the hens to take feed,

where larger particles are more easily recognised than fine dust, and bright colors are

preferred (Blair, 2008). Chemical analysis of silages and vegetables showed these

foraging materials might provide nutrients e.g. energy, protein and amino acids to

some extent and hens seems to prefer certain types of forage materials (Steenfeldt et

al., 2015, accepted). Recent research (Hammershøj and Steenfeldt, 2012) suggests

that laying hens fed a standard organic diet with either maize silage and different

herbs or fresh kale leaves as supplements, increased egg production compared to the

same organic diet fed with maize silage only. Steenfeldt et al. (2007) conducted an

experiment on laying hens performance using maize silage, barley-pea silage and

carrots as foraging materials and observed that 48-33% of the total intake of the

organic diet and forage was silages and carrots, where diets with carrots or maize

silage as supplements obtained the highest egg production. The results indicate that

forage material can be considered as a sort of feed ingredient that has the potential to

contribute with some nutrient to poultry, and as a consequence, organic producers

become more self-sufficient if they grow the forage material, along with other kind of

raw materials, on their own farm. However, since the legumes grown in Northern

Europe are primarily lupin and faba beans with a low methionine content, new

feeding strategies should be considered to facilitate the transition to 100% organic

feed supply. Thus, if the organic poultry production is to maintain its positive

development in the future, it is of utmost importance to find new potential protein

sources rich in methionine which can contribute with nutrients to the diets (Wilson,

2009; Elwinger et al., 2008; Hammershøj & Steenfeldt, 2005; Elwinger &

Wahlström, 2000), combined with feeding strategies that include the nutrient

content of different forage material grown on-farm in the formulation of organic

poultry diets. Further, more research with new soya bean cultivars more suited for

temperate climate, and the development of crop management would be a valuable

contribution towards achieving 100% organic diets from local available feed

ingredients.

22

2.4 Nutrients requirements for protein and amino acids

Protein and amino acids are important building blocks for bird growth as well as

feather development and egg formation hence supply of adequate amount of protein

is very crucial with regard to diet formulation in animal nutrition. The NRC (1994)

requirements for protein in laying hens is 15000 mg/hen/d, methionine 300

mg/hen/d and lysine 690 mg/hen/d, although there are no specific recommendation

for organic laying hens. Tranferring the dietary protein recommendations for

commercial laying hens to organic layers might cause proeblems leading to an

improper nutrient balance. In Sweden it is a common practice to use a 25% more

protein and amino acids for organic laying hens (Elwinger et al., 2008). Since,

synthetic amino acids are not permitted in organic feed, excessive protein is often

included in the diets in order to supply the birds with sufficient amounts of essential

amino acids, which increases feed cost and most likely increase nitrogen excretion to

the environment (Blair, 2008). Body proteins are in a dynamic state with synthesis

and degradation occurring continuously, both for maintenance and production of

meat and eggs; therefore, a constant, adequate intake of dietary amino acids is

required. If one limiting amino acids is present at only 50% of the requirement, the

efficiency of using the other essential amino acids would deacrease to 50% (Blair,

2008). Keeping both dietary protein at an acceptable level and essential amino acids

at a required level is a very difficult task while formulating organic diets for poultry.

Although hens fed with diets rich in protein and essential amino acids can produce

larger eggs (Sohail and Roland, 1997; Schutte et al., 1988), the efficiency of protein

utilisation from a diet depends on the digestibility of available dietary amino acids.

Essential amino acids can influence the feed intake either through their

concentrations in blood reaching the brain, where they influence brain receptors, or

by influencing the metabolism of amino acids in the liver (Angkanaporn et al., 1997).

As an essential amino acid, methionine cannot be synthesised in animal body and

needs regular supply from the diet for the viability of animal, cell development and

feathering in poultry. It is a well-known fact that the deficiency in protein and some

amino acids may be critical for the plumage condition of the hens due to increased

feather pecking (Ambrosen and Petersen, 1997; Elwinger et al., 2002).

23

Protein sources of sufficient quality with regard to the amino acid profiles are limited

in organic production and birds from different stages need different amount of

protein and amino acids for their growth, development and productivity. It was

previously demonstrated that the level of dietaty methionine intake influences

albumen DM which is an important egg quality parameter (Shafer et al., 1996;

Hammershøj and Steenfeldt, 2005). Methionine deficiency in organic diets may be

prevented, in part, by an increased level of dietary protein (Zollitsch and Baumung,

2004), but the methionine or sulphur-containing amino acids to lysine ratio still

remains too low. It was postulated that having access to chicory as forage material,

increased albumen DM, since protein is the major constituent of albumen DM, this

indicates a higher contribution of amino acids from the forage material

supplemented (Horsted, 2006). In recent studies it has been postulated that some

types of silages and vegetables, even though rich in dietary fibre, can provide some

amount of protein and essential amino acids for laying hens (Hammershoj et al.,

2005; Steenfeldt et al., 2007; Hammershoj and Steenfeldt, 2012).

2.4.1 Animal protein

Animal protein is a unique source of protein and amino acids that are widely used in

poultry diets, and the protein and amino acid quality of animal proteins are far better

than that of vegetable proteins. While looking for substitutes for fish meal, insects,

snail and earthworms, available for organic birds on the outdoor area, can contribute

with additional nutrients (Horsted et al., 2007), especially with protein, which is

reported to be in the range from 42-76% (Ravindran and Blair, 1993). Commercial

production of insect meals has received increased attention recently. It was stated it

is possible toproduce insects as an alternative sustainable protein rich ingredient in

organic poultry production on a commertial basis (Veldkamp et al., 2012). The

protein and amino acide content of insect meal is very much similar to fish meal or

soyabean meal, which could substitute 50% of the dietary animal protein supplied by

fish meal without altering egg production and egg shell strength (Agunbiade et al.,

2007). For several reasons, meat from blue mussels (Mytilus edulis) may be an

interesting alternative to fish meal, as mussels are high in protein content (61% of

dry matter), essential amino acids ( Lindahl and Kollberg, 2009; McLaughlan et al.,

2014) and are potential sources of long-chained polyunsaturated fatty acids having

beneficial health effects in humans (Taylor and Savage, 2006). Off-bottom

24

cultivation with long-line production of blue mussels does not require the supply of

additionl nutrients for growth, as fed on phytoplankton and organic materials. They

are used to recycle nitrogen and phosphorus surplus from the surrounding land and

thereby improve the water environment. Under favorable conditions one mussel can

filter 2-3 liters of water per hour (Lindahl et al., 2005). This will pave the way for the

dissemination of important structure-bottom plants such as algaes. These features

make mussels an interesting alternative for commercial use in poultry from both a

production, nutrition and quality point of view. In their gonads, mussels accumulate

carotenoids (Petes et al. 2008) that are deposited in the egg yolk and can influence

the yolk colour positively (Hammershøj et al. 2010). Further, mussels have a high

content of essential minerals (Özden et al. 2010; MacArtain et al, 2007) especially

calcium. Recently, it has been shown that mussel meal has a comparable nutrient

composition as fish meal and can influence egg quality without altering production

parameters in laying hens (Jonsson and Elwinger, 2009). A common problem for

mussel fishermen in the Danish fjords is that starfish (Asteria rubens) which is a

predator, eat very high amounts of the large mussels. However, in poultry diets,

starfish has been considered as a good protein and amino acids source more than 50

years ago. However, in broilers the use of starfish may reduce growth performance

and protein digestibility (Stutz and Matterson, 1964). The main challenge of using

blue mussel and star fish in organic poultry production is to examine their

nutritional value in experimental studies.

2.4.2 Forage material

Use of locally produce silages and vegetables of high quality can reduce imported

protein sources and help to reach the goal of formulating 100% organic layer diets.

Some of the forage materials are very rich in some specific nutrients while others are

not. Therefore, testing the potentials of these forages in feeding experiments is a pre-

requisite for their sustainable use. Silage in general is produced by the controlled

fermentation of crops with high moisture content. Anaerobic conditions and

protection from undesirable microorganisms are the determining factors of a good

quality silage. Legumes are buffered to a higher extent than grasses, and as a

consequence, more difficult to ensile satisfactorily. During ensilation, grass crops

with a dry matter content of about 200 g/kg, normally achieve a pH reduction to 4.0,

25

which will preserve the crop satisfactorily, as long as the silo remains airtight and is

free from penetration by rain (Ibrahim, 2005).

Green forage and grasses have been used as supplements in poultry diets for a long

time, especially in the summer seasons; however during winter the use of fresh

forage is limited due to a low availability (Sundrum et al., 2005; Steenfeldt et al.,

2013). Since beak trimming is not permitted in organic poultry, the hens can easily

handle and eat different kinds of foraging materials. Feeding silages to young meat

geese has been found to have a positive effect on body weight, dressing percentage,

and slaughter value of geese (Faruga et al., 1997; Mazanowski et al., 2000), the

quality of their meat (Kung et al., 2008), the development of the digestive tract

(Vetesi, 1992), and the absorption of nutrients from silages (Wang et al., 2008; Wang

et al., 2009). The nutritive value of silage for poultry may be minimal, because it has

a high content of dietary fibre which, due to the lack of endogenous fibre-degrading

enzymes in the intestine (Scott et al., 1982), is poorly digested by poultry (Steenfeldt

et al., 1998; Lazaro et al., 2003).

Since the use of foraging material in organic egg production is mandatory, there has

been an increased focus on the use of different silages and vegetables as supplements

to layers diets. Results have shown that a higher egg production was recorded when

hens were fed maize silage and carrots as supplements, whereas hens fed barley-pea

silage produces less (Steenfeldt et al., 2007). Birds fed blue lupine showed a

moderate egg production (Hammershøj and Steenfeldt, 2005). The intake of silages

ranged from 33-48% of the total feed intake on an as fed basis, and compared to a

control diet without access to any kind of foraging material, the supplementation of

silages reduced mortality, decreased pecking damage and improved plumage quality

(Steenfeldt et al. 2001; 2007). Thus, it it is suggested that foraging material can

contribute with some nutrients to the hens and at the same time has a positive effect

on feather pecking behavior as the hens exploit their foraging behavior when having

access to a forage material.

2.5 Benefits of forage materials as supplement to poultry diet

Dietary fibre (non-starch polyssacharides (NSP) + lignin), a complex group of

components that differ largely in chemical composition, physical properties and

26

physiological activities (Theander et al., 1989; Bach Knudsen, 1997) is very high in

silages and some vegetables. The content of dietary fibre in poultry diets is

traditionally considered as a diluent of the nutrient content in diets and to have

negative influence on feed intake and overall nutrient digestibility (Mateos et al.,

2002; Rougiere and Carre, 2010). However, recent research has shown that addition

of fibre to the diets to some extent is beneficial for the nutrient digestibility.(Surai

and Sparks, 2001; Kovacs-Nolan et al., 2005; Seuss-Baum, 2005; Abouelezz et al.,

2012) digestive system development (Richter et al., 2002; Gonzalez-Alvarado et al.,

2008; Sacranie et al., 2012), growth (Jimenez-Moreno et al., 2009C; Gonzalez-

Alvarado et al., 2010; Horsted and Hermansen, 2007) and egg production

(Hammershoj and Steenfeldt, 2005; Steenfeldt et al., 2007; Hammershoj et al.,

2010; Horsted et al., 2006; Hammershoj and Steenfeldt, 2012). It has been estimated

that nutrient restricted high producing layers in some periods could cover up to 70%

of their lysine and methionine requirements through different forage material

(Horsted and Hermansen, 2007). The properties of dietary fibre largely depend on

its solubility; the mode of action of soluble fibre in the digestive tract is quite

different than that of insoluble fibres (Choct, 2002; Choct et al., 2006; Kalmendal,

2012).

Feed cost are high, as approximately 70% of total poultry production cost are related

to feed production which has a direct influence on the price of the product in the

supermarket which may affect consumer preference negatively, when it comes to

buying organic eggs. The general utilisation of forage resources that is grown either

on own farm or locally in the same area might have beneficial effects on the

environment due to the proper maintenance of nitrogen and phosphorus load in the

farm area (Dekker et al., 2012). Organic egg production in Europe largely depends on

the imported protein sources from overseas countries. A feasible option to reduce

integral ecological impact of transporting organic feed ingredients from abroad is by

maximising their use of regional grown feed sources (Dekker et al., 2013) which can

reduce the farm’s dependency on imported feed sources (Van de Weerd, 2009).

Further, a higher intake of forage materials of high quality could contribute with

certain amounts of nutrients to the birds and may therefore facilitate the transition

to 100% organic feed supply (Horsted et al, 2007; Steenfeldt et al., 2013).

Supplements of different diets deficient in essential amino acids are susceptible to

27

develop feather pecking behavior (Ambrosen and Petersen, 1997) and in series of

studies with laying hens having access to different kinds of forage materials reduced

feather pecking and improved plumage condition (Wechsler and Huber-Eicher,

1998; Aerni et al., 2000; Kohler et al., 2001; Steenfeldt et al., 2007). Rough materials

are expected to give birds a feeling of satiety and make them occupied by foraging;

consequently, birds get less time for feather pecking. In the future, it can be

speculated that forage materials will not only be considered as being occupying

material for birds but also a source of nutrients as well as having significant

environmental and welfare benefits for birds. It was concluded by Mateos et al.

(2012) that broilers and young pullets require a minimum amount of fibre in the diet

for the proper functioning of the gastro-intestinal tract but the recommended level

will depend on the target species.

2.6 Capacity of hens to digest fiber rich forage

The gastro-intestinal tract (GIT) is the site, where the nutrients of the feed are

digested and absorbed in the body. The purpose of the digestive tract is to break

down the ingested feed and release the nutrients. The paagse of feed through the

digestive tract of animals is a dynamic process, where the necessary changes are

performed by both mechanic and chemical digestion. The digestibility of nutrients is

influenced by many factors such as flow of digesta and mean retention time which

are important parameters of digestion kinetics (Lou et al., 2010).

In poultry ingested feed is swallowed without being chewed into smaller particles due

to the lack of teeth. The feed passes into the crop, which is a diverted part of the

esophagus, and apart from starch hydrolysis (Bolton, 1965), storing and softening of

the ingested feed are the important functions of the crop (Duke, 1986) before it

moves to the next part of the GIT, the proventriculus and gizzard. The storage time of

feed in the crop can be influenced by the structure of the feed, because feed, with a

rough structure and large particle size, it will take a longer time to reduce particle

size by mechanical grinding in the gizzard (Svihus, 2014). Before entering the

gizzard, the feed passes through the proventriculus, the glandular stomach, where

hydrochloric acid, pepsinogens and mucous are secreted in order to initiate digestion

which is continued in the gizzard, the muscular stomach (Larbier et al., 1994;

Sturkie, 2000). In the gizzard the feed is gorund mechanically, which increases the

28

surface of the feed particles for further chemical breakdown, thus increasing the

access for the digestive enzymes in the small intestine (Sturkie, 2000) Amerah et al.,

2007). The gizzard is considered as the pacemaker digestive organ in birds which

regulate particle size of the digesta entering into the small intestine, enzyme and

endogenous juice secretion, gastro-intestinal tract motility including gastro-

duodenal refluxes, and voluntary feed intake (Mateos et al., 2012). A well-developed

gizzard has a high grinding capacity breaking down coarse particles, and gizzard size

can be expanded up to 100% of its original size when structural components are

added to the diet (Sacranie, 2012). It has also been shown that the volume of the

gizzard increases substantially when diets with whole cereals or insoluble fibre are

fed (Hetland et al., 2003; Bjerrum et al., 2005; Amerah et al., 2008).

It was observed previously that hens fed silages have larger gizzards than hens fed

vegetables due the enhancement of mechanical stimulation resulting from fibre

retained in the gizzard (Engberg et al., 2002; 2004; Ide et al., 2005; Steenfeldt et al.,

2007). High content of dietary fibres in poultry diets reduces pH in the gizzard, and a

decreased pH in digesta helps pepsin activation and solubility of the mineral sources

(Guinotte et al., 1995; van der Aar et al., 1983). A low gizzard pH provides a barrier

the acid sensitive bird pathogenic or zoonotic bacaeria (Bjerrum et al., 2005). The

influence of fibres on small intestinal development is minor (Wu et al., 2004). A

reduction of the small intestinal size in relation to soluble fibres has been reported by

(Gabriel et al., 2003).

2.7 Effect of dietary fibre on nutrient digestibility

Dietary fibre (non-starch polyssaccharides (NSP) and lignin) is present in all plant

raw materials to a different extent, and is a complex group of components with high

variations in chemical composition, physiological properties and physiological

activities (Bach Knudsen, 1997), where most the NSPs are associated with the plant

cell walls (Selvendran et al., 1987). NSP can be found in both a soluble and an

insoluble form, where especially the soluble NSP considered an anti-nutritive factor

which can inhibit the digestion and absorption of nutrients in the small intestine due

to its viscosity inducing properties (Langhout et al., 1999; Engberg et al., 2002;

2004). In addition slow passage rate of digesta may cause reduction of the feed

intake and longer transit time which increases the proliferation of bacteria in the

29

small intestine (Guinotte et al., 1995; Steenfeldt et al., 2007). However, the negative

effects of viscous NSP in feed seems to be age dependent, as older birds appear more

capable of transporting viscous material in the GIT (Salih et al., 1991). It has been

suggested that soluble NSP, and smaller components as sugars, are fermented

especially in the ceaca and may contribute with energy to the hens through

production of short chain fatty acids (Jørgensen et al., 1996; Lazaro et al., 2003;

Steenfeldt et al., 1995; 2007). In contrast, insoluble NSP cannot enter the ceaca due

to their size and are only fermented to a small extent in poultry (Choct et al., 1996).

The insoluble NSP fraction acts rather as nutrient diluents and some studies obtain a

negative effect of dietry insoluble NSP on nutrient digestion and absorption in

poultry (Krogdahl, 1986; Kalmendal, 2012). The results of other studies indicate that

insoluble NSP does not decrease nutrient digestibility and nitrogen retention (Cao et

al., 1998; Hetland et al., 2003; Jiménez-Moreno et al., 2009a). It has been reported

previously that a moderate amount of has a beneficial effect on the digestibility of

some nutrients in poultry (Hetland and Svihus, 2001; Steenfeldt et al.,

2007).Ingestion of insoluble NSP increase the contents of bile acids and amylase

activity in digesta by increasing gastroduodenal reflux (Hetland et al., 2003). This

increased the digesta content of bile acids and increased the digestibility of nutrients

of fat and starch in young broiler chickens fed fibrous feed (Jiménez-Moreno et al.,

2009a). Other authors showed that the inclusion of moderate amounts of fibre in the

diet improved nutrient digestibility resulting in improved bird growth and health of

digestive tract (Gonzalez-Alvarado et al., 2008; Sacranie et al., 2012).

In the ceaca, the microbiota, especially bacteria, utilise fibre as a source of energy. As

a consequence, increased energy supply increases microbial metabolism and

microbial population growth. Bacteria produce short chain fatty acids such as

acetate, butyrate and propionate at the time of metabolism and enhance bacterial

populations, this results in further production of short chain fatty acids (Walugembe,

2013) .It was observed that the concentration of short chain fatty acids in digesta is

higher in diets containing soluble NSP compared to insoluble NSP (Jørgensen et al.,

1996). Short chain fatty acids and lactic acids in the intestine have an inhibitory

effect on certain pathogenic and zoonotic bacteria, e.g. Enterobacteriacae including

E. coli and Salmonella (Russell, McHan and Shotts, 1993; Van der Weielen et al.,

30

2000), whereas beneficial bacteria like Lactobacillus are not affected (Van der

Weielen et al., 2000).

2.8 Welfare in organic poultry

Animal welfare may be defined as the concept where animals are kept in a way they

can enjoy freedom to express their natural behavior. Consumers are willing to pay

more for products, when birdshave been reared under good conditions and in

productions systems that ensure high welfare (Dawkins, 2012; Hermansen, 2003).

The condition of the plumage is a good indicator for the welfare status in organic

laying hens, whereas (Bestman and Wagenaar, 2003) and feather pecking indicates

reduced welfare both for the victim and the pecking bird (Bestman and Wagenaar,

2003). Poor quality plumage is associated with feather pecking (Bilcik and

Keeling.,1999) and feather pecking is an undesired behavior of organic layers and is

a major concern in many organic farms (Kjaer and Sorensen, 2002; Bestman 2000),

and a consistent challenge for the organic poultry production. Apart from reduced

animal welfare, poor plumage condition often results in a higher feed intake to

maintain body temperature thus resulting in economic losses. A substantial amount

of evidence suggests that feather pecking is a redirected foraging behavior (Huber-

Eicher and Wechsler, 1997; Aerni et al., 2000) and linked to dietary deficiency of

fibre, protein as well as methionine and cysteine (Kjaer and Sorensen, 2002;

Elwinger et al., 2002). Prohibiting the use of synthetic amino acids in organic

poultry diets affects the possibility to formulate optimal diets which in the end can

have a negative influence on performance and welfare of the birds. Free access to

forage materials in the out-door area allows poultry to perform their natural

foraging behavior, provides sunlight, and reduce stress. Feather pecking can be

prevented by different feeding strategies in the organic poultry production. It has

been observed previously that different kind of roughages as a forage material can

reduce feather pecking in laying hens (Hartini et al., 2002; Steenfeldt et al., 2007;

van Krimpen et al., 2009). Feeding hens with forage has direct influence on

behavior and motivates the hens to spend more time on foraging and less on feather

pecking (Norgaard-Nielsen et al., 1993; Aerni et al., 2000). For the assessment of on

bird welfare, plumage condition, foot health, deformations of keel bone and pubic

bone as weel as colour and wounds of the comb as described by Tauson et al. (1984)

31

and Gunnarson et al. (1995). A gizzard filled with feed makes the hens feel more

satiated, resulting in birds appearing more calm. As a consequence this may

contribute to a lower feather pecking pressure (Wechsler and Huber-Eicher, 1998;

Aerni et al., 2000; Steenfeldt et al., 2007). Further, welfare of birds in forage-based

feeding systems was found to be excellent irrespective to the type of supplementary

feed (Horsted, 2006).

2.9 Environmental issues

A major concern of the society, environmental organisations and organic farmers

that manage part of the agricultural land, is the risks of deteriorating natural

ecosystems and biodiversity, and increasing water and soil pollution (Horrigan et al.,

2002). Although there are many positive aspects of organic egg production, some

aspect needed to be improved (Dekker et al., 2013). Environmental problems of

organic egg production can be classified broadly in 3 categories: 1) transport of

imported feed ingredients over long distances (Meeusen et al., 2003); 2) a high level

of hen house derived ammonia emission (Groenestein et al., 2005) and 3) a high load

of N and P in the outdoor run, resulting in harmful losses to the environment, such

as leaching of nitrate and emission of ammonia and nitrous oxide (Aarnink et al.,

2006). It has been estimated that approximately 57% of acidification is caused by

ammonia emission from organic laying hen farms (Dekker et al., 2008). In the

search for available protein sources for organic poultry diets, not only crop yield and

nutritional value, but also environmental issues have to be taken into account for a

complete judgement of the sustainability of organic egg production. Feed production,

including crop cultivation, feed processing and transport are responsible for about

54–73% of total green house gas (GHG) emissions per kg of animal product (Basset-

Mens and Van Der Werf, 2005).

To ensure the methionine requirement in the practical organic production of egg and

meet, organic diets are often formulated with excessive protein which can result in an

oversupply of nitrogen excreted to the environment (Blair, 2008). Poultry manure is

very rich in nitrogen which means that ammonia emissions from manure

management can be considerable (Sonesson et al., 2009). There is a difference

between poultry manure and manure of other livestock, because in poultry the

majority of the nitrogen is excreted in the form of uric acid (Kalmendal, 2012). The

32

conversion of uric acid to ammonium largely depends on the storage conditions. In

order to determine the fraction of the total nitrogen that is plant-available, both

ammonium and nitrogen content in the uric acid should be considered as it is quickly

converted to plant-available ammonium when in contact with the soil and can thus

be regarded as directly plant-available (Salomon et al., 2006).

Organic egg production should aim at minimising emissions to the environment

throughout the production chain, such as emission of greenhouse or acidifying gases

or eutrophying substrates, and maintaining or enhancing the level and quality of

resources such as fossil energy, inorganic P and land (De Vries and De Boar, 2010).

Organic egg production could be more environmental friendly, when reducing the

import of dietary ingredients and replacing them with locally-grown crops to a higher

extent in the future (Dekker et al., 2013). Steps should be taken to ensure locally

available cereal and protein feed sources and search for new alternatives, especially

with focus on protein. Bos (2005) emphasised that a reduction of the import of

ingredients from overseas countries, by regionalising organic production, could be a

step in the right direction for a more sustainable production, being beneficial for the

environment.

Chapter 3: Aim and Hypothesis

The overall objective of the PhD thesis was to develop alternative feeding strategies

fulfilling 100% organic diet for organic egg production in a sustainable way. In order

to reach the aim, 3 experiments were conducted with following hypothesis and aim:

1. Hypothesis: Foraging materials can contribute energy, protein and amino acids to

some extent in organic diets for laying hen as well as minimise excretion of excess

energy and N into the environment.

Objective: To examine the effect of using different forage materials

supplementation on egg production, welfare condition and N retention and

excretion. In the present study maize, alfalfa, grass, hemp, maize-cob silages and

beetroot, carrot, kale were used as foraging material.

33

2. Hypothesis: It is important to elucidate digestibility of experimental organic layer

diets and forage materials nutrient utilisation of laying hen.

Objective: The specific objective of the present study is to evaluate nutrient

digestibility and microbial activity in laying hens fed different silages and forages

supplemented diets.

3. Hypothesis: Mussel meal and starfish meal can be a potential protein source that

can substitute fish meal in organic poultry production.

Objective: The aim of the study was to evaluate mussel meal and starfish meal in

different dietary concentrations as protein sources in organic layer diets taking

hen production performance, nutrient digestibility and egg quality into

consideration.

Chapter 4: Methods and Materials

The study in this thesis is based on three experiment conducted in different research

facilities situated at Aarhus University, in Foulum, Denmark, in 2012 and 2013.The

first experiment were performed in mobile houses on the outdoor Organic platform

and the second experiment was carried out in digestibility cages placed in an indoor

poultry unit just after the first experiment has been finished. The third experiment

was conducted in floor pens in an indoor poultry unit.

4.1 Experimental design

This represents the overview of experimental design.

34

Table 2 Overview of experimental design of the thesis

Paper I Paper II Paper III

Laying hens × × ×

Outdoor Access ×

Floor System ×

Battery cage × ×

Production Performance

× ×

Egg Quality ×

Microbiology ×

Welfare Assessment ×

Nutrient Digestibility

× ×

Forage supplements × ×

Mussel and Starfish meal

×

The first experiment is represented in Paper І. A Complete randomized block design

was used and 810 white organic laying hens of the genotype Hisex white, were

allocated at the experimental unit, from 16 weeks of age. Eight experimental diets

with different forage supplements and control diet without supplements were

included in the study, with five replicates for each dietary treatment. Thirty outdoor

units with 30 houses contain 27 hens each, where one unit represented one replicate.

Hens had access to outdoor area with or without vegetation (control). This

experiment lasted for 210 days.

Figure 3: Control (Outdoor run without grass) Treatment groups (outdoor run with grass)

35

The second experiment is presented in Paper ІІ. A complete randomized block design

was used with Hisex white hens from the first experiment, which were moved from

the outdoor area to battery cages for the digestibility and balance study. The same

experimental diets and forage supplements were included in the experiment, which

lasted for 21 days.

The third experiment is presented in Paper ІІІ. Six dietary treatments with different

inclusion levels of mussel- and starfish meal, were fed to a total of 300 hens in indoor

floor pens using a complete randomize block design with 5 replicates per treatment.

The experiment lasted 84 days followed by a digestibility and balance trial in battery

cages. According to Danish legislation (Danish Ministry of Justice, 1998:

Bekendtgørelse nr. 210 af 6/4-1998) for organic egg production, the hens were not

beak trimmed in all experiments.

4.2 Experimental Diets

The first experiment was performed to develop a new feeding strategy aiming at

100 % organic diets, taking into account the nutrient content of different forage

supplements. In practice organic soya bean are not grown in Denmark and is

imported from Italy, China and Brazil. However, the soya bean ( cultivar, Merlin) for

this project was grown in south Denmark as an important contribution to the use of

local grown ingredient used in the experimental diets. 4 types of silages such as

maize silage, alfalfa silage, grass-herb silage, hemp silage and maize cob silage and 3

different vegetables such as beetroot, carrot and kale was included in the study.

Maize- , alfalfa- and grass-herb silages were provided as supplement to the laying

hens during the entire experimental period, whereas treatments with hemp silage

(17-34 weeks) and maize cob silage (35-46 weeks) were given in two separate period

due to shortage of ensilaged hemp for entire period. Three types of Danish grown

vegetables being beetroot, carrot and kale were given at different times reflecting the

season of the year during the experimental period. When the hens were shifted from

one type of silage and vegetables to another type, they received one week two

silage/vegetables types for adaptation and to avoid the risk of sudden stopping of

eating silage/vegetables.

The content of nutrient in the forage supplements varied to a high extent (Table 3).

36

Table 3. Chemical composition of silages and vegetables (g/kg DM)

Nutrients Maize silage

Alfalfa silage

Grass silage

Hemp silage

Maizecob silage

Beetroot Carrot Kale

Dry matter 317.2 300.7 367.5 354.7 352.7 78.9 108.9 150.3 Protein (6.25 x N) 95.3 249.7 181.9 189.4 83.8 156.6 102.8 268.4 Ash 34.3 123.8 93.8 130.3 24.6 91.5 88.2 100.1 Starch 259.4 8.8 10.7 7.50 406.8 0.6 ̶ 4.2 Cellulose 187.0 173.0 188.0 173.0 117.0 52.0 76.0 61.0 Total NSP3 405.0 353.0 384.0 369.0 297.0 186.0 228.0 305.0 Soluble NSP 21.0 79.0 71.0 68.0 8.0 73.0 121.0 187.0 Insoluble NSP 384.0 274.0 313.0 301.0 289.0 113.0 107.0 118.0 Lignin 93.0 109.0 119.0 167.0 54.0 16.0 14.0 28.0 Dietary fiber (NSP+Lignin)

498.0 462.0 503.0 536.0 351.0 202.0 242.0 333.0

Cystine 1.2 1.7 1.1 1.7 1.3 0.9 1.0 3.2 Lysine 2.1 8.1 8.3 5.7 1.3 3.4 3.2 13.6 Methionine 1.5 3.6 2.4 2.6 1.3 1.0 0.7 3.6 Threonine 2.8 5.4 7.1 4.9 2.6 2.6 2.7 8.9

Maize silage was obtained from a farmer with help from Midtjysk Mashin Station,

Denmark. Alfalfa- and grass-herb silage were grown and processed at AU-Foulum.

Hemp silage was obtained from a farmer with assistance from the “Knowledge Center

for Agriculture”, SEGES in Denmark. Maizecob silage and kale were bought from

organic producers in Denmark. Carrot and beet root were bought from “Tange Open

Air Gardening”, Denmark. All foraging materials were organically grown.

Figure 4: maize silage, kale, carrot

Figure 5: Hemp silage, beetroot, grass-herb silage and maize cob silage

All layer diets and forage materials were used also included in experiment two.

37

The third experiment was designed to study mussel meal and starfish meal in

different dietary concentrations as protein sources in organic layer diets and the

inclusion level was 4%, 8% and 12% mussel meal and 4% and 8% starfish, replacing

to fish meal, which was included in the control diet. For the mussel meal preparation,

we used raw fresh blue mussel (Mytilus edulis) from Limfjorden in the northern part

of Denmark. The mussels were harvested in March through a standard long-line

production and mussels were approx. 9 months old. The mussel was steamed and

meat was separated from the shell. This technique is used by the seafood industry to

separate shell and meat. The meat was dried between 800C and 900C to about 5%

water content and then ground. The heating from 850C to 900C well fullfils the

hygienic requirements for poultry feed (750C) and grinding using small grind mill.

Starfish (Asterias rubens) were caught in Limfjorden in May. After 1 month of

fishery 1300 t star fish were caught with a specialized starfish purse seine.

The chemical composition of fresh mussel, mussel meal and starfish meal are

presented in the below table 4.

Figure 6: Blue mussel (Mytilus edulis) and star fish (Asterias rubens) (from left to right)

Table 4. Analyzed chemical composition of blue mussel, mussel meal and

starfish meal (g/kg DM).

Mussel Mussel meal Starfish meal DM 254 949 946 Protein(N×6.25) 596 605 700 Fat 161 161 110 Ash 61 81 203 Sand 0 0 4 Phosphorus 9 9 26 Calcium 1 9 47 Cystine 9 9 5 Lysine 43 44 43 Methionine 13 14 17 Valine 28 29 34

38

4.3 Housing of laying hens

In the first experiment, hens were transferred from an organic rearing farm at 16

weeks of age to 30 experimental out-door units at AU-Foulum. Each house had a

size of 6 m2( 2 x 3) made of waterproof ply wood on iron frame with 4 nest boxes, one

feeding trough and one water cup along with pipe placed inside. Windows were

placed at each end of the house to allow daylight. There was no artificial light in the

house until the 15 August, for maintaining a constant day length artificial light was

provided in the house until the end of the experiment, where lighting program of 16h

light: 8h dark was followed.

Figure 7: Housing of laying hens (Experiment І)

One gate was placed along the full length of house in each side, 46 cm high, which

could be opened in order to give the hens free access to the outdoor area. In addition

one door was placed in the front side of the house. The gates were closed on the first

day, so the hens could get adapted to the new environment. There was no floor in the

house, which was placed directly on the ground (grass, dry soil). Two wooden

roughage boxes (height: 40cm, length: 50 cm, width: 40cm) were placed under a

wooden shed outside of the house. The hens were not beak trimmed considering that

this process would hamper hen’s ability to fully exploit the foraging materials.

Figure 8: Housing of laying hens (Experiment І)

39

The second experiment was a continuation of the first experiment which was

performed in 3 Battery cages. The battery cages were placed inside an environmental

controlled house. Each cages contained 12 individual metabolic cages (50cm×

50cm×50cm) with two feeding trough outside, one for experimental layer diets and

another for forage supplements and two water cups inside along to the water pipe.

An easy removal metal collection tray was placed under each meatabolic cage for

excreta collection. Lighting program was same as first experiment, 16 L: 8 D.

Figure 9: digestibility experiment in metabolic multilayer metabolic cages at indoor poultry unit

(Experiment ІІ)

The third experiment was carried out indoor in a poultry unit provided with

automatic ventilation and controlled temperature and light programme. The hens

were randomly allocated to 30 pens with the floor area of 4 m2, each equipped with 4

single nests box, one feed silo and a tape with nipple drinkers. The 30 pens were

separated by 2 m high walls that from the floor to 1.6 m high consisted of thin wood

and with wire mesh above, preventing visual contact between hens from separate

pens. The lighting program was 12L: 12D at 17 weeks of age. The day length was

gradually increased to 16L: 8D at 19 weeks, which continued to the end of the

experiment.

40

4.4 Experimental Procedure

4.4.1. Data Recording

Layer diet and Supplement intake

The layer diet intake data was recorded every fourth week (Paper І) whereas in the

experiment 3 the layer diet was recorded weekly (Paper ІІІ). In experiment two,

during the whole time of experiment the layer diet intake was recorded two times,

beginning and finishing of the experiment, whereas forage consumption was

recorded weekly (Paper І), whereas in experiment two fresh forage materials was

given fresh in minor portions twice a day and weighed left over in same day (Paper

ІІ). Feed intake, forage intake was calculated on hen-d basis in all experiments.

Egg production

Eggs were collected and registered from nests 3 times weekly (Paper І) whereas in

paper ІІ, eggs were collected and registered daily. The egg weight was recorded once

in a week (Paper І and Paper ІІ). Laying rate was calculated on hen-d basis and egg

mass output was calculated based on egg weight and laying rate (Paper І and Paper

ІІ).

4.4.2 Egg quality

Five eggs per treatment were randomly collected from each replicate per treatment

and were analysed for different egg quality parameters e.g. shell strength, yolk color,

yolk weight, albumen dry matter, albumen pH, albumen gel texture. A consumer

sensory evaluation was performed in the 30 week of age (Paper ІІІ).

4.4.3 Chemical analyses

Chemical analyses including dry matter, nitrogen, fat, minerals (Ca and P), ash, gross

energy of layer diets and forage materials (supplement) were performed twice during

the experiment (Paper І, ІІ and ІІІ). Same chemical analyses were performed for

excreta. Dietary fibre, sugar and starch were analysed in paper І. Amino acids except

tryptophan were determined in Paper І, ІІ and ІІІ.

41

4.4.4 Welfare recordings

Plumage, skin and footpad conditions were assessed according to the methods

described by Tauson et al. (2005) with slight modifications. Bumble foot is defined as

a prominent inflammation of the foot pad sole caused by dermatitis (Abrahamsson et

al., 1994). At the end of the experiment (week 44), all birds of each house were

weighed and scored as following the same methods as for 35 weeks of age.

4.4.5 Digesibility experiement

Plumage, skin and footpad conditions were assessed according to the methods

described by Tauson et al. (2005) with slight modifications. Bumble foot is defined as

a prominent inflammation of the foot pad sole caused by dermatitis (Abrahamsson et

al., 1994). At the end of the experiment (week 44), all birds of each house were

weighed and scored as following the same methods as for 35 weeks of age.

4.4.6 Gastrointestinal characteristics and intestinal micro flora

Two dietary groups with maize silage, alfalfa silage and control diet without forage

was performed in this study. The empty relative weight, and pH of gizzard,ileum and

caeca were measured,. The bacterial count of gizzard, cecum and ileum were

performed and short chain fatty acids (SCFA) and lactic acid were also measured by

Canibe et al.(2007).

Chapter 5: Results

This chapter is consisted of three manuscripts.

Manuscripts 1

Effect of organic diets providing silages and vegetables on production performance,

nitrogen and phosphorous excretion and plumage quality of organic layers

Manuscripts 2

Effect of feeding 100% organic diets and forage supplements on apparent nutrient

digestibility, metabolisable energy and microbial activity in laying hens

42

Manuscripts 3

Influence of blue mussel (Mytilus edulis) and starfish (Asterias rubens) meals on

production performance, egg quality and digestibility of nutrients of laying hens

43

5.1 Manuscript 1

Effect of organic diets providing silages and vegetables on production

performance, nitrogen and phosphorous excretion and plumage quality

of organic layers

S. AFROSE1, M. HAMMERSHØJ2, R. M. ENGBERG1 and S. STEENFELDT1*

Prepared for submission to British Poultry Science

44

Effect of organic diets providing silages and vegetables on production

performance, nitrogen and phosphorous excretion and plumage quality

of organic layers

S. AFROSE1, M. HAMMERSHØJ2, R. M. ENGBERG1 and S. STEENFELDT1*

1Department of Animal Science, Aarhus University, Denmark and 2Department of

Food Science, Aarhus University, Denmark

*Corresponding author: Sanna Steenfeldt

Email: Sanna.Steenfeldt@agrsci.dk

Tel: +45 87158074

Short title: Silages and vegetables in organic egg production

45

Abstract:

1. The aim of the present study was to investigate the suitability of using different

silages and vegetables to formulate 100% organic diets considering their nutritive

values.

2. Egg production, egg weight, welfare traits (plumage quality, bumble feet) and

mortality were observed in laying hens during 22-46 weeks. A total of 810 Hisex

white pullets of 16 weeks of age were distributed to 6 experimental diets, including a

control diet without supplements (diet A) and diets supplemented with silages

(maize (diet B), alfalfa (diet C), grass-herb (diet D), hemp/maize-cob (diet E) and

vegetables (beetroot/carrot/kale, diet F).

3. The chemical composition of forage materials and diets varied widely. The protein

content in different silages and vegetables ranged from 83-268 g/kg DM. The protein

content in kale was relatively high (268 g/kg DM) followed by alfalfa silage (249 g/kg

DM) and hemp silage (189 g/kg DM). The starch content was highest in maize-cob

silage (406 g/kg DM) and lowest in kale (4.2 g/kg DM) and carrots where it was not

detected. A high level of total sugar was found in beetroot (610 g/kg DM). Total NSP

was very high in maize silage (405 g/kg DM) and dietary fibre (NSP+lignin) was

highest in hemp silage (536 g/kg DM).

4. Hens having access to diet B + maize silage had a higher egg production (90.1%)

than hens fed with diet C + alfalfa silage (87.2%) and diet E 1 + hemp silage (86.7%).

The egg mass was highest when hens were fed diets supplemented with maize silage

and grass-herb silage. The organic layer diet intake was lower (112g/hen/d as fed

basis) with diet E (E1 and E2) + hemp/maize-cob compared to the control diet A (126

g/hen/d) (P<0.05). The daily highest forage (maize silage) intake was observed in

46

birds fed diet B + maize silage (49 g/hen as fed) compared to grass-herb silage (9 g/

hen as fed)(P<0.05).

5. The calculated daily N and P retentions per hen were significantly higher in diet B

+ maize silage, diet D + grass-herb silage , diet E2 + maize-cob silage and diet F3 +

kale compared to other diets (p<0.05).

6. The plumage quality was improved by feeding silages and vegetables as

supplements. The best plumage quality was found in hens receiving diet B + maize

silage and diet C + alfalfa silage compared to the non-supplemented control diet.

7. The occurrence of bumble feet was lower when hens were supplemented with

forage.

8. Hens receiving forage material tended to have a lower mortality than the non-

supplemented control group. Hens receiving diet F (F1, F2 and F3) +

beetroot/carrot/kale showed the lowest mortality (2.96%) compared to other

treatments.

9. In conclusion, access to forage materials has beneficial effects on egg production,

feather quality and mortality.

47

INTRODUCTION

In Denmark, the market share of organic eggs was 20.4% of all eggs in retail in 2013

(Anonymous, 2014) which reflects the increased consumer interest in organic egg

production. According to the Regulation (EU, 834/2007), organically reared animals

must be fed with organic feed, and at least 20% of the raw materials used in the feed

formulations must be grown primarily on-farm or in the same region in cooperation

with other organic farmers or feed manufacturers (EU,505/2012). However, if

organic feed in terms of quantity and quality is not obtainable, 5% of non-organic

raw materials are still allowed in organic poultry diets until 31 December 2017 (EU,

836/2014). A further provision of the regulation (EU, 834/2007) is that synthetic

amino acids are banned in organic production. Under these circumstances, the main

challenge is to formulate 100% organic feed for poultry nutrients in terms of protein

and amino acids (Crawley, 2015). In conventional poultry industry, soya bean is an

important vegetable protein source which is imported to a large extent from Brazil

and China to Europe (Aramyan et al., 2009). However, from a holistic point of view,

the import of soya bean is costly and associated with environmental issues due to

transportation (Van Krimpen et al., 2013). Locally grown soya bean, grains, legumes

and alfalfa are all promising vegetable protein sources in Europe (Hammershøj and

Steenfeldt, 2005; Van Krimpen et al., 2013).

The EU regulation mandates foraging materials, so that organic layers must have

daily access to vegetation, fresh or dried fodder or silage in addition to the compound

feed. The inclusion level of foraging material is unspecified (EC 1804/1999), but it

has been observed that hens are able to consume ~120g/hen/d (Hammershøj et al.,

2005; Steenfeldt et al., 2007) resulting in reduced consumption of the compound

layer diet of up to 20% (Blair, 2008). A study by Steenfeldt et al. (2007) found that

48

laying hens can consume forage materials without altering egg production which

indicates that forage materials provide a nutrient contribution for hens, although the

nutritive value of forage materials was not considered in the formulation of the

compound diet. The use of different forage materials for organic poultry could

support the principle of increasing utilisation of local resources which could have

positive effects on the cycling of nutrients in the system avoiding the import of soya

beans. Forage materials grown on farm utilise nitrogen (N) and phosphorus (P) from

the soil. Therefore, foraging behavior can reduce the accumulation of N and P in the

soil and hence ensure a desirable balance of N and P in a natural ecosystem which

subsequently will protect the environment from acidification and global warming

(Dekker et al., 2012). Forage materials have welfare benefits in terms of reduced

feather pecking and other undesirable behavior (Van Krimpen et al., 2005;

Steenfeldt et al., 2007). However, little attention has been paid to consider the

nutrients of forage materials to facilitate the transition to 100% organic feed supply

within the stipulated time.

We hypothesise that forage materials given as supplements could be considered a

potential nutrient source contributing energy, protein and amino acids to organic

layer diets to some extent. The main objective of the present study was to provide a

better knowledge on the potentials of utilising the nutrients of forage materials in

diet formulation in order to develop a new feeding strategy for organic laying hens.

Production performance, welfare indicators, including footpad lesions and plumage

condition were studied. Further, an indication of the effect of N and P excretion was

estimated. In the present study, maize, alfalfa, grass-herb, hemp and maize-cob

silages and the vegetables beetroot, carrot and kale were chosen to represent forage

materials with different nutrient compositions.

49

MATERIALS AND METHODS

Experimental design, birds and housing

The genotype used was Hisex white, and a total of 850 pullets were reared on an

organic poultry rearing farm from day-old to 15 weeks of age. At 9 weeks of age, 700

pullets were fed barley silage as a supplement for adaptation to forage materials,

whereas the remaining 150 pullets were allocated in a separate compartment in the

stable with no access to forage materials. The group of pullets without any

supplementation of silage and vegetables was considered the control group in the

study. A total of 810 Hisex white pullets, 16 weeks of age, were moved into 30

outdoor units at the organic facilities at Aarhus University, and 27 hens were

allocated per unit (a total of 119 m2) providing an area of 4.4 m2 per hen which fulfils

the requirements of EU legislation. Each outdoor unit represented one replicate (27

hens). A wooden house of 6 m2 was placed on each unit and equipped with perches, 4

nest boxes, 1 water cup and 1 round feed silo (34 cm diameter). Windows were placed

at each end of the house and small doors (46 cm high) were placed along the full

length of the house on each side which could be opened to allow free access to the

outdoor area. In addition, 1 door (height: 115 cm, width: 61 cm) was placed on the

front side of the house to give access to the house for supplying feed to the silo. The

doors were closed on the first day, so that the hens could adapt to the new

environment. There was no floor in the house which was placed directly on the

ground (grass, dry soil). Two wooden boxes (height: 40 cm, length: 50 cm, width: 40

cm) were placed in a wooden shed outside the house. The outdoor areas were

covered with grass and clover; however, in the outdoor area for the control birds, the

vegetation of grass and clover was completely removed to avoid any forage intake.

The hens were not beak trimmed as this process would hamper the ability of the hens

50

to exploit the forage materials fully. Further, beak trimming is not allowed in organic

egg production in Denmark (Danish Ministry of Justice, 1998: Bekendtgørelse nr.

210 af 6/4-1998). A natural lighting was employed during the experiment. However,

in late autumn (after 15 August) additional artificial light was provided in the house

until the end of the experiment to maintain a constant day length of 16 h. The

experiment lasted from 16 to 46 weeks of age, 210 d.

Diets and treatments

During the experiment, the formulation of the 6 experimental layer diets was based

on 100% organic feed ingredients considering the nutrient content of the forage

material given as a supplement. The composition and calculated nutrient content

and metabolisable energy of the diets are shown in Table 1. Before starting this

experiment, a digestibility trial of the forage materials used in this experiment was

performed. In order to formulate the diets of the present study, daily intake and

digestible nutrient values of different forage materials from the previous study were

taken into account (Steenfeldt, unpublished). During the formulation of the

experimental diets, the calcium content was reduced with 1% in all diets and instead

added on top as a granulated calcium source when feeding the diets every week. This

is common practice in organic egg production in Denmark, as it increases the

possibility to cover the individual calcium requirement. During the present

experiment, the hens, except for diet C + alfalfa silage and diet D + grass silage, ate

forage materials as expected from the pilot study; however hens in the group diet C +

aflalfa silage and diet D + grass silage had a lower intake of the silages than observed

in the previous study. As a consequence, after 3 weeks of study, layer diet C and diet

D were re-formulated according to their actual daily intake. The soya bean (cultivar

Merlin) used in the experimental diets was grown in the Southern part of Denmark,

51

and all other ingredients and forage supplements used were also grown locally in

Denmark in order to develop a sustainable feeding strategy. The hens in the control

treatment received diet A without supplemental forage material. The hens in the

remaining 5 treatments were fed one of the experimental diets B-F together with

different kinds of forage materials as supplements. All hens had access to an outdoor

area covered with vegetation except the diet A group which had access to an outdoor

area without vegetation. The composition of diet A (control), diet B + maize silage,

diet C + grass/herb silage and diet D + alfalfa silage was not changed during the

experimental period, whereas diet E was divided into diet E1 + hemp silage and diet

E2 + maize-cob silage due to the fact that the total amount of harvested and

ensilaged hemp silage was not sufficient to meet the expected demand for the whole

experimental period. Hemp silage was substituted by maize-cob silage halfway

through the experiment, as maize-cob silage is easily available. Diet F was formulated

to be supplemented with different types of Danish grown seasonal vegetables and

formulated according to the vegetable used: F1 + beetroot, F2 + carrot, F3 + kale.

When the hens were shifted from one type of silages/vegetables to another with diet

E (E1 and E2) and diet F (F1, F2 and F3), they received both types of

silages/vegetables during one week to adapt them to the new forage. The dietary

treatments are referred to as diet A without forage material, diet B + maize silage,

diet C + alfalfa silage, diet D + grass/herb silage, diet E (E1 and E2) + hemp, maize-

cob silage and diet F (F1, F2 and F3) + beetroot/carrot/kale.

The layer compound diets were provided as pellets (3 mm) and fed ad libitum. The

silages and vegetable supplements were cleaned and stored in a temperature

controlled feed storage facility in closed containers. All types of silages were from the

same batches, harvested in the summer before starting the experiment, ensilaged

52

and packed in small plastic bags and placed in the freezer to ensure freshness and to

avoid changes in chemical composition. During the first 2 weeks of the experiment,

the hens gradually received increasing amounts of forage materials, so the amount of

offered forage materials was adjusted to avoid wastage and thereby reduce the

quantities of leftovers. The silages were given as fresh materials, and the vegetables

were provided unprocessed. Silage bags were taken out from the freezer and kept in

the cooling room for thawing 1 day before feeding. To ensure freshness, all forage

supplements were provided to the birds in the morning. Beetroots, carrots and kale

were kept in a cooling facility (5oC); either packed in 20 kg bags (beetroot, carrots) or

placed in a small container (kale).

Diet and forage consumption

The consumption of the layer diets was recorded every 4-week, whereas forage

consumption was recorded weekly. Layer diets and forage consumption was

calculated on a hen-d basis taking the actual number of hens housed per floor pen

into account. Mortality was recorded daily, and body weight was recorded at 15, 18,

27, 33 and 42 weeks of age. During the experimental period, forage leftovers were

analysed for DM content. In addition, layer diets and forage samples were collected

for chemical analyses.

Egg production

Eggs were collected 3 times per week, and the number of eggs was registered. Egg

weight was recorded once a week. At the beginning of the laying period, some hens

did not lay eggs in the nest boxes, and eggs collected from the floor were also

recorded. However, floor eggs were reduced gradually before the start of the

experiment. Based on the laying rate and egg weight, the daily egg mass output

(g/hen/day) was calculated.

53

Plumage and footpad conditions

Plumage, skin and footpad conditions were assessed according to the methods

described by Tauson et al. (2005) with slight modifications. At 35 weeks, 12 hens

were selected randomly from each house. Body weight was recorded, and plumage

quality was scored for 5 body parts (Neck, breast, back, wings and tail). The score

was 1, 2, 3, 4 points for each body part; a higher score meant a better plumage

condition. The sum of these body part scores (total score: 5 to 20 points) was used

for analyses of plumage conditions. Cases of bumble foot were observed at the same

time when plumage quality and footpads were evaluated. Bumble foot is defined as a

prominent inflammation of the foot pad sole caused by dermatitis (Abrahamsson et

al., 1994). At week 44, all birds of each house were weighed, and animal welfare

indices (plumage quality, bumble foot, foot pad dermatitis) were scored using the

same score scale as described above.

Chemical analyses

Dry matter (DM) content of the layer diets, forage material was determined by drying

at 1030C for 8 hrs. All samples were freeze-dried before analysis and milled to pass

through a 0.5 mm screen. The nitrogen was analysed by the Dumas method (Hansen,

1989) with a Leco FP 428 nitrogen analyser (Leco Corporation, St. Joseph, MI). Ash

was analyzed according to the method L54/2009(EC, 2009), and fat (HCl-fat) was

extracted with diethyl ether after acid hydrolysis (EC,1998). Amino acids were

analysed by an amino acid analyser (Biochrom 30, BiochromLTd, Cambridge) using

different buffer solutions and ninhydrin. Calcium was determined by method 975.03

(AOAC, 2000), phosphorus by colorimetric (Stuffins, 1967) and energy by a LECO

AC 300 automated calorimeter system 789-500 (LECO, St Joseph, MI, USA). The

sugars (glucose, fructose and sucrose) and the oligosaccharides (raffinose, stachose

54

and versbascose) were analysed by the method of Bach Knudsen and Li (1991). Non-

starch polysaccharides (NSP) and lignin were analysed according to the method

described by Bach Knudsen (1997). Starch was analysed by the enzymatic-

colorimetric method of Bach Knudsen (1997). All analyses were performed in

duplicate. The chemical composition of fresh summer and fall grasses were

performed during the experiment.

Calculations and statistical analyses

The laying rate was calculated as the number of eggs divided by hen day production

and expressed as a percentage. The egg mass was the laying rate multiplied by egg

weight in gram. The values of N and P in egg production and body weight gain were

obtained from the literature (Poulsen et al., 2006; Poulsen, 2014). The daily N and P

excretion per hen were calculated by subtracting daily N and P intake per hen in

gram from N and P retained for egg production and body weight gain. The

experiment followed a randomised complete block design (RCBD) where the single

pen represented the experimental unit (replicate). The statistical analyses were

subjected to an analysis of variance using the MIXED procedure (SAS Institute Inc.

1990). The data were tested for homogeneity. The data were distributed normally,

hence, data were not transferred. The model included treatment as fixed effect and

period and block as random effects. Model control was done by plotting residual

against predicted values. The statistical model used was:

Yijl = ai +bj +cij+eijl

Where Yijl = intake of compound layer diet, supplement intake, protein intake,

methionine intake, lysine intake, cysteine intake, laying rate, egg weight, egg mass,

mortality, body weight, nitrogen and phosphorous excretion in excreta, plumage

condition, bumble feet and cloacal infection; ai = effect of experimental treatment (i

55

= 1,…..,6); bj = effect of periods (j = 1,…….,6); cij = interaction between treatment and

period and eijl = replicate (l = 1,…..,5); cij was excluded when the interaction between

variables were non-significant. The results were presented as means and SEM

(standard error mean). A significant difference between diets was found with

pairwise comparisons; the significance level was defined as a P-value below 0.05.

RESULTS

Chemical compositions of experimental diets and forage materials

The chemical compositions and amino acid contents of different experimental layer

diets are shown in Tables 2a and 2b. The forage materials (silages and vegetables)

used in this experiment were from the same harvest year and the same batch, and

their nutrient values were taken into account to formulate layer diets. As diet C and

diet D were changed halfway through the experiment, the chemical compositions of

the 2 C diets and the 2 D diets were analysed, hence the results of both the C diets

and the D diets are shown in the tables. The DM content of the experimental diets

did not vary widely ranging from 883.4 to 897.9 g/kg DM. Layer diet B, diet E2 and

diet F3 had a higher DM content than the other diets. Layer diet E2 had the highest

protein content (252.19 g/kg DM), whereas the lowest content was observed in diet

E1 (195.62 g/kg DM). Among the diets, the ash content varied widely ranging from

91.55 in diet F3 to 139.9 g/kg DM in diet E2. Except for diet E1, the ash content in the

silage supplemented diets was higher than in the vegetable diets. The content of fat

was highest in diet C (69-74 g/kg DM) and diet D (68-76 g/kg DM), and the lowest

was found in diet F3 (61.7 g/kg DM). The calcium content ranged from 23.3 to 38.4

g/kg DM and was highest in diet E2 and lowest in diet F3. Except for diets E1 and F3,

the calcium level in all other diets was higher than in diet A. The lowest phosphorus

content was found in diet E1 (6.44 g/kg DM) and the highest in diet B (8.30 g/kg

56

DM). The phosphorus content in all other diets was higher than in diet A. The total

NSP content ranged from 120.4 to 138.0 g/kg DM and was highest in diet D and

lowest in diet F1. Diet E2 (136.8 g/kg DM) and diet F2 (135.6 g/kg DM) were also

rich in NSP. The dietary fibre content was high in diet D (185.5 g/kg DM) and diet F2

(181.7 g/kg DM), whereas it was lower in diet E1 (162.1 g/kg DM) and diet F1 (162.9

g/kg DM). The methionine content of the diets exceeded the required concentrations

and ranged from 3.11 to 4.39 g/kg DM with the highest concentration in diet E2. A

high methionine (3.90 g/kg DM) content was found in diet B, although the protein

content of diet B was moderate. The methionine content of diet C and diet D

remained constant, even though they were changed halfway through the experiment.

Except for diet E1, the methionine content was higher in all supplemented diets

compared to the control (diet A). The lysine content ranged from 9.02 (diet A) to

12.61 g/kg DM (diet E2), and all supplemented diets had a higher lysine content than

the control. Diet B (10.92 g/kg DM), diet C (10.69 g/kg DM) and diet F2 (10.05 g/kg

DM) also had relatively high amounts of lysine. As part of this project, the production

of a climate-robust soya bean cultivar (cultivar MERLIN) was an important strategy

towards formulating diets based on 100% organic raw material. The soya beans used

in this experimental was grown in Denmark, and the yield of soya beans was 2.2 t/ha

cleaned and dry seed which was comparable to the average world soya bean

production (2.5t/ha in 2011).

With respect to the content of DM, fat, protein and amino acids, the soya beans used

in the present study were comparable to imported soya beans and provided 937 g

/kg. DM It contained 188 g fat, 488 g protein, 6.3 g methionine, 29.1 lysine and 6.9 g

cysteine per kg DM. The chemical composition of the different silages and vegetables

is shown in Tables 3a and 3b. The dry matter content of silages and vegetables

57

varied to a high extent from 78 to 367 g/kg DM where silages had at least a two-fold

higher DM content compared to vegetables. The lowest DM content was found in

carrots and the highest in grass-herb silage. Varying protein content was observed in

the different silages and vegetables ranging from 83-268 g/kg DM. The protein

content was relatively low in maize silage (95 g/kg DM) and maize-cob silage (83

g/kg DM) and higher in kale (268 g/kg DM) followed by alfalfa (249 g/kg DM) and

hemp silage (189 g/kg DM). The fat content was overall low in all samples ranging

between 3-91 g/kg DM. The starch content in maize-cob and maize silage was 406

g/kg DM and 259.4 g/kg DM, respectively, but was lower in the other silages and not

detectable in carrot. The total sugar content in vegetables was higher than in silages.

A high level of total sugar content was found in beetroot (610 g/kg DM) and carrot

(601 g/kg DM) compared to a very low content in hemp (0.3 g/kg DM) and maize

silage (1.9 g/kg DM). Forage materials differ to a high extent in the content of soluble

and insoluble NSP. The highest content of insoluble NSP was observed in silages and

was much lower in vegetables. Total NSP was very high in maize silage (405 g/kg

DM) and very low in beet root (186 g/kg DM). However the lowest soluble NSP

content was found in maize-cob silage (8 g/kg DM) and the highest in kale (187g/kg

DM). Dietary fibre in silages (462-536g/kg DM) was higher than in the vegetables

(202-351 g/kg DM) with the highest content found in hemp silage (536 g/kg DM)

and the lowest in beetroot (202 g/kg DM). The cellulose content in silages was 3-fold

higher than in vegetables. The highest cellulose amount was found in grass-herb

silage (188 g/kg DM) and the lowest in beetroot (52 g/kg DM). The gross energy

content in all the silages and vegetables was quite similar ranging from 15-19 MJ/kg

DM. The calcium content varied very much ranging from 1 to 27 g/kg DM where the

highest concentrations were found in hemp silage and the lowest in maize silage.

Similarly, the highest phosphorus concentration was found in hemp (7.03 g/kg DM)

58

and the lowest in maize (2.8 g/kg DM). Among the amino acids, the methionine

content ranged from 0.66 to 3.60 g/kg DM. The highest was found in kale and the

lowest in beetroot. Alfalfa also contained a substantial amount of methionine (3.55

g/kg DM). The lysine content ranged from 1.5 to 13.5 g/kg DM. A similar level of

cysteine was found in all the vegetables and silages ranging from 0.91 to 1.71 g/kg

DM, while a high level (3.15 g/kg DM) of cystine was found in kale. However, the DM

content of fresh summer grass was 246 g/kg DM and 322.3 g/kg DM in fall grass. A

substantial amount of protein (127.1 g/kg DM) was found in summer grass, whereas

slightly less protein (124.3 g/kg DM) was found in fall grass. The methionine and

lysine content of summer and fall grass was quite similar, and an average

concentration of 0.22 g/kg DM methionine and 0.65 g/kg DM lysine was analysed

(data not showed).

Production

The results considering egg production and feed consumption are shown in Table 4.

The laying rate of the birds fed diet B + maize silage was significantly (P<0.001)

higher than that of birds receiving diet E (E1 and E2) + hemp/maize-cob silage.

Further, compared to the control, all diets with forage materials were able to

maintain a comparable laying rate level. Although forage materials supplemented in

diet E (E1 and E2) + hemp/maize-cob silage and diet F (F1, F2 and F3) +

beetroot/carrot/kale were replaced several times, the laying rate was still very

comparable to the control diet. Egg weight was influenced positively by the

supplementation of forage materials and was highest when hens were fed diet D +

grass-herb silage. The hens receiving diet B + maize silage and diet D + grass-herb

silage produced a significantly (P<0.001) higher egg mass than those fed diet C +

alfalfa silage, diet E (E1 and E2) + hemp/maize-cob silage and diet F(F1, F2 and F3)

59

+ beetroot/carrot/kale, whereas the egg mass produced by diet E (E1 and E2) +

hemp/maize-cob silage and F (F1, F2 and F3)+ beetroot/carrot/kale was significantly

lower than that produced by diet A.

Forage materials influenced the intake of the compound feed which was reduced

(P<0.0001) significantly when feeding diet B + maize silage, diet C + alfalfa silage,

diet E (E1 and E2) + hemp/maize-cob silages and diet F (F1, F2 and F3) +

beetroot/carrot/kale. The lowest intake was observed with diet E (E1 and E2) +

hemp/maize-cob silage (111.6 g/hen/d). Birds supplemented with maize silage

showed the highest intake of forage material (48.9 g/hen/d) followed by those

receiving vegetables (beetroot, carrot and kale) (40.4 g/hen/d). The intake of birds

receiving grass herb silage was the lowest (9.4 g/hen/d) (P<0.0001).

The feed conversion in terms of g of layer diet/g of egg mass obtained with diet B +

maize silage and diet E (E1 and E2) + hemp/maize-cob silage was better than the

other diets. Comparatively, a better feed conversion was observed with diet D +

grass-herb silage (2.38). The feed conversion ratio was slightly higher in hens fed

layer diets supplemented with forage materials compared to control diet A.

With the exception of diet E (E1 and E2) + hemp/maize-cob silage and diet C +

alfalfa silage, diet B + maize silage (4.4%), diet D + grass-herb silage (5.2%) and diet

F (F1 + F2 + F3) + beetroot/carrot/kale (2.9%) seemed to reduce overall mortality

compared to diet A (7.41%). The lowest mortality rate was found with diet F (F1, F2

and F3) + beet/carrot/kale (2.96%).

Table 5 illustrates the effect of the diets on feed intake and production performance

from period to period during the experiment. The whole experiment consisted of 6

periods where diet E1 + hemp silage was offered from weeks 22 to 34 and diet E2 +

60

maize-cob silage from weeks 34 to 46 weeks. Similarly, diet F1 + beetroot was fed

from 22 to 30 weeks, diet F2 + carrot fed from 30 to 34 weeks and diet F3 + kale

from weeks 34 to 46 .Overall, there was no significant difference between the diets

with respect to laying rate (Table 5). At the beginning of the study, from 22-26

weeks of age, the laying rate with all diets, except with diet C + alfalfa silage, was

almost similar to diet A. During 26-30 weeks, the highest laying rate was observed in

birds fed diet B + maize silage, and it was even higher than that obtained with the

layer diet A. For the period 30-34 weeks, the laying rate remained static and similar

to the period 26-30 weeks in all supplemented diets, thought a slight increase was

observed with diet A. From 34-38 weeks, the general laying rate was improved in all

diets which was most pronounced when diet B + maize silage and diet D + grass

silage were fed. The laying rate of birds fed diet B + maize silage and diet C + alfalfa

silage from 42-46 weeks was higher than that of layers receiving diet A. For the

whole experimental period (22-46 weeks), diet B + maize silage showed a higher

laying rate compared to diet A. Generally, the laying rate increased gradually in a

time dependent manner and was the highest during the last weeks of the experiment.

In the period from 38-42 weeks, the laying rate suddenly declined in all dietary

groups which coincided with a period of harsh, cold weather and heavy rainfall which

did not allow sufficient intake of forage material from the outdoor area. The highest

egg weight values were obtained from 42-46 week of age. Supplementation of forage

materials had a positive effect on egg weight in all the diets during 22-46 weeks of

age compared to diet A. During 30-34 weeks of age, the egg weight differed

significantly (P=0.007) between the dietary groups where the highest egg weight was

found in hens fed diet D + grass-herb silage compared with the other diets. Due to

the addition of forage materials to the diets, the intake of the layer diets was reduced

significantly in all the supplemented diets. During the period from 22-26 weeks, a

61

significantly lower intake of the layer diets was found in all supplemented diets

except for diet D + grass-herb silage compared to diet A (P<0.0001). This difference

continued until the end of the experiment. The feed intake in all periods was the

lowest in birds fed diet E + hemp/maize-cob silage.

During the period from 22-26 weeks, the forage consumption differed significantly

from diet to diet and was the highest when diet F + beet root/carrot/kale was fed and

the lowest when diet D + grass-herb silage (P<0.0001) was fed. However, from 30-34

weeks of age, the highest silage intake (55 g/hen/d as fed) was found with diet B +

maize silage (P<0.0001) and diet E + hemp/maize-cob silage. Overall, a high

variation in silage and vegetable intake was measured between diets and periods.

Maximum intake of forage supplements was observed during the period from 26 to

38 weeks of age.

Figure 1 shows changes in compound feed intake throughout the experimental

period. Although the initial layer diet intake was reduced, a time dependent increase

in layer diet intake was observed in all experimental groups. The layer diet intake

decreased gradually with increasing forage. Among all the diets, the lowest layer diet

intake was observed in diet E1 supplemented with hemp silages.

The daily protein and amino acids intake per hen fed different experimental layer

diets and forage materials as supplements is shown in Table 6. Compared to the

control group, the protein intake was significantly higher (P<0.001) when diet E2 +

maize-cob silage (25.1 g/h/d) was fed. The protein intake of hens receiving diet C +

alfalfa silage (23.6 g/h/d), diet D + grass-herb silage (23.1 g/h/d) and diet F3 + kale

(23.6 g/h/d) tended to be higher than that of hens fed diet A. However, birds fed diet

E1 + hemp silage showed a lower protein intake than birds receiving diet A. Daily

62

methionine intake per hen from diet B + maize silage, diet E2 + maize-cob silage and

diet F3 + kale was significantly higher than the control diet , and the highest daily

methionine intake was found in hens fed diet E2 + maize-cob silage (437.3mg/h/d).

The highest lysine intake was found in birds fed diet E2 + maize-cob silage (1256.1

mg/h/d) followed by diet B + maize silage (1150.49mg/h/d) and diet C + alfalfa

silage (1100.7 mg/h/d). The highest daily protein intake was from supplemental

hemp silage compared to alfalfa silage (2.0 vs 0.1 g/hen/d). Likewise, the highest

methionine intake was provided by hemp silage (27.4mg/hen/d) compared to

beetroot and carrot which provided the lowest intake (4.5 mg/hen/d). Daily lysine

and cystine intake per hen was also highest from hemp silage compared to the other

forage materials.

The calculated N and P intake, excretion and retention per hen per day from birds

fed with different experimental layer diets and forage materials from 20 to 46 weeks

of age are presented in Table 7. The highest daily N intake and excretion were found

with diet E2 + maize-cob silage, whereas the P intake and excretion were highest

with layer diet B + maize silage. The nitrogen excreted ranged from 69.8 to 73.5% of

total N intake, and the N retained from the different diets ranged from 26.6 to

30.6%. The highest N retention was observed when hens were fed diet B + maize

silage, diet D + grass as well as diet F3 + kale, and the lowest N retention was found

with diet F2 + carrot and diet E2 + maize-cob silage. On the other hand, the highest

P retention occurred in birds fed diet D + grass silage and diet F3 + kale. The P

retention ranged from 15.8 to 18.1%.

Forage supplementation improved the plumage condition at 35 weeks of age (Table

8). The total mean score for plumage condition was significantly higher in birds fed

63

diet B + maize silage and diet C + alfalfa silage compared to diet A (P=0.004). The 6

individual body parts were scored separately, and a significant difference was found

between the dietary groups with respect to the plumage of the breast. Diet B + maize

silage resulted in a good plumage quality of the breast part compared to hens fed diet

A. A lower number of hens with bumble feet were found in the groups receiving

supplemental forage as compared to the non-supplemented control group. At 44

weeks of age, the total plumage condition score had increased compared to the values

obtained at 35 weeks. However, except for the group receiving diet D + grass-herb, a

lower total plumage score (P<0.0001) was observed when forage was fed. Overall,

the plumage condition was very good at the end of the experiment, and no feather

pecking behavior was observed. At that time, the feeding of supplemental forage

reduced the incidence of bumble feet significantly compared to the non-

supplemented control diet (P<0.001).

DISCUSSION

Taking into account the nutrients of different forage materials, feeding of 100%

organic diets together with different kinds of foraging materials to hens in the

present experiment had a positive effect on production performance. The highest egg

production observed in hens fed diet B + maize silage (90.11%) clearly indicates that

a combined intake of layer diet and maize silage leads to a higher methionine intake

(433 mg/hen/d) resulting in a higher egg production. Many studies revealed that

methionine and lysine can have a positive effect on egg production (Hammershøj and

Steenfeldt, 2005; Sundrum et al., 2005; Harms et al., 1998). Further, it has been

shown that moderate levels of insoluble fibres with a reduced nutrient concentration

in diets do not have a negative effect on production performance of broilers or layers

(Hetland and Sivhus, 2001; Hetland et al., 2002). In the study by Steenfeldt et al.

64

(2007), the feeding of maize silage resulted in an increased laying rate in organic egg

production. Overall, the positive effect on egg production in the present experiment

was lower in birds fed diet E (E1and E2) + hemp/maize-cob silage as well as diet F

(F1, F2 and F3) + beetroot/carrot/kale, although with diet E1 + hemp silage, the daily

methionine intake was 332 mg/hen/d and with diet E2 + maize-cob silage 458

mg/hen/d which exeeds the level recommended by NRC (1994) which suggests

300mg/hen/d. This result may be due to a higher intake of silages resulting in a

lower intake of both layer diets. Diets supplemented with either carrot (Hammershøj

et al., 2010) or kale (Hammershøj and Steenfeldt, 2012) were able to enhance the

laying rate.

Al-Bustany and Elwinger (1987) and Hammershøj and Kjaer (1999) suggested that

during the laying period, sufficient protein, lysine and methionine intake is essential

for optimal egg production (egg number, egg weight, egg mass and feed efficiency).

Furthermore, Karunajeswa et al. (1987) stated that an increase of the lysine level

from 0.7%-0.8% resulted in a higher egg mass production. In this study, a higher egg

mass was found when diet B + maize silage was fed. This might be a consequence of

the higher intake of methionine (433.38 mg/hen/d) and lysine (1183 mg/hen/d)

from both diet and silage (Table 6). Organic layer diets were in line with the NRC

(1994) recommendation for barn or enriched caged hens; however, in organic

systems, hens must have outdoor access and are thus exposed to high variations in

temperature. Therefore, hens require a higher methionine content than

recommended by NRC for optimal egg production (Elwinger et al., 2008; Al-Saffar

and Rose, 2002; Van Krimpen et al. 2015). A study by Van Krimpen et al. (2015)

found that different factors, e.g. the methionine content of the diets and the ambient

temperature (winter vs. summer periods), could influnce the daily methionine

65

requirement. A major limitation of organic egg production is the unavailability of

locally produced protein sources rich in essential amino acids. Previous results

indicate that some soya bean cultivars might have the potential to grow under

temperate the weather conditions in Northern Europe and could be an important

part of homegrown protein and amino acids source in future organic poultry

production (Vollmann et al., 2000; Edlefsen et al., 2008; Palermo et al., 2012).

However, more research into new cultivars and development of crop management to

achieve a higher and stable yield is necessary. In our study, soya beans grown in the

southern part of Denmark were highly productive yielding 2.2 t/ha cleaned and dry

seed, and the protein content and amino acid profile obtained reflected soya beans of

a high quality comparable to soya beans grown in the major producing countries

such as the USA, Brazil and China (Grieshop and Fahey, 2001).

Recent research found that laying hens can consume up to 120g/hen/d of forage

materials (Steenfeldt et al., 2007; Hammershøj et al.,2005) or 70g/hen/d forage on

DM basis (Horsted et al., 2006). However, the intake of forage materials depends on

the type of forage materials. In addition to this, it was demonstrated that the intake

of forage materials decreased layer diet intake without altering egg production which

suggests that forage materials can supply nutrients to the hens (Hammershøj et al.,

2010; Steenfeldt et al., 2007; Horsted et al., 2006). The intake of forage materials

may in general reduce the feed consumption by up to ~ 20% (Blair, 2008). Steenfeldt

et al. (2007) reported that a 12% reduction in layer diet intake was possible without

altering egg production when birds were fed 108g/hen/d carrot as forage. The results

of the present study show that hens consuming 37g/hen/d hemp silage had a 11%

reduction in layer diet intake, whereas 49g/hen/d of maize silage consumption

caused a 6% reduction of layer diet intake. Bassler et al. (2000) estimated that

66

reducing the quantity of concentrate fed to layers by 15 % has no detrimental effect

on productivity. However, the hens need a certain period of time to increase their

intake of forage materials. In this study, maize and hemp silage as well as vegetables

(beetroot, carrot and kale) intake increased over time, and the highest consumption

was observed at 30 weeks of age. The lower intake of alfalfa silage or grass-herb

silage might be due to high dietary fibre (46 to 50% of DM) that causes the poor

quality of silage in spite of high methionine content (24 to 36% of DM). Moreover,

the experimental outdoor area was covered with fresh grass for all dietary groups

except for the non-supplemented control group, and the chemical composition of the

grass-herb silage provided in diet D was similar to that of fresh grass. As a result,

hens might have preferred to intake fresh grass instead of grass herb silage.

Consequently, hens were urged to cover the nutrient requirement through a high

intake of layer diets (122 to 123 g/hen/d) compared to other diets. The layer diet

intake in all groups increased greatly during weeks 38-42 of the study. This

phenomenon might be explained by environmental factors, as low temperature

together with heavy rain affected forage material intake. Feather pecking is a crucial

issue in organic farming, and layer diets deficient in protein or methionine and

cystine can cause feather pecking and cannibalism (Ambrosen and Petersen, 1997).

Results from the present study demonstrate sufficient intake of protein and amino

acids, hence feather pecking behavior was not observed in any of the dietary groups

during the experiment. In addition, the plumage quality during the experiment was

very appreciable. Compared to diet A, the total plumage condition score was much

better in diet B + maize silage and diet C + alfalfa silage at 35 weeks of age, and at the

end of the study, the total plumage condition score was significantly better in groups

supplemented with foraging materials. This finding is in line with the idea that if

hens spend more time eating and foraging, and if they are feeling satiated longer,

67

they will pay less attention to each other (Vam Krimpen et al., 2005). With access to

an outdoor area, the hens demonstrated the possibility to express their innate

foraging behavior as well as dust bathing activities which reduced the risk for

unwanted behavior such as feather pecking (Knierim, 2006). It was also reported

that chickens are attracted by forage, and birds are motivated to consume forage

even when provided with adequate feed (Cooper and Albentosa, 2003). Much

research has to be done to confirm the importance of forage and roughage from an

animal welfare point of view (Kjaer et al., 2001; Buitenhuis et al., 2006; Steenfeldt et

al., 2007; Horsted et al; 2006).

Imbalanced amino acids and insufficient dietary methionine levels increase the

nitrogen excretion to the environment (Summers, 1993). In the present study, all

diets supplemented with forage materials were balanced with respect to protein and

amino acids of the foraging materials. The calculated N retention values for the

supplemented diets were comparable with the control diet A, except when hens were

fed diets with access to either maize-cob silage or beetroot (P<0.001) indicating that

it was possible with most of the diets to include the foraging material as an

ingredient in the diet formulation and fulfil the requirement for protein and amino

acids. Steenfeldt et al. (2007) found the N retention in laying hens less efficient when

feeding carrot compared to maize silage and barley pea silages. In our experiment,

the hens were less capable of utilising nitrogen from the carrot supplemented diet

which is in line with the findings of Steenfeldt et al. (2007). In general, 25-30%

nitrogen of the total nitrogen intake was retained for egg production on average,

whereas 12-15% total phosphorus was retained for egg production in organic hens

(Kristensen, 1998; Hegelund et al., 2005). The remaining 49% N and 82% P were

found in excreta (Kristensen (1998). In this study, 26-30% N of total intake was

68

found which is in line with laying hens of organic egg production. A study by

Swiatkiewicz et al. (2009) found that organic layers retained 22.5-23.4% nitrogen of

total nitrogen intake which was slightly lower than our results (26.6-30.6 % N

retained as % of N intake). On the other hand, in the ecosystem, nitrogen and

phosphorus in the soil are more or less balanced, because animals take up nitrogen

and phosphorus by feeding on the vegetation and return most of this nitrogen and

phosphorus in faeces and urine (Dekker et al., 2012). However, if N and P retention

is low and excretion is high, there will be unutilised N and P in the nature causing

detrimental effect on the environment. Equal distribution of hens in the outdoor area

may increase foraging which will result in a balanced nitrogen cycle in the organic

production system (Dekker et al., 2012).

Feeding forage materials resulted in reduced mortality rates except with diet E (E1

and E2) + hemp/maize-cob and diet C + alfalfa silage. In diet B + maize silage and

diet F (F1, F2 and F3) + beetroot/carrot/kale, the mortality rate was 4.44% and

2.96%, respectively, whereas in the control group, the mortality was 7.41%. An

improved immune response in laying hens with access to forage materials has been

reported by El-Lethey et al.(2000) which may be the reason for the lower mortality

observed in forage supplemented bird in the present experiment. The mortality rate

of organic laying hens in Denmark was 7.1% in 2013 (Danish poultry council, 2013).

Previously, Steenfeldt et al. (2007) observed a lower mortality rate in hens receiving

supplements of maize silage (1.5%) and carrot (0.5%) compared to non-

supplemented birds control diet (15%) which was slightly lower than our mortality

rate which may be because the hens in our study had outdoor access, while they

performed experiment indoor. It may be concluded that the access to forage

materials has a beneficial effect on feather pecking behavior, plumage condition,

69

body weight gain and mortality. The supply of maize silage and kale has a tendency

to increase egg production indicating that these forage materials had some nutritive

value. Altogether, the supplementation of silages and vegetables, especially maize

silage and kale, to layers may help reach the goal to formulate 100% organic diets.

Thus, it could be recommended for the organic egg production to introduce a new

feeding strategy.

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Table1 Ingredient composition and calculated nutrient content of experimental diets (as fed g/kg)

Ingredients Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3 Wheat 500 393 393(460) 420(490) 573 365 465 480 510 Oat 100 100 100 100 100 80 100 100 10 Barley 50 50 50 50 50 50 50 50 50 Soya bean 120 110 130 130 130 130 130 130 110 Soy bean cake ̶ 90 70(30) 30 ̶ 100 30 30 80 Sunflower meal 80 70 80 80 ̶ 50 50 80 Rapeseed cake 30 30 30 30 30 40 30 30 30 Fishmeal 22 37 25(20) 37.7(22) 31 48 34.5 27 20 Rapeseed Oil 4.0 4.0 4.0 6.0 4.0 6.0 5.0 5.0 4.0 Alfalfa meal 20 20 20 20 20 20 20 20 20 Calcium carbonate 56 75 75(58.3) 75.8(56.7) 49.4 91.6 69.8 62.5 58.8 Monocalcium phosphate

9.7 11 12(9.9) 11.3(9.4) 4.6 10 8.6 8.7 9.2

Sodium bicarbonate 2.5 3 3.3 2.8 2.0 1.9 1.0 1.1 1.8 Sodium chloride 1.0 1.0 1.1 0.9 1.0 2.0 1.1 1.2 1.5 Choline chloride 50% 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Vitamin mineral mixa 4.0 5.0 5.0(4.0) 5.0(4.0) 4.0 5.0 4.5 4.0 4.2 RoxazymeG2 Gb 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Calculated content (g/kg)

ME, MJ per kg 10.9 10.6 10.6(10.6) 10.7(10.9) 11.2 10.6 10.9 11.0 10.9 Protein 178.0 208.0 204.0(179.0) 198.0(171.0) 169.0 217.0 190.0 179.0 162.32 Fat 55.2 58.8 60.5 61.4 51.2 72.2 57.8 61.6 59.6 Starch 348.0 291.8 291.1 304.3 387.4 253.9 329.1 337.5 353.6 Sugar 31.4 37.3 37.4 33.8 30.7 40.0 33.5 33.9 30.7 Lysine 8.3 10.5 10.0(8.7) 9.8(8.2) 8.3 11.6 9.5 8.9 7.6 Methionine 3.1 3.6 3.4(3.0) 3.5(2.9) 2.8 3.8 3.3 3.0 3.3 Methioe + Cystine 6.2 7.3 7.1(6.4) 7.1(6.2) 6.1 7.5 6.8 6.4 6.0 Calcium 25.7 33.9 33.8(26.4) 34.0(25.8) 22.4 40.1 31.0 27.9 26.5 Total Phosphorous 6.9 7.6 7.8(7.0) 7.6(6.9) 5.4 7.7 6.8 6.7 6.7

a The vitamin and mineral premix provided per kg of diet: Vitamin A, 12,000 IU; Vitamin D3, 3000 IU; Vitamin E, 25 mg; Vitamin K3, 2 mg; Vitamin B1; 4 mg; Vitamin B2, 5 mg; Vitamin B6, 8 mg; D-pantothenic acid, 12 mg; Niacin, 45 mg; Choline, 200 mg; Folic acid, 1,4 mg; Vitamin B12 , 0.01 mg; Fe, 60 mg; Zn, 80 mg; Mn, 80 mg; Cu, 15 mg; I, 0.45 mg; Se, 0.2 mg. b Roxazyme: endo-1,4 betaglucanase (IUB nr. 3.2.1.4), endo-1,3:1,4 betaglucanase (IUB nr. 3.2.1.6), xylanase (IUB nr. 3.2.1.8). 2600 units/kg diet. EU nr. E1602.

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Table 2a Chemical composition of experimental layer diets (g/kg dry matter)

Diet A Diet B Diet C* Diet D* Diet E1 Diet E2 Diet F1 Diet F2 DietF3 Dry matter 887.7 890.8 887.7- 889.5 885.4-886.9 885.6 897.9 887.9 883.4 896.3

Protein(N × 6 .25) 205.5 207.7 218.4-218.4 214.4-209.1 195.6 252.19 204.7 209.4 212.5 Ash 104.7 126.4 128.3-111.7 128.1-101.5 96.4 139.9 124.9 117.2 91.6

Fat 68.1 67.1 74.4-68.7 75.9-68.4 63.4 71.4 68.7 68.1 61.7

Ca 27.5 34.7 35.7-31.4 35.0-26.7 24.9 38.4 34.3 31.7 23.3

P 7.4 8.3 8.1-6.8 8.1-6.8 6.4 8.3 7.3 7.6 7.1 NCP1

Rhamnose 1.1 1.1 1.3-0.9 1.2-1.0 1.1 1.3 1.0 1.0 0.8 Fucose 0.6 0.6 0.9-0.4 0.6-0.5 0.5 0.8 0.4 0.5 0.4 Arabinose 22.6 20.6 18.0-21.3 20.5-22.1 23.2 20.9 20.1 20.3 23.2 Xylose 36.4 33.1 27.6-35.5 31.6-38.1 34.8 33.0 31.1 38.2 38.4 Mannose 4.4 4.5 4.5-4.2 4.7-4.4 3.7 4.8 4.0 4.0 4.4 Galactose 9.3 10.7 11.7-9.9 11.0-9.9 9.5 12.5 9.0 9.9 9.5 Glucose 46.1 46.0 46.5-47.8 45.6-53.1 40.6 48.6 42.5 50.0 47.0 Uronic acids 12.6 13.2 14.7-14.7 14.0-8.7 9.3 14.7 12.2 11.8 11.0

Total NSP2 133.1 130.3 125.1-134.9 129.1-138.0 122.6 136.8 120.4 135.6 134.7 Lignin 37.4 37.2 48.5-33.7 56.3-34.1 39.4 31.0 42.6 46.1 31.3 Dietary fiber 3 170.5 167.2 173.6-168.6 185.5-162.1 162.1 167.7 162.9 181.7 166.1

1NCP: Non-cellulosic polysaccharides. 2Total NSP: Soluble NSP + Insoluble NSP. 3 Dietaryfibre: NSP+lignin.

*Diet C and diet D: Re-formulated diet after 2 weeks of experiments

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Table 2b Amino acid content of experimental layer diets (g/kg dry matter)

Diet A Diet B Diet C* Diet D* Diet E1 Diet E2 Diet F1 Diet F2 DietF3 Alanin 8.51 9.76 9.25-9.21 9.18-8.44 8.33 10.99 9.16 9.19 8.98 Arginine 12.52 14.57 14.72-13.99 14.36-13.09 12.46 16.61 12.98 13.01 13.1 Asparagine 15.73 18.94 18.92-17.41 18-15.89 15.38 21.49 16.5 16.71 16.09 Cystine 3.45 3.72 3.54-3.69 3.43-3.6 3.27 4.23 3.38 3.35 3.84 Glutamin 40.25 42.675 40.81-44.99 39.89-43.03 38.49 47.74 39.79 38.86 43.84 Glycine 9.18 10.42 9.99-9.78 9.98-9.14 8.9 11.66 9.72 9.59 9.43 Histidine 4.64 5.23 5.16-5.07 4.98-4.73 4.51 5.85 4.78 4.7 4.8 Isoleucin 8.00 9.21 8.92-8.81 8.61-8.26 7.78 10.34 8.24 8.17 8.37 Lysine 9.02 10.92 10.69-9.92 9.77-9.12 9.22 12.61 9.77 10.05 9.29 Leucin 13.65 15.39 14.79-15.11 14.37-13.95 13.21 17.26 14.29 14.16 14.55 Methionine 3.45 3.90 3.48-3.63 3.55-3.46 3.11 4.39 3.5 3.57 3.65 Phenylalanine 8.97 10.06 9.94-9.85 9.94-9.22 8.78 11.19 9.09 8.99 9.45 Proline 12.72 13.29 12.28-14.34 12.28-13.65 12.5 14.71 12.92 12.58 13.87 Serine 9.56 10.75 10.42-10.57 10.42-9.86 9.18 12.08 9.83 9.77 10.1 Threonine 6.98 8.06 7.7-7.65 7.7-7.07 6.7 9.09 7.28 7.35 7.26 Tyrosine 5.87 6.72 6.58-6.63 6.58-6.1 5.8 7.41 6.23 6.24 6.03 Valine 9.52 10.73 10.61-10.14 10.39-9.51 9.47 11.78 9.5 9.44 9.67 *Diet C and diet D: Re-formulated diet after 2 weeks of experiments

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Table 3a Chemical composition of different silages and vegetables (g/kg DM)

Nutrients Maize silage

Alfalfa silage

Grass-herb silage

Hemp silage

Maizecob silage

Beetroot Carrot Kale

Dry matter 317.2 300.7 367.5 354.7 352.7 108.9 78.9 150.3 Protein (N x 6.25) 95.3 249.7 181.9 189.4 83.8 156.6 102.8 268.4 Ash 34.3 123.8 93.8 130.3 24.6 91.5 88.2 100.1 Fat 19.2 32.9 29.9 91.8 22.6 2.6 6.4 47.20

Gross energy (MJ/kg DM) 19.2 18.8 19.13 19.3 18.3 15.9 17.2 19.0 Calcium 1.4 16.44 10.40 27.25 1.06 1.93 3.83 14.24 Phosphorus 2.80 3.80 3.38 7.03 2.87 3.21 3.00 5.41

Potassium 11.22 29.13 25.87 15.98 7.80 39.09 35.14 24.83 Sodium 0.31 0.77 2.18 0.70 0.14 2.51 2.51 1.2 Starch 259.4 8.80 10.7 7.50 406.8 0.6 4.2

Sugar Glucose 1.4 0.1 13.3 ̶ 3.1 26.5 140.9 47.7

Fructose ̶ ̶ 9.5 ̶ ̶ 19.8 14.72 43.4 Sucrose ̶ 5.0 8.6 ̶ 1.9 556.3 312.5 20.6 Raffinose ̶ ̶ 6.9 40.7

Stachyose ̶ ̶ 1.1 Verbascose ̶ ̶ ̶ ̶ ̶ ̶ Other 1.0 1.0 0.1

Total 1.90 5.40 38.2 0.3 5.4 610.5 601.6 155.5 NSP1 Cellulose 187.0 173.0 188.0 173.0 117.0 52.0 76.0 61.0

NCP2 Rhamnose 1.0 4.0 3.0 4.0 1.0 2.0 4.0 5.0

Fucose 0 1.0 1.0 1.0 0 0 0 2.0 Arabinose 29.0 15.0 23.0 12.0 27.0 46.0 16.0 56.0

Xylose 150 47.0 67.0 65.0 121.0 4.0 3.0 12.0

Mannose 3.0 12.0 7.0 12.0 20.0 3.0 7.0 5.0 Galactose 10.0 13.0 16.0 11.0 9.0 15 17 31.0 Glucose 13.0 4.0 11.0 8.0 10.0 3.0 3.0 3.0

Uronic acids 13.0 84.0 69.0 83.0 10.0 59.0 102.0 131.0 Total NSP3 405.0 353.0 384.0 369.0 297.0 186.0 228.0 305.0

Soluble NSP 21.0 79.0 71.0 68.0 8.0 73.0 121.0 187.0

Insoluble NSP 384.0 274.0 313.0 301.0 289.0 113.0 107.0 118.0 Lignin 93.0 109.0 119.0 167.0 54.0 16.0 14.0 28.0

Dietary fiber (NSP+Lignin) 498.0 462.0 503.0 536.0 351.0 202.0 242.0 333.0 1NSP: Non-starch polysaccharides. 2NCP: Non-cellulosic polysaccharides. 3Total NSP: Soluble NSP + Insoluble NSP.

78

Table 3b Amino acid content of different silages and vegetables (g/kg DM)

Maize

silage Alfalfa silage

Grass -herb silage

Hemp silage

Maizecob ilage

Beetroot Carrot Kale

Alanin 7.60 20.99 10.55 9.61 6.82 2.62 10.27 10.32

Arginine 2.19 3.77 4.98 8.15 1.52 4.63 3.21 17.11

Asparagine 4.30 17.16 16.58 11.05 3.54 8.17 15.64 20.54

Cystine 1.23 1.71 1.09 1.71 1.32 0.95 0.91 3.15

Glutamin 7.99 14.39 14.02 14.64 7.87 42.78 19.85 35.11

Glycine 3.74 10.86 8.32 7.41 3.12 2.30 2.49 10.16

Histidine 1.34 3.53 3.35 2.84 1.51 1.83 1.50 5.56

Isoleucin 3.16 10.77 7.76 6.47 2.67 2.69 2.72 8.23

Leucin 7.46 16.48 12.59 10.29 7.18 3.56 3.33 14.30

Lysine 2.08 8.06 8.32 5.64 1.29 3.37 3.15 13.62

Methionine 1.45 3.55 2.44 2.61 1.34 0.66 0.97 3.60

Phenylalanine 3.72 10.76 8.08 6.75 3.26 1.70 2.80 9.30

Proline 5.38 11.53 8.58 6.54 5.54 2.06 2.23 31.08

Serine 3.01 4.23 7.49 5.89 2.69 3.50 4.15 9.59

Threonine 2.77 5.44 7.11 4.87 2.63 2.67 2.64 8.90

Tyrosine 1.89 3.43 4.65 3.82 1.44 2.39 1.78 6.62

Valine 4.37 12.42 9.55 8.18 3.91 3.56 3.84 11.35

79

Table 4 The effect of experimental diets on egg production, layer diet and forage intake, body weight and mortality (20 to 46 wk.)

Diet A Diet B

Diet C

Diet D

Diet E1 Diet F2 SEM3 P-Value

Maize silage

Alfalfa silage

Grass-herb silage

Hemp/maize-cob silage

Beetroot/car-rot/kale

Egg production

Laying rate, % 89.05ab 90.11a 87.16b 89.26ab 86.68b 87.39ab 0.36 0.0028

Egg weight, g 62.19ab 62.11ab 62.28ab 62.92a 61.89bc 61.28c 0.18 0.0001

Egg mass, g/hen/d 55.41ab 56.02a 54.35bc 56.20a 53.68c 53.59c 0.32 0.0001

Feed consumption

Layer diet, g/hen/d 125.76a 118.27d 121.76bc 123.42ab 111.61e 120.42cd 0.55 0.0001

Forage materials, g/hen/d - 48.89a 13.27d 9.45e 36.93c 40.42b 1.59 0.0001

Feed conversion ratio (g of feed/g of egg)

Layer diet 2.27a 2.12b 2.25a 2.21a 2.09b 2.26a 0.01 0.0001

Forage materials - 0.87a 0.25c 0.17c 0.68b 0.79ab 0.03 0.0001

Layer diet + forage materials 2.27d 2.99a 2.50c 2.38cd 2.77b 2.99a 0.03 0.0001

Initial body weight, g 1168.73a 1149.61ab 1152.77ab 1149.13ab 1151.36ab 1131.27b 2.99 0.0205

Final body weight, g 1829.02 1834.06 1831.87 1832.60 1833.68 1835.19 5.25 0.9996

Mortality, % 7.41 4.44 7.41 5.19

11.11

2.96

0.94 0.1567

1 22-34 wk: Hemp silage: 34-46 wk: Maize-cob silage. 2 22-30 wk: Beetroot; 30-34 wk: Carrot; 34-46 wk: Kale. a,b,c,d,eMeans in each raw followed by different superscript letters differ significantly. 3PooledStandard error of mean

Diet A without forage materials, Diet B + maize silage, Diet C + alfalfa silage, Diet D + grass/herb silage, Diet E + hemp/maize-cob silage and diet F + beetroot/carrot/kale.

80

Table 5 The effect of experimental diets on feed intake and production performance by period (20 to 46 wk)

Diet A Diet B

Diet C

Diet D

Diet E Diet F SEM1 P-Value

Maize silage

Alfalfa silage

Grass-herb silage

xHempy/ymaize-cob silage

pBeetroot/qcarrot/zkale

Laying rate, %

22-26 weeks 86.13 84.31 81.91 84.40 x84.20 p84.79 0.77 0.6754

26-30 weeks 87.45 90.40 84.26 88.62 x85.73 p86.82 0.79 0.2616

30-34 weeks 88.29 89.85 84.49 88.64 x85.97 q86.49 0.81 0.4257

34-38 weeks 90.76 91.80 89.68 91.34 y86.02 z90.31 0.77 0.0607

38-42 weeks 88.61 89.42 88.46 89.00 y86.07 z86.07 0.60 0.3882

42-46 weeks 93.07 94.85 94.14 93.56 y92.11y z90.43 0.73 0.5204

Egg weight, g

22-26 weeks 58.34 58.19 57.89 57.84 57.90 57.61 0.19 0.9242

26-30 weeks 60.68 61.49 61.33 62.10 60.97 59.98 0.23 0.1444

30-34 weeks 62.03ab 62.59ab 62.63ab 63.00a 61.39b 61.29b 0.18 0.0069

34-38 weeks 63.26 63.63 63.71 64.69 63.05 62.79 0.25 0.3398

38-42 weeks 64.31 63.61 64.25 65.07 63.86 63.21 0.21 0.1822

42-46 weeks 64.50 63.17 63.85 64.82 64.16 63.18 0.20 0.0768

Layer diet intake, g/hen/d

22-26 weeks 121.13a 114.83b 117.86ab 122.86a 113.31b 114.41b 0.82 0.0001

26-30 weeks 122.80a 116.74ab 119.48a 123.22a 111.77b 117.84ab 0.97 0.0011

30-34 weeks 121.83a 111.52bc 117.46a 120.15a 107.24c 118.66a 1.13 0.0001

34-38 weeks 124.07a 117.48b 120.19ab 119.13ab 105.96c 117.94b 1.17 0.0001

38-42 weeks 132.32a 120.73b 123.73ab 126.68ab 111.76c 126.22ab 1.41 0.0001

42-46 weeks 132.42a 128.35ab 131.83a 128.47ab 119.63b 127.42ab 1.15 0.0086

Forage materials

81

intake, g/hen/d

22-26 weeks 36.93b 13.02cd 8.58d 18.55c 53.57a 3.42 0.0001

26-30 weeks 53.88b 15.02d 12.10d 39.53c 71.99a 4.72 0.0001

30-34 weeks 54.68a 16.17c 12.35c 31.00b 58.38a 4.05 0.0001

34-38 weeks 52.48a 12.95d 9.27d 42.50b 24.38c 3.47 0.0001

38-42weeks 49.79a 10.22bc 6.39c 43.84a 15.92b 3.57 0.0001

42-46 weeks 45.55a 12.21bc 8.00c 46.21a 18.27b 3.40 0.0001

x22-34 wk: Hemp silage. y 34-46 wk: Maize comb silage. p22- 30 wk: Beetroot. q30-34 wk: Carrot. z 34-46 wk: Kale. a,b,c,d Means in each raw followed by different superscript letters differ significantly. 1Pooled Standard error of mean.

Diet A without forage materials, Diet B + maize silage, Diet C + alfalfa silage, Diet D + grass/herb silage, Diet E + hemp/maize-cob silage and Diet F + beetroot/carrot/kale.

82

Table 6 Protein and amino acid intake with different experimental layer diets and forage materials (silages and vegetables) (20 to 46

weeks)

a,b,c,d,e,f Means in each raw followed by different superscript letters differ significantly. 1Pooled Standard error of mean.

22-34 wk: Hemp silage: 34-46 wk: Maize-cob silage. 22-30 wk: Beetroot; 30-34 wk: Carrot; 34-46 wk: Kale.

Diet A Diet B Diet C

Diet D

Diet E1 Diet E2 Diet F1 DietF2 Diet F3 SEM1 P-value

Maize silage

Alfalfa silage

Grass-herb silage Hemp silage

Maize-cob silage Beetroot Carrot Kale

Layer diet

Protein, g/hen/d 22.94bc 21.88cd 23.64b 23.06bc 19.19e 25.12a 21.10d 21.95cd 23.59b

0.13

0.0001

Meth, mg/hen/d 385.16cd 410.89b 387.61cd 381.79d 305.09f 437.31a 360.87e 374.22de 405.21bc

2.72

0.0001

Lysine, mg/hen/d 1007.00d 1150.49b 1100.70bc 1021.34d 904.48e 1256.13a 1007.34d 1053.49cd 1031.35d

7.69

0.0001

Cystine, mg/hen/d 385.16b 391.93b 394.11b 387.67b 320.79d 421.37a 348.50c 351.16c 426.31a

2.50

0.0001

Forage materials

Protein, g/hen/d ̶ 1.48b 1.00d 0.63ef 2.00a 1.31bc 1.07cd 0.47f 0.79de

0.04

0.0001

Meth, mg/hen/d ̶ 22.49b 14.16c 8.46de 27.43a 20.88b 4.51e 4.47e 10.56cd

0.67

0.0001

Lysine, mg/hen/d ̶ 32.26bc 32.15bc 28.84cd 59.69a 20.10de 23.04cde 14.51e 39.96b

1.18

0.0001

Cystine, mg/hen/d ̶ 19.07a 6.82bc 3.78d 17.96a 20.57a 6.50bcd 4.19cd 9.24b

0.58

0.0001

83

Table 7 N and P balance fed different experimental layer diets and forage (20 to 46 weeks)

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3

SEM1

P-value

Maize silage

Alfalfa silage

Grass-herb silage

Hemp silage

Maize-cob silage Beetroot Carrot Kale

N intake, g/hen/d

Layer diet 3.670bc 3.838b 3.783b 3.694bc 3.071e 4.019a 3.377d 3.512cd 3.775b

0.022

0.0001

Forage - 0.236b 0.159d 0.101ef 0.318a 0.209bc 0.171cd 0.076f 0.126de

0.007

0.0001

Later diet + forage 3.670de 4.074ab 3.942bc 3.795cd 3.389f 4.228a 3.548ef 3.587e 3.900bc

0.022

0.0001

N retention, g/hen/d 1.107ab 1.122a 1.091abc 1.125a 1.036bc 1.123a 1.025c 1.071abc 1.123a

0.006

0.0001

N excretion,g/hen/d 2.563e 2.952b 2.851bc 2.670de 2.353f 3.105a 2.523e 2.516e 2.778cd

0.019

0.0001 N retained as % of N intake 30.178a 27.550bc 27.675bc 29.683a 30.579a 26.618c 28.887ab 29.849a 28.816ab

0.155

0.0001

P intake, g/hen/d

Layer diet 0.827ab 0.871a 0.791b 0.795b 0.632c 0.825ab 0.767b 0.795b 0.783b

0.006

0.0001

Forage 0.043b 0.015cd 0.011d 0.074a 0.044b 0.022c 0.013cd 0.016cd

0.002

0.0001

Layer diet + forage 0.827bc 0.915a 0.806c 0.806c 0.706d 0.869ab 0.789c 0.808c 0.799c

0.006

0.0001

P retention, g/hen/d 0.135ab 0.137a 0.134ab 0.138a 0.128b 0.137a 0.127b 0.132ab 0.138a

0.001

0.0002

P excretion, g/hen/d 0.692bc 0.777a 0.672c 0.669c 0.578d 0.732ab 0.662c 0.676c 0.661c

0.005

0.0001 P retained as % of N intake 16.350bc 15.016c 16.714ab 17.232ab 18.097a 15.825bc 16.077bc 16.322bc 17.261ab

0.121

0.0001

a,b,c,d,eMeans in each raw followed by different superscript letters differ significantly. 1Pooled Standard error of mean.

22-34 wk: Hemp silage: 34-46 wk: Maize-cob silage 22-30 wk: Beetroot; 30-34 wk: Carrot; 34-46 wk: Kale.

84

Table 8 Plumage scoring, bumble feet, cloacal infection fed different experimental layer diets and forages (35 and 44 weeks)

Diet A Diet B

Diet C

Diet D

Diet E1 Diet F2 SEM3 P-Value

35 week Maize silage

Alfalfa silage

Grass silage

Hemp/maize-cob silage

Beetroot/carrot/kale

Plumage condition4

Total scores (0-3)5 0.292a 0.100b 0.117b 0.200ab 0.152ab 0.157ab 0.153 0.0039 Separate body parts

Neck 0.008 0.000 0.000 0.000 0.000 0.000 0.001 0.4176

Breast 0.233a 0.085b 0.117ab 0.192ab 0.138ab 0.157ab 0.013 0.0210

Rump 0.050 0.069 0.000 0.008 0.014 0.000 0.006 0.1203

Bumble feet (yes/no)6 0.333 0.220 0.166 0.117 0.188 0.235 0.021 0.0809

Clocal infection (yes/no)6 0.050 0.085 0.050 0.068 0.058 0.078 0.013 0.9598

44 weeks

Plumage condition 4

Total scores (0-3)5 0.628a 0.375bc 0.302c 0.504ab 0.384bc 0.395bc 0.019 0.0001

Separate body parts

Neck 0.004 0.000 0.000 0.004 0.000 0.004 0.001 0.7108

Breast 0.384ab 0.345b 0.302b 0.492a 0.333b 0.355b 0.014 0.0013

Rump 0.233a 0.030b 0.000b 0.000b 0.000b 0.027b 0.011 0.0001

Bumble feet (yes/no)6 0.085a 0.053ab 0.008b 0.008b 0.009b 0.016b 0.006 0.0003

Clocal infection (yes/no)6 0.008 0.031 0.000 0.000 0.009 0.031 0.004 0.0728

a,b,c Means in each raw followed by different superscript letters differ significantly. 3Pooled Standard error of mean. 122-34 wk: Hemp silage: 34-46 wk: Maize-cob silage. 222-30 wk: Beetroot; 30-34 wk: Carrot; 34-46 wk: Kale. 4A maximum of 0 points for good quality plumage, a minimum of 3 points for a very poor plumage. 5Total score is the sum of body part scores, max 18 and min 6 points. 6 Both bumble feet and cloacal infection were evaluated by the present and absent.

Diet A without forage materials, Diet B + maize silage, Diet C + alfalfa silage, Diet D + grass/herb silage, Diet E + hemp/maize-cob silage and Diet F+ beetroot/carrot/kale.

85

Figure1 Average intake of the layer diets (g/hen/d) calculated at a 4 week basis

80.00

90.00

100.00

110.00

120.00

130.00

140.00

1819202122232425262728293031323334353637383940414243444546

A-Control

B-Maize

C-Alfalfa

D-Grass-herb

E1-Hemp

F1-Beetroot

E2-Maize comb

F2-Carrots

F3-Kale

86

5.2 Manuscript 2

Effect of feeding 100% organic diets and forage supplements on apparent

nutrient digestibility, metabolisable energy and microbial activity in laying

hens

S. AFROSE1, R. M. ENGBERG1 and S. STEENFELDT1

Prepared for submission to British Poultry Science

87

Effect of feeding 100% organic diets and forage supplements on apparent

nutrient digestibility, metabolisable energy and microbial activity in laying

hens

S. AFROSE1, R. M. ENGBERG1 and S. STEENFELDT1

1 Dept. of Animal Science, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark

Corresponding author: Sanna Steenfeldt

E-mail: sanna.steenfeldt@anis.au.dk

Short title: Digestibility of diets and forage materials of laying hens

88

Abstract

1. The objective of the present study was to evaluate nutrient digestibility and

microbial activity in laying hens fed different silage and forage supplemented diets

with the intention of developing a 100% organic layer diet.

2. Birds were allocated into 9 diet groups. Except for the control diet, all experimental

diets were balanced nutritionally by considering the nutrients of supplemented

silages and home grown vegetables.

3. The dietary gross energy values ranged from 17.44 to 18.25 MJ/kg DM, and the

methionine (4.0~g/kg) content in analysed diets was higher than that of calculated

diets which is close to the requirement. Starch digestibility of the coefficient was

higher in all diets compared to diet A. Nitrogen corrected apparent metabolisable

energy (AMEn) varied significantly between the diets, and the highest AMEn was

found in diet F3 + kale (14.19 MJ/kg). The N retention varied from 0.380 to 0.488.

There was a significantly higher N retention in birds fed diet D + Grass silage

(0.485) and diet F3 + kale (0.488) compared to diet B + maize silage, diet C +

alfalfa, diet E2 + maize-cob silage and diet F2 + carrot (P<0.05). There were no

significant differences between diets for P and Ca retention. The digestibility of

methionine (0.884) and lysine (0.862) was the highest (P<0.0001) in diet F3 + kale

compared with diet E2 + maize-cob silage and diet F1 + beetroot. The amount of

lactic acid and count of lactic acid bacteria were higher in the birds fed diets

supplemented with maize silage than the other hens.

4. Thus, it might be concluded that grass-herb, hemp silage and kale supplemented

diets are digestible by the laying hens and contain appreciable amounts of AMEn

89

and N retention which might be considered to develop a forage based 100% organic

feeding strategy.

INTRODUCTION

In general, dietary fibre (NSP and lignin) is considered to be anti-nutritive (Mateos et al.,

2012) and poorly digestible by hens due to the lack of endogenous enzymes in the small

intestine (Scott et al.,1982; Larbier and Leclerq, 1994). However, recent studies have

shown that the physio-chemical structure of dietary fibre, including particle size, solubility,

water holding capacity and their intestinal fermentation are key factors affecting nutrient

digestibility and production performances of birds (Engberg et al., 2004;Mateos et

al.,2012; Svihus et al., 2011; Jimenez-Moreno et al., 2009). The action of soluble NSP in

the digestive tract is considered to be very critical creating a viscous environment within

the intestinal lumen (Choct and Annison, 1992; Choct et aI., 1996).

The access to soluble fibre might contribute with energy to hens by increased microbial

fermentation in caeca, whereas insoluble fibre is poorly fermented (Duke, 1986; Choct et

al., 1996). High amounts of soluble NSP increase intestinal viscosity which reduces digesta

passage rate and increases microbial activity in the small intestine and increases the

competition of nutrients between the bird and the microflora. Further, a number of

intestinal bacteria are able to de-conjugate bile acids (Knarreborg et al., 200) which in turn

decrease dietary fat digestion and absorption (Maisonnier et al., 2003; Knarreborg et al.,

2004) and thus reduce growth performance especially in growing birds (Choct and

Annison, 1992; Choct, 2002; Choct et al., 2004; Hetland et al., 2003;Jimenez-Moreno et

al., 2010; 2013). On the other hand, a high dietary content of insoluble NSP may result in

prolonged mechanical feed digestion in the gizzard which consequently increases gizzard

90

activity and weight. Mechanical stimulation by a fibrous feed structure increases HCl

secretion in the proventriculus which reduces gizzard pH and thereby supports the barrier

function of the gizzard against acid sensitive pathogenic bacteria and prevents them from

entering the small intestine (Engberg et al., 2002, 2004). A moderate amount of insoluble

fibre has also been shown to enhance starch digestibility (Hetland et al., 2003) and to

increase the apparent metabolisable energy (AMEn) (Choct et al., 1996).

Foraging materials usually contain significant amounts of dietary fibre (Steenfeldt et al.,

2001, 2007; Hammershøj and Steenfeldt, 2005). However, results from related studies

indicate that laying hens can eat certain amounts of different silages and vegetables

resulting in a reduced intake of the layer compound diet without compromising production

performance and bird welfare (Steenfeldt et al. 2007; Afrose et al., 2015). However, the

sustainability of using these ingredients in organic layer diets largely depends on the

digestibility of nutrients and their utilisation in the digestive tract. The composition of

silages and vegetables varies to a large extent (Steenfeldt et al., 2007; Hammershoj and

Steenfeldt, 2012). Limited information is available about the nutritional value of forage

materials, particularly when considering their potential use as new ingredients in bird

diets. Further, the effect of these forage materials used as dietary supplements on nutrient

retention and excretion has not been investigated.

Thus, the objective of the present study was to evaluate feeding value, nutrient

digestibility, apparent metabolisable energy, nitrogen corrected (AMEn) and composition

and activity of the microflora of laying hens fed diets supplemented with different silages

(maize, alfalfa, grass, hemp, maize-cob) and vegetables (beetroot, carrot, kale).

91

MATERIALS AND METHODS

Diets and experimental design

The present study was part of a larger experiment performed at outdoor facilities with

organic laying hens from 20-46 weeks of age fed organic diets where the nutrient

composition of different foraging materials was considered when formulating the feed f

(Afrose et al. 2015). Since the expected daily intake of forage material was taken into

account in the feed formulation, the calculated ingredient composition and calculated

nutrient content of the experimental diets were different (Table 1). At 46 weeks of age, a

total of 36 hens representing the different treatments were selected randomly from the

outdoor experimental units and placed in individual battery cages (4 replicates per

treatments) located indoor in a poultry unit with automatic ventilation and controlled

temperature and light. A three-tier battery cage consisted of 12 individual cages (50

x50x50 cm) with raised wire floors and with 2 feeding troughs outside for layer diets and

forage materials. Two water cups were placed inside each cage. In the digestibility

experiment, the same diets and foraging materials as used in the outdoor experiment were

studied. The composition of the experimental diets is shown in Table 1. During the

experiment, all birds were healthy and ate their allocated daily feed. The control diet (diet

A) was fed without any forage material, and the other 8 experimental diets were fed

together with 5 different silages (maize, alfalfa, grass-herb, hemp and maize-cob) and 3

vegetables (beetroot, carrot, kale). The diets are referred to as diet A (control), diet B +

maize silage, diet C + alfalfa, diet D + grass-herb silage, diet E1 + hemp silage, diet E2 +

maize-cob silage, diet F1 + beetroot, diet F2 + carrot, diet F3 + kale. Detailed information

about diets, feed formulation and foraging materials in the main experiment have been

reported by Afrose et al. (2015, manuscript). The layer diets were provided as pellets (3

mm) and fed ad libitum, and forage materials were given fresh in small portions, once in

92

the morning and again in the afternoon. After a 2 week adaptation period, the digestibility

and AMEn experiments were carried out. The bird genotype used was Hisex white, and

according to the organic egg production legislations, hens were not beak trimmed (Danish

Ministry of Justice, 1998: Bekendtgørelse nr. 210 af 6/4-1998). The lighting programme

used in this study was 16L: 8D during the whole experiment. The experiment was carried

out according to the guidelines of the Danish Ministry of Justice for animal

experimentation and care of birds. No hens died during the digestibility experiment.

Collection of excreta

During three consecutive days, total excreta were collected 3 times per day from trays

under each cage and from 4 cages per treatment. All contamination like feathers and scales

were removed from excreta which was placed in closed plastic containers kept at -200C

until analysis to prevent microbial degradation. The forage residuals were weighed

separately every day, whereas the layer diet was weighed at the beginning and at the end of

the experiment in order to calculate total daily forage intake per hen (replicate). The hens

used in this study were weighed at the beginning and the end of the experiment.

Gastrointestinal characteristics and intestinal microflora

At 46 weeks, a total of 45 hens from the main experiment, representing 15 hens from diet

A, diet B + maize silage and diet C + alfalfa silage, respectively, were selected to study the

composition and activity of the intestinal microflora. The hens were killed by cervical

dislocation, and the contents of gizzard, ileum and caeca from 3 hens per replicate (5

replicates) were collected quantitatively and pooled by segment before further analyses.

Jejunum and ileum were defined as the intestinal segments cranial and caudal to Meckel’s

diverticulum. The weight of the empty gizzard was measured for all the hens, and the

relative gizzard weight per kg body weight was calculated. Within 5 minutes of slaughter,

93

the pH of the contents of gizzard, ileum and cecum was measured using a combined

glass/reference electrode. Digesta (10g) from these segments were obtained for the

determination of short chain fatty acids (SCFA) and lactic acid (Canibe et al., 2007).

Gastrointestinal contents, coliform bacteria, lactose negative enterobacteria, lactic acid

bacteria, enterococci and total anaerobic bacteria were enumerated following the method

described by Engberg et al. (2004).

Chemical analyses

Following the digestibility experiment, chemical analyses were carried out on layer diets,

forage materials and excreta samples in duplicate. The DM content was determined by

drying at 1030C for 8 h. All samples were freeze dried before analysis and milled to pass

through 1mm and 0.5 mm sieves. The nitrogen content was analysed by the Dumas

method with a Leco FP 428 nitrogen analyser (Leco Corporation, St. Joseph, MI) (Hansen,

1989). The protein content was calculated by multiplying the nitrogen content by 6.25. Ash

was analysed according to method L54/50(EC, 2009), and fat (HCl-fat) was extracted with

diethyl ether after acid hydrolysis (EC,1998). Amino acids were determined as described by

the EU method (EC 152/2009 2009). Calcium was determined by method 975.03 (AOAC,

2000) and phosphorus by the colorimetric method described by Stuffins (1967). The

sugars (glucose, fructose and sucrose) and the oligosaccharides (raffinose, stachyose and

verbascose) were analysed by the method of Bach Knudsen and Li (1991). Non-starch

polysaccharides (NSP) and lignin were analysed according to the method described by

Bach Knudsen (1997). The concentration of sugars, NSP + lignin was only analysed in diets

and forage materials. Starch was analysed by the enzymatic-colorimetric method of

Knudsen (1997).

94

Calculations and statistical analyses

The total tract apparent digestibility coefficient of nutrients and the retention of nitrogen

were calculated using the concentration of nutrients in diets and excreta according to the

following formula:

Apparent digestibility = nutrient in diet – nutrient in excreta/nutrient intake.

Nitrogen retention = nitrogen in diet – nitrogen in excreta/diet intake.

The nitrogen corrected apparent metabolisable energy (AMEn) was calculated as: AMEn =

energy in diet – energy in excreta/diet intake, and for the correction to zero nitrogen

retention, a value of 34 kJ/g retained nitrogen was used (Hill and Anderson,1958).

The experiment was designed as a randomised complete block design (RCBD) where the

single cage represented the experimental unit (replicate). The results are presented as LS

means and standard error of means (SEM) calculated by standard procedures. Analysis of

variance by the general linear model (GLM) procedure (SAS Institute, 1990) was used to

determine the significance of treatment effects on the dependent variables (apparent

digestibility, AMEn, layer diet intake, forage intake, nitrogen intake from layer diet and

forage and methionine intake from layer diet and forage). Normality of data was tested by

an univariate procedure. An outlier test was performed. The LS means were calculated,

and differences were regarded as significant at P <0.05. With respect to intake, apparent

digestibility and AMEn, differences were classified by the Ryan-Einot-Gabriel-Welsch

(REGW) multiple range test (SAS Institute, 1990).

RESULTS

Chemical analyses of diets and foraging materials

The results of the chemical analyses of the 9 experimental diets used in the digestibility

study are given in Tables 2a and 2b, and the results confirm a different nutrient

95

composition as expected. The dietary gross energy value ranged from 17.4 to 18.3 MJ/kg

DM and was highest in diet F3. The DM content of the diets did not vary much with values

from 903 to 915 g/kg DM. The dietary ash content varied widely ranging from 91.8 to 143.7

g/kg DM and being highest in diet E2. The fat content was lower in all diets with

supplements compared to diet A (66.3 g/kg DM) except for diet E2 (69.4 g/kg DM). The

dietary calcium level ranged from 22.6 to 39.5 g/kg DM. With the exception of diet E1 and

diet F3, the diets with supplements contained higher calcium amounts than diet A where

the highest calcium content was found in diet E2 (39.5g/ kg DM). The phosphorous

content was very similar in the diets, however, highest in diet E2 (8.1 g/kg DM) and lowest

in diet E1 (6.7 g/kg DM). The diets were formulated with different amounts of protein.

However, the total protein content of diets and forage materials was supposed to be similar

according to the feed formulation strategy used. The lowest protein content (196.6 g/kg

DM) was found in diet E1 and the highest in diet E2 (252.8 g/kg DM). Except for diet E1,

the NSP content of diets was almost similar to each other ranging from 138.9 to 154.2 g/kg

DM and being highest in diet E2. The dietary fibre (NSP + Lignin) concentration ranged

from 155.8 to 193.2 g/kg DM. Among the amino acids, the highest concentrations of

methionine (4.49 g/kg DM) and lysine (12.57 g/kg DM) were found in diet E2 followed by

diet B (methionine, 4.17 g/kg DM, lysine11.05 g/kg DM).

The chemical composition of different forage materials (silages and vegetables) used in this

experiment is shown in Tables 3a and 3b. The 5 silages and 3 vegetables were analysed,

and the DM content varied to a high extent when comparing the silages to the vegetables.

The DM content of silages ranged from 297.7 to 369.1 g/kg and that of vegetables from

110.9 to 162.6 g/kg DM. The protein content ranged from 54.1 to 275.6 g/kg DM, and the

highest content was observed in kale (275.6 g/kg DM) and the lowest in maize-cob silage

96

(54.1 g/kg DM). Alfalfa silage is also a good source of protein with a protein content of

235.3 g/kg DM. The methionine content ranged from 3.80 to 0.58 g/kg DM and lysine

from 13.78 to 1.10 g/kg DM; alfalfa, grass silage and kale are very rich in methionine and

lysine. The highest ash content (134.9 g/kg DM) was found in hemp silage, whereas the

lowest was found in maize-cob silage (25.0 g/kg DM). The fat content varied widely and

was highest is in hemp silage (80.7 g/kg DM) and lowest in beetroot (2.7 g/kg DM). The

gross energy content was almost similar in all the supplements (~19.0 MJ/kg DM) except

in beetroot (16.6 g/kg DM) and was in general higher than the values found for the diets.

Compared to the other forage materials, the calcium content was higher in hemp silage

(28.8 g/kg DM), followed by alfalfa silage (17.9 g/kg DM) and carrot (12.6 g/kg DM).

Except for maize and maize-cob silage (257.1 and 431.5 g/kg DM, respectively), the starch

content in all other forage materials was very low (~ 10 g/kg DM). The content of NSP and

dietary fibre (DF: NSP + lignin) were higher in silages compared to the vegetables where

DF constituted more than 500g/kg DM in grass-herb and hemp silages. The DF in the

vegetables ranged from 184.9-352.7g/kg DM and was highest in kale. The lignin content

was also highest in silages, especially in alfalfa, grass-herb and hemp silages.

Diet and forage intake

The intake of layer diets and forage materials in laying hens (as fed) as well as the

calculated nitrogen and methionine intake are shown in Table 4. The daily intake of the

different layer diets was not significantly different. The supplementation of silages and

vegetables resulted in a reduced layer diet intake which was most pronounced when diets B

(106.4 g/hen/d), E2 (113 g/hen/d) and F1 (113.47 g/hen/d) were fed. With regard to the

forage materials, the intake of maize silage (67.12 g/hen/d) was the highest among the

silages, and the intake of beet root (104.84 g/hen/h) was the highest among the vegetables

97

(P <0.001). There was no significant difference in daily nitrogen intake from the layer

diets; however, due to the contribution from the different foraging materials, the total

nitrogen intake was higher in the diets + supplements compared to the control. Especially

the nitrogen intake from alfalfa silage and kale increased the total nitrogen intake due to

the higher protein content in these 2 forage types. The methionine intake from layer diets

did not differ significantly between diets. Among the silage supplementations, the highest

methionine intake was found in hemp silage (44.96 mg/hen/d), whereas among

vegetables, the highest methionine intake was found in kale (64.26 mg/hen/d).

Digestibility and AMEn

The nitrogen corrected apparent metabolisable energy (AMEn) and the total tract apparent

digestibility coefficient (DC) of nutrients are presented in the Table 5. The DC of organic

matter varied to a high extent (P <0.001) between the diets where the highest value was

found for diet E1 + hemp silage as compared to the diet F3 + carrot (0.775 vs 0.661). The

DC of fat was generally high with an average DC of 0.881, however, lowest (0.846) when

the hens were fed diet F2 + carrots and it was significantly different from most of the other

diets (P <0.05). The nitrogen retention was found to be in the range of 0.380 (diet F1 +

maize-cob silage) to 0.488 (diet F3 + kale) (P <0.05). There was a significant higher

nitrogen retention in birds fed with diet C + grass-herb silage and diet F3 + kale than with

diet A. The DC of starch was numerically higher in all diets compared to diet A (P = 0.06).

The retention of calcium did not differ significantly among the diets, even though the

values obtained with hens fed diet E1+hemp silage were much higher compared to the

other diets (P = 0.09) due to a high variation in the data. The retention of phosphorus

differed largely between diets (P = 0.0767), and the highest values were found when diet

E1 + hemp silage (0.339) was fed followed by diet D + grass-herb silage and diet F3 + kale

98

(0.268). The AMEn varied between the diets (P <0.001) where the highest AMEn was

observed for diet F3 + kale (14.189 MJ/kg DM) and the lowest for diet F1 + beet root

(12.867) and diet F2 + carrot (12.955). A high AMEn was also found with diet E1 + hemp

silage and diet D + grass-herb silage. The lowest DM in excreta was found with diet E1 +

hemp silage and diet F1 + beet root and was the highest with diet F2 + carrot. The results

indicate that the kale and the hemp silage supplements to some extent contribute with

more nutrients, especially energy, to the hens.

The average DC of total AA was 0.834 (Table 6). The values observed were quite similar for

most of the treatments except for diets F1 and F2 with the vegetable supplements. The

treatment with diet F3 + kale resulted in the highest DC of the total AA (0.868) and it was

significantly higher than for diets F1 and F2 with beetroot and carrots (0.804 and 0.809,

respectively), (P <0.05). In general, diet F3 + kale achieved the highest DC of most amino

acids except for histidine, and overall, the lowest DC of the different amino acids was

found with diets F1 + beetroot and F2 + carrots. The DC of methionine, cysteine and lysine

obtained with diet F3 + kale was 0.884, 0.844 and 0.862 which was significantly improved

compared to the control diet A and some of the other diets (P ≤0.001). Overall, the results

indicated that not only kale, but also some of the silages, especially alfalfa silage,

contributed with amino acids to the hens, whereas the lower content of protein and amino

acids in beetroot and carrots influenced the DC of AA due to the high intake of particularly

beetroot.

Gastrointestinal characteristics and intestinal micro flora

The relative weight of the empty gizzard, small intestine and caecum was determined in

diet A, diet B + maize silage and diet C + alfalfa silage (data not shown). No dietary effect

was found on the relative gizzard, small intestine and caecum weight, whereas a slightly

99

higher relative gizzard weight was found in birds fed diet B + maize silage (18.01 g/kg BW)

than birds fed diet A (17.56 g/kg BW) and diet C + alfalfa silage (17.48 g/kg BW). The

bacterial population and pH of the digestive tract is shown in Table 7. There was no

significant effect on bacterial populations in gizzard, ileum and caeca contents. However,

the highest coliform count was in caeca of hens fed diet B supplemented with maize silage

compared with diet A (P <0.05). The slightly lower pH was measured in the contents of the

gizzard and ileum of hens fed silage diets than hens fed diet A which was not significant,

whereas, in the caeca, the numerically higher pH in the caecum was found in maize silage

and alfalfa silage supplemented diets compared to diet A. In ileum, the most abundant

organic acids were lactic and acetic acid, and no significant difference was found when

hens were fed experimental layer diets supplemented with maize and alfalfa silage

compared to diet A. There was no significant influence of the treatments on concentrations

of organic acids in caeca.

DISCUSSION

In the present study, a comprehensive chemical analysis of different foraging materials

revealed a huge variation in nutrient compositions where the highest content of protein

and methionine was found in alfalfa silage and kale followed by grass-herb and hemp

silage, whereas maize and maize-cob silage were characterised by high starch content. The

hens showed in general interest in the foraging materials; however, the daily intake of the

silages and vegetables differed to a large extent (P <0.001) in agreement with other studies

(Steenfeldt et al. 2007; Hammershøj and Steenfeldt, 2012). The intake of beetroot and kale

was very high with a daily intake of more than 100 g/hen compared with a much lower

intake of alfalfa, grass-herb and maize-cob silages which had an average daily intake of 30

100

g/hen. The intake of forage materials did not affect the intake of the layer diets

significantly, although a difference of almost 35 g/hen/day was observed between diet B +

maize silage (106 g) and diet F1 + carrot (142 g). Compared to the control without access to

forage materials, the feed intake was numerically lower for diet B + maize silage (12%), diet

E2 + maize-cob silage (7%) and diet F1 + beetroot (7%) and higher for diet F2 + carrot

(14%). It has been reported that the consumption of foraging materials can reduce the

intake of the layer feed by up to 20% (Blair 2008), and recent studies showed results where

the access to forage materials reduced feed intake significantly (between 9-17%),

(Steenfeldt et al., 2007; 2015; Afrose et al., 2015). However, in some studies, no effect on

diet intake was found when using maize silage or kale as supplements (Hammershøj and

Steenfeldt, 2005; 2012) which indicates that several factors such as fibre content and

possibly also the flavour of the forage type can affect the hens.

Based on the chemical analysis and daily feed intake of both layer diets and forage

materials, the daily methionine intake per hen ranged from 380 mg (diet F1 + beetroot) to

567 mg (diet F3 + kale). The results indicate that the different forage materials contribute

with some methionine to the hens, and especially kale and hemp silage can be considered

valuable supplements in terms of protein and essential amino acids to organic diets which

was also found in recent studies (Hammershøj and Steenfeldt, 2012; Afrose et al., 2015,

manuscript). On the other hand, despite the low methionine content in maize silage and

beetroot, their contribution to the methionine intake was high due to a higher

consumption of maize silage and beetroot (67 and 113.5 g/hen/d). The daily nitrogen

intake (layer diet + forage) was on average 6.50 g per hen in all forage supplemented diets

compared to diet A (3.79) (P <0.05), and the highest intake was observed with diet F3 +

kale (9.18 g/hen/d). A significantly higher daily nitrogen intake was found when hens were

101

fed diets providing alfalfa or maize silages and carrots as supplement (Steenfeldt and

Hammershøj, 2015). Previous work revealed that hens can ferment soluble NSP partly by

microbial bacteria in the caeca and produce short chain fatty acids (SCFA) (Jørgensen et

al., 1996; Carre et al., 1990) and thus contribute with energy to hens. In the present study,

the highest AMEn was found in diet F3 + kale (14.19 MJ/kg DM) which was 5.3% higher

than the control diet. The observed increased AMEn with diet F3 might be due to an

increased protein intake resulting from the increased intake of kale. The DC of fat was

higher in all diets compared to diet F3 + carrot (P <0.05) which is in line with the findings

of Steenfeldt et al. (2007). The DC of organic matter in all forage supplemented diets was

on an average of 0.719 which is slightly lower than the value obtained by Steenfeldt et al.

(2007).

It is well known that starch contributes with the major part of energy in poultry diets. The

results of the present study also indicate that starch in all forage supplemented diets was

digested to a high extent by the hens. In recent studies, the inclusion of insoluble fibre (oat,

wood shavings, cellulose) was found to have a positive effect on starch digestibility in both

broilers and laying hens (Svihus and Hetland, 2001; Hetland et al., 2003; Steenfeldt et al.,

2007) which is in line with the present study. In general, the results from different studies

indicate that silages, vegetables or other ingredients with high fibre content may supply

nutrients to poultry (Choct, 2002, van Krimpen et al., 2008; 2009). In organic poultry

production, the formulation of diets balanced with respect to energy, protein and amino

acid compositions that meets the hens’ daily requirements as closely as possible is a major

concern. Excessive protein is often included in organic diets to supply sufficient levels of

essential amino acids, in particular sulphur containing amino acids, which increases the

risk of nitrogen excretion to the environment (Scholtyssek et al., 1991; Summer, 1993). In

102

the present study, the forage materials were considered as ingredients in the total feed

formulation in order to take the nutrient composition of the forage into account with the

purpose of formulating more balanced diets. The challenge with this new feeding concept

is to predict the hens’ daily intake of forage which is an important parameter as described

by Afrose et al. (2015).

In this experiment, the nitrogen (N) retention ranged from 0.380 to 0.488 which was

approximately the same levels as reported previously by Steenfeldt et al. (2007) where

hens were fed layer diets supplemented with maize, barley pea silage or carrot. A higher N

retention was found in both diet F3 + kale (0.488) and diet D + grass-herb silage (0.485)

compared to diet A, whereas the N retention in hens fed diets supplemented with carrot,

maize and alfalfa silage was much lower. Steenfeldt et al. (2007) also found a similar N

retention when supplementing maize silage. Many studies reported that diets with a lower

protein content but supplied with synthetic amino acids improved N retention (Summers,

1993; Meluzzi et al., 2001; Keshavarz and Austic, 2004). However, synthetic amino acids

are not allowed in organic poultry diet (EU, 2007).

It is well known that phytase supplementation can increase P availability from plant

ingredients to diets, which have a positive effect on egg production and P retention

(Gordon and Roland, 1997; Scott et al, 2001; Lim et al., 2003; Keshavarz, 2003).

According to the EU legislation a limited number of exogenous enzymes is permitted in

organic poultry production, however, phytases is not permitted though it can improve P

retention, (EU, 2007). In this study P retention in diets with forage supplements was 0.227

on an average compared to a value of 0.245 with the control (without forage). The value of

P retention was not significantly different between diets. This might be explained by the

large variation of data or the use of the total collection excretion method, which might not

103

be accurate compared to the pre-caecal measurement method as described by

Rodehutscord et al. (2002). Steenfeldt and Hammershøj (2015) recently reported that the

P retention in forage was 0.264 on average when maize and carrot silages were used as

forage supplement to organic diets, which is in line with our study. Steenfeldt and

Hammershøj (2015) stated that a higher calcium content have a positive effect on the Ca

retention. There was no significant difference on Ca retention between diets in our study.

However, numerically higher values were found in diet E1 + hemp silage compared to the

other dietary treatments. Many studies found that increasing the Ca level in layer diets

negatively influence both Ca and P retention (Rodehustscord et al., 2002; Lim et al.,

2003). Härtel (1990) reported that dietary interactions between Ca and P influence hen

performance to a high extent and observed depressed egg production and increased

mortality when a low P content was combined with high Ca in the diet. When Ca is high in

the diet, it affects the availability of P, magnesium, manganese and zinc. Inversely, a high P

content may hamper utilisation of Ca (Pelicia et al., 2009).

The analysed content of methionine and lysine was lowest in maize (1.45 g/kg DM) and

maize-cob (1.34 g/kg DM) silages, beetroot (0.97 g/kg DM) and carrot (0.66g/kg DM)

compared to kale and alfalfa silage. It has been reported previously by Hammershøj and

Steenfeldt (2005) that the content of methionine and lysine was low in maize silage

(methionine: 1.30 g/kg DM; lysine: 2.57 g/kg DM) and carrot (methionine: 0.84 g/kg DM;

lysine: 2.65 g/kg DM), and it was concluded that the contribution of methionine and lysine

from these kinds of forage was minor. Even though the methionine digestibility of maize

silage was low, the higher intake of maize silage enabled the birds to take up some amounts

of methionine. Except for diet F1 + beetroot and diet E2 + maize-cob silage, all other diets

obtained a higher DC of methionine and lysine, and diet F3 + kale had the highest DC of

104

most amino acids indicating that the high content of methionine in kale contributes to with

essential amino acids to the hen. This result might be taken into account in the feed

formulation and some kinds of forage, especially kale, could contribute protein and

methionine to hens.

The study considering the influence of silages on gastro intestinal size as well as microbial

composition and activity was part of the production study which was performed in the

outdoor area. The gizzard weight was slightly increased in diet B + maize silage (18.01 g/kg

BW) and diet C + alfalfa silage (17.9 g/kg BW) compared to diet A (17.6 g/kg BW. In a

previous study, the feeding of silages significantly increased the gizzard weight (Steenfeldt

et al., 2007). Hens fed silage had a higher gizzard weight than hens receiving carrots or no

forage supplementation which is due to an increased mechanical stimulation by the

increased amounts of dietary fibre retained in the gizzard and to a coarser feed structure

(Steenfeldt et al., 2007; Engberg et al., 2002, 2004; Hetland et al., 2003; Idi et al., 2005).

The small effect of silages on gizzard development compared to the control diet observed in

our study might be related to that control hens having an access to the outdoor run which

enabled hens to pick up coarse feed items other than green grasses. Although the grass

from the outdoor run was removed regularly, the hens still had access to grassroots, stones

and other unidentified fibre sources which potentially helped to develop the gizzard of the

control hens. The results obtained by Shrestha (2013) support this explanation. Shrestha

(2013) performed an experiment with hens from the same outdoor study, but moved to an

environmentally controlled indoor facility, and observed significant differences in relative

gizzard weight between birds fed the control and the maize silage supplemented group

(14.9 vs 17.4 g/kg). The pH of gizzard content from birds fed silages in the present study

was slightly lower than that of birds fed the control diet suggesting an increased secretion

105

of hydrochloric acid as a response to the coarse structure of the silage (Engberg et al.,

2004; Idi et al., 2005). Steenfeldt et al. (2007) also observed a similar tendency

concerning gizzard pH when hens were fed silages. Feeding animals with diets high in

dietary fibre, in particular soluble fibre, alters the rate of feed passage, the composition

and activity of the microbiota and efficacy of digestion (Bach Knudsen, 2001). Beneficial

bacteria in the caeca utilise fibre as a source of energy. Generally, the effect of the dietary

treatments on bacterial numbers and SCFA concentrations was quite limited in the present

study. In caeca, the number of coliform bacteria was higher in birds receiving maize silage

than in birds fed the control (Table 7). Lactose-negative enterobacteria and lactic acid

bacteria in the caeca were also slightly increased when birds were fed maize silage.

Steenfeldt et al. (2007) found reduced counts of coliform bacteria in caeca of laying hens

fed with silages. Compared to the other gastrointestinal segments, the caeca harbours the

highest numbers of bacteria (Barnes, 1979; Jamroz et al., 1998) which are responsible for

the fermentation of non-starch polysaccharides that have escaped from enzymatic

degradation in the small intestine. However, the contribution of energy in the form of

SCAFA is suggested to be quite limited and in the range of 2-5% of the total energy. In

contrast to the present results, Steenfeldt et al. (2007) found that the feeding of silages

decreases caecal bacterial populations and similar observations were reported by Engberg

et al. (2004) and Bjerrum et al. (2005). In this experiment, the concentration of organic

acids in ileum and caeca increased slightly when feeding supplemental maize silage which

indicates the fermentation of NSP at this location. This result is in accordance with the

findings of Shrestha (2013) who also observed a tendency to an increased organic acid

concentration in cecum.

106

CONCLUSION

It can be concluded that forage supplementations to diets improved the nutritional value of

the diet as indicated by a higher nutrient digestibility. In particular, hemp silage and kale

supplemented diets showed appreciable AMEn and nitrogen retention in haying hens. The

results also demonstrate that the forage supplementation has influence on gastrointestinal

characteristics; however, it depends on the type of forage material. Thus, the present study

reinforces previous findings that forage supplemented diets can be digested to some extent

to provide required nutrients to enhance production performances in laying hen.

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111

Table1 Composition of the experimental compound layer diets (as fed g/kg)

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3 Wheat 500 393 460 490 573 365 465 480 510 Oat 100 100 100 100 100 80 100 100 10 Barley 50 50 50 50 50 50 50 50 50 Soya bean 120 110 130 130 130 130 130 130 110 Soya bean cake ̶ 90 30 30 ̶ 100 30 30 80 Sunflower cake 80 70 80 80 ̶ 50 50 80 Rape seed cake 30 30 30 30 30 40 30 30 30 Fishmeal 22 37 20 22 31 48 35 27 20 Rapeseed oil 4 4 4 6 4 6 5 5 4 Alfalfa meal 20 20 20 20 20 20 20 20 20 Calcium carbonate 56 75 58.3 56.7 49.4 91.6 69.8 62.5 58.8 Monocalcium phosphate 9.7 11 9.9 9.4 4.6 10 8.6 8.7 9.2 Sodium bicarbonate 2.5 3 3.3 2.8 2.0 1.9 1.0 1.1 1.8 Sodium chloride 1.0 1.0 1.1 0.9 1.0 2.0 1.1 1.2 1.5 Choline chloride 50% 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 Vitamin mineral mixa 4.0 5.0 4.0 54.0 4.0 5.0 4.5 4.0 4.2 RoxazymeG2 Gb 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Calculated Nutrients

ME,MJ/kg 10.9 10.6 10.6 10.9 11.2 10.6 10.9 11.0 10.9 Protein 178.0 208.0 179.0 171.0 169.0 217.0 190.0 179.0 162.3 Fat 55.1 58.8 60.5 61.4 51.2 72.2 57.8 61.6 59.6 Starch 348.0 291.8 291.1 304.3 387.4 253.9 329.1 337.5 353.6 Sugar 31.4 37.3 37.4 33.8 30.7 40.0 33.5 33.9 30.7 Lysine 8.3 10.5 8.7 8.2 8.3 11.6 9.5 8.9 7.6 Methionine 3.1 3.6 3.0 2.9 2.8 3.8 3.3 3.0 3.3 Methioe+Cystine 6.2 7.3 6.4 6.2 6.1 7.5 6.8 6.4 6.0 Calcium 25.7 33.9 26.4 25.8 22.4 40.1 31.0 27.9 26.5 Total Phosphorous 6.9 7.6 7.0 6.9 5.4 7.7 6.8 6.7 6.7

a The vitamin and mineral premix provided per kg of diet: vitamin A, 12,000 IU; vitamin D3, 3000 IU; vitamin E, 25 mg; vitamin K3, 2 mg; vitamin B1; 4 mg; vitamin B2, 5 mg; vitamin B6, 8 mg; D-pantothenic acid,

12 mg; niacin, 45 mg; choline, 200 mg; folic acid, 1,4 mg; vitamin B12 , 0.01 mg; Fe, 60 mg; Zn, 80 mg; Mn, 80 mg; Cu, 15 mg; I, 0.45 mg; Se, 0.2 mg.

b Roxazyme: endo-1,4 betaglucanase (IUB nr. 3.2.1.4), endo-1,3:1,4 betaglucanase (IUB nr. 3.2.1.6), xylanase (IUB nr. 3.2.1.8). 2600 units/kg diet. EU nr. E1602.

112

Table 2a Chemical composition of the experimental compound layer diets (g/kg DM)

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3 Drymatter 913.2 914.4 913.6 910.2 905.8 903.7 913.8 915 912.5 Protein(N ×6 .25) 213.1 231.9 218.8 209.7 196.6 252.8 208.4 211.9 222.5 Ash 104.0 127.0 115.2 104.9 97.2 143.7 126.9 121.7 91.8 Fat 66.3 55.1 63.8 61.7 54.2 69.4 56.9 58.2 61.9 Starch 379.3 332.1 416.0 380.2 425.5 300.1 372.8 350.1 399.4 Calcium 26.5 33.3 31.6 28.7 25.9 39.5 33.6 32.0 22.6 Phosphorus 7.3 7.7 7.1 7.0 6.7 8.1 7.4 7.8 7.1 Gross energy(MJ/kg) 17.9 17.5 17.8 18.0 17.8 17.7 17.4 17.7 18.3 NCP1

Rhamnose 1.4 0.8 1.5 1.4 1.3 1.6 0.8 1.1 1.4 Fucose 0.7 0.5 0.7 0.6 0.6 1.0 0.4 0.9 0.7 Arabinose 21.1 20.9 19.7 20.3 20.8 19.9 20.7 20.1 22.0 Xylose 38.6 37.3 36.1 38.0 33.1 32.6 31.9 43.4 36.6 Mannose 4.9 5.4 4.8 4.9 4.1 5.6 5.2 4.7 4.7 Galactose 11.0 13.2 11.8 11.1 10.6 13.8 11.9 11.2 10.9 Glucose 62.4 65.3 60.4 60.8 47.9 59.1 56.2 56.2 58.7 Uronic acids 9.9 12.8 13.9 12.7 10.1 14.0 12.8 14.7 12.6

NSP2 149.9 145.0 149.4 150.2 128.2 148.6 138.9 154.2 146.6 Lignin 39.7 35.2 31.0 34.1 27.6 31.4 34.2 39.0 37.9 DF3 189.7 191.3 180.4 184.3 155.8 180.0 173.1 193.2 184.5

1NCP: Non-cellulosic polysaccharides. 2NSP: Non-starch polysacchardes. 3DF: NSP+Lignin.

113

Table 2b Amino acid concentrations in the experimental compound layer diets (g/kg DM)

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3 Alanine 8.84 10.01 9.10 8.52 8.19 11.05 8.89 9.36 9.34 Arginine 12.80 14.61 13.67 13.29 12.41 16.66 12.73 13.09 13.30 Asparagine 16.30 19.30 17.44 16.35 15.46 21.51 16.45 17.20 16.96 Cystine 3.88 3.97 3.88 3.86 3.49 4.34 3.67 3.68 3.97 Glutamine 43.73 44.87 44.01 43.03 37.77 47.52 38.51 38.81 43.40 Glycine 9.58 10.70 9.62 9.24 8.79 11.69 9.49 9.68 9.53 Histidine 9.31 10.29 9.88 4.74 4.37 5.78 4.62 4.70 4.86 Isoleucine 8.33 9.32 8.60 8.20 7.57 10.44 8.11 8.36 8.58 Leucine 14.31 15.86 14.85 13.99 12.98 17.26 13.88 14.42 15.14 Lysine 9.23 11.05 9.53 9.00 9.08 12.57 9.36 10.05 9.36 Methionine 3.82 4.17 3.76 3.58 3.22 4.49 3.53 3.72 3.88 Phenylalanine 9.42 10.25 9.70 9.23 8.44 11.13 8.75 8.99 9.46 Proline 13.33 13.80 13.71 13.39 12.22 14.38 12.47 12.37 13.83 Serine 10.01 10.87 10.4 9.93 9.12 12.00 9.51 9.71 10.19 Threonine 7.21 8.05 7.49 7.08 6.62 8.94 7.03 7.33 7.32 Valine 9.94 11.11 10.08 9.75 8.92 11.98 9.44 9.78 10.03

114

Table 3a Chemical composition of the expiremental forage materials (silages and vegetables) (g/kg DM)

Maize silage

Alfalfa silage

Grass silage

Hemp silage

Maize-cob silage

Beetroot Carrot Kale

Dry matter 323.0 297.7 369.1 348.6 338.4 130.1 110.9 162.6 Protein (N x 6.25) 84.7 235.3 164.4 172.5 75.9 94.1 54.1 275.6 Ash 35.0 126.4 98.2 134.9 25.0 52.2 46.6 101.9 Fat 21.5 34.5 34.8 80.7 28.7 3.4 2.7 26.5 Gross energy(MJ/kg DM) 19.0 19.3 19.1 19.4 18.7 16.6 17.0 18.9 Calcium 0.8 17.9 10.3 28.8 0.4 2.0 12.6 2.8 Phosphorus 2.7 4.2 3.5 7.4 3.1 2.5 2.1 5.8 Starch 257.1 9.2 11.2 8.0 431.5 0.7 t 6.9 NCP1 Rhamnose 1.0 4.1 3.4 4.1 0.9 2.4 3.5 4.1 Fucose 0.3 1.5 1.4 1.4 0.3 0.4 0.7 2.3 Arabinose 23.9 10.8 20.7 10.5 23.7 44.1 23.5 38.6 Xylose 154.5 49.2 71.7 63.2 123.8 2.2 3.3 14.3 Mannose 3.3 11.7 9.5 13.1 2.7 4.4 7.0 8.6 Galactose 9.2 10.7 15.2 11.5 8.3 12.6 38.1 31.3 Glucose 197.0 172.2 192.1 181.5 131.2 51.2 71.9 100.6 Uronic acids 12.6 85.2 69.3 78.2 9.3 51.4 81.0 113.9 NSP2 401.8 345.4 383.4 363.5 300.3 168.6 229.0 313.7 Lignin 88.4 106.7 117.2 168.6 51.4 16.3 16.2 39.0 Dietary fibre (NSP + Lignin)

490.2 452.0 500.6 531.7 351.7 184.9 245.2 352.7

1NCP: Non-cellulosic polysaccharides. 2NSP: Non-starch polysaccharides.

115

Table 3b Amino acid comcentrations in the expiremental forage materials (g/kg DM)

Nutrients Maize silage

Alfalfa silage

Grass silage

Hemp silage

Maize-cob silage

Beetroot Carrot Kale

Alanine 7.41 24.86 10.15 9.90 6.90 2.69 4.09 12.26 Arginin 1.97 3.70 4.94 8.60 1.31 2.20 1.76 15.84 Asparagine 4.11 12.58 15.96 11.49 3.34 5.30 6.22 20.79 Cystine 1.16 1.68 1.07 1.78 1.25 0.60 0.43 3.37 Glutamine 6.95 13.29 13.67 15.42 6.35 28.77 8.54 33.44 Glycine 3.74 10.69 7.97 7.45 3.11 1.77 1.69 11.04 Histidine 1.05 2.99 3.07 2.61 1.47 1.20 0.76 5.68 Isoleucine 3.24 11.22 7.32 6.79 2.79 2.12 1.82 9.49 Lysine 1.79 6.17 7.79 5.56 1.10 2.65 2.22 13.78 Leucine 7.34 16.84 12.08 10.62 7.13 2.63 2.54 15.67 Methionine 1.38 3.53 2.35 2.64 1.30 0.64 0.58 3.80 Phenylalanine 3.57 10.38 7.61 6.58 3.13 1.34 1.71 11.36 Proline 5.41 11.67 8.44 6.66 5.61 1.55 1.39 25.20 Serine 2.92 4.15 7.3 6.12 2.69 2.24 2.11 13.10 Threonine 2.68 4.27 6.84 5.07 2.65 1.82 1.74 10.43 Tyrosine 1.89 3.43 4.65 3.82 1.44 1.78 2.39 6.62 Valine 4.55 13.84 9.28 8.91 3.99 2.54 13.95 2.40

116

Table 4 Intake of the compound layer diets, forage and nitrogen (g/hen/d) as well as methinion intake (mg/hen/d) in laying hens

fed different experimental layer diets and forage materials

abcde Means in each row followed by different superscript letters differ significantly. 1 Pooled Standard error of mean.

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3

SEM1

P-value

g/hen/d

Maize silage

Alfalfa silage

Grass-herb silage

Hemp silage

Maize-cob

silage Beetroot Carrot Kale

Layer diet 121.58 106.42 124.08 127.52 126.80 113.11 113.47 142.11 126.22 3.15 0.2758

Forage intake 67.12ab 31.80b 27.18b 48.86b 30.45b 104.84a 45.48b 104.01a 6.18 0.0001

N intake Layer diet 3.79 3.61 3.97 3.89 3.61 4.13 3.46 4.41 4.10 0.10 0.4159 Forage 1.32c 4.27a 3.05b 3.17b 1.24c 1.56c 1.12c 5.08a 0.28 0.0001

Layer diet + forage

3.79d 4.93cd 8.24ab 6.95bc 6.78bc 5.38cd 5.02cd 5.53cd 9.18a 0.32 0.0001

Methionine intake, mg/hen/d Layer diet 424.04 405.77 426.22 415.53 369.83 458.95 366.01 429.64 503.12 11.25 0.1245

Forage 29.92bcd 33.42bc 23.57cd 44.96b 13.40de 13.74de 2.93e 64.26a 3.55 0.0001

Layer diet + forage

424.04ab 435.68ab 459.64ab 439.10ab 414.79b 472.34ab 379.75ab 432.57ab 567.39a 12.23 0.0220

117

Table 5 Nitrogen corrected apparent metabolisable energy (AMEn) (MJ/kg DM), apparent digestibility of nutrients and nitrogen and mineral retention in laying hens fed different experimental layer diets and forage materials

abcd Means in each row followed by different superscript letters differ significantly. 1Pooled standard error of mean. 2 Nitrogen corrected apparent metabolisable energy.

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Diet F2 Diet F3

SEM1

P-value

Maize silage

Alfalfa silage

Grass-herb silage

Hemp silage

Maize-cob

silage Beetroot Carrot Kale

Organic matter 0.722abc 0.709abc 0.700bc 0.735ab 0.775a 0.694bc 0.716abc 0.661c 0.765ab 0.01 0.0006

Fat 0.895a 0.893a 0.887a 0.895a 0.876ab 0.892a 0.862ab 0.846b 0.889a 0.004 0.0388

Starch 0.984 0.993 0.991 0.992 0.994 0.999 0.992 0.992 0.992 0.001 0.0619 N retention 0.448ab 0.396b 0.378b 0.485a 0.439ab 0.380b 0.417ab 0.390b 0.488a 0.010 0.0135 Ca retention 0.482 0.447 0.235 0.344 0.609 0.290 0.378 0.331 0.430 0.029 0.0900

P retention 0.245 0.165 0.193 0.311 0.339 0.185 0.197 0.156 0.268 0.017 0.0767

AMEn2, MJ/kg 13.476bcd 13.234de 13.319cde 13.854ab 13.724bc 13.141de 12.867e 12.955e 14.189a 0.078 0.0001 DM in excreta, % 25.33 24.86 27.79 25.01 20.60 24.63 23.88 28.59 24.31 0.64 0.1433

118

Table 6 Apparent digestibility of amino acids in laying hens fed different experimental layer diets and forage materials (silages and vegetables)

abcdMean in each row followed by different superscript letters differ significantly.

1 Pooled standard error of mean.

Diet A Diet B Diet C Diet D Diet E1 Diet E2 Diet F1 Die tF2 Diet F3

SEM1

P-value

Maize silage

Alfalfa silage

Grass-herb silage

Hemp silage

Maize-cob silage Beetroot Carrot Kale

Alanine 0.776bc 0.815abc 0.827ab 0.803abc 0.784abc 0.806abc 0.760c 0.774bc 0.842a 0.005 0.0001

Arginine 0.893c 0.909b 0.908b 0.909b 0.892c 0.908b 0.884c 0.889c 0.925a 0.002 0.0001

Asparagine 0.798ab 0.804ab 0.825ab 0.827ab 0.825ab 0.818ab 0.775b 0.786b 0.852a 0.005 0.0107

Cystine 0.766bc 0.737c 0.744bc 0.776abc 0.817ab 0.809abc 0.761bc 0.768bc 0.844a 0.008 0.0011

Glutamine 0.911ab 0.908ab 0.913ab 0.918ab 0.912ab 0.902ab 0.896b 0.888b 0.930a 0.003 0.0563

Glycine 0.641 0.652 0.628 0.716 0.700 0.636 0.663 0.618 0.706 0.009 0.0578

Histidine 0.924a 0.927a 0.930a 0.868bc 0.864bc 0.864bc 0.824c 0.836c 0.889ab 0.007 0.0001

Isoleucine 0.834bc 0.853ab 0.857ab 0.851ab 0.854ab 0.849ab 0.825b 0.828b 0.880a 0.004 0.0480

Leucine 0.856bc 0.878ab 0.879ab 0.875ab 0.871ab 0.867ab 0.843c 0.849bc 0.898a 0.004 0.0144

Lysine 0.812bc 0.831bc 0.829bc 0.840ab 0.806ab 0.830c 0.799c 0.808bc 0.862a 0.004 0.0001

Methionine 0.839bc 0.846bc 0.864ab 0.846bc 0.834abc 0.857c 0.834c 0.838bc 0.884a 0.003 0.0001

Phenylalanine 0.879ab 0.899ab 0.902a 0.896ab 0.888ab 0.882ab 0.860b 0.861b 0.912a 0.004 0.0016

Serine 0.838ab

c 0.845abc 0.858ab 0.863ab 0.848abc 0.848abc 0.798c 0.817bc 0.879a 0.005 0.0062

Threonine 0.784ab 0.795ab 0.809ab 0.810ab 0.804ab 0.807ab 0.747b 0.776ab 0.839a 0.006 0.0369

Valine 0.821bcd 0.846ab 0.850ab 0.847ab 0.811cd 0.835bc 0.793d 0.803cd 0.870a 0.005 0.0001

Totalamino

acids

0.825ab 0.836ab 0.842ab 0.843ab 0.843ab 0.835ab 0.804b 0.809b 0.868a 0.004 0.0271

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Table 7 The plate count of selected bacteria (log CFU/g digesta) in laying hens fed different experimental

diets and forage materials (46 weeks of age)

Diet A Diet B Diet C SEM1 P-Value

Maize silage Alfalfa silage

Total anaerobic

Gizzard 7.82 7.92 7.91 0.09 0.9097

Ileum 7.86 7.71 7.94 0.09 0.6359

Caecum 8.75 9.24 8.95 0.11 0.1997

Coliform bacteria

Gizzard 2.82 3.22 2.85 0.12 0.3603

Ileum 3.75 3.86 3.51 0.11 0.4602

Caecum 4.17b 5.27a 4.94ab 0.18 0.0276

Lactose-negative enterobacteria

Gizzard 2.82 3.16 2.85 0.11 0.3745

Ileum 3.47 3.43 3.42 0.03 0.7343

Caecum 3.97 4.35 4.21 0.09 0.2499

Lactic acid bacteria

Gizzard 7.52 7.61 7.79 0.17 0.8267

Ileum 7.89 7.70 7.82 0.09 0.6968

Caecum 8.46 8.50 8.44 0.08 0.9658

Enterococci

Gizzard 4.08 4.53 4.33 0.15 0.5305

Ileum 4.85 5.18 4.83 0.20 0.7462

Caecum 4.69 5.48 4.94 0.16 0.1215

pH

Gizzard 4.87 4.65 4.63 0.08 0.4093

Ileum 6.89 6.83 6.93 0.09 0.9162

Caecum 5.89 6.25 6.14 0.10 0.3446

a,b Means in each raw followed by different superscript letters differ significantly. 1 Pooled standard error of mean.

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Table 8 Concentrations of SCFA and lactic acid (mmol/kg) in ileal and caecal contents of laying hens

fed different experimental diets and forage materials (46 weeks of age)

Diet A Diet B Diet C SEM1 P-value

Maize silage Alfalfa silage

Ileum

Lactic acid 1.37 2.40 3.73 0.49 0.1458

Acetic acid 10.44 17.81 10.87 2.03 0.2671

Caecum

Acetic acid 70.12 77.15 65.09 4.16 0.5699

Propionic acid 28.94 30.24 26.10 1.74 0.6648

Iso butyric acid 0.92 0.99 0.62 0.11 0.3691

nbutyric acid 11.37 11.74 9.91 0.88 0.4932 1Pooled standard error of mean.

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5.3 Manuscript 3

Influence of blue mussel (Mytilus edulis) and starfish (Asterias rubens)

meals on production performance, egg quality and digestibility of nutrients of

laying hens

Sadia Afrose1, Marianne Hammershøj2, Jan Værum Nørgaard1, Ricarda Greuel Engberg1 and Sanna

Steenfeldt1*

Submitted to Animal Feed Science and Technology

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Influence of Blue mussel and common starfish meals on production

performance, egg quality and digestibility of nutrients of laying hens

Sadia Afrose1, Marianne Hammershøj2, Jan Værum Nørgaard1, Ricarda Greuel Engberg1

and Sanna Steenfeldt1*

1 Dept. of Animal Science, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark

2 Dept. of Food Science, Aarhus University, P.O. Box 50, DK-8830 Tjele, Denmark

*Corresponding author: Tel. +45 87158074; E-mail:sanna.steenfeldt@anis.au.dk

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Abstract

The aim of the study was to evaluate mussel meal and starfish meals as protein sources for

organic layers by studying the effect on production performance, nutrient digestibility and

egg quality. A total of 300 Hyline white laying hens between the age of 20 and 31 weeks

were distributed randomly to 6 dietary treatment groups, each with five replicates,

including a control diet providing fishmeal, 3 diets providing mussel meal (4, 8 and 12%)

and 2 diets providing starfish meal (4 and 8%). The feed intake was higher (P<0.05) for

hens fed 4% starfish meal compared to the control and 8% mussel meal diets. The egg

weight was not different from the control diets, but the 4% mussel meal resulted in lower

(P<0.05) egg weight than the 8 and 12% mussel meals. Laying rate, egg mass, feed

conversion ratio, mortality and live weight of the hens did not differ significantly. The egg

shell strength was not affected by any of the diets. The egg yolk colour was lower (P<0.05)

in lightness (L*) and higher (P<0.05) in redness (a*) for each increase in mussel meal

concentration, but was not affected by starfish meal. The albumen dry matter content was

not significantly different among diets, whereas the albumen gel fracture stress was lower

(P<0.05) in eggs from hens fed the 4% mussel meal compared to the starfish diets. Eggs

from hens fed the 12% mussel meal showed a fishy smell in the sensory evaluations. The

retention of nitrogen was significantly higher in hens receiving the 12% mussel meal and

8% starfish meal than in control hens (P<0.05). Increasing dietary mussel meal

concentrations increased the nitrogen corrected apparent metabolisable energy (AMEn)

(P<0.001). Diets with 8 and 12% mussel meal showed the highest apparent metabolisable

energy, whereas the lowest was observed in 4 and 8% starfish meals. The apparent total

amino acid digestibility was higher (P<0.05) in the diets with 8 and 12% mussel meal and

4% starfish meal compared with the control diet. Compared to the control diet, the

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apparent digestibility of methionine was higher (P<0.05) in hens fed with 8 and 12%

mussel meals, and the apparent digestibility of lysine was higher in hens fed 4, 8 and 12%

mussel meals and 4% starfish meal. Fat digestibility was higher (P<0.05) in diets with 4, 8

and 12% mussel meal compared with the control diet. In conclusion, up to 8% mussel meal

and starfish meal can be used in diets as a high quality protein source for egg production.

These meals can replace fish meal; however, a higher concentration may result in a fishy

smell.

Keywords: Digestibility, mussel meal, organic egg production, starfish meal, yolk colour

Abbreviations: AMEn, nitrogen corrected apparent metabolizable energy; C, control; DC,

digestibility coefficient; DM, dry matter; GLM, general linear model; RCBD, randomised

complete block design; SEM, standard error mean; TMA, trimethylamine.

1. Introduction

It is prohibited to use crystalline amino acids in diets for organic layers (EC, 2007).

Therefore, the requirement of hens for essential amino acids, especially sulphur containing

amino acids, has to be covered by the available feed ingredients. Deficient dietary

methionine has a negative effect on poultry welfare by increasing the risk of feather

pecking and cannibalism in floor rearing systems (Tiller et al., 2001). Furthermore,

methionine deficiency can lead to poor feather quality which results in increased feed

intake and reduced egg weight (Elwinger et al., 2008). Since 2005, the transition to use

100% organic feed ingredients in organic poultry production has been discussed

intensively within the European Union, and the date for implementing this has been

postponed until 1 January 2018 (EC, 2014).

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Approved organic high quality protein sources are limited in European countries, since

only a few countries grow soya beans which have a good amino acid profile for poultry

(Willis, 2003). Most soya beans are imported from overseas countries which are in

contrast to the idea of organic production based on local feedstuffs. Today, it is allowed to

use conventional ingredients at levels of maximum 5% in organic poultry diets, and items

such as potato protein concentrate and corn gluten meal are sometimes used as protein

sources to cover especially the methionine requirement of the birds. With the introduction

of 100% organic diets by 1 January 2018, potato protein concentrate and corn gluten meal

will most likely be phased out, since an organic production of these ingredients will be

limited and very expensive. Fish meal is rich in sulphur amino acids and is today used

frequently in organic poultry diets. However, the sustainability of using fishmeal in animal

feeds may be questioned, and increasing market prices may limit the availability of fish

meal in the future. If the organic poultry production is going to use diets based on 100%

organically produced ingredients, it is necessary to find new high value protein sources

rich in methionine (Elwinger et al., 2008).

Blue mussel (Mytilus edulis) cultivation as a marine nutrient mitigating tool has

emerged during recent years (Lindahl and Kollberg, 2009; Petersen et al., 2014). Off-

bottom cultivation with long-line production of blue mussels can be used to recycle

nitrogen and phosphorus surplus from the surrounding land and thereby improve the

water environment. The size of the mitigation mussels makes them inappropriate for use

in human consumption, and the enormous quantities would require new markets to be

developed. Furthermore, the blue mussels contain carotenoids and chlorophyll as they are

filter feeders and consume algae (Matsuno, 1989). The xanthophylls, which are oxy-

carotenoids, are known to be efficient egg yolk pigmenters (Hammershoj et al., 2010);

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hence, the inclusion of mussel meal in a layer diet is hypothesised to potentially affect the

egg yolk colour.

The common starfish (Asteria rubens) is a predator which has been a major problem

for mussel fishermen in the fjords for many years. Starfish was considered a good source of

protein and amino acids for poultry more than 50 years ago. However, the high calcium

carbonate concentration of starfish meal could reduce growth performance and protein

digestibility in broilers (Stutz and Matterson, 1964).

The aim of the study was to evaluate mussel meal and starfish meal in different dietary

concentrations as protein sources in organic layer diets taking hen production

performance, nutrient digestibility and egg quality into consideration.

2. Materials and methods

2.1. Hens, housing and experimental design

A total of 300 Hisex white pullets, 17 weeks of age, were allocated at random to 6 diets

in 30 floor pens, i.e. 5 replicates per treatment. The floor pens were placed indoor in one

poultry unit provided with automatic ventilation and controlled temperature and light.

Each replicate pen contained 10 hens, and the floor area (4 m2) was equipped with 4 single

nest boxes, 1 feed silo and a tape with nipple drinkers. To prevent visual contact between

hens from separate pens, the pens were separated by 2 m high wooden walls from the floor

up to 1.6 m and with wire mesh. According to legislation (Danish Ministry of Justice, 1998:

No. 210 of 6/4-1998) for organic egg production, the hens were not beak-trimmed. The

lighting programme included 12 h of light and 12 h of darkness at 17 weeks of age. The day

length was increased gradually to 16 h of light and 8 h of darkness at 19 weeks which

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continued to the end of the experiment. The experiment lasted 12 weeks and started when

the hens were 20 weeks of age.

2.2. Diets, ingredients and feeding

Mussels from off-bottom cultivation with long-line production were harvested at an

age of approx. 9 months from Skive Fjord (Denmark) in March 2013 and were de-shelled

by boiling (Vildsund Blue, Nykøbing Mors, Denmark) whereafter the meat was frozen.

Frozen mussel meat was loaded into a drum dryer for defrosting and heated so that the

mussel meat reached a temperature over 85°C for at least 1 h. The dried meat was cooled to

room temperature and then grinded into meal by using a high speed rotating grain mail

with a mesh size of 2.5 mm. Starfish were caught in the same fjord in May 2013, and the

production of starfish meal was performed at a Danish fish meal factory. Both types of

meal were stored at -20ºC, until the diets were prepared.

During the pre-laying period from 17 to 19 weeks, all hens were fed a commercial

organic pre-layer diet. From 20 weeks of age, the hens received the experimental diets

(Table 1) which were fed ad libitum. A commercial organic layer diet was used as control

diet (C) and contained fish meal as one of the protein sources. The other experimental

diets consisted of 3 diets with 4%, 8% and 12% mussel meal, respectively, and 2 diets

containing 4% and 8% starfish meal. The diets with mussel and starfish meals did not

contain fish meal. The formulation of the diets was based on the analysed nutrient content

of the mussel and starfish meals (Table 2) to meet the nutrient recommendations of NRC

(NRC, 1994). The calculated concentration of the experimental diets was 176 g crude

protein and 3.3 g methionine/kg DM. The feed was pelleted and subsequently made into

pellet-cross.

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2.3. Recordings

2.3.1 Production parameters

During the experimental period of 12 weeks, eggs were collected per pen, the number

of eggs was recorded every day, and all eggs collected 1 day per week were weighed. Feed

consumption was recorded every second week on the same day as registration of egg

weight. Egg production and feed consumption were calculated on hen day basis, and the

feed conversion ratio was calculated as g feed per g egg. Mortality was recorded daily. The

individual hen body weight was recorded at the beginning and at the end of the

experiment.

2.3.2. Egg quality

The egg quality parameters were analysed at 20, 25 and 30 weeks of age. A total of 150

fresh egg shells (6 experimental diets × 5 eggs × 5 replicates) were collected randomly and

stored at 4C for 1 day before analysis. The eggs were weighed, and the analysis of shell

strengths was carried out by compression analysis of each egg using a TaHdi Texture

Analyser (Stable Micro Systems Ltd., Surrey, England) as described by Hammershoj et al.

(2012). Recordings of force (N) and displacement (m) were obtained until fracture of the

shell. The maximum force recorded was used as shell strength value (N). The position

(mm) of maximum force detection was used as a measure of shell elasticity.

After that, the eggs were broken and egg yolk and albumen were separated. The egg

yolk weight was measured separately. The yolk colour was measured as described by

Hammershoj et al. (2010). The individual eggs were weighed, broken and the albumen was

removed from the yolk. The yolk colour was measured by a Minolta chroma Meter CR-300

(Minolta Co. Ltd., Osaka, Japan) using the CIE (Commission Internationale de

L’Enclairage) Lab scale with standardised daylight (D65) as reference. The L*, a* and b*

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values reflect lightness (0 = black, 100 = white), redness (-100 = green, 100 = red) and

yellowness (-100 = blue, 100 = yellow), respectively. The dry matter content of the egg

albumen was measured in duplicate by drying a 3 g homogenised albumen sample for 18 h

at 98C in a heating cabinet. The pH of the egg albumen was measured by a MeterLab TM

PHM220 pH meter (Radiometer, Copenhagen, Denmark).

For the analysis of the egg albumen gel texture, albumen gels were prepared by heating

30 ml of homogenised egg albumen in polyamide tubes in a water bath at 90C for 20 min

and cooled at 4C for 18 h. Four cylindrical gel samples (0.015 m height, 0.015 m diameter)

were cut from each sample. The egg albumen gel texture was analysed by uniaxial

compression using a TAHdi Texture Analyser (Stable Micro Systems Ltd., Surrey, England)

with a 100 kg load cell, an 0.001 N detection range, an 0.075 m diameter plate probe and a

compression speed of 0.8 mm/s. Recordings of force (N), displacement (m) and initial gel

height (m) before fracture were used to calculate axial stress σ (Pa) and Hencky strain ε ( ̶ )

at the gel fracture point according to Hammershoj et al. (2001).

2.4. Sensory evaluation of eggs by consumers

A total of 185 eggs from each of the 6 diets were collected at 30 weeks of age and used

in a sensory consumer evaluation by 37 persons who each evaluated 5 eggs per dietary

treatment. The test persons were asked to give a score in relation to taste of the egg and

colour of the egg yolk. There was no quantified scale for test persons, however; they were

asked to describe the taste of the eggs using different descriptors. The taste of the eggs was

evaluated using different descriptors such as fresh, creamy, dry yolk and fishy taint where

‘creamy taste’ was considered positive and ‘fishy taste’ was considered a negative attribute.

The yolk colour was quantified by a 10 point scale, where a low score indicates a light

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yellow colour, and a high score indicates a dark yellow/reddish colour. The test persons

were instructed to evaluate the eggs as hard-boiled, i.e. boiling for 10 min.

2.5. Digestibility experiment

At 32 weeks of age, a digestibility and AMEn trial was conducted in three-tier battery

cages with raised wire floors, where each battery cage consisted of 12 cages (50 × 50 × 50

cm) with 1 feeding trough outside the cage and a water pipe with 2 water cups inside the

cage along the wire wall. One hen from each of the 30 floor pens was selected randomly

and placed in individual cages (5 replicates per treatment). The lighting programme and

temperature were the same as in the main experiment. After a 1 week adaptation period,

total excreta were collected 3 times per day for 3 consecutive days from trays under each

cage. Contaminations such as feathers and scales were removed from the daily collection

which was then weighed and stored in closed plastic containers at 20C. Before analysis,

excreta were thawed and freeze-dried. After that, all the samples were ground finely with a

0.5 mm sieve and stored for chemical analyses. Feed intake was recorded per pen, and the

birds were weighed at the beginning and the end of the experiment.

The apparent digestibility coefficient of nutrients in excreta and the retention of nitrogen

were calculated using the nutrients in the diet and excreta according to the following

formula: apparent digestibility = nutrient in feed - nutrient in excreta/nutrient intake. The

nitrogen corrected apparent metabolisable energy (AMEn) was calculated as: AMEn =

energy in feed - energy in excreta/feed intake and corrected to 0 nitrogen retention using a

value of 34 kJ/g retained nitrogen (Hill and Anderson,1958), where the nitrogen retention

was determined as: nitrogen retention = nitrogen in feed - nitrogen in excreta/feed intake.

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2.6. Chemical analyses

The DM content of the layer diets and excreta was determined by drying duplicate

samples at 103C for 8 h. The nitrogen content was analysed by the Dumas method with a

Leco FP 428 Nitrogen analyser (Leco Corporation, St. Joseph, MI) (Hansen, 1989). The

crude protein content of the diets was calculated by multiplying the nitrogen content by

6.25. Amino acids were determined by the EU method (EC 152/2009, 2009). Ash was

analysed according to the method L54/50 (EC, 2009), and fat (HCl-fat) was extracted with

diethyl ether after acid hydrolysis (EC, 1998). Gross energy was determined by a LECO AC

300 automated calorimeter system 789-500 (LECO, St Joseph, MI, USA). Calcium was

determined by method 975.03 (AOAC, 2000) and phosphorus by colorimetric (Stuffins,

1967).

2.7. Statistical analyses

The experiment was designed as a randomised complete block design (RCBD)

considering the single floor pen with 10 hens as the experimental unit (replicate). Data

were subjected to a one-way analysis of variance using a GLM procedure (SAS ver. 9.2, SAS

Institute Inc., Cary, NC, USA) with the response variables as fixed effect. The univariate

procedure was used for the normality test of data. The following statistical model was used:

Yij = µ + ai +eij, where Yij =the j-th observation (j = intake of layer diet, laying rates, egg

weights, egg mass, mortality, body weight, AMEn, apparent digestibility (DC) of nutrients,

nitrogen retention on the i-th diets (I =1,………….,6 levels), µ = Mean; ai = i-th diet effect and

eij = the random error which is j-th observation on the i-th treatment. Model control was

done by plotting residuals against predicted values. The results were presented as means

and SEM (standard error of mean). The means were compared pairwise, and the

significance level was defined as P < 0.05.

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3. Results

3.1. Chemical analysis of diets

The results of the chemical analyses of the 6 diets are given in Tables 2 and 3. The

dietary gross energy values ranged from 17.09 to 17.58 MJ/kg DM and DM content from

883 to 904 g/kg. The ash content ranged from 121.3 to 143.8 g/kg DM and calcium content

from 35.8 to 45.5 g/kg DM with the highest amount found in starfish meal-based diets.

The phosphorus content was higher than expected in both the 8% and 12% mussel meal

diets being 7.1 g/kg DM (6.3 g/kg as fed) and 7.0 g/kg DM (6.7 g/kg as fed), respectively.

The fat content in all diets was lower than the calculated values except in diet 8% M (60.9

g/kg DM as fed). The diets were formulated to contain the same amounts of protein (Table

1); however, the protein content of the control diet was higher (203 g/kg DM) than that of

the other diets ranging from 173.3 g/kg DM (155.3 g/kg as fed) in the diet with 4% starfish

meal to 192.5 g/kg (172.4 g/kg “as fed) DM in the 12% mussel meal; the latter being closest

to the expected value. Especially in the diet with 4% mussel meal and the 2 diets with

starfish meal, the protein content was much lower than the calculated content seen in

Table 1, being in the range from 155.3 to 158.4 g/kg as fed. The analysed contents of

methionine and lysine in the control, 8% and 12% mussel meal diets were closest to the

expected value, whereas in the other experimental diets, these amino acids were lower than

the calculated values.

3.2. Hen performance

Hens fed with different levels of mussel and starfish meals from 20 to 31 weeks of age

showed an average laying rate of 92.9% (Table 4). The egg weight for the different diets

ranged from 51.7 to 53.6 g for the entire experimental period of 12 weeks. However, lower

egg weight was found for hens fed with 4% mussel meal and 8% starfish meal compared to

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the other diets (P<0.05). An effect of diet on feed consumption was observed (P<0.004).

Hens fed with the control and the 8% mussel meal diets consumed less layer diet

compared with hens fed 4% starfish meal (P<0.05). No effect of the diets was found with

respect to egg mass and feed conversion ratio. Mortality was generally low throughout the

experiment with an average of 3%. Further, no difference in body weight was found

between the dietary groups. The daily methionine intake was different (P=0.0001) among

diets; the highest value was found in the group receiving the control and the 12% mussel

meal diets and the lowest for the 4% mussel meal diet. However, the daily lysine intake was

highest (P<0.05) in the hens fed the 12% mussel meal diet and lowest (P<0.05) with the

8% starfish meal diet. Nitrogen intake differed among diets (P<0.001) with the highest

nitrogen intake for the hens fed the control diet and the lowest when fed the 8% mussel

meal diet.

3.3. Egg quality

The egg yolk colour differed (P<0.001) among diets and was clearly affected by the

concentration of mussel meal, but not by starfish meal (Table 5). Lower egg lightness (L*)

values (P<0.05) and higher redness (a*) values (P<0.05) were observed in all groups

receiving mussel meal compared with the control and the starfish meal diets. Neither yolk

weight, albumen DM, albumen pH nor shell quality were affected by the dietary

treatments. The albumen gel stress was observed to be reduced only in eggs from hens fed

the 4% mussel meal compared to the other diets (P<0.05).

3.4. Consumer sensory evaluation

The results showed that the test persons gave the most negative evaluations (6%

consumers) (e.g. fishy taint) of eggs from hens fed the 12% mussel meal diet, whereas

the highest (32% consumers) positive evaluation was observed in the 4% mussel meal

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diet. Eggs from 8% mussel meal and 4% starfish meal diets were accepted by 14% of

consumers. Further, eggs from hens fed the 12% mussel meal diet had the highest

score with regard to yolk colour, which was described as ‘dark yellow/red’, which was

in agreement with the yolk colour measurements.

3.5. Digestibility and apparent metabolisable energy

The DM content in excreta was affected by diet (P=0.02) with a higher DM for the 4%

starfish meal diet compared to the control diet (P<0.05). The apparent digestibility

coefficient (DC) of organic matter (Table 6) was on average 0.70 and did not differ among

the diets (P<0.08). The DC of fat was higher (P<0.05) when the hens were fed 4% mussel

meal as compared to the control diet (0.86 vs. 0.88). The AMEn varied among diets

(P<0.001) and increased compared to the control diet when the hens were fed mussel meal

– the highest values being found for the diets providing 8% (12.6MJ/kg) and 12% mussel

meals (12.5 MJ/kg). However, the AMEn was lower in the hens fed starfish meal compared

with mussel meal diets. The nitrogen retention varied from 0.36 to 0.46 (P=0.01) and was

improved in the hens fed the mussel and starfish meals compared to birds fed the control

diet. The nitrogen retention increased with increasing levels of mussel meal and starfish

meal in the diets. The apparent total amino acid digestibility was higher (P<0.05) in the

diets with 8 and 12% mussel meals and 4% starfish meal compared with the control diet.

Compared to the control diet, the apparent digestibility of methionine was higher (P<0.05)

in hens fed with 8 and 12% mussel meals, and the apparent digestibility of lysine was

higher in hens fed 4, 8 and 12% mussel meals and 4% starfish meal.

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4. Discussion

In the present study, production performance was in general not affected significantly

by feeding different levels of mussel meal and starfish meal compared to the control diets

with fishmeal. However, the daily feed intake varied between the diets (P<0.004) where

the highest feed intake was found when the birds were fed starfish meal. This may be

explained by the lower protein content in the starfish diets. It has been shown previously

that laying hens receiving low protein diets increase their feed intake in order to cover their

amino acid requirement (Hurwitz et al., 1998). Van Krimpen et al. (2009) speculated that

hens eat continuously until they have fulfilled their nutrient requirements. Other authors

have reported that an adequate amount of methionine in layer diets is important in

relation to egg production, egg weight, egg mass and body weight (Harms et al., 1998). In

the present experiment, the average laying rate was high (92.9%) when the hens were fed

different levels of mussel meal and starfish meal which indicates that mussel meal and

starfish meal at the chosen levels provided adequate amounts of methionine. The

methionine intake from the mussel meal and starfish diets ranged from 318 mg/hen/d to

380 mg/hen/d and from 326 mg/hen/d to 348 mg/hen/d, respectively, which fulfils the

National Research Council recommendations. It has been revealed that a high egg

production is associated with the optimum level of methionine in the diet. An increased

level of mussel meal increased the methionine and lysine intake, and the same trend was

found for starfish meal diets. As crystalline amino acids were not supplied in this study, the

hens were able to cover their recommended daily methionine and lysine requirements

from the experimental diets. The protein fraction of mussel and starfish meals contains an

essential amino acid profile very similar to fish meal, except for starfish meal being lower

in lysine and leucine compared to fish meal, but the seasonal variation in chemical

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composition can be great (Nørgaard et al., 2015). Jönsson et al. (2011) showed that up to

7% mussel meal in organic diets did not have any negative effect on the production

performance of laying hens compared with a control diet with fish meal. Jönsson and

Elwinger (2009) fed mussel meal at levels of 3, 6 and 9% and found that 6% mussel meal

improved laying rate and egg mass. The growth performance improved when broilers were

fed up to 12% starfish meal (Heuser and McGinnis, 1946); however, 18% starfish meal

impaired growth and induced high mortality (Ringrose, 1946). Overall, the eggs of the hens

in the present experiment had a lower weight than expected between 20 and 31 weeks of

age according to the recommendations for Hisex White hens. This may be explained by the

lower average body weight at the onset of lay (1160 g) compared to the recommended

standard body weight (1225 g) of Hisex hens. The reduced feed intake in the beginning of

the production period could have resulted in low egg weights which is in agreement with

previous reports (Lesson et al., 1987).

The DM content of the egg albumen has a significant influence on egg albumen gel

strength (Hammershøj et al., 2001). Our results showed that there was no significant

difference for albumen DM between the diets, but showed a tendency to increase the

albumen DM, when the hens were fed the 12% mussel meal diet. This may be caused by an

increased methionine and lysine intake associated with the increased level of mussel meal.

On the other hand, the DM of albumen was numerically higher in the 4% starfish meal diet

compared to the 8% starfish meal diet. Other studies have reported that an increase in

methionine and lysine intake of laying hens increases the albumen dry matter content

(Prochaska et al.,1996) which is in agreement with this experiment.

Egg gel strength was higher at both levels of starfish meal compared to the mussel

meal diets. It was revealed that an increased L-lysine intake increases the albumen DM,

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protein content and the albumen gel hardness of laying hens (Prochaska et al., 1996).

However, in this study, the lysine intake was lower in the starfish meal diets than the

mussel meal diets. The discrepancy in these results is inexplicable and would therefore be

interesting to study further. The inclusion of different levels of mussel meal and starfish

meal has a positive effect on egg shell strength. The high calcium content of the

experimental diets in the present study was expected to influence egg shell strength

positively. In our experiment, the daily calcium intake per hen was between 4979 mg and

3709 mg which fulfils the calcium requirement for laying hens which is approx. 3500

mg/hen per day. Further, McLaughlan et al. (2014) found that zebra mussel meal has a

positive effect on egg shell strength.

The yolk colour is considered a major factor influencing the product acceptability of

the consumer. Beardsworth and Hernandez (2004) reported that the egg yolk colour is

derived from yellow (e.g., lutein, zeaxanthin, apo-ester) and red (e.g., canthaxanthin,

citraxanthin, astaxanthin) xanthophylls in the diet. Carotenoids cannot be synthesised by

the hens and must be derived from the diet. Due to the consumption of micro algae,

mussels are rich in carotenoids. The carotenoid content in blue mussels is on average 80

mg/kg (corresponding to 400 mg/kg DM) depending on season and maturity (Campbell,

1969). In our study, the carotenoid content was not analysed; however, the results of the

yolk colour intensity showed that mussel meal diets increased redness (a*) and reduced

lightness (L*) values significantly compared to the control diet. The lightness (L*) values

increased when the hens were fed starfish meal compared to the mussel meal diet. The

yolk redness increased and yolk lightness (L*) decreased with increasing concentrations of

dietary mussel meal which could be due to the subsequent increase of xanthophylls in the

138

diet. Jönsson and Elwinger (2009) reported that increased dietary mussel meal content

increases egg yolk colour which is in agreement with our results.

The sensory preference of the consumer mainly depends on culture and food habits.

Therefore, the sensory egg quality is an important parameter for the egg producer. In the

present study, the perception of the eggs by the test persons decreased with increasing

levels of dietary mussel meal due to a fishy taint. This fishy taint may be generated by an

accumulation of trimethylamine (TMA) in the yolk. Some researchers showed that marine

products like fish meal or fish oil in layer diets reduce the sensory quality of eggs

(Gonzalez-Esquerra and Leeson, 2000) which is in line with our results. The egg yolks of

hens receiving mussel meal at a level of 12% had a very dark yolk colour; however, adding

12% mussel meal caused an off-flavour in eggs. The fatty acid proportion of the total fat

content is 49% for mussel meal and 81% for starfish meal (Nørgaard et al., 2015), and the

fatty acids will increase the concentration of n-3 fatty acids in the egg yolk.

The highest concentrations of mussel meal and starfish meal in the diets had a positive

effect on the nitrogen retention compared with the control diet with fish meal. This result

is comparable with findings of Blair et al. (1999) who observed that diets balanced with

essential amino acids require less crude protein in the layer diet. Thus, better nitrogen

retention is an indicator of an amino acid balanced ration. McLaughlan et al. (2014) fed

7.5% and 15% whole zebra mussel (Dreissena Polymorpha) (shell and meat) in order to

investigate the effect of protein and energy on the nutrient digestibility and production

performance in laying hens. They concluded that zebra mussels were highly digestible for

poultry, and that the nitrogen retention increased with increasing concentrations of mussel

which is in the line with the present findings. Diets with unbalanced amino acid contents

can result in an increased amount of nitrogen excreted to the environment. Since the

139

highest nitrogen retention was observed for diets with 12% mussel meal and 8% starfish

meal, it may be concluded that these diets provided an amino acid balance superior to that

of the other diets resulting in a lower amount of excreted nitrogen.

As compared to the control diet, the apparent amino acid digestibility was higher when

hens were fed diets containing mussel meal and starfish meal. The digestibility of lysine

and methionine from the mussel meal diets containing 8 and 12% was higher than with the

control diet which means that blue mussels can be used as a valuable source of amino acids

in layer diets. McLaughlan et al. (2014) fed 7.5 and 15% zebra mussel meals to laying hens

and found that methionine and lysine digestibility coefficients were 0.96 and 0.88 in the

7.5% diet and 0.96 and 0.89 in the control diet, respectively which is in line with our study.

The diet containing 8% starfish meal resulted in a lower AMEn compared to the diets

containing 8 and 12% mussel meals which could be caused by the lower fat content in

starfish meal diets compared to mussel meal diets. The tendency to obtain a higher AMEn

due to increased fat content agrees with a previous report (Aziza et al., 2013) where the

AMEn increased when 10% camelina meal with a high fat content (26.5%) was included in

a layer diet. Mateos and Sell (1980) also found that a higher ratio of unsaturated fatty acids

at the expense of saturated fatty acids enhances dietary AMEn and the utilisation of energy

from dietary lipid constituents. An increased digestibility of fat may be caused by an

increased dietary level of mussel meal which is supported by findings of Hellwing et al.

(2007). Atteh et al. (1985) observed that the fat digestibility of laying hens increased when

they were fed a diet with an increased animal-vegetable fat level of laying hens.

140

5. Conclusion

In conclusion, the present study showed that both starfish meal and mussel meal can

maintain the production performances of layers at an acceptable level. To avoid off-flavour

in eggs, up 8% mussel and starfish meals can be included. The ingredients can be used as

protein sources in organic layer diets and replace fish meal effectively. Further, the

inclusion of mussel and starfish meals may reduce the excretion of nitrogen which, from an

environmental point of view, is of great advantage and thus can improve the sustainability

of the organic egg production.

Acknowledgement

The authors thank technician Kirsten Balthzersen, Danish Institute of Agricultural

Sciences (DIAS) for daily hen management together with the Growth Forum–North

Denmark Region (grant number 2012-148001), Danish AgriFish Agency under the Danish

Ministry of Food, Agriculture and Fisheries (grant number 33010-12-p-0249; funds from

the European Union) and the Department of Animal Science, Aarhus University for

financial support of the study.

Conflict of interest

We hereby confirm that there is no known conflict of interest regarding this

publication. We also confirm that the manuscript has been read and approved by all

authors.

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145

Table 1

Composition of experimental layer diets (as fed g/kg)

Ingredients C 4% M 8% M 12% M 4% S 8% S

Wheat 300 306 300 336 301 316

Wheat bran 200 178 142 89 169 153

Oat 83 101 150 150 120 150

Maize 50 50 50 80 50 50

Soya bean cake, toasted 57 75 21 0 73 0

Soya bean, toasted 23 0 0 0 0 0

Sunflower cake 70 67 70 70 70 70

Rapeseed cake 10 40 40 0 40 40

Mussel meal 0 40 80 120 0 0

Starfish meal 0 0 0 0 40 80

Fish meal 37 0 0 0 0 0

Rapeseed oil 8 10 15 12 10 10

Alfalfa meal 30 30 30 40 30 40

Calcium carbonate 57 55 55 58 53 52

Oyster shell 30 30 30 30 30 30

Mono calcium phosphate 8 11 11 11 8 5

Sodium bicarbonate 2 0 0 0 0 0

Sodium chloride 1 2 1 0 2 0

Vitamin mineral mixa 4 4 4 4 4 4

Cholin chloride 50% 0.4 0.4 0.4 0.4 0.4 0.4

Roxazyme G2 Gb 0.1 0.1 0.1 0.1 0.1 0.1

Calculated Nutrients

ME, MJ/kg 10.6 10.6 11.0 11.1 10.5 10.7

Dry matter 886.1 887.5 895.3 890.5 886.7 885.4

Ash 111.0 109.6 108.6 109.6 109.5 108.9

Protein 179.2 175.2 170.6 175.9 178.7 176.9

Fat 52.1 52.5 56.3 61.1 51.4 53.1

Starch 359.0 356.7 344.9 356.8 356.2 363.7

Sugar 32.2 32.7 27.1 22.4 32.7 26.4

Lysine 8.0 7.6 7.8 8.4 7.7 7.8

Methionine 3.3 3.1 3.2 3.4 3.2 3.5

Calcium 36.0 35.0 35.0 35.8 35.0 36.0

Total Phosphorus 6.5 6.4 6.3 6.0 6.5 6.4

Sodium 1.5 1.4 1.6 1.7 1.4 1.2

Potassium 6.5 6.4 5.8 5.5 6.4 5.6

Chloride 1.7 2.3 2.3 2.3 2.3 1.8

a The vitamin and mineral premix provided per kg of diet: Vitamin A, 12,000 IU; Vitamin D3, 3000 IU; Vitamin E, 25 mg; Vitamin K3, 2 mg; Vitamin B1; 4 mg; Vitamin B2, 5 mg; Vitamin B6, 8 mg; D-pantothenic acid, 12 mg; Niacin, 45 mg; Choline, 200 mg; Folic acid, 1,4 mg; Vitamin B12 , 0.01 mg; Fe, 60 mg; Zn, 80 mg; Mn, 80 mg;Cu, 15 mg; I, 0.45 mg; Se, 0.2 mg. b Roxazyme: endo-1,4 betaglucanase (IUB nr. 3.2.1.4), endo-1,3:1,4 betaglucanase (IUB nr. 3.2.1.6), xylanase (IUB nr. 3.2.1.8). 2600 units/kg diet. EU nr. E1602.

146

Table 2

Chemical composition of layer diets with fishmeal (C), mussel meal (M) or starfish meal (S),

and in mussel and starfish meals (g/kg DM)

C 4% M 8% M 12% M 4% S 8% S Mussel

meal

Starfish

meal

Dry matter 903.5 883.2 890.1 895.7 896.2 892.4 949.1 946.1 Ash 121.3 128.9 132.6 135.5 143.8 141.6 81.0 203.4

Protein

(N×6.25) 203 179.4 180.8 192.5 173.3 175.9 605.0 699.7

Fat 51.6 58.7 68.4 65.7 56.5 55.8 160.5 109.6

Calcium 35.8 41.2 42.6 43.6 45.5 45.5 9.2 47.1

Phosphorus 6.5 6.9 7.1 7 6.6 6.4 8.9 25.9

Gross energy,

MJ/kg 17.52 17.39 17.58 17.54 17.09 17.17 - -

C= Control; 4% M= Experimental diet + 4% mussel meal; 8% M= Experimental diet + 8% mussel meal;

12% M= Experimental diet + 12% mussel meal; % S= Experimental diet + 4% starfish meal;

8% S= Experimental diet + 8% starfish meal.

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Table 3

Content of amino acids in layer diets with fishmeal (C), mussel meal (M) or starfish meal (S), and

in mussel and starfish meals (g/kg DM)

C 4% M 8% M 12% M 4% S 8% S Mussel

meal

Starfish

meal

Alanine 8.8 7.6 7.9 8.5 7.7 8.2 29.4 45.3

Arginine 11.4 10.8 10.8 11.2 10.5 9.8 38.9 39.3 Asparagine 14.6 14.4 14.2 15.1 13.7 12.3 59.2 57.4 Cystine 3.6 3.2 3.3 3.3 3.1 2.8 8.9 5.3 Glutamine 43.5 35.4 33.8 33.3 34.7 32.6 70.9 80.5 Glycine 9.1 8.4 8.9 9.7 9.1 10.3 35.8 61.0 Histidine 4.6 4.1 4.0 4.0 4.0 3.8 11.7 13.9 Isoleucine 7.8 7.1 7.1 7.4 6.8 6.3 27.2 27.2 Leucine 14.0 12.1 12.0 12.5 11.6 11.1 40.5 44.1 Lysine 8.1 8.0 8.3 9.2 7.5 7.2 43.5 43.4 Methionine 3.6 3.1 3.4 3.7 3.1 3.4 14.1 17.5 Phenylalanine 9.2 8.2 8.0 8.2 7.9 7.4 23.6 24.4 Proline 13.9 11.1 10.7 10.7 11.2 11.2 23.0 34.8 Serine 9.5 8.5 8.4 8.7 8.2 7.7 28.4 28.7 Threonine 6.8 6.5 6.7 7.1 6.1 5.9 27.8 26.4 Valine 9.7 8.5 8.6 8.9 8.3 8.1 29.1 33.6

C= Control; 4% M= Experimental diet + 4% mussel meal; 8% M= Experimental diet + 8% mussel meal;

12% M= Experimental diet + 12% mussel meal; 4% S= Experimental diet + 4% starfish meal;

8% S= Experimental diet + 8% starfish meal.

148

Table 4

Production performance of laying hens fed diets with fishmeal (C), mussel meal (M) or starfish meal (S)

C 4% M 8% M 12% M 4% S 8% S SEM1 P-value

Feed consumption, g/hen/d 113.3b 115.6ab 112.3b 114.6ab 119.2a 116.4ab 0.59 0.004 Egg weight, g 52.8ab 51.7b 53.6a 53.2a 52.9ab 51.9b 0.21 0.04 Number of eggs/hen 63 65 62 65 64 63 0.63 0.86 Laying rate, % 93.1 92.5 92.1 92.5 94.5 92.9 0.45 0.73 Egg mass, g/hen/d 49.3 48.0 49.5 49.4 50.1 48.3 0.30 0.29 FCR, g feed/g egg 2.30 2.41 2.27 2.32 2.38 2.41 0.02 0.07 Body weight, kg 17 weeks 1.16 1.14 1.18 1.16 1.18 1.16 0.02 0.27 32 weeks 1.50 1.61 1.56 1.67 1.58 1.56 0.06 0.26 Mortality, % 6 0 6 0 4 4 4 0.54 N intake, g/hen/d 3.32a 2.93cd 2.89d 3.16b 3.03c 2.92cd 0.03 0.001 Methionine intake, mg/hen/d 368.57a 318.56d 336.81bc 379.75a 325.78cd 348.06b 4.27 0.0001 Lysine intake, mg/hen/d 827.23bc 816.81bc 833.53b 943.21a 797.89c 746.00d 11.42 0.0001

a,b,c,d Means in each row followed by different superscript letters differ significantly. 1 Pooled Standard error of mean. C= Control; 4% M= Experimental diet + 4% mussel meal; 8% M= Experimental diet + 8% mussel meal; 12% M= Experimental diet + 12% mussel

meal; 4% S= Experimental diet + 4% starfish meal; 8% S= Experimental diet + 8% starfish meal.

149

Table 5

Egg quality parameters of laying hens fed diets with fishmeal (C), mussel meal (M) or starfish meal (S)

C 4% M 8% M 12% M 4% S 8% S SEM1 P-value

Egg weight, g 53.12 52.41 53.24 53.29 52.77 52.50 0.38 0.76 Yolk weight, g 13.71 13.94 13.93 14.05 13.63 13.57 0.34 0.82 Yolk colour Lightness, L* 67.39a 66.06b 64.07c 62.33d 68.71a 68.71a 0.52 0.0001 Redness, a* -3.89d -0.80c 2.79b 4.82a -4.76d -4.76d 0.43 0.0001 Yellowness, b* 49.71 48.29 48.75 48.27 47.73 47.71 0.73 0.37 Albumen DM, g/kg 129.6 129.6 128.7 131.9 131.2 127.9 0.53 0.10 pH 9.30 9.26 9.26 9.28 9.27 9.29 0.01 0.22 Shell Fracture strength, N 40.16 41.03 40.57 40.32 40.56 41.55 0.81 0.83 Fracture point, µm 223 217 221 218 222 218 0.004 0.90 Gel Gel fracture stress, kPa 21.06ab 19.66b 20.33ab 20.84a

b 21.29a 21.27a 0.38 0.02

Gel fracture strain, ( ̶ ) 0.93 0.90 0.93 0.96 0.97 0.92 0.02 0.29

a, b,c,d Means in each row followed by different superscript letters differ significantly. .1 Pooled Standard error of mean. C= Control; 4% M= Experimental diet + 4% mussel meal; 8% M= Experimental diet + 8% mussel meal; 12% M= Experimental diet + 12% mussel

meal; 4% S= Experimental diet + 4% starfish meal; 8% S= Experimental diet + 8% starfish meal.

150

Table 6

Nitrogen-corrected apparent metabolisable energy (AMEn) (MJ/kg DM), apparent digestibility of

nutrients in the excreta and nitrogen retention in laying hens fed diets with fishmeal (C), mussel

meal (M) or starfish meal (S)

C 4% M 8% M 12% M 4% S 8% S SEM1 P-value

Organic matter 0.691 0.708 0.711 0.711 0.702 0.693 0.01 0.08

Fat 0.859c 0.886a 0.882ab 0.884ab 0.864bc 0.868abc 0.01 0.002

N-retention 0.364b 0.413ab 0.423ab 0.456a 0.425ab 0.454a 0.02 0.01

Amino acids

Alanine 0.760bc 0.751c 0.788ab 0.805a 0.776abc 0.788ab 0.01 0.0001

Arginine 0.868c 0.879bc 0.891ab 0.899a 0.887ab 0.885ab 0.01 0.0001

Asparagine 0.868d 0.879c 0.891ab 0.899a 0.887bc 0.885bc 0.01 0.0001

Cystine 0.794 0.793 0.808 0.809 0.801 0.791 0.01 0.07

Glutamine 0.905 0.898 0.910 0.911 0.905 0.902 0.00 0.04

Glycine 0.612c 0.731b 0.792a 0.805a 0.784ab 0.810a 0.03 0.0001

Histidine 0.827b 0.834ab 0.848a 0.851a 0.846ab 0.836ab 0.01 0.004

Isoleucine 0.819bc 0.816c 0.843ab 0.854a 0.829bc 0.821bc 0.01 0.0002

Leucine 0.851bc 0.843c 0.865ab 0.875a 0.855abc 0.854abc 0.01 0.0009

Lysine 0.745d 0.774c 0.812ab 0.832a 0.785bc 0.771cd 0.01 0.0001

Methionine 0.842c 0.837c 0.871ab 0.879a 0.851bc 0.857abc 0.01 0.0001

Phenylalanine 0.853 0.855 0.870 0.876 0.866 0.860 0.01 0.06

Proline 0.885ab 0.876b 0.885ab 0.892a 0.881ab 0.883ab 0.00 0.02

Serine 0.827ab 0.823b 0.839ab 0.848a 0.832ab 0.827ab 0.01 0.009

Threonine 0.760c 0.763c 0.798ab 0.812a 0.775bc 0.770bc 0.01 0.0001

Valine 0.777c 0.776c 0.806ab 0.820a 0.793bc 0.790bc 0.01 0.0001

Total amino

acids

0.806d 0.815cd 0.840ab 0.850a 0.829abc 0.826bcd 0.01 0.0001

AMEn, MJ/kg2 12.20abc 12.29ab 12.55a 12.52a 12.06bc 11.89c 0.06 0.0003

DM in excreta, %

20.41b 19.47b 19.42b 20.75ab 25.02a 21.16ab 0.54 0.02

a,b,c,d Means in each row followed by different superscript letters differ significantly. 1 Pooled Standard error of mean. 2 Nitrogen-corrected apparent metabolisable energ

151

Chapter 6: General Discussion

This section of the thesis is structured according to the results obtained in three individual

studies. The aim of this work was to find alternative feed ingredients of both plant and

animal origin with focus on protein to formulate 100% organic diets. The purpose of

paper I was to evaluate whether foraging materials of high quality can be considered as a

nutrient source in addition to their use as occupying material, which could facilitate the

transition to 100% organic diets that cover the nutritional requirements of laying hens.

Further, the use of locally grown feed ingredients supports the organic production systems

in a sustainable way by reducing the need for imported protein sources. The effect on

apparent nutrient digestibility, apparent metabolisable energy and microbial activities in

hens fed different organic diets including forage material were examined in paper II. In

paper III, mussel and star fish meal were evaluated as alternative animal protein sources

considering their ability to replace fishmeal.

6.1 Production performance

Based on the hypothesis that forage materials and marine animals have the potential to

supply certain amounts of nutrients, it has been expected that their use as supplements or

inclusion in the experimental diets would positively influence egg production, hen welfare

and the environment.

The results in paper I showed that eight different forage materials (maize silage, alfalfa

silage, grass-herb silage, hemp silage, maize cob silage, and beetroot, carrot and kale) had

no negative effect on egg production performances and some of the diets numerically

improved laying rate, egg mass and egg weight compared to a control diet without access to

forage material. Hens fed diet B + maize silage achieved the most efficient laying rate,

which might be due to the higher total methionine uptake resulting from a higher silage

intake. The total methionine intake from diet B + maize silage was higher compared to

some of the other diets including the control without access to forage material and it had

been previously demonstrated that a higher methionine intake increases laying rate and

that methionine deficiency reduces egg production (Harms et al., 1998; Keshavarz, 2003;

152

Moghaddam et al., 2012). A similar trend of laying rate was observed, when feeding maize

silage to laying hens in the study by Steenfeldt et al. (2007). Further, hens fed the kale

supplemented diet showed an average egg production of 88.94%, which was comparable

with the findings by Hammershøj and Steenfeldt (2012) who found an egg production of

84% when layer diet supplemented with kale were fed. The high methionine and lysine

content of kale might explain this increase in the egg production. The egg weight, egg mass

and laying rate tended to be positively influenced when feeding diet D +grass-herb silage.

In agreement with our findings, egg production, egg weight and egg mass significantly

increased following dietary inclusion of 3% grass clipping waste (Nobakht, 2014). This

could be caused by the increased layer diet intake might lead to increased fat intake, as

other research found that egg weight increased with increasing dietary fat content (Grobas

et al., 2001). Although the underlying mechanism by which silage improved egg

production is difficult to elucidate completely, diet D+grass-herb silage, the high

concentration of dietary fibers may play a role. The dietary supplementation of moderate

amounts of fibers has been previously reported to enhance egg production parameters

(Mohiti-Asli et al., 2012; Steenfeldt et al., 2013). Therefore, because of an overall positive

effect on all the production parameters, grass-herb silage might be a potential candidate

for future research.

In order to reach the goal of 100% organic feeding in egg production in a sustainable way,

alternative animal protein sources from the marine environment were evaluated with

respect to their potential to replace fish meal in layer diets. In Study III, it was shown that

mussel meal and starfish meal can be used as protein source and can replace fish meal in

layer diets.

The average laying rate was high (93%) when the hens were fed different levels of mussel

meal and starfish meal, which indicates that mussel meal and starfish meal at the chosen

levels (up to 8%) provided adequate amounts of methionine. The methionine intake from

the mussel and starfish meal supplemented diets ranged from 318 to 380 mg/h/d and

from 326 to 348 mg/h/d, respectively (Table 4), which fulfils the NRC recommendations

(NRC, 1994). The laying rate, egg mass and egg weight resulting from mussel and starfish

meal is very similar to that of the fishmeal supplemented control. It has been revealed that

a high egg production is associated with the optimum level of methionine in the diet

(Keshavarz, 2003; Bunchasak, 2009; Moghaddam et al., 2012). An increased level of

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mussel meal increased the methionine and lysine intake and the same trend was found

with starfish meal supplemented diets. Jönsson et al. (2011) showed that up to 7% mussel

meal in organic diets did not have any negative effect on the production performance of

laying hens compared with a control diet with fish meal. Again, Jönsson and Elwinger

(2009) fed mussel meal at the levels of 3, 6 and 9% and found that 6% mussel meal

improved laying rate and egg mass. The growth performance improved when broilers were

fed up to 12% starfish meal (Heuser and McGinnis, 1946); however, 18% starfish meal

impaired growth and induced high mortality (Ringrose, 1946).

6.2 Feed and forage intake

There are many variables that potentially influence the amount of feed consumed by

poultry, mostly palatability, feed availability, plant species, nutrient content of forage and

diets, temperature, the stage of growth/production and therefore the actual requirement of

the birds. It has been shown that layers can consume up to 120g/hen/d of forage materials

(Steenfeldt et al., 2007; Hammershøj et al.,2005) or 70g/hen/d forage in dry matter

(Horsted et al., 2006). In Study I, total forage intake from diets ranged from 10-50

g/hen/d which was lower than observed in the previous studies with forages as

supplements (Steenfeldt et al., 2007; Hammershøj and Steenfeldt, 2012). Different poultry

breeds have a different capacity of foraging and their efficiency with respect to balancing

their intake in order to cover their specific nutritional requirements in free choice feeding

system is also regulated by the genetic make-up of the bird (Pousga et al, 2005; Steenfeldt

and Hammershøj, 2015, in press). The intake of forage materials also depends on the

forage type and on the time of the day, when it is offered (Dawkins et al., 2003; Horsted et

al., 2007). In the rearing phase it is important that organic pullets are introduced to forage

material as it will influence gizzard development, which increase the capacity to eat forage

material of a rough structure in adult hens. Maize silage has been known to be very

palatable to hens (Steenfeldt et al., 2007), which is probably due to the fermented aroma

motivating the hens to increase their forage intake. However, the silage quality is very

important. In particular, the fibre content should not be too high (Hammershøj and

Steenfeldt, 2005). In the present study, it was observed that the forage supplementation

decreased the intake of the compound layer diet without affecting egg production which

implies that forage can provide some of the required nutrients to the hens as found in

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other studies (Hammershøj et al., 2010; Steenfeldt et al., 2007; Horsted et al., 2006).

Further, this result indicates that the feeding strategy providing diets formulated under

consideration of the nutrient content in the forage has been successful and should be

evaluated in further studies. The results on productivity and egg quality suggest that laying

hens consume large amounts of foraging material when accessible (Horsted et al., 2006).

The intake of forage material may in general reduce the feed consumption by up to ~ 20%

(Blair, 2008), indicating that digestion and absorption of the foraging material take place

(Steenfeldt et al., 2015, in press). Steenfeldt et al. (2007) reported a 12% reduction of layer

diet intake, while we observed a reduction of 11% when birds fed hemp silage and a 6%

reduction with maize silage. Bassler et al. (2000) estimated that reducing the quantity of

concentrate fed to layers by 15 % has no detrimental effect on productivity. However, the

hens need a certain period of time to adapt to the intake of forage material. In this study,

the intake of maize and hemp silage, as well as vegetables (beetroot, carrot and kale)

increased over time and the highest consumption was observed at 30 weeks of age. The

lower intake of alfalfa silage or grass-herb silage might be due to the high concentration of

dietary fiber (46 to 50% of dry matter) that affects the quality of the silage in spite of a high

methionine content (24 to 36% of DM), or the flavor of the alfalfa- and grass-herb silages

might not be acceptable for laying hens. The increased layer diet intake during 38-42

weeks of age might be due to decreasing outdoor temperature and harsh weather in the

autumn period. To cope with the changing environment, birds often consume more feed,

thus producers are concerned about higher feed intakes and lower efficiencies of feed

utilization during cold periods (Blair, 2008). On the other hand, the access to forage

material seem to have a positive effect on behaviour as the hens are occupied with foraging

and use less time on feather pecking behaviour (Aerni et al., 2000; Steenfeldt et al., 2007;

Kalmendal and Wall, 2012).

In organic poultry production maximum reliance is placed on locally produced feed or

farm derived renewable resources to reduce cost, and in Study III an initiative was taken

to assess potentials of mussel and starfish meal on productivity. It was observed that hens

showed a positive effect on feed intake increased in a dose depended manner. The feed

intake was 5.2% higher in the diet with 4% starfish meal compared to the control diet. It

has been shown previously that laying hens receiving low protein diets increase their feed

intake in order to cover their nutrient requirements (Hurwitz et al., 1998; Van Krimpen et

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al. 2009). The daily feed intake varied between the diets having highest feed intake with

the birds fed starfish meal and this variation might be due to the lower protein content in

the starfish diets. Hurwitz et al.(1998) stated that laying hens receiving low protein diets

increase their feed intake in order to cover their amino acid requirement .

6.3 Egg quality

The experiments were designed on basis of the hypothesis that forage materials and

marine animals might supply the laying hens differently with nutrients depending on the

type of supplementary feed, and that such effects are reflected in the production as well as

egg quality parameters. It is important that a feeding strategy based on foraging does not

compromise the egg quality and thereby reduces the number of outlets for the organic egg

producers (Hammershoj et al., 2010). Over all, the inclusion level of mussel meal or forage

material has positive effect on the egg quality parameters. Egg yolk colour is a very

important egg quality parameter and it can be difficult to obtain an attractive colour of the

egg yolk without adding feed additives. However, in Study III all inclusion levels of

mussel meal significantly improved the yolk colour compared to the control with fishmeal.

The higher inclusion of mussel meal resulted in a darker and a more red egg yolk colour.

The intensity of the yolk colour largely depends on the carotenoid accumulation in the

yolk. Most consumers have a preference for a darker, yellow yolk colour. Laying hens

cannot synthesize carotenoids and are thus dependent on a dietary supply of these

pigments (Nys, 2000). Lightness of egg was reduced with increasing inclusion levels of

mussel meal, however due to the lack of pigments e.g. carotenoids, starfish meal did not

change the yolk colour. Mussels consume algae rich in carotenoids and it has been

reported that the total carotenoid content in fresh mussels is approximately 80 mg/kg

resulting in 400 mg/kg dry matter (Campbell, 1969; Jönsson, 2009) and is above the

carotenoid content in corn gluten meal (Belyavin & Marangos, 1989). Inclusion of forage

materials are supposed to increase yellowness/redness of egg yolk and a darker yolk colour

has been found to be related with forage intake it has been seen that (Ringrose & Morgan,

1939; Sipe & Polk, 1941; Hammershøj & Steenfeldt, 2005).

A high DM content of the egg albumen is desirable when the albumen is heated to obtain a

strong gel (Hammershøj et al., 2001; Hammershøj et al., 2010), or is processed to dried

powder products. The DM of albumen was not increased by the supplementation of up to

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8% mussel meal, however, the highest concentration of mussel meal addition numerically

increased albumen DM. The albumen DM content was not significantly different between

diets, whereas the albumen gel fracture stress decreased in eggs from hens fed the low level

of dietary mussel meal (Jönsson, 2009). The DM of albumen was numerically higher when

the 4% starfish meal diet was fed. As observed from the chemical composition, starfish

meal is rich in methionine which has been shown to increase the albumen DM content

(Prochaska et al., 1996).

The inclusion of different levels of mussel meal and starfish meal had a positive effect on

egg shell strength. Fracture strength of eggs from all the mussel- and starfish meal

treatments was higher than the control. The high calcium content of the experimental diets

in the present study was expected to influence egg shell strength positively. The mussel

meal contained high levels of calcium, essential for egg shell formation, which was

absorbed and retained by the hens. The calcium content in the starfish meal supplemented

diets was even higher than in the mussel meal supplemented diets. A positive effect of

mussel meal on egg shell strength has also been observed previously, and it has been

reported there is 10 folds more calcium in mussel meal compared to a basal diet (Jonsson,

2009; McLaughlan, 2014). Calcium carbonate makes up 97 % of the eggshell why calcium

is very essential for shell formation (Hunton 1995).

6.4 Environment

Production of safe food in a sustainable production system with a low environmental

impact and high animal welfare is the ultimate goal of organic egg production (Tauson et

al., 2005; Abouelezz et al., 2012; Dekker et al., 2012). The retention of N and P is a great

concern in organic egg production, since an excess of these nutrients in the nature is

related to environmental damage such as water eutrophication (EC, 1991; Steinfeld et al.,

2006), which is against the spirit of organic poultry production systems. To ensure

optimum supply of methionine and lysine in the layer diets, the use of excess amounts of

protein in the diet is a common practice, which increases the risk of nutrient leaching from

the out-door area, where toxicity and acidification of soil enhance greenhouse gas emission

(Elwinger et al., 2008; Zeltner and Hirt, 2008). As shown in Study III, nitrogen retention

remarkably increased in a dose dependent manner by the supplementation of mussel and

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starfish meal. It is always desirable to formulate diets, which facilitate the maximum

retention of nutrients, reducing the excretion of nutrients (Aletor et al., 2000). There was a

numerical increase in nitrogen retention, when increased inclusion of zebra mussel meal

was added to the diet in a study by McLaughlan et al. (2014). Aletor et al. (2000) stated

that lower protein intake causes higher nitrogen retention to regain some of what was lost.

The highest nitrogen retention with 12% mussel meal in our study indicates that this diet

provided a more balanced amino acids profile for poultry. The use of mussel meal in

poultry diets is expected to lead to an increase of commercial mussel cultivation

(McLaughlan et al., 2014), which effectively cleans sea waters from agricultural wastes

such as nitrogen and phosphorus which consequently will protect the environment from

pollution and simultaneously produce an valuable protein source in a sustainable way.

In Study I, although the N-retention was less efficient when hens were fed with carrots as

supplements, Steenfeldt et al. (2007) concluded that high intake of silages or carrots did

not increase excretion of nitrogen to the environment. A similar tendency of nitrogen

excretion was also observed in our study. In Study II, where nitrogen retention was

measured in a balance experiment, diet F3+kale and diet E1+hemp silage showed the

highest N retention, though differences among diets were only numerical. Proportionately,

phosphorus retention was also higher in these diets. Although certain fibres may improve

mineral retention in broiler chickens (Ortiz et al., 2009), the effects of different fibres on

different minerals are equivocal (van der Aar et al., 1983). Feeding dietary fibers did not

change nitrogen retention in growing chicks (Akiba and Matsumoto, 1980). Cultivation of

forage material improves soil quality by fixating nitrogen and other organic matters

(Racheal, 2014) and legumes maintain the soil fertility and reduce the need of chemical

fertilizer.

Improved N-retention with decreasing protein content in the diets and without negative

effect on egg production is found in other studies, however, the lower protein diets were

often supplemented with synthetic amino acids (Summers, 1993; Meluzzi et al., 2001;

Keshavarz and Austic, 2004), which was not used in the present study, as they are not

permitted in organic animal production. However, Steenfeldt et al. (2015) observed

increased nitrogen retention when hens were fed organic diets together with forage

materials. A high excretion of P can also cause environmental hazard. In our study, P

retention with diet D+grass-herb silage, dietE1+hemp silage and diet F3+kale was higher

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than with the non-supplemented control diet, though a recent study by Steenfeldt et al.

(2015) found that P retention was generally low when feeding organic diets and foraging

material. Enhanced availability of P from plants will reduce the uses of inorganic feed

phosphates (Gordon and Roland, 1997; Scott et al, 2001; Lim et al., 2003; Keshavarz,

2003).

6.5 Nutrient digestibility

As shown in Study II, the coefficient of total tract digestibility (DC) of different nutrients

varied widely between the experimental diets. The DC of organic matter was found to be

highest with diet E1+hemp silage and lowest with diet F2+carrot. Steenfeldt et al., (2007)

also observed higher DC of organic matter, where carrots were used as supplements,

though diets fed together with maize silage showed that the DC of organic matter was

somewhat lower than that of our present study. Except for diet F2+ carrot, the DC of fat

was high and quite similar in all the diets including the control diet. This might be

explained that insoluble NSP may reflect enhanced conditions for fat emulsification due to

the suggested stimulatory effects of insoluble fiber on gut motility and bile contents in

digesta (Hetland et al., 2003). Inclusion of up to 30% high fiber sunflower meal in a corn

based broiler diets resulted in significant linear increases in apparent ileal digestibility of

fat and protein but the DM and energy digestibility decreased (Kalmendal et al., 2011).

Diets with mussel and starfish meal improved the DC of fat compared to control diet with

fish meal (Study III), which might be due to a better energy to protein balance of this diet.

Mussel and star fish meal are good sources of fat and long chain fatty acids (Jonsson,

2009; Nørgaard et al., 2015) which ultimately can contribute to AMEn to provide energy

and unsaturated fatty acids for egg production. In our study the hens fed diets

supplemented with forage materials digested starch very efficiently in all groups compared

to control diet without forage supplement. Interestingly, though starch content of both

maize cob and maize silage was high, the highest digestibility of starch of all treatments

was found with diet E2+maize-cob silage, being significantly different from the control

(P<0.04). A similar effect on starch digestibility was observed when birds were fed

different forage materials in the study by Steenfeldt et al., (2007). It was reported that

inclusion of 6% wood shavings in a wheat, soya bean meal diet increased ileal starch

digestibility from 98.5 to 99.4% (Mateos et al., 2012). Comparable data on the effects of

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dietary fiber on starch digestibility was obtained by others (Hetland et al., 2003; Rogel et

al., 1987; Svihus et al., 2001) in both broilers and laying hens.

Nitrogen retention was significantly higher with diet E1+hemp silage and diet F3+kale

compared to the others treatments, where the lowest values was found with the diets

supplemented with carrot, maize and alfalfa silage ( Paper II). Steenfeldt et al. (2007) also

found a similar N retention when supplementing maize silage and carrot. Although it is a

great concern in organic production that diets should have a nutrient composition that

reflects balanced diets in order to increase the N-retention, the nitrogen retention of diet

B+maize silage in the present study was lower (0.40) than that obtained (0.52) in the study

by Steenfeldt et al. (2007). This difference might be due to a higher protein contribution

from silage, as the amount of silage eaten by the hens was higher in their experiment than

that observed in the present study. All though the calculated nitrogen intake from both diet

C+alfalfa silage and diet F3+kale was high, surprisingly, the nitrogen retention was

significantly lower with C+alfalfa silage compared to diet F3+kale (P<0.01). The chemical

composition in terms of dietary fiber, DM, starch and fat of diet C was comparable with

other experimental diets, whereas content of dietary fibre was higher in the silage than in

kale, which could explain the difference. It has been shown in other studies that diets high

in fibrous ingredients result in low nitrogen retention (Hogberg and Lindberg, 2004; Holt

et al., 2006; Roberts et al., 2007). In Study III, diets with mussel and starfish meal

increased N-retention in all treatments and indicate that both products contribute with

protein of a high quality. The N-retention increased dose dependently, which is similar to

the findings of McLaughlan et al. (2014).

The Ca retention in most of the diets + supplements was lower than the control diet except

with diet E1+hemp silage, where a higher Ca retention was observed (0.609), however due

to a large variation in the data there were no significant difference between any of the

treatments. There was no significant difference on Ca retention between diets in our study. It has

been stated that a higher calcium content have a positive effect on the Ca retention (Steenfeldt and

Hammershøj, 2015). The P retention was very low in all treatments and the difference

between treatments was not significant. The large variation of data or the use of the total

collection excretion method might be responsible for the variation, also the method used might not

be sufficiently preceise compared to the pre-caecal measurement method as described by

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Rodehutscord et al. (2002). A metabolic interaction between Ca and P occurs in laying hens

(Härtel, 1989) and a high dietary Ca content suppresses the absorption of inorganic

phosphorus (Hurwitz and Bar, 1965). In other studies low values of P retention were able

to give positive results on egg production, egg-weight, eggshell quality and P retention

(Gordon and Roland, 1997; Scott et al, 2001; Lim et al., 2003; Keshavarz, 2003).

In Study II, the amino acid digestibility with all diets, except with diet F1+beet root and

diet F2+carrot was higher than with the non-supplemented control diet. The digestibility

of sulfur containing amino acids was significantly higher with diet F3+kale, which might be

a consequence of a higher N-retention of that diet. It is a big challenge when formulating

diets for organic poultry production to meet the essential amino acid requirements of the

birds while avoiding excessive dietary protein. Essential amino acids can influence the feed

intake, as , as deficiencies or excesses of certain essential amino acids cause feed intake to

decline by influencing the areas of the brain controlling feed intake (Blair, 2008; O’Connell

and Lynch, 2004). As shown in Study III, the digestibility of amino acids in mussel meal

supplemented diets increased dose dependently, however starfish meal with 8% in the diet

reduced amino acids digestibility. The digestibility of amino acids observed in the present

study is comparable to that of McLaughlan et al. (2014).

In Study I, the supplementation of forage material enhanced AME in most of the diets

and a significantly higher AME was observed with diet F+ kale (14.2 MJ/kg DM). Diets

formulated in this study provided more energy than the forage material supplemented

diets of Steenfeldt et al. (2007). Compared to insoluble fibres, the access to soluble fibre

might contribute with energy to hens via an increased microbial fermentation in the caeca

which provides short chain fatty acids, which can be absorbed (Duke, 1986; Choct et al.,

1996). However, AME of diets supplemented with different mussel and starfish meal

(Study III) is comparable with the findings of McLaughlan et al (2014). Poultry tend to

eat to satisfy their energy requirements if fed ad libitum. An absolute requirement for

energy in terms of kilojoules per kilogram of diet cannot be stated because poultry adjust

their intake to obtain their necessary daily requirements. Poultry will increase feed intake

when diets provide low energy content to maintain their nutrients requirement (Scott,

2001). Although fibrous diets tend to limit digestive capacity resulting in low AMEn, even

if fibres escaping digestion by endogenous enzymes (Jørgensen et al., 1996). However, in

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our study diet F3+kale showed an increased AMEn which was higher than that of control

diet, which could reflects the lower fibre content in kale compared to silages.

6.6 Animal welfare

The results of study I showed that hens fed different forages as supplements had a good

plumage quality compared with hens fed layer diet without forage supplement. However,

no feather pecking behavior was found during the experiment in any of the treatments.

From a nutritional point of view this might be due to the fact that the diets were able to

cover the protein, methionine and lysine requirement of hens; hence hens a had good

quality plumage during the experimental period. Ambrosen and Petersen (1997) revealed

that protein or methionine and cysteine deficiency can impair plumage condition.

Additionally, it seems that access to forage keeps the hens occupied allowing them to

perform their foraging behavior (Vam Krimpen et al., 2005; Cooper and Albentosa, 2003).

Further, forage material is suggested to give hens a feeling of satiety decreasing feather

pecking and cannibalism which consequently improves plumage condition (Aerni et al.,

2000; Steenfeldt et al 2007; Van Krimpen et al., 2005; 2009; Kalmendal and Wall, 2012).

6.7 Gastro intestinal characteristics and microbiology

Two forage supplemented diets and the non-supplemented control from Study I were

selected to study the composition and activity of the gastrointestinal microbiota. Hens

receiving diet B+maize silage and diet C+alfalfa silage showed only a tendency to increase

gizzard size. It has been shown that high amounts of insoluble NSP in silage stimulate

gizzard activity and increase the retention time of the feed and increase gizzard weight

(Steenfeldt et al., 2007; Engberg et al., 2002, 2004; Hetland et al., 2003; Idi et al., 2005).

In our experiment a numerically higher gizzard weight was found in diets with maize silage

and alfalfa silages compared to control diet without silage, but the difference was not

significant in contrast to the study by Steenfeldt et al. (2007), who found heavier empty

gizzard weights when hens fed maize silage, barley pea silage and carrots as supplement

than control diets without forage supplement. This might be explained by control hens

having an access to the outdoor run which enabled hens to pick up coarse feed items other

than green grasses. Although the grass from the outdoor run was removed regularly, the

hens still had access to grassroots, stones and other unidentified fibre sources which

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potentially helped to develop the gizzard of the control hens. A study by Shrestha (2013)

also found a significantly higher relative gizzard weight, when maize silage was added to

the diet as compared to a non-supplemented control diet. The access to silages has been

shown to decrease gizzard pH via an increased secretion of HCl in the proventriculus and

increased mechanical activity of the gizzard (Engberg et al., 2004); Idi et al., 2005;

Steenfeldt et al., 2007). In our study gizzard pH was numerically low compared to control

diet. The influence of the diets on the number of selected dominant bacteria and organic

acid concentrations in the gastrointestinal tract was very limited. Although not significant,

the concentration of organic acids in the caeca was numerically increased when feeding the

maize silage supplemented diet, which might indicate a higher fermentation activity at this

location.

Chapter 7: Conclusions

This PhD thesis demonstrated that alternative feeding strategies to formulate 100%

organic layer diets support egg production and welfare of organic laying hens. A holistic

view on utilizing the nutrient from locally available forage material in term of energy,

protein and amino acids or marine feed ingredients as alternative protein sources is

expected to facilitate the transition to 100% organic feed supply for laying hens. The

following conclusions were drawn from the studies.

The different foraging material varied in nutrient composition to a high extent, where

the highest content of protein and methionine was found with alfalfa silage and kale

followed by grass-herb- and hemp silage, whereas maize- and maizecob silage were

characterized with high content of starch. Formulating organic layer diets considering

nutrients from different forage materials did not show any negative effect on egg

production compared to the control group without forage and diets supplemented with

maize silage and kale tended to increase egg production and improve some egg quality

parameters.

Increasing intake of different forage materials reduced layer diet intake without

altering production parameters which have beneficial effects on feed cost.

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In relation to the welfare of laying hens, there was no incidence of feather pecking in

forage material supplemented diets. Other welfare parameters and mortality rate were

also improved in some of the groups diets supplemented with forage.

The calculated daily nitrogen (N) and phosphorus (P) retentions per hen were

improved in diets with maize-, grass-herb-, maize cob- silage and kale , however diets

with alfalfa-, hemp silage, beeroot or carrot did not have positive influence on N and P

retentions..

Different layer diets supplemented with silages and vegetables in apparent nutrient

digestibiliy, where increased fat and starch digestibility were obtained in spite of the

high content of dietary fibre. Layer diet supplemented with especially kale provide

protein, amino acids and some extent energy to laying hens, contributing to the hens

nutrient requirement for maintaining their egg production.

Marine alternative feed sources, e.g. mussel and starfish meal can replace fishmeal in

organic poultry production, as the content of protein and the amino acid profile are

comparable to fishmeal. The 12% mussel meal or 8% starfish meal inclusion in layer

diet have positive effects on the production performances of laying hens, even though a

higher level of the star fish meal and mussel meal in the diets may cause off-flavour in

the eggs. In relation to the environment, excretion of N, Ca and P may be reduced by

the use of mussel meal as protein source in organic layer diets.

Increasing amount of mussel meal but not the star fish meal improved egg yolk colour,

compared to diets with fishmeal, which is an important parameter for the consumer.

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Chapter 8: Perspectives

The EU regulation impose organic farmers to fed their animals with diets, where at least

20% of the raw materials used in the feed formulations have to be grown primarily on-

farm or in the same region in cooperation with other organic farmers or feed

manufacturers. The main challenge in this circumstance is to formulate 100% organic feed

for poultry that fulfill the nutrient requirements based on available organic ingredients.

The motivation for this thesis was to provide a holistic view on the potential of utilizing the

locally available forage materials to hens in relation to the development of an alternative

feeding strategy. Organic vegetable protein sources if high quality grown within EU is very

limited, however it should be possible to formulate diets based on 100% organic

ingredients if new protein sources of both animal and vegetable origin can be approved as

organic feed within the EU. Further, nutrients from foraging material and feed items from

the outdoor area should also be considered as part of the daily nutrient intake of organic

poultry. Chemical analyses of different foraging materials as well as mussel- and starfish

meal showed that the protein and amino acid contents could contribute to the nutrient

requirement of organic poultry. By recognising the nutritional value of foraging material, it

would be possible to optimize organic feed more appropriately. However, before

implementing foraging materials as an ingredient in feed formulation, there is a need,

through digestibility and performance trials, to validate the digestible nutrient content and

the effect of fibre content. The result from the present study indicated that some types of

forage material contribute with nutrients, however, additional studies would be valuable

for a further understanding of the utilization of nutrient in foraging material and the effect

of high content of fibres. The challenge in relation to safeguarding adequate nutrition in

organic poultry is related to the fact that a majority of the organic poultry production

systems of today is based on the same rationale as in the conventional production systems,

expecting a highly efficient production. The results of the present study indicate that some

of the forage materials are very efficiently consumed by hens without altering their

production potentials, even diet containing forage material like maize silage, grass-herb

silage and kale can improve production parameters. Between the two animal protein

sources, mussel meal had a profound impact on improving egg production and egg yolk

color. Hens are capable to digest efficiently different nutrients from forage materials as

well as from mussel and starfish meal. Several steps are needed before it would be possible

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to go from the results obtained in this thesis, to an actual implementation of the new

feeding strategies at the farmer level. Interpretations of the impact and applicability of the

results will largely depend on the factors like breed, housing condition and feeding

strategy. The following issues deserve attention for future research:

From our study it was revealed that some soybean cultivars can be grown in the temperate

climate in Denmark and, the quality with regard to protein content and amino acids profile

were comparable to world standard. In future, focus should be made on the development

of a cold tolerant soya bean cultivar to facilitate a sustainable supply of vegetable protein

for organic laying hens. The possibility to increase the use of local grown soybeans and

other legumes will reduce the need for imported soybeans from overseas countries, which

also will have a positive effect on the environment due to reduced transportation from long

distances.

Hemp silage have some potentials to be used in the organic diet, however, difficulty of

harvesting hemp is another practical issue that should be addressed for any future

research.

If organic egg production can be integrated with other organic production systems like

fisheries and agriculture, production cost will be reduced as well as it will ensure cycling of

nutrient resources, which ultimately can result in less environmental impact.

Although in the present study mussel meal without shell was used, preparing mussel meal

with flesh plus shell could meet the Ca requirement of the laying hens together with

protein and amino acids.

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Chapter 9: References

1. Aarnink, A. J. A., Hol, J. M. G. & Beurskens, A. G. C. (2006) Ammonia emission and nutrient load in outdoor runs of laying hens. NJAS-Wagen. Journal of Life Science, 54: 223-234.

2. Abouelezz, F.M.K., Sarmiento-Franco, L., Santos-Ricalde R. & Solorio-Sanchez, F. (2012) Outdoor egg production using local forages in the tropics. World,s Poultry Science Journal, 68: 679-692.

3. Aerni, V., El-Lethey, H. & Wechsler, B. (2000) Effect of foraging material and food

form on feather pecking in laying hens. British Poultry Science, 41: 16-21.

4. Agunbiade, J. A., Adeyemi, O. A., Ashiru, O. M., Awojobi, H. A., Taiwo, A. A., Oke, D. B.

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