STARFISH AS FEEDINGREDIENT FOR PIGLETS

SØSTJERNER SOM FODERMIDDEL TIL SMÅGRISE

PIA SØRENSEN

STUDENT ID: 20107218

MASTER THESIS · AGROBIOLOGY · AUGUST 2015

Supervisor:

Jan Værum Nørgaard, Department of Animal Science, Aarhus University

I

PREFACE

This current thesis is completed as the final part of the master degree in Agrobiology with area of

specialisation in animal health and welfare, at Science and Technology, Aarhus University. The

thesis corresponds to 45 ECTS and includes a review of published literature and a practical

experiment. It was a privilege, within the scope of the budget and what was realistic in relation to

the objective of the study, to be the one who should design the study appropriate. The experimental

work was carried out during November and December 2014 at the facilities of Aarhus University,

Research Centre Foulum. The practical experiment and results are presented in a manuscript, and

are intended to be submitted to the Animal Feed Science and Technology. As the manuscript only

presents some results and a discussion of the corresponding results, a separate section will present

additional results and discus the results in general.

The project was financed by the Green Development and Demonstration Programme under the

Ministry of Food, Agriculture and Fisheries, Denmark, in cooperation with the Danish Shellfish

Centre, Foreningen Muslingeerhvervet and Agro Korn.

This thesis aims to clarify perspectives for feeding newly weaned pigs with a new alternative source

of animal protein. The target audience of this master thesis includes students, pig farmers, advisors

and scientists within the field of pig nutrition with special interest in feeding of newly weaned pigs.

I would like to express my gratitude to my supervisor associate professor Jan Værum Nørgaard,

Department of Animal Science – Animal Nutrition and Environmental Impact, Aarhus University,

for support, useful discussions and feedback through all parts of the process.

Also a great thanks to the personnel at the pig facilities at Research Centre Foulum, Aarhus

University, for helping with collection of data during the practical experiments.

Foulum, August 2015 Pia Sørensen

II

III

SUMMARY

Starfish in high concentrations are considered pests by the commercial mussel fishermen in

Denmark because starfish are predators to mussels that are otherwise used for human consumption.

A regulation of the population size is therefore needed. Concurrently, the organic feeding business

wants new protein sources, which can contribute in supplying the livestock production with

essential amino acids. The need for such protein sources is reinforced in a time, where feed for

organic animals are determined to be of 100% percent organic origin in a foreseeable future.

Starfish meal is not a new product, and was evaluated as feed ingredient in studies with poultry

several decades ago. It was concluded that the protein fraction in starfish meal (SM) was

comparable to fish meal (FM) in quality. The objective of the present study was to investigate

whether piglets fed a diet containing SM as a high-quality protein source would perform on equal

terms with pigs fed traditional diets for piglets weighing 9 to 15 kg of bodyweight (BW).

The effects of including SM as an alternative protein source in diets for piglets on performance,

faeces characteristics and plasma parameters were investigated. Four diets were formulated to

contain different protein sources; FM, soy protein concentrate (SPC) and two levels of SM (SM5%

and SM10%). All diets contained 174.4 g soybean meal (SBM)/kg and were supplemented up to

166-172 g SID CP/kg with either FM, SPC, SM or a combination giving raise to two SM + SPC

diets with different levels of SM and SPC. One week after weaning, 96 pigs (BW 9.6 ± 0.4 kg) were

individually housed and allocated to one of the four diets (n=24) and feed ad libitum for a 14-d

period. Average daily feed intake (ADFI), average daily gain (ADG) and gain to feed ratio (G:F)

were determined. Pigs fed the SM10% diet had an ADG 23-28% lower (P<0.001) than pigs fed the

other diets, despite that they ate the same amount of feed. Faeces characteristics were evaluated by

visual judgement during five days and no differences were observed, indicating no effect of the diet

on diarrhoea. Blood samples were collected on d 15. Plasma Ca concentration was higher (P<0.001)

in pigs receiving SM10% compared to FM, SPC and SM5%. The opposite was the case with P

where the concentration was lower (P<0.001) in SM10% compared to FM, SPC and SM5%.

In conclusion, feeding diet SM5% resulted in performance equal to pigs fed the control diets. The

negative effect on performance when feeding the SM10% diet, may be the high Ca level and wide

Ca:P that affects digestibility and absorption of P negatively. Moreover, formation of mineral-

phytate complexes, may reduce mineral bioavailability. The determining factor for the maximum

inclusion level of SM in diets for piglets may be the dietary Ca level and the resulting Ca:P.

IV

SAMMENDRAG (DANISH SUMMARY)

Muslingefiskeriet i Danmark er plaget af enorme mængder søstjerner, som æder muslingerne på

havbunden, der ellers fiskes og bruges til human konsum. En regulering af søstjernebestanden er

derfor ønsket. Samtidig, efterspørger den økologiske foderstofbranche nye proteinkilder, der kan

bridrage med essentielle aminosyrer til husdyrproduktionen. Behovet for sådanne proteinkilder er

forstærket i en tid, hvor foder til økologiske dyr indenfor en årrække skal være af 100% økologisk

oprindelse. Søstjernemel (SM) anses ikke for værende et nyt produkt, da det for flere årtier siden

blev evalueret som foderingrediens i studier med fjerkræ. Det blev konkluderet, at kvaliteten af

proteinet i SM var sammenlignelig med den i fiskemel (FM). Formålet med studiet var, at

undersøge om smågrise fodret med en blanding indeholdende SM som høj-kvalitets proteinkilde, vil

udvise samme produktivitet som hos smågrise fodret med traditionelle blandinger til smågrise i

vægtintervallet 9-15 kg.

Effekten af at inkludere SM i foderblandinger til smågrise blev bestemt ud fra produktivitet, fæces

karakteristik og plasma parametre. Fire blandinger blev sammensat til at indeholde forskellige

proteinkilder; FM, sojaprotein koncentrat (SPK) og to niveauer af SM, henholdsvis 5 og 10%. Alle

blandinger indeholdt 174,4 g sojaskråfoder/kg, og FM, SPK eller SM, eller en kombination af SM

og SPK i forskellige mængder, resulterende i 166-172 g standardiseret ileal fordøjeligt råprotein/kg

foder. En uge efter fravænning, blev 96 smågrise (9,6 ± 0,4 kg) individuelt opstaldet og tildelt en af

de fire blandinger (n=24) og fodret ad libitum i 14 dage. Gennemsnitlig daglig foderindtag og

tilvækst, og tilvækst til foder ratio blev bestemt. Tilvæksten ved tildeling af foder med 10% SM var

23-28% lavere (P<0,001) end hos grise tildelt de andre blandinger, trods samme foderoptag. Fæces

karakteristik blev evalueret vha. visuel vurdering henover 5 dage. Ingen forskel blev observeret,

betydende at blandingerne ingen effekt havde på forekomsten af diarré. Blodprøver blev taget på

dag 15. Calcium koncentrationen i plasma var højere (P<0,001) hos grise tildelt 10% SM

sammenlignet med de andre blandinger. Det modsatte var tilfældet med P, hvor plasmaindholdet

var lavere (P<0,001) hos grise tildelt 10% SM sammenlignet med de andre blandinger.

En blanding med 5% SM giver en produktivitet som hos smågrise tildelt kontrolblandingerne. Den

negative effekt af 10 % SM på produktivitet kan være det høje Ca indhold og brede Ca:P, der

påvirker fordøjelse og absorption af P negativt, sammen med reduceret mineraltilgængelighed

grundet dannelse af mineral-fytate komplekser. Den bestemmende faktor for maksimum iblanding

af SM i blandinger til smågrise er indholdet af Ca i foderet og den resulterende Ca:P.

V

TABLE OF CONTENTS

PREFACE ............................................................................................................................................. I

SUMMARY ....................................................................................................................................... III

SAMMENDRAG (DANISH SUMMARY) ...................................................................................... IV

ABBREVATION KEY .................................................................................................................... VII

INTRODUCTION ............................................................................................................................... 1

Hypothesis and objective.................................................................................................................. 2

LITTERATURE REVIEW .................................................................................................................. 3

1 The need for alternative protein sources ....................................................................................... 3

1.1 Constraints in protein sources ............................................................................................... 3

1.2 Demands in relation to feeding organic pigs ......................................................................... 3

1.3 Environmental benefit ........................................................................................................... 6

2 Starfish .......................................................................................................................................... 8

2.1 Occurrence............................................................................................................................. 8

2.2 Handling and use of starfish .................................................................................................. 9

2.3 Chemical composition of starfish ........................................................................................ 10

2.4 Starfish as feed ingredient ................................................................................................... 13

3 Nutrient requirement of piglets ................................................................................................... 16

3.1 Energy requirement ............................................................................................................. 16

3.2 Protein and amino acids requirement .................................................................................. 17

3.3 Mineral requirement ............................................................................................................ 18

4 Practical feeding around weaning ............................................................................................... 19

4.1 The transition of weaning .................................................................................................... 19

4.2 Diet characteristic and protein sources ................................................................................ 19

5 Final remarks and conclusion of literature review ..................................................................... 23

GENERAL DISCUSSION OF RESULTS AND THE STUDY ....................................................... 53

6 Discussion and future development ............................................................................................ 53

6.1 Design of the study .............................................................................................................. 53

6.2 Faeces score method ............................................................................................................ 54

6.3 Results from the study ......................................................................................................... 55

6.4 Possibly same features in starfish meal and fish meal ........................................................ 56

6.5 Possibility of further development of the product ............................................................... 58

VI

CONCLUSION .................................................................................................................................. 59

IMPLICATATIONS AND PERSPECTIVES ................................................................................... 60

LITERATURE CITED ...................................................................................................................... 61

VII

ABBREVATION KEY

AA Amino acids

ADFI Average daily feed intake

ADG Average daily gain

ANF Anti-nutritional factors

BW Body weight

Ca Calcium

CP Crude protein

CAA Crystalline amino acids

DHA Docosahexaenoic acid

EPA Eicosapentaenoic acid

FA Fatty acids

FCR Fed conversion ratio

G:F Gain to feed ratio

ME Metabolisable energy

N Nitrogen

NE Net energy

P Phosphorus

PSP Paralytic shellfish poison

PUFA Polyunsaturated fatty acids

SBM Soybean meal

SID Standardized ileal digestibility

SPC Soy protein concentrate

VFI Voluntary feed intake

VIII

Introduction

1

INTRODUCTION

Mussel fishermen in Limfjorden, Lillebælt and Isefjorden in Denmark, have reported that the

occurrence of starfish in the inner Danish waters have been increasing the past years, and the

mussel fishery has been plagued by substantial amounts of starfish that negatively affect the fishery

of mussels (Kjølhede, personal communication, 2015). Likewise, the Danish Shellfish Centre has

documented that mussels in many relaying areas have been eaten by starfish and confirms that the

density of starfish in areas of Limfjorden is increasing (Nielsen et al., 2014). Therefore, there is a

need to regulate the population of starfish. Concurrently, the organic feeding business needs new

protein sources, which can contribute in supplying the livestock production with indispensable

amino acids (AA). Because starfish has been proven to have a potential AA and protein profile,

which can lead to a reduction in nitrogen excretion and emission, it will help making organic

production more sustainable. The need for such protein sources is reinforced in a time, where feed

for organic animals are determined to be of 100% percent organic origin by December 31 2017

(Smith et al., 2014).

In 2014 the project “Starfish as New Source to Marine Proteins” (STARPRO) was financed by the

Green Development and Demonstration Programme under the Ministry of Food, Agriculture and

Fisheries, Denmark, a project cooperation between the Danish Shellfish Centre, Foreningen

Muslingeerhvervet, Agro Korn, and Aarhus University, Foulum. The purpose of STARPRO is to

establish a sustainable fishery for starfish in preparation for producing a 100% organic feed

ingredient for monogastric livestock. The project includes the whole value chain with the concrete

goal to develop cost-effective methods for production of starfish meal (SM) and within a

foreseeable future establish a fishery of 10,000 t of starfish a year amounting a production of 2,500 t

of SM a year. The activities in the project STARPRO will constitute determination of the

population size, testing of developed methods for production of SM constituting the preparatory

processing e.g. washing sand of the caught starfish to grinding of the dried starfish, designing of

diets containing SM and test the diets including SM in feeding studies with poultry and pigs. It is

expected that it will generate employment through the establishment of a Danish production of feed

for monogastric livestock containing SM. The environment will also benefit from the removal of

starfish, since the fishery of starfish will remove nutrients from the coastal waters, and the emission

of nutrients from organic farming will be less due to improved feed efficiency. Furthermore, the

production of mussels for human consumption will be more profitable as the result of reduced

predation on mussels. On the basis of existing analysis of SM on the chemical characteristics and

Introduction

2

the results of ileal digestibility in pigs (Nørgaard et al., 2015), it is assessed that the product is

suitable as feed ingredient for piglets, by which the possibility of SM as feed ingredient for piglets

were requested to be examined.

Hypothesis and objective

The hypothesis of the thesis:

It is hypothesised, that SM as a high-quality protein source can be used as feed ingredient for

piglets.

The objective of the thesis:

The objective of this master thesis was to describe the need to investigate new protein sources for

piglets due to the increasing need for alternative protein sources, together with requested

sustainability and local origin of feed ingredients. Moreover, the increasing problems experienced

with starfish in the Danish waters leads to description of the occurrence of starfish and the possible

use as feed ingredient for piglets. The objective was, furthermore, to describe the piglets need for

nutrients post-weaning and the high-quality protein sources used at the present. Finally, a study was

conducted using four diet of which two contained SM adjusted for piglets with body weight 9 to 15

kg. In the study it was investigated, whether the use of SM as feed ingredient in diets for piglets will

give the same average daily feed intake (ADFI), average daily gain (ADG) and gain to feed ratio

(G:F) as traditional diets.

Literature review

3

LITTERATURE REVIEW

1 The need for alternative protein sources

1.1 Constraints in protein sources

Protein feedstuff used earlier, have either become a limited resource as fish for the production of

fish meal (FM), or banned in many countries as animal by-products like meat and bone meal on

account of the risk of transferring transmissible spongiform prion transferred diseases such as BSE.

Moreover, vegetable protein sources often used are to a great extend genetically modified. Sieradzki

et al. (2006) found that about 98% of the soybean samples collected contained transgenic soya, a

feed ingredient used for the production of both supplementary and compound feedstuffs. In many

countries there is still strong public opposition against using genetic modified plants and is

considered unsuitable as feed for food producing animals.

Despite the fact that soybean meal (SBM) is often genetically modified, SBM is the most important

protein source in pig feed, and is generally known as a protein source with a consistent and high

quality (Jezierny et al., 2010). It contains a comparable high portion of indispensable AA that are

easily digestible for monogastric animals. In temperate environments, soybeans can be difficult to

cultivate and the pig industry relies heavily on the import of soybeans. The concerns of the

sustainability and security of pig production have been increasing, due to the amounts of the raw

material used (e.g. soybean) currently, and if it continues to increase in the same magnitude. Also,

the increasing demand for SBM is associated with increasing demands for land use change lead to

environmental concerns (Fearnside, 2001). Thus, there is a need to find other applicable protein

sources to use as feed for pigs. In temperate environments, vegetable protein sources such as grain

legumes have been taken into consideration, while potential marine protein sources often have been

neglected, but could be considered as a feed ingredient for monogastric livestock. In a country like

Denmark, with a proportionally small area of cultivated land compared to the number of animals,

the surrounding sea could contribute with interesting protein sources such as seaweed, mussels,

crabs and starfish.

1.2 Demands in relation to feeding organic pigs

Organic farming is experiencing difficulties in feeding with 100% organic feed (Sundrum et al.,

2005; Nicholas et al., 2007; Grela et al., 2012). At the end of December 2014, the delegation from

the EU Organic Regulatory Board that allowed organic poultry and pig producers to include up to

Literature review

4

5% non-organic feed in the diets was supposed to cease. From then on, it would have been required

by all producers to feed a 100% organic diet for monogastric animals. Due to difficulties in

implementing this, the deadline has been extended to 31 December 2017 (Smith et al., 2014). Thus,

there is a need for possible strategies on how to cover the nutritional needs with organically

produced feed ingredients at different phases of production, ensuring high animal welfare and

health. The main problem is the scarcity of organic feed ingredients in Europe in proportion to the

demand of both energy and protein in concentrated feed ingredients that at the present are essential

when feeding monogastric animals (Smith et al., 2014). Furthermore, organic farming does not

accept the use of processed oilseed products, when subjected to solvent extraction processes (e.g.

hexane), or the use of genetically modified feed ingredients (e.g. SBM). In addition, it is prohibited

to supplement with crystalline amino acids (CAA) to optimise the diet for pigs according to their

AA requirements (Ministry of Food, 2015b). It is therefore crucial to generate suitable alternatives

to meet the pigs’ protein and AA requirements in organic production system according to the

Danish recommendations set by Tybirk et al. (2014).

The European organic research project ICOPP (Smith et al., 2014) looked at data on the organic

crop production, due to the request for using entirely organically feed for livestock. It was

concluded, that based on the protein supply and demand in Europe, it seems unrealistic that the

ICOPP countries (10 European countries) will be able to fulfil the organic protein demand in the

foreseeable future. Even if the countries try to produce protein crops relevant for the country in

question, it will be difficult to meet the indispensable AA requirements for the individual animal

category. The climate is the determining factor and the area of arable land for high-protein crops is

limited in the northern part of Europe, and considered as not suited for cultivation of the most

valuable high-protein crop; soybeans. In addition, the European Commission envisions more strict

regulation for feed, where a greater proportion of the feed should be produced on the farm. This

means, increased self-sufficiency for the organic farm or the product should be produced in the

region where the feed is to be used. For some producers, this can be complicated to achieve (Smith

et al., 2014). Factors such as metabolisable energy (ME) content, AA availability, digestibility, fibre

content and type and quantity of anti-nutritional factors (ANF), depending on the product in

question, will have an influence on the possible maximum inclusion rate of home grown protein

sources. For instance, a feed ingredient having a low protein content or deficient in one of the

indispensable AA, may be considered a valuable feed ingredient if other qualities are present, such

as being high in an indispensable AA important for the animal intended (Smith et al., 2014).

Literature review

5

The fundamental basis of the breeding material in conventional and organic pig herds are the same.

Therefore, the metabolic processes do not differ between the organic and conventionally reared

farm animals, and the pigs requirement for nutrients are equal in both rearing systems (Nicholas et

al., 2007). The Danish Energy Assessment System (abbreviated FE; 1 FE = approx. 7,380 kJ NE) is

designed so that the feed intake per kg growth is independent of the combination of feed ingredients

in the diet, provided that there is the same level of essential nutrients per FE. For growing pigs,

including piglets, growing and finisher pigs, the energy value FEsv is used. However, the

recommendations for an adequate nutrient supply generally follow similar guidelines in organic and

conventional feeding; the outdoor access required in organic farm systems leads to higher energy

requirements by the pigs but not necessarily higher micronutrient requirement. Concurrently, with

the restricted availability of indispensable AA in organic pig production, pigs may not be able to

fulfil their genetic potential for protein accretion. Even if they are offered a standard optimized feed

ration ad libitum, the living conditions can affect the performance level negatively (Nicholas et al.,

2007).

It has been pointed out by Grela et al. (2012), that organic feeding of pigs is mainly limited by the

supply of AA and some specific compounds as calcium (Ca), not often found in feed ingredients

grown at the farm. The chemical composition of organic feed ingredients may differ substantially

from the conventionally grown feed ingredients, especially regarding crude protein (CP) and

minerals, but also the energy value is slightly lower as compared to the conventional feeds (Grela et

al., 2012). Some organic feed ingredients have a higher crude fibre level and detergent fibre fraction

(ADF and NDF) than conventional ones, related to e.g. the small-sized seeds in grain that contain

more fibre and less starch. This can also refer to the depressed digestibility when having high crude

fibre content, that is inversely proportional to feed energy value (Grela et al., 2012). Furthermore,

roughage, fresh or dried fodder, or silage must be added to the daily ration for pigs, contributing to

the intake of crude fibre in pigs (Ministry of Food, 2015b).

To fulfil the need of AA, especially for piglets, it is proposed that it only can be achieved by

feeding with some natural non-agricultural feed ingredients like FM, that is not considered organic,

but is still approved with up to 5% in an organic diet (Ministry of Food, 2015b). An experiment

conducted to compare pigs fed with plant feedstuffs of organic origin and a diet enriched with FM

showed that the addition of FM significantly improved average daily gain (ADG), feed conversion

ratio (FCR) and carcass characteristics in the body weight (BW) interval 25-70 kg. The conclusion

Literature review

6

was that it was found reasonable to fortify typical organic mixtures with FM (Grela et al., 2012).

Though, despite the good effects, in Denmark, it is recommended not to feed pigs weighing more

than 40 kg with FM. This is due to the risk of rancidity and reduced storage stability of the pork

caused by high content of polyunsaturated fatty acids (PUFA) (Bejerholm et al., 1990).

Conventional and organically produced mussel meal are found to provide highly digestible AA

(Smith et al., 2014; Nørgaard et al., 2015), which can help improve the AA balance in feed for

organic piglets and is a potential substitute to FM. Mussel meal was found to be used as a

replacement for common protein sources in feed for growing/finishing pigs with maintained

production results regarding growth, feed efficiency and carcass quality (Smith et al., 2014).

Corresponding to the recommended maximum inclusion rate of FM, inclusion rate of mussel meal

should not exceed 5% in accordance with the allowed inclusion rate of non-organically feedstuffs,

as allowed only until 31 December 2017 (Smith et al., 2014).

An EU founded research project (EGTOP, 2015) with the objective to provide more knowledge on

how to achieve 100% organic rations in diets for organic livestock, has found it relevant to include

novel animal species such as insects, blue mussels and starfish as feed ingredients in the evaluation.

In same context, the Danish research project, “Biofouling and pest animals: sea stars” used starfish

as a protein rich feed ingredient for pigs, poultry and fish, and the results were promising

(Holtegaard et al., 2008). Starfish is not registered in the Community Catalogue of feed materials as

is the case with mussel meal. In the report by EGTOP (2015), it is shown that starfish may fit under

the code numbers 10.1.1 and 10.2.1 and can be used as feed ingredient in animal feed according to

Reg. (EC) 767/2009. Furthermore, according to Reg. (EC) 889/2008, Article 22 (e) as amended by

Reg. (EC) 505/2012 products from sustainable fisheries may be used as an organic feed ingredient.

It was concluded that feeding pigs with starfish should be included in the legislations, since starfish

fishery fulfil the environmental requirements for sustainable fishery. At the present, starfish is not

allowed as feed ingredient for pigs in EU, but the Danish authorities comprising

NaturErhvervstyrelsen, are working on getting starfish licensed as feed ingredient in the EU.

1.3 Environmental benefit

One of the largest pollution problems to the marine environment worldwide is the excess loading of

nutrients. Excess run-off primarily from agriculture of especially nitrogen (N) and phosphorus (P)

caused by e.g. low digestibility of protein in livestock, unbalance and oversupply of nutrients for

livestock, has accelerated the fluxes of these nutrients to coastal waters. Thus, fertilization of

Literature review

7

coastal ecosystems is a serious environmental problem (Petersen et al., 2014). An alternative to

terrestrial reduction measures, such as winter green fields, restricted period for applying fertilisers

and reduced fertilisation below economic optimum, is mussel production (Petersen et al., 2012).

Mussel farming is primarily used for producing mussels for human consumption, but can likewise

be used as a measure to remove nutrients from the marine environment (Edebo et al., 2000). The

concept of mussel farming as a tool for mitigation is that by harvesting the cultured mussels, the

mineral nutrients that are incorporated in the mussels will be removed and recycled when bound in

mussels and brought back from sea to land (Edebo et al., 2000; Petersen et al., 2014). Petersen et al.

(2014) concluded that the production of 50-60 t of mussels ha-1

mussel farm per year corresponds to

a potential nutrient removal of 0.6-0.9 t N and 0.03-0.05 t P per ha mussel farm per year. The cost

for nutrient removal in the study done by Petersen et al. (2014) was 105-150 DKK per kg N, which

did not include the potential of having an income by selling the mussels. If compared to e.g. sowing

catch crops in the spring, which has a price of 26-79 DKK per kg N and fertilization below

optimum with a price of 60-242 DKK per kg N, the cost for nutrient removed by mitigation mussels

is comparable to other land-based mitigation measures, making it a cost-efficient measure (Petersen

et al. 2014).

On equal terms with mussels, it can be suggested that starfish could also be used as a mitigation

tool, since they, contrary to fish, do not move over long distances, but stay in the same area most of

the time. Starfish have not yet been permitted as a mitigation tool, but have a great potential equally

with mussel farming, even without impact on the environment, e.g. sedimentation of organic matter

as is the case with mussel farms. Living starfish contain on average 1.65% N and 0.15% P of wet

weight. The removal of 10,000 t of starfish, would directly remove approximately 165 t N and 15 t

P from the coastal water (Petersen, personal communication, 2015). Such removal could be part of

achieving “good ecological status” in the Danish coastal waters, as is the goal for the European

Union Water Framework Directive (Anonymous, 2000). The usage of starfish in diets for

monogastric animals would therefore not only contribute with dietary protein, but also reduce the

environmental impact of the animal production by recycling of nutrients.

Literature review

8

2 Starfish

Starfish (Asterias rubens) are probably the single most significant predator of blue mussels living

on the seabed in natural mussel beds, bottom cultures or relaying areas. In areas like these, starfish

can cause significant decreases in the number of live mussels that are otherwise fished and used for

human consumption (Holtegaard et al., 2008). The occurrence of starfish in the inner Danish waters

has been increasing (Holtegaard et al., 2008) and still is (Kjølhede, personal communication, 2015).

Hence, the mussel fishermen in Limfjorden, Lillebælt and Isefjorden, have reported that the mussel

fishery has been plagued by substantial amounts of starfish that negatively affect the fishery. The

fishermen have suggested that if they fish the starfish, they could be of benefit elsewhere (Kjølhede,

personal communication, 2015).

2.1 Occurrence

The observed density of starfish fluctuate and is dependent upon the seabed, where in fine sand 3-

31 individuals have been observed per m2 to 324-809 individuals/ m

2 on alga carpets (Holtegaard et

al., 2008). Dare (1982) showed in his examinations in the Irish Sea, that in certain periods starfish

attained swarms of 300 to 400 individuals/m2, equivalent to a wet weight biomass of around 12 to

16 kg/m2. In relation to a translocation trial in Nissum Bredning in a part of Limfjorden, a swarm of

starfish devoured 72.7 t of blue mussels during a period of 7 months, indicating that starfish in

substantial extent can reduce the mussel population (Holtegaard et al., 2008). The size of the

population of starfish in all Danish waters is not known, but examination done in 1996 in the central

part of Limfjorden showed an estimated population size of 10,000 t (Holtegaard et al., 2008). The

impact study done by Nielsen et al. (2014) on request of NaturErhvervstyrelsen in Denmark,

showed that a fishery of 7,000 t starfish in the period 2014-2015 as proposed in the fishing plans

would constitute 18-27% of the population in Løgstør Bredning, the greatest part of Limfjorden,

depending on the method used to determine the population size. DTU Aqua assessed, in proportion

to the total population of starfish in Limfjorden, that fishing 7,000 t along with fishing 2,000 t in

Lovns Bredning, the south-eastern part of Limfjorden, would be sustainable compared to the whole

population in Limfjorden (Nielsen et al., 2014). There is no well-documented data related to the

population size in Limfjorden, and the estimate of the population size is therefore considered with a

good amount of uncertainty. The increase in population size during the latest years entail that the

assessment of the impact of fishing starfish is not related to any insecurity when taking the whole

population in consideration (Nielsen et al., 2014). Therefore, a fishery of 10,000 t of starfish per

Literature review

9

year would be possible. For the fishermen to have an economically sustainable fishery of starfish,

the estimated density of starfish on the seabed should be 1.5 kg/m2 (Nielsen et al., 2014).

The predation on starfish is rather limited and the population seems primarily controlled by

environmental considerations and amount of feed available. Starfish have not been characterised as

a species worth for preservation, and can on the contrary affect biogenic reefs, constituted by thick

layers of mussels, negatively (Nielsen et al., 2014). In Denmark, the natural mussel beds are

considered as a part of these biogenic reefs. With the documented predation on the natural mussel

beds, the starfish pose a potential threat to biogenic reefs (Nielsen et al., 2014).

2.2 Handling and use of starfish

During the first 30 hours after the starfish have been caught, depending on the handling of the

starfish, it can lose up to half of its bodyweight in fluid (Holtegaard et al., 2008). The colour of the

fluid that is lost indicates that the starfish undergoes a process with running alterations in which of

the ingredients in starfish is lost with the fluid. The initial bright fluid might indicate that it is water

from the hydrocoel (a complex water vascular system unique for Echinodermata) that is lost

together with fluid from the body cavity. The increasing proportion of brown particulate material in

the fluid indicates an increasing compression and decay of the digestive glands that are in the same

brown colour. This stage is reached not more than 5 hours after catch, where the starfish has lost a

fourth of its bodyweight. 30 hours after catch, a vivid red-orange colouring of the lost fluid

indicates that a part of the pigmentation of the starfish is lost through the fluid. The amount of fluid

lost is greatest immediate after catch and drops until about five hours after collection. Afterwards,

the loss of fluid is rather constant over the next 24 hours (Holtegaard et al., 2008). Because of the

unknown knowledge about the accurate loss of nutrient from the starfish after catch, it is important

to take that in consideration when storing before and after transportation to the processing of the

starfish if the product is intended to use as a feed ingredient.

It has been well-known for decades and around the world, that starfish are a serious pest of both

mussels and oyster-beds. Hutchinson et al. (1946) reported that starfish was a serious pest of oyster-

beds in the coastal waters of the north-eastern United States, and were captured in considerable

quantities by fishermen engaged in oyster culture. The starfish were here grounded into meal and

sold for either food for farm animals or used as fertilizer. In the same study, it was also reported that

during the First World War in Europe, starfish were mixed with oyster-shells and used as fertilizer

for acid soils in France. In Denmark, during the period 1959 to 1980 there was a small fishery for

Literature review

10

starfish in Limfjorden, where the starfish were grounded into meal and some mixed with herring

meal and fed for pigs in Denmark (Kjølhede, personal communication, 2015). The greatest part of

the SM was, however, exported to West-Germany, where it was mixed with FM and fed for poultry

(Holtegaard et al., 2008). Unfortunately, all existing data about this has gone lost. Due to more

accessible resources available, together with the fear of mad cows disease when mixed with other

sources of bone meal, the production was terminated. As other marine resources have been scarce,

the interest for alternative, unutilized resources have increased. The use of starfish is not necessarily

limited to feed for monogastric animals, since it has also been speculated as fertilizer, product for

biogas plant, utilization of their spawn and extraction of antibacterial peptides as found in

invertebrates. Levin et al. (1960) pointed out that the economic problems of utilizing starfish

involved low-cost fishery providing an inexpensive raw material, comparable processing costs per

unit of protein like the conversion of fish into FM and practical use of SM in for instance feed for

animals.

For more than three years, the mussel fishermen in Limfjorden have been using a starfish seine to

remove the starfish from the seabed, to reduce the occurrence of starfish and their predation on

mussels. Some of these starfish were carried to Cux-haven in Germany and processed into meal,

while others were pressed in a clamp, by which a fluid fraction came, giving two different products

of starfish. Chemical analyses of the products led to a project that was to determine the chemical

composition and digestibility of SM in pigs (Nørgaard et al., 2015) and poultry.

2.3 Chemical composition of starfish

The biochemical composition of the starfish changes during the annual reproductive cycle and in

general, the biochemical composition of the ovaries gives a good indication of the changes in

nutrients of the total starfish. In the ovary of starfish, relatively low levels of carbohydrates and high

levels of protein and lipid are found (Oudejans and Vandersluis, 1979). During yolk deposition

(vitellogenesis), the process of yolk formation through nutrients being deposited in the oocyt, the

main weight increase of the ovary occurs. This increase starts in the early autumn and continues

until maturity in late spring (April/May) when the starfish spawn. In contrast, the ovarian growth is

relatively small during pre-vitellogenesis (June to September) (Oudejans and Vandersluis, 1979).

Oudejans and Vandersluis (1979), found that the contents of total lipids, total carbohydrates, free

AA and proteins shows a rectilinearly increase, contributing to ovarian growth. In relation to the

size of the ovaries, the free AA level is rather constant during the development (Oudejans and

Literature review

11

Vandersluis, 1979). Levin et al. (1960) found indication of that all the constituents of starfish are

variable and depend largely on size, freshness, season and environmental factors associated with the

specific samples. The knowledge of the biochemical composition of the starfish tells when it is most

favourable to fish them, for instance in relation to use as feed ingredient for monogastric livestock,

where it is desirable to have a high CP content. Table 1 confirms the findings done by Oudejans and

Vandersluis (1979), that the total AA level and relative distribution of AA in proportion to CP is

rather constant during the year, while the content of CP varies the lowest in June until autumn and

greatest in May following the starfish reproduction cycle. Fishing starfish in May where the CP

level is greatest, will in relation to using starfish as a feed ingredient be of greatest interest.

Table 1 - Chemical composition, total amino acid (AA) content and relative distribution of AA in proportion to crude protein (CP) in

g/kg dry matter in fresh starfish and starfish meal (SM). The data from the fresh starfish is collected on 6 different days throughout

year 2007 and modified from Holtegaard et al. (2008). The data from SM is on sample date 050107 from Holtegaard et al. (2008) and

sample date 110514 and 040115 is data from the current STARPRO project.

Fresh starfish SM

Sample date Nov

06

Jan

07

Feb

07

Mar

07

May

07

Jun

07

Aug

07 Average

May

07

Nov

14

Apr

15

Item, g/kg dry

matter

Dry matter 242 239 243 222 233 243 284

936 961 935

CP 361 446 459 423 536 347 379

520 383 452

Crude fat 53 52 75 65 89 59 50

68 91 96

Crude ash 470 395 374 455 313 483 430

325 421 350

Ca 137 117 130 137 84 157 146

104 130 106

P 4,6 6,1 7 5,9 9,6 4,3 4,3

19,2 6,3 7,3

Total amount of

AA

Cys 4,7 5,8 5,7 5,7 6,1 4,6 5 5,4

4,8 4,7 5,4

His 5,6 7,9 7,1 6,7 7,4 5,69 6,3 6,7

8,9 6,1 7,3

Ile 12,4 16,6 15,9 14,6 16,9 12 13,5 14,6

19 14,5 14,8

Leu 18,1 24,1 23,1 21,5 25,5 17,6 19,6 21,4

30,4 21,4 24,2

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12

Lys 20,8 28,2 28,3 26,6 31,4 20,3 21,3 25,3

30,6 20,6 26,2

Met 6,6 8,3 8,5 7,5 8,7 6,2 7,5 7,6

11,3 8,1 9

Phe 10,5 13,8 13,4 14 16,3 10 12,1 12,9

17,1 12,5 15

Thr 15,6 19,8 19 17,6 20,3 14,7 18 17,9

20,6 18 19,6

Trp 3,3 4,3 4,1 3,9 4,3 3,3 3,7 3,8

3,9 3,8 4,8

Tyr 9,8 12,9 12,4 12,7 14 9,3 10,8 11,7

13,5 13,1

Val 15,8 20,7 18,7 19,7 22,5 14 16,5 18,3

22,7 17,8 18,9

Relative

distribution of

AA in proportion

to CP

His 1,6 1,8 1,5 1,6 1,4 1,6 1,7 1,6

1,6 1,6 1,6

Ile 3,4 3,7 3,5 3,5 3,2 3,5 3,6 3,5

3,4 3,8 3,3

Leu 5 5,4 5 5,1 4,8 5,1 5,2 5,1

5,5 5,6 5,4

Lys 5,8 6,3 6,2 6,3 5,9 5,9 5,6 6

5,5 5,4 5,8

Met 1,8 1,9 1,9 1,8 1,6 1,8 2 1,8

2 2,1 2

Met+Cys 3,1 3,2 3,1 3,1 2,7 3,1 3,3 3,1

2,9 1,2 1,2

Phe 2,9 3,1 2,9 3,3 3 2,9 3,2 3

3,1 3,3 3,3

Phe+Tyr 5,6 6 5,6 6,3 5,6 5,6 6 5,8

5,5

Thr 4,3 4,4 4,1 4,2 3,8 4,2 4,7 4,2

3,7 4,7 4,3

Trp 0,9 1 0,9 0,9 0,8 1 1 0,9

0,7 1 1,1

Val 4,4 4,6 4,1 4,7 4,2 4 4,4 4,3

4,1 4,6 4,2

Determinations of certain biologically important elements have been made on different species of

the genus Asterias, found in their respective area in the world. Starfish contains various secondary

metabolites including steroids, storoidal glycosides, anthraquinones, alkaloids, phospholipids,

peptides, and fatty acids (FA). These chemical constituents exhibit cytotoxic, hemolytic, antiviral,

antifungal and antimicrobial activities. Therefore, starfish is of great interest as a natural bioactive

marine product and presumably in a wide variety of pharmacological activities (Dong et al., 2011).

Asterias rubens is native to the north-east of Atlantic Ocean, and is the most common starfish found

in Danish waters. Asterias rubens produces the secondary metabolite asterosaponins, that are

pentaglycoside or hexaglycoside sulfated steroids. These molecules are interesting because of their

Literature review

13

hemolytic, cytotoxic, anti-bacterial, anti-fungal, anti-viral and anti-tumor properties (Demeyer et al.,

2014). Secondary metabolites of interest have also been found in Asterias rollestoni Bell, one of the

most commonly distributed starfish in China (Zhang et al., 2013). Gudbjarnason (1999) mentions

that extract from the starfish Ctenodiscus crispatus showed considerable cytotoxic activity. The

pharmacological activities the literature of this area indicate that starfish have, can be of great value,

for instance if used as feed ingredient to livestock, e.g. if the cytotoxic effect would be transferred

to the animal. Therefore, starfish not only in Scandinavia can be of great interest for the animal feed

industry.

Furthermore, toxicity of starfish has been described. Asakawa et al. (1997) found that Asterias

amurensis inhabiting the Hiroshima Bay in Japan was toxified with paralytic shellfish poison (PSP).

Paralytic shellfish poison is a most hazardous marine toxin and cause sporadic food poisoning in

humans around the world, and could potentially cause the same condition in pigs. The toxification

of starfish with PSP happens through predation on mussels. Paralytic shellfish poison is produced

by phytoplankton such as Alexandrium tamarense, accumulated in filter feeding bivalves such as

mussels, and then transferred to carnivorous invertebrates. The phytoplankton has several times

been the cause of closed areas for commercial fishery on blue mussels at the east coast in Denmark,

e.g. Limfjorden. The occurrence of phytoplankton in Danish waters is under constant surveillance

by the Danish monitoring program on toxic and potentially toxic algae/phytoplankton and algae

toxins in bivalve molluscan shellfish in relation to the Danish mussel fishery, and therefore there is

no risk of any toxicity of starfish in Denmark (Ministry of Food, 2015a).

2.4 Starfish as feed ingredient

Several decades ago, as mentioned previously, experiments were conducted to determine if starfish

was a possible substitute for other well-known protein sources in poultry. This was done both

because of the abundance of starfish, but also because of the need for alternative protein sources as

a replacer for e.g. FM. Growth has been used as a criterion of measurement when determining the

practical value of SM (Bird, 1946; Heuser and McGinnis, 1946; Morse et al., 1946; Ringrose, 1946;

Stuart and Hart, 1946; Whitson and Titus, 1946; Levin et al., 1960). A summary of the published

papers on the chemical composition of SM and experiments determining the practical value of SM

in feeding poultry is presented in Table 2. It is rather difficult to compare data on chemical

composition of SM from different experiments because of the difference in species of the genius

Asterias, time of fishing in the reproductive cycle of the starfish and difference in the biochemical

Literature review

14

analyses. The same goes for experiments related to growth performance because of the difference in

chemical composition of SM, experimental conditions, breed and age of the animal, management,

etc. SM as product varies also in preparation method. Some have used air- or sun-dried starfish

grounded into meal (Stuart and Hart, 1946; Whitson and Titus, 1946) and others, defatted SM

(Levin et al., 1960). The effect of SM in diets for pigs on performance has not been reported.

Table 2 - Chemical composition and practical value of starfish meal (SM) in poultry feeding using growth as a criterion of

measurement.

Chemical

composition

SM

SM% in diet Age of

chickens

(weeks)

Average

final BW

(g)

Average

feed

consumed

Gain to

feed

Reference

CP: 24.9% 0 (reference rationa)

10

20

40

2-4 594

533

482

376

0.654

0.621

0.579

0.542

0.539

0.468

0.415

0.247

(Stutz and

Matterson,

1964)

CP: 34%

Ca: 16.9%

P: 0.43%

0

3

6

0-9 846

949

934

0.336

0.350

0.337

(Bird,

1946)

CP: 34%

Ca: 16.9%

P: 0.43%

0 (3% fish meal)

3

6

12

0-8 699

700

673

441

(Heuser

and

McGinnis,

1946)

CP: 33.63%

Ca: 15.14%

P: 0.48%

0 (2.5% fish meal)

4

8

0-8 854

803

816

2.35

2.13

2.12

0.346

0.348

0.350

(Morse et

al., 1946)

CP: 34%

Ca: 16.9%

P: 0.43%

0 (4% fish meal)

9

18

0-6 254

209

170

0.209

0.171

0.140

(Ringrose,

1946)

CP: 34%

Ca: 16.9%

P: 0.43%

0

4

0-12 1,283

1,332

5.99

6.27

0.467

0.471

(Stuart

and Hart,

1946)

CP: 30.7%

Ca: 17.6%

P: 0.35%

2.5

4

7.5

8

12

3.5-6 450

399

351

297

243

(Whitson

and Titus,

1946)

aSemi-purified reference ration - described in paper.

Literature review

15

Though older literatures rather limited information of how the experiments have been conducted,

and some ostensibly important results have been led out, the findings in Table 2 gives an impression

of that SM is an acceptable alternative protein source for poultry.

The amount of SM tested in the experiments range from 2.5% to 40% of the diet. Bird (1946)

recommended 3% SM inclusion in the diet, Whitson and Titus (1946) recommended 2.5-5%

inclusion, where Morse et al. (1946) recommended 4% inclusion level of SM. Moreover, Heuser

and McGinnis (1946) found that the maximum inclusion level was 6%, and 3% SM was equally as

good as 3% FM. While on a 12% SM ration, very poor results were obtained. It was reported by

Ringrose (1946) that SM is 83% as effective as FM when 9% of SM was used in a 13% protein

ration. The same author found that depressed growth and high mortality was the result of 18% SM

in an 18.5% protein ration. Ringrose (1946) also concluded that the depressed growth and high

mortality were undoubtedly caused by the high Ca compared to the low P level, giving a wide

calcium to phosphorus ratio (Ca:P) in the feed used. Furthermore, it was noted in a metabolism

study done by Stutz and Matterson (1964) that when the level of SM increased from 10% to 40%,

greater growth depression was experienced with an average BW for the whole experimental period

of 533 and 376 grams, respectively (see Table 2). Levin et al. (1960) concluded that a protein

concentrate prepared from a defatted, HCl washed SM exhibits high protein quality values,

comparable to good quality FM. Moreover, the Ca level of the HCl washed SM was sufficiently

reduced to allow incorporation into the diet, providing 10% protein without raising the dietary Ca

level (Levin et al., 1960). Their experiment showed that mixtures of 20% SM and 80% FM, and

30% SM and 70% FM, respectively, had higher protein quality values than FM alone in a ration

with a 10% dietary protein level. When added at levels of 3-6% to purified (less variable nutrient

concentrations) or practical diets, while maintaining the same dietary Ca level, defatted SM is on a

protein basis equal to FM, maintaining the same dietary Ca level.

In a digestibility study done by Nørgaard et al. (2015), SM was analysed for sand (acid-insoluble

ash) to explain the high ash content of SM. They found SM to contain 47 g Ca, 26 g P, 24 g Cl and

18 g Na per kg DM, meaning that only about half of the ash content in SM is constituted by Ca, P,

Cl and Na. The remaining fraction of the ash content was not identified, but could be silica (SiO2; a

major constitute of sand) (Nørgaard et al., 2015). The high ash content was also noted by Stuart and

Hart (1946), Levin et al. (1960), and Stutz and Matterson (1964), but the actual composition of

minerals in the ash and possible effect on the animal fed a diet high in ash, was not reported.

Literature review

16

The preceding indicates that starfish can be a possible substitute for other well-known protein

sources because of their abundance, their biochemical composition and possible pharmacological

activities. It can be questioned if it will be possible to use starfish as the sole protein source as an

alternative to the most common used protein sources as SBM, due to the high Ca content that

apparently limits the possible inclusion level. Thus, there is a need for further investigation of

starfish as an alternative protein source for monogastric livestock, especially pigs.

3 Nutrient requirement of piglets

Over the years, genetic selection of pigs has increased capacity for lean tissue growth (Tausen,

2012). Simultaneously, the rate of fat deposition has been reduced, and capacity for feed intake

declined. A high voluntary feed intake (VFI) is necessary for making use of the capacity for rapid

lean tissue growth, whereas limited fat deposition and efficient feed utilization requires a low VFI.

The pig’s inherent maintenance requirement and desire to retain body protein and fat is linked to its

VFI. The genetic selection for lower fat deposition in pigs has not changed the genetic drive to

consume the required amounts of nutrients and energy, but the pigs having lower energy

requirements, has led to decreased VFI (Tausen, 2012).

3.1 Energy requirement

The energy content of feed for piglets has been reduced during recent years - mainly due to the

higher barley content and less addition of fat in the compound feed compared previously. Milk and

fish products are also used to a lesser extent than earlier (Rasmussen and Vinther, 2015). Piglets are

in an energy dependent phase of growth, and their requirement for ME can roughly be ascribed to

the sum of energy requirement for maintenance, thermoregulaion, and energy deposition in body

tissue. The energy requirement for piglets are normally stated for both maintenance and production

combined. A function of BW (maintenance) and the proportion of protein and lipid in gained

tissues, determinates the energy need for growth (NRC, 2012).

As estimated by Rasmussen and Vinther (2015), piglets achieve the best performance, i.e. maximal

growth and feed efficiency, at an energy content of 1.08 FEsv/kg diet at a BW of 9.5-31 kg. The

maximal average daily feed intake (ADFI) is achieved at an energy content of 1.17 FEsv/kg feed,

whereas the highest ADG is achieved at 1.12 FEsv/kg feed. Indicating, that different energy

contents is needed for different parameters. An increment in the energy content with 1 FEsv/100 kg

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feed in the interval 100 to 123 FEsv/100 kg feed gave a significantly reduced feed efficiency of

0.006 FEsv/kg growth. The price of a FEsv determines what energy content the pigs should be fed,

and the cheaper the feed is the more shifting towards higher energy content. The recommended

energy content in feed for piglets with BW of 9.5-31 kg was found to be between 1.07 and 1.12

FEsv/kg feed (Rasmussen and Vinther, 2015).

3.2 Protein and amino acids requirement

A piglet’s requirement for N is expressed on basis of net energy (feed units; FE) in the Danish feed

evaluation system. Beside requirement for N, the dietary supply of N or CP also needs to meet the

requirement for AA. The physiological use of AA is divided in two distinct, but dependent uses;

AA for maintenance and AA for protein accretion. The indispensable AA are a main determinant of

the level of production that can be obtained, and are therefore of great importance. Sufficient energy

supply through carbohydrates and fat from the diet is a prerequisite for optimal utilization of dietary

proteins. The Danish recommendations are based on having an appropriate balance between energy

and protein expressed as the pig’s need for protein and AA on an energy basis; in other words, as

gram digestible product per FEsv (Nørgaard et al., 2012). If there is an imbalance in the AA profile

or if the pigs are fed in excess amount of N, the surplus will be secreted as urea in the urine.

Commercial herds and controlled experiment conditions ascertain that the risk of diarrhoea after

weaning increases with increasing protein level in the diet. Together with both source and level of

dietary protein, these are known to influence enteric health in piglets (Heo et al., 2013; Tybirk et al.,

2014). In Denmark, so-called low-protein diets for piglets have a total protein content of 18-20%

(Maribo, 2010), amounting 145-158 g/FEsv, 140-152 g/FEs and 140-154 g/FEsv digestible CP,

respectively, for pigs of 6-9 kg, 9-15 kg and 9-30 kg BW, respectively. Normal simple protein diets

for piglets contains 20-22% of protein, amounting 158-170 g/FEsv and 150-162 g/FEsv digestible

CP, respectively, for pigs of 6-9 kg and 9-30 kg BW (Maribo, 2010). The dietary CP reduction with

CAA inclusion can effectively reduce N excretion, ammonia emission and precisely match the pig’s

AA requirement (Zervas and Zijlstra, 2002; Norgaard et al., 2014).

To optimize the use of limited resources, especially protein sources, it is necessary to adapt the level

of AA given to the pig followed the protein accretion capacity. A suboptimal supply will reduce the

performance of the pig while excess supply of AA will not have an increasing effect on

performance (Sundrum et al., 2005). The Danish recommendations for a piglet’s requirement for

individual indispensable AA is given by Tybirk et al. (2014).

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18

3.3 Mineral requirement

Calcium and P are minerals that have an important role in the development and maintenance of the

skeleton, together with many other physiologic functions in the body. When formulating diets for

pigs, it is necessary to consider an appropriate Ca:P for an adequate absorption and utilization of

both minerals, since excess or deficiency in one of the minerals will cause impaired utilization of

the other (Gonzalez-Vega and Stein, 2014). However, dietary Ca and P levels are generally

formulated to achieve optimum growth instead of maximum bone mineralization, which is, of great

importance for the pig’s health. The amount of P needed for optimum growth is lower than for

maximum bone mineralization (Maxson and Mahan, 1983; Koch et al., 1984; Varley et al., 2011).

Furthermore, the P level in diets is often low in the attempt to reduce emission of P to the

environment (Lenis and Jongbloed, 1999; Metzler et al., 2008). Calcium and P share a mutual

antagonism and both have antagonistic effects on other elements (Peo, 1991). It was indicated by

Peo (1991) that adequate level of Ca and P for pigs in every growth phase is dependent upon: a) an

adequate level of both Ca and P in an available form in the diet, b) an appropriate ratio of available

Ca and P in the diet, and c) adequate vitamin D present for Ca to be absorbed. In many commercial

diets Ca is frequently found in excess and the various Ca:P may result in different effects depending

on the dietary P level.

The Danish Pig Research Centres’ recommendations in diets for piglets with BW from 6 to 9 kg

and 9 to 15 kg is, respectively, 7.0 g and 8.5 g Ca/FEsv without phytase added to the diet, and when

phytase is added, 6.5 g and 8.0 g Ca/FEsv (Tybirk et al., 2014). The content of digestible P in feed

cannot be controlled, therefore, the diets content of digestible P must be considered in the context of

the total content of P. The recommended minimum content of total P for pigs with a BW from 9 to

30 kg in diets added phytase is 5.2 g tP/FEsv when phytase is added equalling 100% dose, and 4.9 g

tP/FEsv when phytase is added equalling 200% dose in a diet containing grain and SBM, where the

source of P is monocalcium phosphate (Tybirk et al., 2014). So, the requirement for P is lower

when phytase is added to the diet. The figures are given as the pigs’ requirement, plus a safety

margin. It is therefore not recommended to supply these nutrients in surplus, since e.g. Ca interacts

with some micro and macro minerals and can impede absorption of these minerals.

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19

4 Practical feeding around weaning

4.1 The transition of weaning

Weaning from the sow is one of the most stressful events in the pig’s life, where the pigs experience

nutritional, psychological, environmental, and social challenges that can predispose the pig to

subsequent diseases and production losses (Pluske et al., 1997; Campbell et al., 2013; Heo et al.,

2013). When the piglet is weaned, the piglet must adapt abruptly from the sow’s milk that is highly

digestible, palatable and high in protein, fat and lactose to a less digestible, palatable starch-based,

solid dry diet containing complex protein and carbohydrate including various anti-nutritional factors

(Heo et al., 2013). The consequence is usually that VFI is sharply reduced initially after weaning

and the piglet becomes malnourished with reduced transient growth rate (Pluske et al., 1997).

Approximately 50% of weaned piglets consume their first feed within 24 hours post-weaning;

unfortunately, in about 10% of the weaned piglets, weaning anorexia persists for up to 48 hours

(Heo et al., 2013). By the end of the first week post-weaning, it is estimated that ME intake is about

60-70% of pre-weaning milk intake and it takes approximately 8-14 days for the piglet to recover

the pre-weaning ME intake level (Campbell et al., 2013). As reviewed by Lalles et al. (2007), even

a minimal consumption of feed in the suckling period will stimulate VFI after weaning. The

positive relationship between cumulative feed intake during day 0-3 post weaning and villous

height confirms the importance of VFI in this period. Thus, to stimulate VFI and thereby energy

intake post-weaning, the ingredients in the feed must be highly palatable to stimulate feed intake,

highly digestible, and contain a high net energy (NE) concentration.

4.2 Diet characteristic and protein sources

The overall aim in the grower period is to produce healthy pigs with a high daily gain at the lowest

expense for feed and medicine (Maribo, 2010). Feed for piglets should be compounded so that the

piglet’s requirements for nutrients and energy are fulfilled. Furthermore, to achieve maximum

growth performance the dietary ingredients selected, should match to the pig’s digestive capacity.

In growing pigs, the feed cost account for more than 60% of total production costs. A change in the

feed composition should be assessed in proportion to if it will pay in the form of better performance

and health in relation to the price of the feed (Maribo, 2010).

Commercial diets for piglets with a BW of 9-15 kg differ among feed companies in content and

amount of the different feed ingredients. The Danish Pig Research Centre has set a line of

Literature review

20

recommendations for the maximum amount of feedstuffs in diets for 5 week old piglets. Principally,

the diet should contain highly digestible protein sources such as FM, soy protein, potato protein,

whey powder, shimmed milk powder and similar products (Maribo, 2010). Table 3 gives an

overview of the guiding figures from the Danish Pig Research Centre (Jørgensen, 2012) for

maximum use of the most used feedstuff comprising grain and protein sources together with their

content of CP and the standardized ileal digestibility (SID) of CP.

Table 3 - Extract of guiding figures from the Danish Pig Research Centre for maximum use of feedstuff in diets (percent of kg feed)

for piglets from 5 weeks of age (Jørgensen, 2012). Only main ingredients as grain and protein sources often used in diets for

weanlings pigs is presented. Crude protein (CP) (% of dry matter) and standardized ileal digestibility (SID) of CP is presented to

show difference between feed ingredients (VSP, 2015).

From 5 weeks CP SID of CP

Barley 70 10 75

Wheat 70 10 82

Oat 50 11 66

Soy bean meal, dehulled toasted 20 48-56 86-88

Potato protein concentrate 5 85-88 89

Fish meal, all types 12 76 90

Salmon protein concentrate 10 70 92

Skimmed milk powder 25 34-37 94

Whey powder 25 9-13 85-92

Soy protein concentrate 25 56-66 82-90

Palm oil 7 0 0

As indicated in Table 3, protein is available in a variety of dietary sources. These include

ingredients of vegetable and animal origin. Evaluation of how good a protein source is is

accomplished by determining the quality and digestibility of the protein (Hoffman and Falvo,

2004). The quality refers to the availability of the AA that the protein supplies and digestibility deal

with the proteins capacity of being easy to digest. All dietary protein sources of animal origin are

considered to be complete proteins, which mean that the protein contains all of the indispensable

AA. Vegetable protein sources are considered incomplete since they generally are deficient in one

or more indispensable AA. Thus, to get all the protein needed solely from vegetable sources, it is

Literature review

21

needed to consume a variety of legumes, fruits, grains and vegetables to ensure the right

consumption of all the indispensable AA (Hoffman and Falvo, 2004). This is a difficult task when

composing diets for piglets. Digestibility of protein involves rating how well the body can

efficiently utilize protein of dietary sources. Typically, animal protein has a higher digestibility than

vegetable protein sources (Hoffman and Falvo, 2004). Animal proteins like FM, whey and sprey-

dried plasma provide the highest quality, primarily due to the composition of indispensable AA and,

therefore, the completeness of the protein source. Despite that these proteins are associated with

high intakes of saturated fat, there have been a number of studies that have demonstrated the

positive benefits of animal proteins. On the contrary, intake of vegetable proteins will reduce the

intake of saturated fats and provide other nutrients such as fibre and phytochemicals. However,

some vegetable sources of protein are recommended not to use for piglets such as rye, beet pellets,

peas and sunflower meal (Maribo, 2010).

As described earlier, SBM is a commonly used high-quality protein source for pigs. However, when

SBM is fed for piglets, digestive disturbances can be observed. Presence of trypsin inhibitors,

antigenic soy proteins, and indigestible carbohydrates as oligosaccharides, may be contributors to

the problem often experienced as diarrhoea (Pluske et al., 1997; Cervantes-Pahm and Stein, 2010).

Other ANF present in SBM beside trypsin inhibitors, such as lectins and tannins, have been

reported to increase losses of endogenous proteins at the terminal ileum. Furthermore, decreased

hydrolysis of protein and absorption of AA may be related to these ANF. Therefore, only limited

quantities of SBM are included in diets for piglets, by which animal-derived alternatives as milk

proteins and FM are widely used (Cervantes-Pahm and Stein, 2010).

Piglets profit from dairy products known to have a beneficial effect on VFI, growth performance,

feed efficiency and health, due to the high palatability and high SID of energy and protein (Lalles et

al., 2007; Cervantes-Pahm and Stein, 2010).

Spray-dried plasma is a good protein source for piglets and is commonly used in countries where

they wean piglets earlier than 24-31 days of age. Spray-dried plasma is a highly digestible protein

source and it is presumed that the immunoglobulins in plasma contribute to the maintenance of the

mucosal integrity and the reduction of inflammatory response in the intestine, helping the piglet to

resist bacterial presence (Torrallardona et al., 2003; Nofrarias et al., 2006). Spray-dried plasma has

not been included in Table 3 since The Danish Pig Research Centre recommend phasing out the use

Literature review

22

of spray-dried plasma with regard to the potential risk of Porcine Epidemic Diarrhoea (PED)

followed the use of the product (VSP, 2014).

Fish meal is considered a very digestible protein source with a high content of vitamins, minerals,

and favourable AA composition (Jonsdottir et al., 2003). However, the quality of FM varies

depending on the species of fish, the freshness before processing and the way the fish is processed

into meal (Kim and Easter, 2001). A study found that gain to feed ratio (G:F) was higher in pigs fed

a diet containing 5 rather than 2.5% mackerel FM (Kim and Easter, 2001). Fish meal has a high

content of PUFA, including the n-3 FA C20:5 (EPA; Eicosapentaenoic acid), and C22:6 (DHA;

Docosahexaenoic acid), that can lead to higher content of PUFA in the carcass fat. PUFA’s can give

rise to rancidity and reduced storage stability leading to undesirable off-flavours and off-odours in

the meat (Hertzman et al., 1988; Jonsdottir et al., 2003; Hallenstvedt et al., 2010). A study

conducted with pigs having an initial BW of 25 kg, found that supplementation of FM significantly

increased the content of both n-6 (linoleic, eicosadienoic, and docosadienic acids) as well as n-3

(linolenic acid) in the longissimus dorsi muscle and at the same time decreased the content of

stearic acid (Grela et al., 2012). Cho and Kim (2011) reported that the n-3 PUFA has anti-

inflammatory properties and benefits to the immune system, whereas Thies et al. (1999) reported

that n-3 PUFA have been shown to downregulate immune function in humans, laboratory animals

and also pigs. Nevertheless, FM is considered one of the best protein sources for piglets (Kim and

Easter, 2001), and no adverse effect on immunity on piglets have been reported. Jørgensen (2004)

reported that a diet for piglets without added FM, did not significantly affect productivity or feed

costs. Diets containing a composition of soy protein concentrate, potato protein concentrate, whey

powder and/or skimmed milk powder, were good alternatives to diets containing FM. It is worth a

mention that diets without FM were added several different protein sources compared to only a few

when FM was used, thereby increasing the need for several different feed ingredients when not

using FM. Fish meal supply with a good composition of indispensable AA and possibly improve

palatability (Jørgensen, 2004).

As mentioned before, SM can be considered as a new and alternative high-quality source of protein.

Both the chemical characteristics and the results of SID of SM obtained by Nørgaard et al. (2015),

points toward a potential use of SM in pig diets. It was concluded that CP content (700 g/kg DM)

and indispensable AA except lysine and leucine in SM were comparable to that of FM. Moreover,

Literature review

23

the SID of CP (0.80) and AA (0.84) were higher than in commercial fish silage and could be of

interest as feed ingredient for piglets.

5 Final remarks and conclusion of literature review

- Starfish in diets for monogastric animals will both contribute with dietary protein but also

reduce the need for increasing land use change and minimise the environmental impact of

the animal production by recycling of nutrients.

- Animal proteins e.g. SM provides a high quality of protein, primarily due to the composition

of indispensable AA and, therefore, of great interest as alternative source of protein

compared to vegetable sources.

- Starfish meal is comparable to FM in quality and can be a realistic alternative to many well-

known protein sources.

- The numerous chemical components that exhibit e.g. antimicrobial, antiviral and antifungal

activities found in starfish can be of great interest, if they show any significant bioactivity

effect on pig performance.

- Starfish meal can contribute with a potential organic feed ingredient that meets the organic

production standards and concept and help to accomplish the implementation of a 100%

organic diet for monogastric animals by the end of 31 December 2017.

- There may be a potential of feeding organically reared piglets with SM, especially owing to

the highly digestible AA, which can contribute to improve the AA balance in feed for

organic piglets.

The literature review indicates that there is a need for, but also a possibility of producing new high-

quality protein alternatives to piglets comprising starfish. Starfish is a promising protein source,

however, it is not tested in pigs yet.

24

Manuscript to be submitted

25

Starfish (Asterias rubens) as feed ingredient for piglets 1

2

P. Sørensena*

3

4

a Department of Animal Science, Aarhus University, Foulum, P.O. Box 50, DK-8830 Tjele, 5

Denmark 6

7

Abbreviations: AA, amino acids; ADFI, average daily feed intake; ADG, average daily gain; 8

BW, body weight; Ca:P, calcium to phosphorus ratio; CP, crude protein; DM, dry matter; FM, 9

fish meal; G:F, gain to feed ratio; PUN, plasma urea nitrogen; SBM, soy bean meal; SID, 10

standardized ileal digestibility; SM, starfish meal; SPC, soy protein concentrate. 11

12

*Corresponding author: E-mail address: pia.sorensen@anis.au.dk 13

14

ABSTRACT 15

The effects of including starfish meal (SM) as an alternative protein source in diets for piglets on 16

performance, faeces characteristics and plasma parameters were investigated. Four diets were 17

formulated to contain different protein sources; fish meal (FM), soy protein concentrate (SPC) 18

and two levels of SM (SM5% and SM10%). All diets contained 174.4 g soybean meal (SBM)/kg 19

and were supplemented up to 166-172 g SID CP/kg with either FM, SPC, SM or a combination 20

giving raise to two SM + SPC diets with different levels of SM and SPC. One week after 21

weaning, 96 pigs (body weight (BW) 9.6 ± 0.4 kg) were individually housed and allocated to one 22

of the four diets (n=24) and feed ad libitum for a 14-d period. Average daily feed intake (ADFI), 23

average daily gain (ADG) and gain to feed ratio (G:F) were determined. Pigs fed the SM10% 24

Manuscript to be submitted

26

diet had an ADG 23-28% lower (P<0.001) than pigs fed the FM, SPC and SM5% diets, despite 25

that they ate the same amount of feed. Faeces characteristics were evaluated by visual judgement 26

during five days and no differences were observed, indicting no effect of the diet on diarrhoea. 27

Blood samples were collected on d 15. Plasma urea nitrogen (PUN) in SM10% was higher 28

(P<0.003) than the concentrations in SPC and SM5% but not FM. Plasma Ca concentration was 29

higher (P<0.001) in pigs receiving SM10% compared to FM, SPC and SM5%. The opposite was 30

the case with P where the concentration was lower (P<0.001) in SM10% compared to FM, SPC 31

and SM5%. In conclusion, feeding diet SM5% resulted in performance equal to pigs fed the 32

control diets. Inclusion of 10% SM in the diet affects performance negatively, due to the wide 33

Ca:P in the SM10% diet that affects digestibility and absorption of P negatively. Moreover, the 34

possible formation of mineral-phytate complexes may reduce mineral bioavailability. The 35

negative effects increases as the dietary Ca:P increases. The determining factor for the maximum 36

inclusion level of SM in diets for piglets may be the dietary Ca level and the resulting Ca:P. 37

38

Keywords: Dietary protein source, nutrition, performance, piglets, starfish. 39

40

1. Introduction 41

The global demand for high-quality protein feedstuffs for use in animal nutrition is 42

increasing. Together with variability in the price development of agricultural commodities on the 43

world market along with the cost and availability of protein sources, the livestock production is 44

driven to explore alternative feedstuffs for animal nutrition in order to maintain the 45

competitiveness of livestock products (Trostle, 2008; Jezierny et al., 2010). Therefore, the 46

interest to maximise the use of sustainable and locally produced feed ingredients is increasing. 47

Manuscript to be submitted

27

Starfish in high concentrations is considered as pests by the commercial mussel farmers 48

because starfish predate mussels that are otherwise used for human consumption (Holtegaard et 49

al., 2008). Starfish meal (SM) is not a new product, and was evaluated as feed ingredient in 50

studies with poultry several decades ago. It was concluded that the protein fraction in SM was 51

comparable to fish meal (FM) in quality (Bird, 1946; Whitson and Titus, 1946; Levin et al., 52

1960). However, the high calcium (Ca) content in SM was reported to result in poor growth 53

performance (Heuser and McGinnis, 1946; Morse et al., 1946; Whitson and Titus, 1946) and 54

reduce protein digestibility (Stutz and Matterson, 1964) when fed in high amounts to poultry. 55

The chemical composition and standardized ileal digestibility (SID) of crude protein (CP) and 56

amino acids (AA) in SM has been evaluated in pigs (Nørgaard et al., 2015), and their findings 57

showed chemical characteristics making SM potential for use in commercial diets for piglets. 58

Starfish show numerous chemical components that exhibit e.g. antimicrobial, antiviral and 59

antifungal activities (Dong et al., 2011) and can also of this reason be of great interest, if SM 60

shows any significant bioactivity effect on pig performance. This point toward a potential use of 61

SM in pig diets and as a serious competitor to well-known high-quality protein sources in 62

piglets, both due to its sustainability and local origin and nutrient composition. However, the use 63

of SM as feed ingredient in diets for pigs has not been reported. Based on this, it was 64

hypothesised, that feeding a diet containing SM will generate the same average daily feed intake 65

(ADFI), average daily gain (ADG) and gain to feed ratio (G:F) as traditional diets. 66

The objective of the present study was to evaluate whether piglets fed a diet containing SM 67

as a protein source perform on equal terms with pigs fed traditional diets for newly weaned pigs. 68

69

2. Materials and methods 70

Manuscript to be submitted

28

The experiment complied with the Danish Ministry of Justice, Act no. 253 of March 2013 71

concerning experiments with animals and care of experimental animals and a license issued by 72

the Danish Animal Experiments Inspectorate, the Ministry of Food, Agriculture and Fisheries, 73

Danish Veterinary and Food Administration. 74

75

2.1 Animals and diets 76

A total of 96 cross-bred (Danish Landrace/Yorkshire x Duroc) female and male castrated 77

pigs at the same proportion were included in the experiment. Seven days after weaning the pigs 78

were housed individually in pens (1.0 × 2.2 m) with visual and physical contact to neighbouring 79

pigs. The pens comprised two-thirds concrete floor and one-third cast iron slatted floor which 80

permitted drainage. The pigs were placed in three identical rooms of 16 pens, 4 from each 81

treatment per room. During the experimental period, room environment was controlled: 82

temperature 23°C, humidity 67% and the electrical light was on for 12 hours a day. 83

Starfish (Asterias rubens) were caught in Limfjorden in Denmark in October 2014 using a 84

specialized starfish purse serine. In two days, approximately 32 t of starfish was caught by three 85

36-45 feet boats at a speed of 1.5-2 knot. The starfish was transported from the harbour in 86

Nykøbing Mors to a fish meal plant (Hanstholm Fiskemelsfabrik, Denmark) where they were 87

processed into meal. The SM was prepared by mincing the starfish followed by drainage in the 88

drying facility under normal pressure in contiguity with rotating heat surfaces. The retention time 89

in the drying facility was 2-2½ hour depending on the moisture content. The meal reached a 90

temperature of approximately 100°C and a water content of 5-10% was achieved in the final 91

product. During the process, the water content was controlled every hour by using a dry matter 92

analyser. The dried starfish was grinded into meal by using a 10 mm griddle and sealed into big 93

bags. 94

Manuscript to be submitted

29

The pigs were allocated to one of four diets formulated to contain different protein sources; 95

fish meal (FM; FF classic, FF Skagen, Skagen, Denmark), soy protein concentrate (SPC; 96

AlphaSoy PIG 530, Agro Korn, Videbæk, Denmark) and two levels of starfish meal (SM; 97

starfish meal). The chemical composition of the main ingredients (Table 1) was determined 98

before the diets were optimized and diets were planned to be isoenergetic (9.4 MJ NE/kg as-fed) 99

and to fulfil the pigs minimum requirement for indispensable AA, Ca and P according to the 100

Danish recommendations for pigs weighing 9 to 15 kg (Tybirk et al., 2014). The composition 101

and, calculated and analysed chemical composition of the diets is presented in Table 2. All four 102

diets contained 174.4 g dehulled toasted soybean meal (SBM)/kg and were supplemented up to 103

166-172 g SID CP/kg with either FM, SPC, SM or a combination of these protein sources giving 104

raise to two SM + SPC diets with different levels of the two protein sources. Since all diets were 105

formulated to contain approximately 20% CP, SM as such, could not be fed as a sole source of 106

dietary protein besides SBM, without appreciably increasing the Ca level of the diet. Therefore, 107

SM was mixed with SPC, leading to a diet containing 5% SM (SM5%) and one containing10% 108

SM (SM10%). Diets were provided in meal form. 109

110

2.2 Experimental design 111

Pigs were weaned at day 25-32 of age. During the first week after weaning, the pigs were 112

fed a commercial diet containing 2500 ppm Zn. The experiment was conducted in two 113

periods/replicates with 48 pigs in each period, each with duration of two weeks. The experiment 114

started 7 days after weaning, i.e. without an adaption period, with an average initial body weight 115

(BW) of 9.6 ± 0.4 kg (9.3 ± 0.3 kg in period 1 and 9.8 ± 0.4 kg in period 2). The experimental 116

diet was designed to be a potential substitute for commercial diets for pigs with BW 9 to15 kg, 117

therefore, the experiment started one week after weaning. The pigs were allocated to one of four 118

Manuscript to be submitted

30

experimental diets and balanced according to sex, weight and room. The pigs were provided feed 119

ad libitum for 14 days. The feed was supplied twice daily to ensure fresh feed, with feed 120

dispensers adjusted on a regular basis to minimize feed spillage. Feed spillage was collected and 121

weighed daily and stored (4°C) for analysis of dry matter. The pigs had permanent access to 122

fresh water from drinking nipples. 123

Pigs were weighed on day 0, 7 and 14 of the experimental period. Average daily feed intake 124

and ADG were determined day 7 and 14. Faeces characteristics were evaluated during five 125

consecutive days from day 5 to 9 of the experiment by visual judgement in both periods. Faeces 126

were given one of four scores: 1, firm and shaped; 2, soft and shaped; 3, loose/often shining 127

surface, or 4, watery/flows through slatted floors (Pedersen and Toft, 2011). Piglets showing 128

score 4 for two consecutive days were treated for diarrhoea with injection of antibiotics 129

(Engemycin Vet, 100 mg/ml oxytetracyclin, MSD Animal Health, Ballerup, Denmark) for three 130

days as prescribed by the veterinarian. The pigs were monitored daily and the general health 131

status was recorded. 132

Blood samples were collected on day 15 of the first period of the experiment after an 133

overnight fasting starting at 1800 h. On the day of sampling, pigs received 25 g/kg BW0.75

of 134

feed at 0730 h. Blood samples were collected starting at 0930 h. The meal size and feeding time 135

before blood sampling was fixed to reduce variation. Blood samples were collected by jugular 136

vein puncture into 9 mL heparinized tubes (Greiner BioOne GmbH, Kremsmünster, Austria) and 137

immediately placed on ice. Samples were mixed and centrifuged at 2000 × g at 4°C for 10 min. 138

Plasma was immediately harvested and stored at -80°C until laboratory analysis. 139

140

2.3 Chemical analysis 141

Manuscript to be submitted

31

Dry matter content was determined for wheat, barley, SBM, FM, SPC, SM and feed 142

residuals by oven drying at 103°C for 20 hours. Nitrogen content for wheat, barley, SBM, FM, 143

SPC and SM was analysed by a modified Kjeldahl method (AOAC Int., 2000; 984.13), and CP 144

was estimated as total nitrogen × 6.25. Crude fat was extracted with petroleum ether after a 145

hydrochloric acid hydrolysis (European Commission, 2009; Procedure B). Representative 146

samples of each of the six before mentioned ingredients were hydrolysed for 23 hours at 110°C 147

with (Cys and Met) or without (Arg, His, Ile, Leu, Lys, Phe, Tyr, Thr, Trp, Val) performic acid 148

oxidation, and AA were separated by ion exchange chromatography and quantified by 149

photometric detection after ninhydrin reaction (European Commission, 1998). 150

Blood plasma were analysed for plasma urea nitrogen (PUN), AA, Ca and inorganic P. 151

Blood plasma free AA and PUN were analysed using an Amino Acid Analyser fitted to a lithium 152

high performance system for physiological amino acids (Biochrome 30+ Amino Acid Analyser; 153

Biochrome, Cambridge, England). The Amino Acid Analyser was calibrated using a standard for 154

acidic, neutral, and basic AA (Sigma Aldrich, St. Louis, MO). Calcium and inorganic P (plasma 155

only) concentrations were measured photometrically on a Roche/cobas c 111 analyser (Roche 156

Diagnostics International Ltd., Rotkreuz, Switzerland) using commercial reagents (Roche 157

Diagnostics GmbH, Mannheim, Germany). The analyser was calibrated using a lyophilized 158

human serum (Roche Diagnostics GmbH, Mannheim, Germany) as well as 0.9% NaCl. 159

Precinorm U and Precipath U based on lyophilized human serum (Roche Diagnostics GmbH, 160

Mannheim, Germany) were used as controls. 161

162

2.4 Calculations and statistical analyses 163

Gain to feed ratio was calculated by ADG divided by ADFI. All statistical processes were 164

performed in SAS (Version 9.3, SAS Inst. Inc., Cary, NC, US), using the individual pig as 165

Manuscript to be submitted

32

experimental unit. Animal performance data (ADFI, ADG and G:F) and plasma AA, Ca, 166

inorganic P and PUN were analysed by the MIXED procedure (Littell et al., 2002), as all 167

variables showed normal distribution by histogram plots. The models all included diet (diet 1 to 168

4) and sex (female, castrate) as fixed effects, and initial BW as a covariate. Room (room 1 to 3) 169

and period (1, 2; rooms were used twice) were handled as random variables, though period was 170

not included in the models for plasma parameters, since blood samples were only taken by the 171

end of the first period. All models have been tested for three-way and two-way interactions, but 172

the interactions were not significant for any of the response variables and were left out of the 173

final models. 174

Faeces score data are presented as the distribution of scores during the evaluation period 175

from day 5 to 9 in both periods. The distribution of days on the four faeces scores were analysed 176

using the GLIMMIX procedure (Littell et al., 2002) with a multinomial distribution (a 177

generalization of the binomial distribution) and a cumulative logit link function (i.e. the response 178

was restricted to one of a finite number of ordinal values). The model included diet (diet 1 to 4), 179

score day (d 5 to 9), the interaction term diet × score day, and the initial weight as covariate. 180

Room was handled as random effect. 181

Applied to all models, residual variance has been examined by plotting the residuals against 182

the predicted values. Applicable for all the models, the residuals were evenly distributed around 183

zero, and the models were therefore accepted. Presented data are least squares means and 184

standard error of mean (SEM). Statistically significance was accepted at P<0.05. 185

186

3. Results 187

3.1 Chemical composition of the protein sources and diets 188

Manuscript to be submitted

33

The SM was characterized by containing half the CP/kg DM of FM, and more than double 189

the ash content. The protein fraction of SBM, FM, SPC and SM was analysed for 11 AA, and 190

difference was found between the content. However, the content of the individual AA in relation 191

to CP, i.e. the AA profile, was similar among the protein sources. Starfish meal was lower in 192

Trp, Phe and His compared to the other protein sources. Starfish meal was comparable to FM in 193

AA composition, but had a higher content of Met and Cys. Soybean meal and SPC were very 194

similar in their composition of AA, but the content of Met was only about half of the content in 195

SM and FM. The SM was analysed for sand (acid-insoluble ash) to explain the high ash content 196

(data not shown), but the content was negligible. Approximately one third of the ash content in 197

SM consisted of Ca (130 g/kg DM) and the content of P was 63 g/kg DM. 198

The calculated chemical composition of the FM, SPC and SM5% diets were similar between 199

treatment, whereas the SM10% diet had a higher CP, ash, fat, Ca and Na content, compared to 200

the other diets. The analysed values for CP were more similar among diets, and in general, the 201

analysed values were higher than the calculated. The Ca:P was in the range of 1.5:1 to 2.6:1, 202

where SPC and SM5% had the narrowest and SM10% had the widest ratio. 203

204

3.2 Animal performance, faeces score and plasma samples 205

In general, pigs maintained good health status during the experiment. Average daily feed 206

intake, ADG and G:F performance of animals divided in d 1-7, d 7-14 and d 1-14 are given in 207

Table 3. Average daily feed intake did not differ significant among diets in either one of the 208

periods. No significant differences of diet on ADG and G:F were observed among pigs fed FM, 209

SPC and SM5% in any of the periods. In all periods, pigs receiving SM10% had lower (P<0.001) 210

ADG and G:F compared to FM, SPC and SM5%. From d 1 to 14, pigs fed the SM10% diet had 211

Manuscript to be submitted

34

an ADG, which was 23-28% lower (P < 0.001) than pigs fed the FM, SPC and SM5% diets, 212

despite that they ate the same amount of feed. 213

Faeces characteristics were evaluated from day 5 to 9 in both periods. The data from the 214

evaluation periods, showed no significant differences among diets (Table 4). 215

There was no significant difference in PUN concentration between pigs fed diet FM, SPC 216

and SM5%, but the concentration of PUN was higher (P<0.003) for SM10% compared with the 217

plasma concentration in SPC and SM5%, but not FM (Table 5). Plasma concentration of Thr in 218

SPC was higher (P<0.001) than the plasma concentration in SM5% and SM10%, but not FM. 219

The Met concentration in plasma from pigs fed SPC was higher (P<0.003) than in plasma from 220

FM and SM10%, but not SM5%. Regarding Ca, there was a higher (P<0.001) concentration of 221

Ca in plasma from pigs receiving SM10% compared to FM, SPC and SM5%. The opposite was 222

the case with P where the concentration was lower (P<0.001) in SM10% compared to FM, SPC 223

and SM5%. 224

225

4. Discussion 226

Data from the present study supported the hypothesis that feeding a diet containing SM to 227

piglets from BW 7 to 15 kg, will give the same ADFI, ADG and G:F as diets containing 228

traditional protein sources. This was, however, only the case when the inclusion level of starfish 229

meal was 5% and not 10%. Despite it has been concluded that the protein fraction in SM is 230

comparable to that of FM in quality (Bird, 1946; Whitson and Titus, 1946; Levin et al., 1960), 231

the poor growth results obtained on high amounts of SM in studies with poultry, was suggested 232

to be caused by the excess dietary Ca, which also widened the Ca:P of the diet (Heuser and 233

McGinnis, 1946; Morse et al., 1946; Whitson and Titus, 1946). In the present experiment, pigs 234

offered the diet containing 10% SM with a Ca:P of 2.6:1 (14.8 g Ca and 5.8 g P per kg feed) 235

Manuscript to be submitted

35

(Table 2) had no detrimental effect on ADFI, but ADG and G:F were lower than in pigs offered 236

diets containing FM, SPC and SM5%. The dietary Ca and P levels in the SM10% diet was above 237

the Danish recommendations (Tybirk et al., 2014) for pigs with BW of 9 to 15 kg (8.7 g Ca and 238

5.7 g total P per kg feed when phytase is added). Due to the design of the experiment, to see the 239

response of different levels of SM in pigs, it was necessary to have a level of Ca higher than 240

recommended. 241

Phosphorus has been shown to exert greater influence on growth characteristics than Ca 242

(Maxson and Mahan, 1983) as P plays a major role in the development and maintenance of the 243

skeletal system and is also involved in muscle tissue deposition (Varley et al., 2011). In the 244

SM10% diet, there could be a possible interaction among the feed ingredients. Starfish do not 245

contain phytate, but plant ingredients contain phytic acid. Digestion and utilization of dietary 246

phytate-P by monogastric animals requires hydrolysis of phytate by phytase (Fisher, 1992). The 247

apparent negative impact of high Ca level and wide Ca:P, when feeding the SM10% diet on 248

ADG and G:F, may be a detrimental effect on phytase activity. The negative effects of extra Ca 249

in the diet on the efficacy of phytase, may be explained by three mechanisms: 1) High Ca level 250

may lower P absorption as a result of the formation of insoluble phytate-Ca complexes in the 251

small intestine that is not accessible for hydrolysis by phytase (Wise, 1983; Fisher, 1992), 2) 252

high Ca levels increases the pH of the intestinal content, which decrease the exogenous phytase 253

activity (Sandberg et al., 1993) and 3) high Ca levels in the diet may directly reduce the activity 254

of the phytase by competing for the active sites of the enzyme, limiting phytase efficacy in 255

hydrolysing phytate-P (Wise, 1983; Qian et al., 1996). In the SM10% diet there was a high 256

dietary Ca level, and it is found that increasing the dietary Ca level, decreases the apparent totalt 257

tract digetibility (ATTD) of P in a phytase-supplemented diet (Liu et al., 1998). This was 258

confirmed by Qian et al. (1996), who found that reducing the Ca:P from 2.0:1 to 1.2:1 increased 259

Manuscript to be submitted

36

phytase efficiency by approximately 16% and at the same time improved performance. 260

Moreover, Lei et al. (1994) demonstrated that the ability of phytate to improve phytate-P 261

availability was greatly reduced when widening the Ca:P from 1.3:1 (4.8 g Ca/kg, 3.2 g P/kg) to 262

3.0:1 (8.8 g Ca/kg, 2.9 g P/kg). The high Ca level in the SM10% diet may, therefore, may have 263

inhibited phytase activity and lowered the ATTD of P, hence, the level of P to maximize muscle 264

tissue deposition may have been insufficient (Varley et al., 2011). The effect of the wide Ca:P in 265

the SM10% diet is substantiated by the plasma concentrations of Ca and P (Table 5), which 266

showed higher Ca level and lower P level compared to pigs fed the other diets. As Ca:P is 267

increased, an inverse relationship of serum Ca and P has been reported, where serum Ca linearly 268

increases and serum inorganic P decreases. This substantiate that the digestibility and absorption 269

of P decreases as the dietary Ca:P increases. That is, increased dietary Ca levels results in 270

decreased ATTD of P (Stein et al., 2011). Furthermore, divalent minerals can readily bind to 271

phytic acid and thus form mineral-phytate complexes that may be resistant to hydrolysis by 272

phytase. Formation of these complexes is associated with a reduction in mineral bioavailability 273

(Maenz et al., 1999). Mineral-phytate complexes are mainly formed at a pH that is above, or the 274

upper limit of the microbial phytase activity spectrum (Selle et al., 2000). Thus, the pH in the gut 275

may have an influence on the efficiency of phytase, where the high Ca level most likely create a 276

buffering effect thereby increasing pH (Stein, 2002). Hence, availability of both P and minerals 277

involved in growth performance can be reduced by high Ca levels. 278

Pigs receiving diet SM10% had same ADFI as did pigs on the other diets. A palatability 279

study done by Falkowski et al. (1998) found that pigs preferred a high NaCl content (0.57%) in 280

the diet. Palatability may have driven the pigs to eat more of the SM10% diet containing a higher 281

level of Na (Table 2), therefore having the same ADFI as the other diets. But the lower ADG of 282

pigs fed the SM10% diet indicates that the pigs have not been capable of utilizing the consumed 283

Manuscript to be submitted

37

feed. Dietary NaCl enhance P absorption and improve its digestibility due to the active transport 284

of P from the intestinal lumen via the Na+/Pi co-transport (Yin et al., 2008). Furthermore, Na 285

plays a role in the intestinal absorption of AA. The missing apparent power of Na, can be a result 286

of the wide Ca:P as discussed above. 287

Plasma urea nitrogen is an indicator of AA catabolism and the concentration can be related 288

to protein requirement, an unbalance in the AA profile and/or over supply of protein (Zervas and 289

Zijlstra, 2002). Since the four diets only slightly differed in CP level (Table 2) and were 290

optimised to have the same content of SID AA (Table 2), the higher PUN concentration found in 291

the SM10% diet, indicates that the requirement for CP was met at or before the CP content of the 292

SM10% diet. No explanation was found for the significant differences observed in both Thr and 293

Met concentration in plasma (Table 5) in the experiment. 294

The registration of faeces characteristics indicated no diet dependent effect on the incidence 295

of diarrhoea, and in general the incidence of diarrhoea was low. One of the reasons may be the 296

seven days pre-experimental period from weaning to the beginning of the experiment. This 297

period was included to secure that all pigs ate from the beginning of the experimental period and 298

thus had been adapted to the shift from sow milk to dry feed. Another reason could be that the 299

diets contained only 166-172 g SID CP/kg, which is lower than the diets reported to cause 300

diarrhoea. Diets formulated to less than 180 g CP/kg and fortified with crystalline AA to supply 301

adequate indispensable AA levels for growth, can minimise the expression of diarrhoea (Heo et 302

al., 2009; Kim et al., 2012). High levels of dietary Ca can create a buffering effect in the 303

digestive tract, which can cause diarrhoea in piglets (Stein, 2002). However, no increase in 304

faecal score was observed in pigs fed SM10% diet. 305

The chemical composition of SM is promising in relation to pig feed, but the literature 306

shows variable level of CP varying from 27-36% (Levin et al., 1960), 34% (Bird, 1946; 307

Manuscript to be submitted

38

Ringrose, 1946) to 70% (Nørgaard et al., 2015). Levin et al. (1960) found indication of that all 308

the constituents of starfish are variable and depends largely on size, freshness, season and 309

environmental factors associated with the specific samples. The knowledge of the biochemical 310

composition of the starfish tells when it is most favourable to fish them. It was reported by 311

Oudejans and Vandersluis (1979) that during the year, the total AA content and relative 312

distribution of AA in proportion to CP is rather constant, while the content of CP varies; lowest 313

in June until autumn and greatest in May following the starfish reproduction cycle. Fishing 314

starfish in May, which was the case in the study by Nørgaard et al. (2015), will in relation to 315

using starfish as a feed ingredient for pigs be of greatest interest. In the present study the starfish 316

was caught in November, amounting a CP level of 38%. 317

318

5. Conclusion 319

Inclusion of 5% SM in the diet for piglets with BW 9-15 kg resulted in pig performance equal to 320

diets containing either FM or SPC. Starfish meal is therefore a potential competitor to high-321

quality protein sources for piglets. Pigs fed 10% SM had lower ADG and G:F than pigs fed the 322

other diets, but results on ADFI was not significantly different among treatments. The negative 323

effect on performance when feeding the SM10% diet, may be the high Ca level and wide Ca:P 324

that affects digestibility and absorption of P negatively. Moreover, binding of divalent minerals 325

to phytic acid and thus formation of mineral-phytate complexes, may reduce mineral 326

bioavailability. The negative effects increases as the dietary Ca:P increases. The determining 327

factor for the maximum inclusion level of SM in diets for piglets may be the dietary Ca level and 328

the resulting Ca:P. 329

330

Conflict of interest statement 331

Manuscript to be submitted

39

The author has no conflict of interest regarding this work. 332

333

Acknowledgements 334

The research project was founded by the Green Development and Demonstration 335

Programme under the Ministry of Food, Agriculture and Fisheries, Denmark, grant number 336

34009-14-0845. 337

338

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affect the apparent total tract digestibility of calcium, but decreases digestibility of 427

phosphorus by growing pigs. Journal of Animal Science 89, 2139-2144. 428

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and phytase addition on performance, bone parameters, apparent nutrient digestibility, 441

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Yin, Y.I., Huang, C.H., Wu, X., Li, T.J., Huang, R.L., Kang, P., Hu, Q., Chu, W.Y., Kong, X.F., 451

2008. Nutrient digestibility response to graded dietary levels of sodium chloride in weanling 452

pigs. Journal of the Science of Food and Agriculture 88, 940-944. 453

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patterns and postprandial plasma urea profiles in grower pigs. Journal of Animal Science 80, 455

3238-3246. 456

457

458

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45

Table 1 459

Analysed chemical composition (g/kg DM) of wheat, barley, dehulled toasted soy bean meal 460

(SBM), fish meal (FM), soy protein concentrate (SPC) and starfish meal (SM). 461

Item Wheat Barley SBM FM SPC SM

Dry matter 886 898 909 950 916 961

Crude protein (CP) 108 118 496 735 552 383

Crude ash 17 21 76 171 71 421

Crude fat 22 29 19 90 30 91

Ca 0.29 0.28 3.0 36 3.4 130

P 3.0 3.3 7.5 24 7.2 63

Cys 2.3 2.6 7.6 6.5 8.0 4.7

His 2.4 2.5 13.3 14.4 14.0 6.1

Ile 3.6 4.3 24.1 32.0 27.6 14.5

Leu 6.8 7.8 38.0 52.8 42.8 21.4

Lys 3.1 4.2 30.7 56.2 32.5 20.6

Met 1.6 1.9 6.7 20.7 7.2 8.1

Phe 4.7 5.6 25.7 28.4 29.2 12.5

Thr 3.0 3.8 19.9 29.8 21.6 18.0

Trp 1.3 1.4 6.8 7.3 7.1 3.8

Val 4.7 6.1 25.4 37.0 27.8 17.8

462

463

Manuscript to be submitted

46

Table 2 464

Composition, and calculated and analysed chemical composition (g/kg, as-fed basis) of 465

experimental diets containing fish meal (FM), soy protein concentrate (SPC) and 5 or 10% 466

starfish meal (SM). 467

FM SPC SM5% SM10%

Ingredient

Wheat 536 504 500 469

Barley 200 200 200 200

SBM 174 174 174 174

SM

- - 50 100

SPCa - 75 52 33

FMb

55 - - -

Calcium carbonate 17.2 15.6 9.1 -

Mono-calcium phosphate 5.6 9.9 9.1 8.4

Soy oil - 6.0 1.4 4.0

NaCl 2.3 3.6 1.8 -

Vitamin-mineral premixc 4.0 4.0 4.0 4.0

L-Lysin HCl (780 g/kg) 3.5 4.4 4.2 3.9

DL-Methionine (990 g/kg) 0.8 1.4 1.2 1.0

L-Threonine (985 g/kg) 1.2 1.4 1.1 0.8

L-Tryptophan (980 g/kg) 0.3 0.2 0.2 0.2

Microgritsd

0.5 0.5 0.5 0.5

Phytase (Natuphos 5000)e 0.2 0.2 0.2 0.2

Calculated chemical composition

Manuscript to be submitted

47

Dry matter 865 853 877 881

Crude ash 60.8 60.5 61.2 77.2

Crude fat 23.3 26.0 25.0 30.9

Crude protein (CP) 194 192 198 203

SID CPf

167 166 170 172

SID lys 11.4 11.4 11.4 11.4

Ca 10.2 8.7 8.7 14.8

P 5.7 5.9 5.8 5.8

Ca:P 1.8:1 1.5:1 1.5:1 2.6:1

Na 1.6 1.6 2.2 2.8

Analysed chemical composition

Dry matter 903 903 902 903

Crude ash 63.9 58.9 65.0 80.3

Crude fat 26.9 28.0 30.0 34.9

CP 206 202 204 207

a AlphaSoy PIG 530 (Agro Korn, Videbæk, Denmark). 468

b FF classic (FF Skagen, Skagen, Denmark). 469

c Danish Vilomix A/S. Provided per kg diet: 11,000 IU vitamin A, 1,000 IU vitamin D3, 16.9 IU 470

vitamin E, 4.4 mg vitamin K3, 4.4 mg vitamin B1, 8.8 mg vitamin B2, 6.6 mg vitamin B6, 0.04 471

mg vitamin B12, 22 mg Ca-D-panthotenic acid, 44 mg niacin, 0.44 mg biotin, 330 mg Fe (FE (II) 472

sulphate), 260 mg Cu (Cu (II) sulphate), 200 mg Zn (Zn oxide), 88 mg Mn (Mn oxide), 0.44 mg 473

I (calciumjodat), and 0.50 mg Se (natriumselenite). 474

d Jadis Additiva (Haarlem, NL). Corn bran in various colours to identify diets. 475

e Natuphos 5000 (BASF, Ludwigshafen, Germany). 476

Manuscript to be submitted

48

f SID, standardised ileal digestible. 477

Manuscript to be submitted

49

Table 3 478

Average daily feed intake (ADFI), average daily gain (ADG) and gain to feed ratio (G:F) of pigs 479

fed diets containing fish meal (FM), soy protein concentrate (SPC) and 5 or 10% starfish meal 480

(SM).1

481

Item FM SPC SM5% SM10% SEM P-value

ADFI, g, d 1-7 717 681 705 647 25 0.06

ADG, g, d 1-7 593a 557

a 591

a 438

b 22 <0.001

G:F, d 1-7 0.829a

0.832a

0.843a

0.667b

0.04 <0.001

ADFI, g, d 7-14 1023 997 1064 1066 36 0.25

ADG, g, d 7-14 756a

736a

744a

615b

33 <0.001

G:F, d 7-14 0.738a

0.744a

0.701a

0.579b

0.02 <0.001

ADFI, g, d 1-14 861 836 884 862 31 0.55

ADG, g, d 1-14 675a

650a

669a

526b

20 <0.001

G:F, d 1-14 0.770a

0.774a

0.760a

0.614b

0.01 <0.001

a,b Means within rows without a common superscript differ (P≤0.05). 482

1 Values are least squares means and standard errors of the means (SEM). n = 24. 483

484

Manuscript to be submitted

50

Table 4 485

Distribution of faeces scores of pigs fed diets containing fish meal (FM), soy protein concentrate 486

(SPC) and 5 or 10% starfish meal (SM).1

487

FM SPC SM5% SM10% P-value2

Score 1 26 34 19 25 0.47

Score 2 51 40 47 50

Score 3 39 43 44 37

Score 4 4 3 5 8

1 Faeces score: 1, firm and shaped; 2, soft and shaped; 3, loose/often shining surface, or 4, 488

watery/flows through slatted floors. 489

2 The P-value is based on a F-test for differences in the distribution of days with score 1-4 among 490

the diets. 491

492

Manuscript to be submitted

51

Table 5 493

Plasma urea nitrogen (PUN) and amino acid concentrations (µmol/L), and calcium and 494

phosphorus plasma concentrations (mg/L) in pigs 120 minutes after feeding 25 g diet/kg BW0.75

495

on day 15 in period 1 after an overnight fasting. The experimental diets containing either fish 496

meal (FM), soy protein concentrate (SPC) and 5 or 10% starfish meal (SM).1 497

Item FM SPC SM5% SM10% SEM

P-value

PUN 3254ab

2594b

2914b

3919a

246 0.003

Ile 127 158 141 126 23 0.23

Leu 141 167 147 130 23 0.18

Lys 238 276 224 220 36 0.22

Met 42b

61a 56

ab 41

b 7 0.003

Phe 82 99 88 82 14 0.15

Thr 154ab

203a

145b

105b

25 <0.001

Trp 44 48 43 45 7 0.88

Tyr 90 115 100 88 15 0.07

Val 222 240 226 227 33 0.86

Ca 113b

114b

114b

128a 1 <0.001

P 100a

103a

103a

72b

2 <0.001

a,b Means within rows without a common superscript differ (P≤0.05).

498

1 Values are least squares means and standard errors of the means (SEM). n = 12. 499

52

General discussion of results and the study

53

GENERAL DISCUSSION OF RESULTS AND THE STUDY

6 Discussion and future development

This section of the thesis will discuss the results in general and the methods of the study. It will be

discussed whether the study could have been improved, and if so, how. Moreover, supplementary

results on FA composition of SM left out of the manuscript will be presented and discussed, and

finally, the possibility of further development of SM. Discussion in manuscript will not be repeated.

6.1 Design of the study

It was determined beforehand that the piglets should be individually housed, but the remaining

elements of the study were yet to be chosen. These elements included the target group of the

experimental diets, number and composition of the diets, number of piglets necessary to have

enough repetitions per diet, parameters to be measured and the whole procedure during the study.

When choosing the number and composition of the experimental diets, simple diets with few

ingredients were composed, which is seldom the case with diets for the specific target group. They

often contain a number of different high-quality protein sources. The purpose was to make diets that

more clearly identified what affected piglet performance, and relatively be able to rule out any

affecting interactions among the feed ingredients. It was a difficulty to balance a diet containing

10% SM, according to the recommendations especially regarding Ca. The content of Ca is high in

SM (Stutz and Matterson, 1964; Nørgaard et al., 2015), hence, the maximum level of Ca in the diet

is met rapidly, by which the possible inclusion level of SM was limited. Consequently, that

precluded the possibility to fulfil the need for the other nutrients such as AA requirements. Well

aware that the high Ca level in the SM10% diet could negatively affect performance in pigs, since it

has been reported in poultry (Heuser and McGinnis, 1946; Ringrose, 1946; Stutz and Matterson,

1964), it was more relevant to see the result of a higher level of SM included in the diet than

possible, if to follow the nutrient recommendations. This was due to several reasons, both that

performance studies with SM and pigs have never been conducted, and also the possibility to see

differences in performance, if any, among the diets. The study was intended to be on performance

in relation to SM, and the most important measures were found to be feed intake, weight gain,

faeces characteristics and blood parameters. Another parameter brought into play was having the

pigs in metabolism cages and collect faeces and urine. If the Ca:P is greater than that needed for

bone tissue synthesis, excretion of Ca in the urine is increased due to low availability of P

General discussion of results and the study

54

(Gonzalez-Vega and Stein, 2014). Moreover, calculation of absorption and retention of both Ca and

P would be possible giving an indication of the availability of the two minerals (Gonzalez-Vega et

al., 2013). Dissection was also brought into play to evaluate bone composition. The activity of cells

involved in bone tissue degradation and synthesis is regulated by parathyroid hormone and

calcitonin, in which the secretion of these hormones is dependent on the concentration of Ca in

plasma (Gonzalez-Vega and Stein, 2014). When concentrations of Ca in plasma are lower than

normal, parathyroid hormone is secreted, resulting in increased resorption of Ca from bones. The

results from the study conducted by Reinhart and Mahan (1986) suggested that high Ca:P can

adversely affect bone development, including percent bone ash, bone wall thickness and bone-

bending moment. Hence, urine, faeces and bone tissue could contribute in explaining the results

observed. This was, however, more than the budget could handle, and moreover, would be too far-

reaching in relation to the objective of the study.

A neglected factor in the study was the relative high content of Na in the SM10% diet. It could have

been interesting to record the water consumption, since many of the pigs receiving diet SM10%

“moved” water from the drinking nipple to the container, making the feed wet. Pigs can tolerate

high levels of NaCl (Todd et al., 1964; Mason and Scott, 1974; Chittavong et al., 2013), provided

that water is freely available. However, no indication of a negative effect of NaCl content was

found. Furthermore, it would have been interesting to record the water consumption, if there were

any difference among piglets.

6.2 Faeces score method

A study with observations requires a test for intra-observer reliability to be done to make sure that

the observations also resemble each other throughout the entire period. Faeces characteristics are

highly subjective measurements and what is observed can be very different from person to person.

To ensure that the observations in the first period of the study resembled the observations obtained

in the second period, pictures of the different scores of faeces were taken in the first period to have

something to resemble the second period. Normally, resembling can be done by making some of the

early observations later in the period and comparing the result. This was not possible in this case,

since these observations were done in a specific period of the study and the observations could not

be repeated. A trained eye would maybe have obtained other results, which could have changed the

outcome of the faeces characteristics. This is not the case, however, since the observations have

been compared to the record of health among pigs and no incongruence was found.

General discussion of results and the study

55

6.3 Results from the study

The time of fishing the starfish was not optimal in relation to having the best possible product. It

was known that the CP content could be higher than at the time, given that the CP content in

starfish follow the reproduction cycle, i.e. the ovarian growth that is only at its beginning in the late

autumn (Oudejans and Vandersluis, 1979). However, due to a fixed time frame for the thesis, the

product used was having a lower CP content than known possible. This was not thought of as a

liability on the result of the study, since it was not an experiment done to see the results of the best

possible product, but rather the response on performance when feeding with the product well

knowing that it varies in quality. Using starfish fished when the amount of CP is high, however,

could have affected the interpretation of the results. A higher CP content would have made it

possible to include less SM and thereby the amount of Ca in the diet. On the contrary, the inclusion

level of SM in the diet would still have been put to a maximum, to see the piglet’s response on the

feed ingredient and the problem with high Ca level in the diet would possibly still be there.

No results in the present study can be considered immaterial since it is a new field of study, but

some results say more than others. It was rather difficult to interpret the difference observed on

some of the plasma parameters. Of the chosen blood parameters, only Ca and P contributed in

explaining the performance results. The concentration of Ca and P in plasma and the effect on

performance are well described in the literature (Maxson and Mahan, 1983; Koch et al., 1984;

Reinhart and Mahan, 1986; Gonzalez-Vega and Stein, 2014), thereby contributing to the

interpretation of the results. Contrary, the differences observed in both PUN and some of the AA

were rather difficult to interpret. The analysis on PUN and AA in plasma aimed to display no

difference in the protein sources, since the four diets were optimised to have the same content of

SID AA and only slightly differed in CP level. However, after viewing the results, this was not the

case. If blood samples had been taken before the beginning of the experiment, it could have given

appreciation in the individual pig’s PUN and plasma AA concentration. But doing that would be

considered as going too far, since it was not within the scope of the study. It can be speculated if

other blood parameters such as parathyroid hormone and calcitonin related to Ca homeostasis could

be of greater relevance and contribute to the explanation of the observed performance. However,

more blood parameters would most likely not have helped to interpret the results. It would,

however, have been appropriate to collect faeces samples as discussed above. On reflection,

collection of faeces and urine would maybe have helped in clarifying e.g. the bioavailability of P

and the possible formation of mineral complexes.

General discussion of results and the study

56

The study was financed by the project STARPRO, whose objective among other things was to

produce a 100% organic feed ingredient for pigs and poultry. The present study was conducted with

conventionally reared piglets and a conventionally designed diet containing conventional feed

ingredients and CAA. Interpretation of the results in relation to organic livestock production, must

therefore be considered in proportion to the differences in the production systems and the

constraints there are in rearing organic livestock, among these prohibition of supplementation to the

feed with CAA (Ministry of Food, 2015b). However, it is presumed, that as with any other feed

ingredient, SM can be included in diets for organic livestock, due to the content of CP and

composition of indispensable AA in SM.

6.4 Possibly same features in starfish meal and fish meal

What characteristics of SM that lead to the positive results of 5% SM in the diet for piglets is an

interesting question. It was reported by Whitson and Titus (1946) that SM contained the same

growth stimulating action as sardine FM when SM was used to supply the same amount of protein.

It was suggested that the growth stimulating action was due to the quantity of certain AA or

vitamins present in SM. It can be speculated if SM contains unknown positive factors on

performance as do FM. Traditionally, FM is recognised as a feed ingredient high in protein, energy,

vitamins and minerals with positive effect on performance (Cho and Kim, 2011). An increase in

ADG was presumably a result of increased ADFI as reported by Stoner et al. (1990). The increased

ADFI was suggested to be due to a preference by the pig for FM, where preference was thought to

be a function of taste or palatability. Though, other data illustrating what characteristic of taste or

palatability that makes pigs prefer FM have to my knowledge not been reported. Fish meal is a

major dietary source of n-3 PUFA (Cho and Kim, 2011). The perception of the n-3 PUFA in FM is

divided; some have reported that the n-3 PUFA has anti-inflammatory properties and benefits to the

immune system (Cho and Kim, 2011) while others have reported that n-3 PUFA made pigs more

susceptible to bacterial infections (Thies et al., 1999). In the present study, SM was analysed for FA

to compare the content of FA in SM with them of FM (Table 4). The idea was that if the two

products had similar FA composition, SM could have the same features as FM. Cho and Kim

(2011) reported that it is EPA and DHA of FM that benefits to the immune system. It has not been

possible to find a complete analysis of the composition of FA in FM, but it could be hypothesised

that SM and FM have similar contents of EPA and DHA. Therefore, SM may share the benefits or

poor effect of FA found on the immune system. In the light of these findings, it is definitely an area

worth to look into.

General discussion of results and the study

57

Table 4 - Fatty acid (FA) content (g FA/ 100 g FA) and iodine value (g/100 g fat) in starfish meal (SM) and in fish meal (FM).

g FA/100 g FA

Formula Name SM1

FM2

Sat

ura

ted

C14:0 Myristic acid 11.4 6.0

C15:0 Pentadecylic acid 1.5

C16:0 Palmitic acid 14.7 17.8

C18:0 Stearic acid 6.8 3.6

C20:0 Arachidic acid 0.4

C22:0 Behenic acid 0.2

C24:0 Lignoceric acid 0.0

Total saturated fatty acids 34.9

Monounsa

tura

ted

C16:1 ω-9 Hexadecenoic acid 0.6

C16:1 ω-7 Palmitoleic acid 2.0 7.2

C18:1 ω-9 Oleic acid 2.0 12.3

Trans vaccenic acid 6.9

C18:1 ω-7 Vaccenic acid 0.7

C20:1 ω-9 Eicosenoic acid 21.7 6.6

C22:1 ω-11 Docosanoic acid 0.8

C22:1 ω-9 Erucic acid 1.5 7.7

C24:1 ω-9 Nervonic acid 2.0

Total monounsaturated fatty acids 38.1

Di-

and t

ri u

nsa

tura

ted

C18:2 ω-6 Linoleic acid 0.4 2.1

C18:3 ω-6 γ-Linolenic acid 0.5

C18:3 ω-3 α-Linolenic acid 0.2 1.93

C20:2 ω-6 Eicosadienoic acid 0.7

C20:3 ω-6 Dihomo-γ-linolenic acid 0.1

C20:3 ω-3 Eicosatrienoic acid 0.2

Total di- and tri unsaturated fatty acids 2.0

≥ T

etra

unsa

tura

ted

C18:4 ω-3 Octadecatetraenoic acid 0.5

C20:4 ω-6 Arachidonic acid 4.0 2.4

C20:5 ω-3 Eicosapentaenoic acid 10.8 9.0

General discussion of results and the study

58

C22:5 ω-6 Osbond acid 1.1

C22:5 ω-3 Clupanodonic acid 1.2 2.6

C22:6 ω-3 Docosahexaenoic acid 7.0 6.6

Total ≥ tetra unsaturated fatty acids 24.5

Omega-6 fatty acids 6.7

Omega-3 fatty acids 19.8

Omega-6/omega-3 0.3

Iodine value (g/100g fat) 132.0

1 Caught November 5

th 2014 in relation to the STARPRO project. The analysis was done by the Bligh and Dyer

extraction procedure (Jensen, 2008).

2 The values are from Sauvant et al. (2004).

3 Distribution between ω-6 and ω-3 is not known.

6.5 Possibility of further development of the product

Supplementation with chelators to the diet such as EDTA, citrate and phthalate have been found to

increase the efficiency of microbial phytase in hydrolysing phytic acid in vitro (Maenz et al., 1999).

It is thought that EDTA functions as a competitive chelator and minimise the formation of enzyme-

resistant mineral-phytate complexes. This observation is interesting, and could be of great interest

to follow up, since it maybe could contribute in solving the problem with the high Ca content in

SM. However, EDTA is not authorised in the EU to be used as additive in feed ingredients for

animals, and this can therefore only be considered as speculations and ideas for future studies. More

studies also need to be performed with the objective to find the maximum inclusion level of SM. In

this context, it could be appropriate to further develop the product and possibly in that way elevate

the inclusion level of SM. Levin et al. (1960) produced defatted SM made by the VioBin azeotropic

process (see paper) and found that protein concentrates prepared from defatted SM either by

pancreatic digestion or by washing with HCl, had high protein quality values, compared to good

quality FM. Further processing of SM could be of interest, but the cost of the processing must be

considered, so that the product can compete on price with e.g. FM.

Conclusion

59

CONCLUSION

From a theoretical aspect, the literature review stated that there is a need for alternative high-quality

protein sources, that is both sustainable and of local origin, and can fit the demands of organic feed

ingredients. Moreover, the increasing problems experienced with starfish in the Danish waters and

the chemical composition found in SM, together with the results found in studies with poultry,

make SM a realistic potential as feed ingredient for both conventional and organic reared piglets.

The level of energy, protein, vitamins and minerals are the main determinants of the level of

production that can be obtained, and are therefore of great importance. Protein sources of animal

origin provide the highest quality, primarily due to the composition of indispensable AA and,

therefore, the completeness of the protein source, making marine sources as SM more interesting as

feed ingredient.

Inclusion of 5% SM in the diet for piglets with BW 9-15 kg resulted in pig performance equal to

diets containing either FM or SPC. Starfish meal is therefore a potential competitor to high-quality

protein sources for piglets. Pigs fed 10% SM had lower ADG and G:F than pigs fed the other diets,

but results on ADFI was not significantly different among treatments. The negative effect on

performance when feeding the SM10% diet may be the high Ca level and wide Ca:P that affects

digestibility and absorption of P negatively. Moreover, binding of divalent minerals to phytic acid

and thus form mineral-phytate complexes, may reduce mineral bioavailability. The negative effects

increases as the dietary Ca:P increases. The determining factor for the maximum inclusion level of

SM in diets for piglets may be the dietary Ca level and the resulting Ca:P.

It is therefore concluded, that SM can be implemented in diets for piglets, and take part in the

compliance of the claim, that diets for organic reared livestock should constitute feed ingredients of

100% organic origin before the deadline in December 2017- assuming that it is legitimised due to

the promising results found until now. It is, however, still important to look into the effect on piglet

performance and possibly further develop the product, so higher inclusion level could be possible,

and moreover, for the product to win acceptance with farmers and secure a foothold as a high-

quality protein source.

Implications and perspectives

60

IMPLICATATIONS AND PERSPECTIVES

Inclusion of 5% SM in the diet for piglets with a BW of 9 to 15 kg, offers the same growth potential

as inclusion of traditional high-quality protein sources as FM and SPC. The performance response

to SM was similar in magnitude to that obtained with diets containing either FM or SPC, suggesting

that SM is a good alternative to known protein sources. Feeding a diet containing 10% SM to

piglets affected growth negatively, compared to feeding a diet containing either 5% SM, FM or

SPC. The current situation in which the need for alternative protein sources is requested should

encourage further investigation into the use of marine protein sources comprising SM as a mean of

achieving the goal of feed of 100% organic origin for organic livestock before the deadline in

December 2017.

A new study with SM for piglets in larger scale at a private conventional farm was planned to begin

in May 2015 as a dose response relationship. However, it was discontinued for want of

authorisation for the use of starfish as feed ingredient in the EU. In the first draft for the study, it

was planned to have three similar diets only differing in the content of FM and SM; one control diet

containing FM and two diets containing different levels of SM. These diets were to be feed for

piglets after weaning when the BW is expected to be 6 to 9 kg and in the following period (BW 9 to

16 kg). This study, when it becomes a reality, will hopefully answer questions in connection to SM,

about the products ability to sustain the positive effect on growth, also when fed to piglets that are

group stabled and therefore more challenged on health. Moreover, if the high G:F can be sustained.

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