Contents AGRICULTURAL AND FOOD SCIENCE - MTTAGRICULTURAL AND FOOD SCIENCE Salputra, G. et al. Policy...

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ContentsSalputra, G., Chantreuil, F., Hanrahan, K., Donnellan, T., van Leeuwen, M. and Erjavec, E.Policy harmonized approach for the EU agricultural sector modelling

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Vauhkonen, V., Lauhanen, R., Ventelä, S., Suojaranta, J., Pasila, A., Kuokkanen, T., Prokkola, H. and Syväjärvi, S.The phytotoxic effects and biodegradability of stored rapeseed oil and rapeseed oil methyl ester

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Sironen, A., Uimari, P. and Vilkki, J. Comparison of different DNA extraction methods from hair root follicles to genotype Finnish Landrace boars with the Illumina PorcineSNP60 BeadChip.

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Manninen, M., Honkavaara, M., Jauhiainen, L., Nykänen, A. and Heikkilä, A.-M. Effects of grass-red clover silage digestibility and concentrate protein concentration on performance, carcass value, eating quality and economy of finishing Hereford bulls reared in cold conditions

151

Wiander, B. and Palva, A. Sauerkraut and sauerkraut juice fermented spontaneously using mineral salt, garlic and algae.

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Wiander, B. and Korhonen, H.J.T. Preliminary studies on using LAB strains isolated from spontaneous sauerkraut fermentation in combination with mineral salt, herbs and spices in sauerkraut and sauerkraut juice fermentations.

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Hyvönen, T. Impact of temperature and germination time on the success of a C4 weed in a C3 crop: Amaranthus retro-flexus and spring barley.

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© Agricultural and Food Science Manuscript received March 2010

Policy harmonized approach for the EU agricultural sector modelling

Guna Salputra1*, Frédéric Chantreuil2, Kevin Hanrahan3, Trevor Donnellan3, Myrna van Leeuwen4 and Emil Erjavec5

1Latvian State Institute of Agrarian Economics (LSIAE), Struktoru 14, LV-1039, Riga, Latvia,

2INRA, 4 allée Adolphe Bobierre, CS 61103, 35011 Rennes cedex, France,3Rural Economy Research Centre (RERC), Teagasc, Athenry, Co. Galway, Ireland,

4LEI, Alexanderveld 5, 2585 DB Den Haag, The Netherlands,5University of Ljubljana (LJUB), Groblje 3, 1230 Domzale, Slovenia,

*e-mail [email protected]

Policy harmonized (PH) approach allows for the quantitative assessment of the impact of various elements of EU CAP direct support schemes, where the production effects of direct payments are accounted through reaction prices formed by producer price and policy price add-ons. Using the AGMEMOD model the impacts of two possible EU agricultural policy scenarios upon beef production have been analysed – full decoupling with a switch from historical to regional Single Payment scheme or alternatively with re-distribution of country direct payment envelopes via introduction of EU-wide flat area payment. The PH approach, by systematizing and harmonizing the management and use of policy data, ensures that projected differential policy impacts arising from changes in common EU policies reflect the likely actual differential impact as opposed to differences in how “common” policies are implemented within analytical models. In the second section of the paper the AGMEMOD model’s structure is explained. The policy harmonized evaluation method is presented in the third section. Results from an application of the PH approach are presented and discussed in the paper’s penultimate section, while section 5 concludes.

Key-words: modelling, CAP, direct support, decoupling, policy evaluation


Salputra, G. et al. Policy harmonized approach for the EU agricultural sector modelling

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Introduction

The European Union’s Common Agricultural Policy (CAP) and the sectoral modelling of agriculture in the EU are closely intertwined in the literature. EU decision makers have traditionally used the results of a wide range of quantitative tools in framing their choices among alternative policy instruments. Quantitative assessments of the impact of policy changes and policy effectiveness can be catalysts for public debate. Through the provision of quantita-tive analysis of alternative policies such economic research influences the direction and tone of policy debates by defining the quantitative range of possible future alternatives (Bartosova et al. 2008).

Since its origins, the CAP has been under con-stant internal and external reform pressures (Yr-jölä et al. 2001; Garzon 2006; Swinnen 2008). The 1992 MacSharry CAP reform, in an attempt to ar-rest growing production and budgetary costs, in-troduced direct payments to the CAP. These direct payments were introduced as compensation for in-come losses expected to result from reduced inter-vention prices. Expenditure on such compensatory direct payments, in subsequent reforms described as coupled payments or premiums, was limited by restrictions on the total number of premiums claims allowable per Member State. The pattern of inter-vention price reductions being “compensated” for by increases in coupled direct payments was again observed in the 2000 reforms when the decisions on increases in the value of direct payments and reductions in intervention prices were adopted by the European Council along with an agreement on the multi-year EU budgetary programme called Agenda 2000 (Daugbjerg et al. 2007).

The Fischler reform in 2003 changed the form of CAP direct income support payments by intro-ducing decoupled direct income supports, though it largely preserved the scope and distribution of funds across Member States and types of agricul-tural holdings (Swinnen 2008). Policy modifica-tions under the CAP Health Check (HC) agreement of 2008 followed the direction established in 2003 by further decoupling direct payments, increasing the rate at which payments are modulated and al-

lowing Member States to switch from historical to regional flat area payment regimes. Despite the almost continuous reform process the pressure for further CAP reform has not abated, with the up-coming EU Budget review likely to lead to further agricultural policy change in the medium term.

Within the parameters of the 2003 Fischler re-form (and subsequent CAP Health Check agree-ment) EU Member States have some flexibility in the degree to which they must decouple direct pay-ments and in the choice of model used to implement the decoupled Single Payment (SP) scheme. The Old Member States (OMS) can implement the SP scheme by either granting historical support level to farmers or using a regional flat area payment di-rect income support scheme. OMS also have some flexibility in the degree to which the link between production and receipt of direct income support is retained. The New Member States (NMS) are still allowed to use the transition support system – Single Area Payment (SAP) scheme - one of the advantages of which is the flexibility to provide national top-ups to agriculture in coupled and de-coupled forms until 2013 in line with phasing-in of EU direct payments, while EU support within SAP scheme must be totally decoupled. Thus the accession of NMS in 2004 and 2007, when com-bined with the Fischler reforms of 2003, introduced a considerable degree of policy heterogeneity to the CAP by comparison to Agenda 2000 policy framework.

In line with the HC decisions, the diverse agri-cultural policy systems permissible under the CAP may be gradually equalized over the period 2010 to 2013 through the mandatory decoupling of direct payments that under the Fischler reforms could be retained by Member States as coupled direct pay-ments and by the voluntary switch from historical SP schemes to a regional SP scheme in OMS.

The effects of the decoupling of direct payments have been analysed for the arable crop sector using the Policy Evaluation Model (PEM) developed at OECD (2006). The implementation of increasingly diverse and complex policies in European agricul-ture argues for the linking of models from differ-ent disciplines that operate across different spatial and other analytic scales. The resulting model



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conglomerates have the advantage of being able to exploit the synergies of diverse methodological approaches and tools (Britz et al. 2008). However, Esposti (2008) in a review of recent model based economic analysis of the CAP concluded that the long-term perspective of CAP after the year 2013 at the EU27 level, in the dimensions being currently publicly discussed (Buckwell 2007; Swinnen 2008; Begg et al. 2008; ECORYS 2008), and even the current shape of CAP under its different schemes, is not completely and systematically covered in any model application, because the existing modelling approaches may still miss the correct representa-tion of some detail of the policy itself. These de-ficiencies are largely due to the heterogeneity and complexity of the current CAP system as well as the nature of the questions posed by the possible future development of the CAP.

Considering the analytic challenges faced, a unified methodological approach that allows for quantitative assessments of the impact of various elements of dynamic CAP direct support systems is required (Salputra et al. 2007). Such an analytical approach was developed as the Policy harmonized evaluation for the AGMEMOD agricultural par-tial equilibrium sector model1 that allows for the systematic examination of the impact of existing direct support policies as well as those proposed and expected in the future. Using the AGMEMOD model, the aim of this paper is to demonstrate the usefulness of the PH approach to the economic analysis of agricultural policy changes in the sec-tor modelling.

The AGMEMOD partial equilibrium sector model

The AGMEMOD model is a dynamic, multi-product partial equilibrium model. The modelling strategy used is to build an EU aggregate model by com-bining separate country models, where commodity market sub-models are the basic components in each country-level model. The commodity market sub-models endogenously determine supply and demand, international trade and prices. Each country model captures the behavioural response of economic agents (farmers/producers and consumers/users) to changes in prices, exogenous macroeconomic variables and policy instruments, as well as the response to the previous years’ outcome according to the dynamic structure of the model. Model’s parameters are mostly econometrically estimated, however, considering limited opportunity for econometric estimations due to data constraints in NMS, synthetic or calibrated parameters are applied as well. Using the parameters, exogenous data and lagged endogenous data, it is possible to generate projections for the model’s endogenous variables over a set of alternative policy scenarios, for a given projection period. The model is solved with endogenous prices balancing supply and use of each modelled commodity at both Member State and EU-27 levels. Price linkage equations are used to capture the relationships between market clearing prices in Member State and EU markets, and between the EU market and the Rest of the World market. Greater detail on the AGMEMOD modelling approach can be found in Erjavec et al. (2006), Chantreuil et al. (2005) and Bartova et al. (2008).

As in econometric approach the AGMEMOD model’s evaluation of policy change is based on the reaction of agri-food markets to other policy and market changes during the sample period over which the model’s parameters were estimated. When the original AGMEMOD model was devel-oped in FP5 project the main analytic focus was on the responses of agricultural supply and demand to changes in key European market prices and changes in the value of coupled direct payments.

1The AGMEMOD model was developed under the auspices of two FP projects the FP6 project “Agricultural Member States Modelling for the EU and Eastern European Countries (AGMEMOD 2020)” and the FP5 project “Agricultural Sec-tor in the EU Member States and Newly Associated States in Central and Eastern Europe (AG-MEMOD)”. See Chantreuil et al. (2005) and Salamon et.al. (2008) for further details.



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Beginning with the MacSharry reforms of 1992, the CAP evolved with a focus on production related direct support (payments per area and per animal head). Up until 2004, the modelling approach used to examine CAP support under “Agenda 2000” was in general also appropriate for the evaluation of policies in the NMS. In these countries pre-acces-sion support was mostly coupled to agricultural production, crop area or animals. Following the 2003 Fischler reform and the enlargement of the EU in 2004 direct income support to farmers was made available without an obligation to produce a specific volume of production. This change ne-cessitated some changes in how the effect of ag-ricultural policy on production was modelled and led to the development of the policy harmonized evaluation approach.

As the motivation for production, in a com-mon market such as the EU, depends not only on the direct support system applied by an individual country, but also on the support system applied by other countries, all of the different types of direct payments that were allowed under the CAP were included in the structure of the AGMEMOD model through the implementation of the policy harmo-nized approach. Under the PH approach these direct payments were recalculated in the form of policy add-ons to market prices increasing the margin between the producer price and input costs. CAP market support has an impact on EU prices affect-ing supply-demand balance and external trade. The modelling structure used in the AGMEMOD model is reflected in.

Price

Harvested area/ animal inventories

Yield/ per animal production

Imports

Beginning stocks

Production

Ending stocks

Feed use

Non feed use

Domestic uses

Exports

Policy add -on price

CAP Direct support CAP Marke t support

GDP, GDP deflator, Population, Exchange rate

Legend

Exogenous

Estimated

Derived

Closure

Input costs

Fig.1. General modelling structure of AGMEMOD model. Source: AGMEMOD Partnership.



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Policy harmonized evaluation approach

OECD studies have identified three channels through which government support policies affect produc-tion – market effects, risk effects, and dynamic effects, which can occur simultaneously and are cumulative (OECD 2006). The methodological basis for the grouping of direct support measures under different systems used in this paper is the two way classification of support applied by the OECD (2008) in the calculation of its Producer Support Estimates (PSE):

• Coupled support – payments based on output, on area planted or animal numbers where production is required;

• Decoupled support – payments based on non-current area and the number of ani-mals where production is required or not required (historical entitlements).

Decoupled payments as a concept are interpret-ed differently by economists and decision-makers. The theoretical assumption that decoupled pay-ments do not have production, market and redis-tributive effects has many advocates, but also many opponents (Conforti 2005). The assertion that de-coupled payments do not affect production should hold in the event of production neutrality and when markets are complete. However, Key and Roberts (2008) list numerous studies (USDA 2003; USDA 2004), which establish that in the event of incom-plete markets, decoupled payments can have pro-duction effects. These effects are, according to the literature cited by Key et al. (2008), insignificant on average, but could have important consequences for the structure of individual agricultural holdings. Decoupled payments affect production decisions in the following ways (Bhaskar et al. 2007): they affect the producers’ risks, i.e. they either decrease the level of risk non-benevolence (“wealth effects”) or they reduce the risk (“insurance effects”), allevi-ate the conditions for obtaining loans, affect the decisions in allocation of labour on agricultural holdings, change the value of land, rents and prices

of land, and affect production decisions in view of expected payments. Cumulatively these effects, together with the effects of remaining coupled pay-ments, could have important production effects.

In the implementation of the PH approach with-in the AGMEMOD model all direct payments are recalculated as a policy price add-on to the relevant producer price to form a reaction price. These pol-icy based price add-ons are used in the assessment of the impact of total budgetary support on agricul-tural production. The reaction price accounts for the effect of decoupled direct payments through the application of coefficients – the multipliers, which adjust the share of budgetary support in the reaction price. It is assumed that support related to a product or production factor associated with a particular product has a direct impact on production. Support granted to land, irrespective of the type of product produced, can also act as a stimulating factor. The magnitude of the multipliers applied to different types of decoupled subsidies depends on the nature of these support payments.

The value of the regional multiplier is set equal to 0.3 and the value of the historical multiplier is set equal to 0.5. They are assumed based on OECD (2006) results. The historical payment provides a greater production incentive than the regional pay-ment since the appropriate production technologies have already been established on farms. If the pay-ment is fully coupled to production then the multi-plier used is set equal to 1.

Reaction price, when deflated by input costs indices is the economic variable that drives the sup-ply decisions of farmers within the model’s struc-ture incorporating both the policy and market based signals to producers. Thus the supply response of farmers to decoupled payment is positive and changes in the value of decoupled payments will lead to responses by farmers that are analogous to but smaller than farmers’ responses to changes in agricultural output prices.The similar effects of de-coupled payments have been discovered by Gohin (2008), Rude (2008) and Balkhausen et.al (2007).

To make the AGMEMOD model capable of incorporating the switches in agricultural policy regimes, first, all applicable direct support meas-ures that form part of the CAP (under the 2003



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CAP reform and the more recent HC decisions) are implemented in the policy block through the envelopes which reflect the total amount of budg-etary resources allocated to the sector (see Fig. 2). The links between different policy measures in the model ensure that the evaluation of policy changes involving switches between policy schemes (his-torical to regional) and changes in the objects of policy, e.g. the switch from per animal direct pay-ments to per hectare supports are feasible. Second, in each country model equations were added that calculate the country specific reaction prices. Final-ly, the equations on the supply sides of each of the country level commodity sub-models, where the reaction prices should be used, were specified.

The first set of equations allocates, Member State by Member State, the Pillar I budget between different types of envelopes (coupled, historical and regional). This set of equations is formulated in the same way for all countries and is implemented at the level of the combined AGMEMOD model. The reaction prices for a commodity j are simulated as endogenous variables. The policy price add-on adjusts depending on assumptions made concern-ing exogenous policy input variables. These exoge-

nous policy variables include modulation, coupling rates and multipliers, as well as variables control-ling the allocation of budgetary envelopes between coupled payments, regional and historical payment schemes. The relationship between AGMEMOD policy variables and the various CAP instruments are set out in the following equations.

Effective national envelope of 1st Pillar direct payments envt is a part of National ceiling remain-ing after reduction by the effective modulation rate calculated according to the farm structure in each country:

envt = ENVt · (1– cmot – vmot) (1)

where ENVt is National ceiling defined by Regulations of European Commission, cmot is compulsory modulation rate and vmot is voluntary modulation rate.

The ceiling for total coupled payments enve-lope cptt is limited by premiums p for commodity j com p j , maximum coupling rates defined for each premium p and commodity j crt p tj ; and country spe-cific reference numbers for each premium p and commodity j ref p j .

1. ENV National ceiling

Regulation (EC) 73/2009

3 .HPT Historical payments

2 .CPT Coupled payments

Sector 1. ... Sector n

4 .RPT Regional payments

5 .A G T Arable regional payments

-

-

= -

= 6 .GRT Grassland regional payments

Res

ourc

es

Allo

catio

n C

oupl

ed

Dec

oupl

ed

MOD Effective modulation

rate (depending on country farm structure)

In case the re is no difference between arable and grassland regional payments

Bala

nce

Fig.2. Allocation of total direct support.



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8

The first set of equations allocates, Member State by Member State, the Pillar I 223

budget between different types of envelopes (coupled, historical and regional). This 224

set of equations is formulated in the same way for all countries and is implemented at 225

the level of the combined AGMEMOD model. The reaction prices for a commodity j 226

are simulated as endogenous variables. The policy price add-on adjusts depending on 227

assumptions made concerning exogenous policy input variables. These exogenous 228

policy variables include modulation, coupling rates and multipliers, as well as 229

variables controlling the allocation of budgetary envelopes between coupled 230

payments, regional and historical payment schemes. The relationship between 231

AGMEMOD policy variables and the various CAP instruments are set out in the 232

following equations. 233

Effective national envelope of 1st Pillar direct payments tenv is a part of National 234

ceiling remaining after reduction by the effective modulation rate calculated 235

according to the farm structure in each country: 236

)1( tttt vmocmoENVenv (1) 237

where tENV is National ceiling defined by Regulations of European Commission, 238

tcmo is compulsory modulation rate and tvmo is voluntary modulation rate. 239

The ceiling for total coupled payments envelope tcpt is limited by premiums p for 240

commodity j pjcom , maximum coupling rates defined for each premium p and 241

commodity j p

tjcrt ; and country specific reference numbers for each premium p and 242

commodity j p

jref .243

l

ljtt

pjj

pj

ptj

s

p

l

j

pj

pj

ptjt

vmocmoahrydrcomcrt

refcomcrtcpt

1

1 1

)]1()(

)([

244

;,...,1;,1;,1 splllj (2) 245

Reference numbers for premium p are defined for livestock products and by 246

reference areas and reference yields for crop products .247

llj ,1248

Decoupled historical payments thpt :249

(2)

Reference numbers for premium p are defined for livestock products and by reference areas and reference yields for crop products

Decoupled historical payments hptt:hptt = ENVt · hrtt · (1 – cmot – vmot) (3)

where hrtt is a share of historical payments in national ceiling chosen by each country.

Decoupled regional payments rptt : (4)

The second set of equations that are added in the implementation of the policy harmonization ap-proach within the AGMEMOD model is country specific and is implemented in the country models directly. These equations calculate the policy add-ons to be included in reaction prices which are then used as explanatory variable in supply side equa-tions of the country level AGMEMOD models. In following equations we present the specifications for arable crops and meat. Policy price add-on for arable crop j is modelled as

(5)

where cpm, hpm, rpm are multipliers of cou-pled, historical and regional payments defining the weight of impact of different type of direct pay-ments on production decisions. Policy add-on for meat i:

(6)

where utr is average livestock density.

Application of the methodology – policy scenarios results

The use of the PH approach in the AGMEMOD model allows us to define and analyse detailed policy scenarios that involve changes to CAP policy instruments such as modulation rates, coupled direct payments values, budgetary shares of regional and historical SP schemes.

Under the terms of the 2008 CAP Health Check agreement the degree of decoupling of direct pay-ments will be deepened with the full decoupling of the direct payments that, in some Member States, remained coupled under the 2003 Fischler reforms. The HC agreement also includes a provision for a voluntary move by OMS that currently apply the historical payment scheme to a regional flat area payment system. The policy environment set out in the HC with its incorporation of possible SP scheme regime switches, increases in modula-tion rates or redirection of certain amount of Pillar I funds for specific country rural, environmental and quality support; and decoupling of remaining coupled direct payments can be analysed using the AGMEMOD model with its PH component. In the broader sense with respect of rural devel-opment and review of other studies HC has been recently analysed by Nowicki et.al (2009). More radical reform scenarios than those agreed in the recent Health Check can also be analysed. Such scenario analysis illustrates the usefulness of the PH approach as well as allowing for an assessment of the effects of agreed and possible future policy changes on individual Member States.

To illustrate the capacity of the PH approach as implemented within the AGMEMOD model we specify Baseline and two alternative scenarios:• Baseline scenario – which involves the con-

tinuation of policy as agreed under the HC. Under the Baseline the mix of historic, regional, and dynamic hybrid direct aid schemes with coupled payments (where EU Member States have chosen them) will continue along with the mandatory elements of the Health Check decisions implemented through to the end of

8

The first set of equations allocates, Member State by Member State, the Pillar I 223

budget between different types of envelopes (coupled, historical and regional). This 224

set of equations is formulated in the same way for all countries and is implemented at 225

the level of the combined AGMEMOD model. The reaction prices for a commodity j 226

are simulated as endogenous variables. The policy price add-on adjusts depending on 227

assumptions made concerning exogenous policy input variables. These exogenous 228

policy variables include modulation, coupling rates and multipliers, as well as 229

variables controlling the allocation of budgetary envelopes between coupled 230

payments, regional and historical payment schemes. The relationship between 231

AGMEMOD policy variables and the various CAP instruments are set out in the 232

following equations. 233

Effective national envelope of 1st Pillar direct payments tenv is a part of National 234

ceiling remaining after reduction by the effective modulation rate calculated 235

according to the farm structure in each country: 236

)1( tttt vmocmoENVenv (1) 237

where tENV is National ceiling defined by Regulations of European Commission, 238

tcmo is compulsory modulation rate and tvmo is voluntary modulation rate. 239

The ceiling for total coupled payments envelope tcpt is limited by premiums p for 240

commodity j pjcom , maximum coupling rates defined for each premium p and 241

commodity j p

tjcrt ; and country specific reference numbers for each premium p and 242

commodity j p

jref .243

l

ljtt

pjj

pj

ptj

s

p

l

j

pj

pj

ptjt

vmocmoahrydrcomcrt

refcomcrtcpt

1

1 1

)]1()(

)([

244

;,...,1;,1;,1 splllj (2) 245

Reference numbers for premium p are defined for livestock products and by 246

reference areas and reference yields for crop products .247

llj ,1248

Decoupled historical payments thpt :249

9

)1( ttttt vmocmohrtENVhpt (3) 250

where thrt is a share of historical payments in national ceiling chosen by each 251

country.252

Decoupled regional payments trpt :253

thptcptenvrpt tttt , (4) 254

The second set of equations that are added in the implementation of the policy 255

harmonization approach within the AGMEMOD model is country specific and is 256

implemented in the country models directly. These equations calculate the policy add-257

ons to be included in reaction prices which are then used as explanatory variable in 258

supply side equations of the country level AGMEMOD models. In following 259

equations we present the specifications for arable crops and meat. Policy price add-on 260

for arable crop j is modelled as 261

jtttt

jttjtjtj

yieldahrptrpmhpthpm

ahahrcptcpmprc

,11

1

/)/)(

),max(/(

(5) 262

where rpmhpmcpm ,, are multipliers of coupled, historical and regional payments 263

defining the weight of impact of different type of direct payments on production 264

decisions. Policy add-on for meat i: 265

tjtttt

jttjtj

slwutrahrptrpmhpthpm

cctcptcpmprc

/)//)(

/(

11

,1

(6) 266

where utr is average livestock density. 267

Application of the methodology – policy scenarios results268

The use of the PH approach in the AGMEMOD model allows us to define and 269

analyse detailed policy scenarios that involve changes to CAP policy instruments such 270

as modulation rates, coupled direct payments values, budgetary shares of regional and 271

historical SP schemes. 272

Under the terms of the 2008 CAP Health Check agreement the degree of decoupling 273

of direct payments will be deepened with the full decoupling of the direct payments 274

that, in some Member States, remained coupled under the 2003 Fischler reforms. The 275

HC agreement also includes a provision for a voluntary move by OMS that currently 276

apply the historical payment scheme to a regional flat area payment system. The 277

policy environment set out in the HC with its incorporation of possible SP scheme 278

regime switches, increases in modulation rates or redirection of certain amount of 279

9



country.252










jtttt

jttjtjtj

yieldahrptrpmhpthpm

ahahrcptcpmprc

,11

1

/)/)(

),max(/(

(5) 262




tjtttt

jttjtj


cctcptcpmprc

/)//)(

/(

11

,1

(6) 266














9



country.252










jtttt

jttjtjtj

yieldahrptrpmhpthpm

ahahrcptcpmprc

,11

1

/)/)(

),max(/(

(5) 262




tjtttt

jttjtj


cctcptcpmprc

/)//)(

/(

11

,1

(6) 266
















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Vol. 20(2011): 119–130.

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the projection period in 2020. Rates of modu-lation are increased, milk quota and set aside are abolished, and direct supports related to production are to be fully decoupled with the exception of some beef and sheep payments. The additional funds raised through the increase in the rate of modulation are used to fund Pil-lar II measures and thus reduce the effective national envelopes. The CAP budget National ceilings remain at their current level.

• Regional Flat Rate (RFR) scenario – this sce-nario assumes that all Member States currently using a historical payment model move to a regional flat area payment model from 2010 onwards, with the transition to this regional payment model occurring through a series of three annual changes in the total value of exist-ing entitlements. The annual reductions in the total value of historical entitlements will be 25 percent in the first year and 50 percent of the rest of historical envelope in the second year. In the third year national flat area payments are assumed to be implemented, where these are defined as the National ceilings divided the total eligible area. In addition all coupled payments that remain under the HC agreement are assumed to be fully decoupled;

• European Union Flat Rate (EUFR) scenario – this scenario assumes that an EU wide flat regional rate payment will be introduced instead of the historical and various Member State specific regional payment models. The EU flat area payment rate per hectare is set at the level of the average per hectare entitlement in 2014 (the first year following the completion of the transition period for those Member States that acceded to the EU in 2004). This rate 247 EUR/ha is calculated as the sum of all Member States’ National Ceilings in 2014 divided by the sum of all Member States’ eligible areas according to the EUROSTAT data. All other agricultural policies are the same as those ap-plying under the RFR scenario, i.e. all coupled direct payments are fully decoupled.

In this paper analysis focuses on the results for a selection of Member States that represent the

range of different implementations of direct sup-port schemes within the EU-27: France – a Mem-ber State which retained the maximum number of coupled payments and applied the historical SP scheme; Finland – a Member State that retained some coupled payments but applied the hybrid dynamics SP scheme (where the payment scheme changes from the historical to the regional scheme by 2013); Ireland: a Member State that retained no coupled payments but applied the historical pay-ment scheme; Latvia – a Member State applying the SAP scheme with coupled Complementary Na-tional Direct Payments (CNDP). Only results for the beef sectors in the individual Member States are presented. Bureau et al. (2008) among others have identified the EU beef sector as particularly vulnerable to policy reform. This and the wide-spread prevalence of coupled direct payments that are linked to beef production under the current CAP motivates our analytic focus on this sector. Full details of the impact on other commodity mar-kets and other Member States impacts are available from the authors.

Table 1 presents the Baseline projections of beef producer prices, the value of the beef policy add-on, beef reaction price and projections for beef production in France, Finland, Ireland and Latvia. The policy add-on to beef prices incorporates all of the different coupled cattle payments and decou-pled historical and regional payments that affect cattle production in EU Member States as adjusted by the historical and regional payment multipliers.

For France existing coupled payments for suck-ler cows and cattle slaughter premiums as well as decoupled historical payments are incorporated in the supply inducing policy add-on. In Ireland only the decoupled historical payments contribute to the policy add-on given the absence of any coupled di-rect payments. In Finland coupled male bovine pre-miums and the dynamic decoupled payments, with a declining share of historical and increasing share of regional payments, are included. For Latvia the coupled CNDP for suckler cows and slaughter pre-miums, as well as the decoupled regional payments are incorporated in the supply inducing policy price add-on c.



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In 2008 the policy price add-on in each of the four countries adds up to 20% of the beef producer price to the reaction price. Under the Baseline the decrease in the value of the policy price add-on in Finland between 2008 and 2020 arises because of the shift from a historical to regional payment model that occurs under the Finnish hybrid SP model and the compulsory decoupling of special male premium agreed under the Health Check. In Latvia the decrease in the value of the policy add-on for beef is due to the phasing out of the coupled CNDP. These payments are totally phased out by 2012 in all of the member states that joined the EU in 2004. Given that under the Baseline no change occurs in the SP model used in Ireland and that all direct payments in Ireland were decoupled under the 2003 Fischler reform, as expected, the policy add-on does not change under the Baseline between 2008 and 2020. There are no changes also for France due to the reason that disappearance of coupled slaughter premiums is compensated by emerging availability of significant amounts of decoupled arable crops premiums. Producer price developments reflect the past tendencies and elas-ticities in connection with EU market, country self-sufficiency level and quality of beef.

Despite the projected increase in the Latvia beef price under the Baseline, the reduction in the policy price add-on and increases in the costs of produc-tion augmented by ongoing increases in dairy cow yields, leads to a decline in total Latvian beef pro-

duction. Comparing with other countries analysed, dairy herd in Latvia is of exceptional dominance as even increasing share of suckler cows in total number of cows in 2020 is projected to comprise only 11%. In Finland, where suckler cow beef pro-duction (as in Latvia) is of limited importance, the decline in the policy add-on when combined with increased costs and declining dairy cow numbers offsets the positive market price impact on total beef production. By 2020, under the Baseline, production is projection to decline by almost 15 percent compared with 2008. In Ireland, despite the constant nominal value of the policy add-on over the Baseline projection period, production of beef in Ireland declines. This arises due to project-ed decrease in cattle prices, declines in dairy cow numbers and increases in production costs over the projection period. In France, under the Baseline, the more or less constant policy add-on in combina-tion with increased beef producer prices leads to a small increase in production. This result illustrates the role the positive effect of the remaining cou-pled premiums in France on beef production. In addition French dairy cow numbers following the removal of milk quota do not decline by as much as in Ireland.

Scenario results presented in Table 2 show the effect of the analysed policy changes on beef pro-duction and prices. The main policy change under RFR scenario is the decoupling of all direct pay-ments and the switch from historical to regional

Table 1. Baseline scenario results for beef.

Beef producer price, EUR/100 kg

Policy add-on, EUR/100 kg

Beef reaction price, EUR/100kg

Beef production, 1000 t

(1) (2) (3)=(1)+(2)France 2008 160 29 189 1 462

2020 179 30 209 1 533Ireland 2008 173 24 197 554

2020 141 25 166 400Latvia 2008 155 22 177 25

2020 176 11 187 19Finland 2008 192 44 236 89

2020 190 17 207 76Source: AGMEMOD combined model, Version 4.0.



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schemes. The EUFR scenario assumes, in addition to the decoupling of all direct payments, that all country direct payment envelopes or national ceil-ings are re-distributed across the EU through the payment of an EU flat area payment. Under this scenario some countries will see total budgetary support decline while others will see total budget-ary support increase.

The switch from the historical to regional scheme and the decoupling of all direct payments has negative effects on French and Irish beef pro-duction. The magnitude of the negative impact on production in Ireland is smaller than in France. This is due to the fact that all direct payments in Ireland are decoupled under the Baseline, however, the negative impact appears because historical pay-ments granted for beef producers will be equally distributed between all eligible hectars. The strong reaction of France beef producers is the result of dynamic effect in the long term following to the shock of total decoupling of payments in 2014 rein-forced by switch to regional payments. The change to a regional flat area payment system is expected to have minor though positive effects on beef pro-duction in Latvia and Finland. These small positive impacts arise due to the increase in cattle prices that follows from the decline in total EU27 beef produc-tion. The negative production impact of the move to a regional payments model in France and Ireland does not arise in either Latvia or Finland since in both Member States regional payments models (the SAP scheme in the case of Latvia) are being used under the Baseline.

The introduction of an EU wide flat area pay-ment (under the EUFR scenario) does not signifi-

cantly change the level of the policy add-ons for France when compared with the outcome under the RFR scenario. This result is due to the fact that the French regional flat rate payment is very close to the average EU27 rate. The larger change in the Irish policy add-on occurs because the Irish re-gional flat area payment is larger than the average EU27 flat rate payment. The Finnish regional flat rate payment is marginally lower than the aver-age EU27 rate and consequently the policy price add-on component for beef increases and this leads to a small increase in beef production relative to the Baseline. In contrast the Latvian policy price add-on increases dramatically under the EUFR sce-nario. The Latvian regional flat rate payment is the lowest in the EU27 and the move to a EU27 flat rate payment increases the policy price add-on by almost 160 percent. This dramatic increase gives rise to a large increase in Latvian beef production. The small scale of Latvian beef production in an EU context ensures that the large change in produc-tion in Latvia (and other EU Member States) does not negatively affect either Latvian or EU27 cattle prices, which under the EUFR scenario increase relative to the Baseline.

Conclusions

The Fischler reforms of the CAP in 2003 and the CAP Health Check of 2008 together with the up-coming EU budget review will mean that analysis of the impact of changes in agricultural policy

Table 2. Regional flat rate (RFR) and EU wide flat rate (EUFR) scenario results for beef for 2020 in % changes.

2020, RFR via Baseline 2020, EUFR via Baseline

Beef producer price

Policy add-on Beef production

Beef producer price

Policy add-on Beef production

France 5.05 -61.50 -10.38 5.51 -66.34 -11.46Ireland 0.25 -40.76 -2.61 -0.53 -52.84 -3.86Latvia 5.59 -4.14 2.73 6.11 158.92 11.19Finland 0.10 -4.13 0.03 0.13 0.38 0.08

Source: AGMEMOD combined model, Version 4.0.



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at Member State and EU levels will continue to be demanded. Changes in the level of budgetary support to EU agriculture, changes in the method of distributing such budgetary support between farmers and amongst Member States, and changes in the distribution of support between Pillar I and Pillar II measures are all possible over the medium term. The policy harmonized approach presented in this paper allows for the systematic analysis of these and other agricultural policy issues at both the Member State and EU levels.

The usefulness of the PH approach has been illustrated through its implementation in the AG-MEMOD model and its use in analysing a series of EU agricultural policy reform scenarios. The scenarios examined involved the full decoupling of all direct payments and a movement to a regional payment model (the RFR scenario) and the full de-coupling of direct payments and the introduction of an EU wide flat area payment model (the EUFR scenario).

The complexity of these reforms scenarios in the European context arises from i) the differing nature of baseline or current agricultural policy en-vironments that exist in different EU member states under the Common Agricultural Policy and ii) from the fact that policy changes and associated market changes in one member state can affect the market environment in other Member States.

The results presented in this paper illustrate the capacity of the policy harmonized approach (as outlined in section 3) to both reflect the com-plexity of the implementation of existing EU ag-ricultural policy across different Member States and analyse changes in the EU agricultural policy environment.

The projections on the impact of both the Re-gional Flat rate scenario and the EU wide flat rate scenario underline the importance of the Baseline or status quo ante policy position of different mem-ber states in determining the impact of a common policy change. Because Latvia under the Baseline already has a regional flat area payment system, the Single Area Payment scheme, beef produc-tion in Latvia is only marginally affected by the move to a regional payment model. The projected growth in Latvian beef production occurs because

of the increase in market prices that results from the declines in total EU beef production under the RFR scenario. The projected decline in EU pro-duction is as a result of the negative impact of the RFR on production in other Member States such as France. Under the Baseline France retains coupled beef premiums and utilises a historical decoupled payments model, as a result production of beef is projected to decline under the RFR scenario.

The large increase in Latvian beef production under the EU wide flat area payment scenario con-trasts with the minor impact projected under the regional flat rate scenario. Budgetary support per hectare to Latvian agriculture under the Baseline is the lowest in the EU27 and the move to an EU wide flat rate dramatically increases the support provided to Latvian agriculture and leads to an increase in Latvian beef production. In contrast the move to an EU wide flat rate payment model under the EUFR scenario significantly reduces the budgetary support to Irish agriculture and leads to a reduction in Irish beef production.

The PH approach, by systematizing and har-monizing the management and use of policy data, ensures that projected differential policy impacts arising from changes in common EU policies reflect the likely actual differential impact as op-posed to differences in how “common” policies are implemented within analytical models. However, the importance of the policy harmonized approach presented in this paper extends beyond the assur-ance of such analytic coherence within a specific partial equilibrium policy modelling tool such as the AGMEMOD model and could be extended to other policy modelling contexts where economic models are used in conjunction with expert knowl-edge on policy issues.

Acknowledgement.This work was supported by EU FP6 research funding, contract SSPE-CT-2005-021543, by contribution from partner’s institutes throughout the EU and through associated projects for the Institute for Prospective and Technological Studies.The authors would like to acknowledge the work of the AGMEMOD members and affiliates in the development of the model used for this study.



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Vauhkonen, V. et al. The phytotoxicity and biodegradation of RSO and RME Vol. 20(2011): 131–142.

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© Agricultural and Food Science Manuscript received August 2010

The phytotoxic effects and biodegradability of stored rapeseed oil and rapeseed oil methyl ester

Ville Vauhkonen1*, Risto Lauhanen1, Sarita Ventelä1, Juhani Suojaranta1, Antti Pasila1, Toivo Kuokkanen2, Hanna Prokkola2 and Sanna Syväjärvi2

1Seinäjoki University of Applied Sciences, School of Agriculture and Forestry, FI-60800 Ilmajoki, Finland2University of Oulu, Department of Chemistry, PL 3000, FI-90014 Oulu, Finland

*Current address: UPM Research Center, FI-53200 Lappeenranta, Finland email: [email protected]

The aims of this study were to determine the phytotoxicity of stored rapeseed (Brassica rapa) oil (RSO) and rapeseed oil methyl ester (RME) after “spill like” contamination on the growth of barley (Hordeum vulgare) and the biodegradability of these substances in OECD 301F test conditions and in ground water. Rapeseed oil and rapeseed oil methyl ester were both stored for a period of time and their fuel characteristics (e.g. acid number) had changed from those set by the fuel standards and are considered to have an effect on its biodegradation. The phytotoxicity was tested using two different types of barley cultivars: ‘Saana’ and ‘Vilde’. The phytotoxic effect on the barley varieties was determined, after the growth season, by measuring the total biomass growth and the mass of 1000 kernels taken from the tests plots. Also visual inspection was used to determine what the effects on the barley growth were. These measurements suggest that both RSO and RME have a negative impact on barley sprouts and therefore the total growth of the barley. RSO and RME both decreased the total amount of harvested phytomass. The weight of 1000 kernels increased with low concentrations of these contaminants and high contamination levels reduced the mass of the kernels. The results of these experiments suggest that the stored rapeseed oil and rapeseed oil methyl ester are both phytotoxic materials and therefore will cause substantial loss of vegetation in the case of a fuel spill. The RSO and RME biodegraded effectively in the measurement period of 28 days under OECD test conditions: the degree of biodegradation being over 60%. The biodegradation in the ground water was much slower: the degree of biodegradation being about 10% after 28 days.

Key-words: barley, biodegradation, biodiesel, contamination, phytotoxicity, rapeseed oil

A G R I C U L T U R A L A N D F O O D S C I E N C E A G R I C U L T U R A L A N D F O O D S C I E N C E

Vauhkonen, V. et al. The phytotoxicity and biodegradation of RSO and RME

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Introduction

The use of diesel-like renewable fuels is increasing due to the decisions made by the European Union. The amount of renewable fuel used by the end of 2010 should have reached 5.75% of all transportation fuel used. The next goals are 10% by the year 2020 and 25% by the year 2030. These goals have been set to decrease the total CO2 emissions produced by vehicles.

These goals mean that the use of renewable fuels will also increase in agriculture. Agriculture will continue to use mainly diesel-like fuels in the near future. The renewable fuels used in agricul-tural diesel engines are at the moment vegetable oils, certain animal fats and biodiesels.

This study focuses on two main renewable fuel types used in Finnish agriculture; rapeseed oil and rapeseed oil methyl ester. Esters produced from various vegetable oils and animal fats are com-monly known as biodiesel (Demirbas and Kars-lioglu 2007). Biodiesel and vegetable oils are the only liquid transportation fuels that it is possible to produce on a farm scale. The production of other transportation fuels such as ethanol, pyrolysis oil and renewable diesel (Knothe 2009) need much larger facilities and more complex methods.

The use of neat oils and fats means that the diesel engine is run with a fuel that it hasn’t been designed to use. The vegetable oils and fats have a greater viscosity which can cause problems when using the engine. The transesterification process is done to improve the physical properties of the fuel to make them similar to those of crude oil based diesel fuel (Demirbas 2008). This process also changes the characteristics of the fuel with the result that the produced fuel can actually weaken the fuel lines and gaskets of the engine. These char-acteristics increase the risk of engine failure and the probability of a fuel leakage and therefore con-tamination of the soil or water systems. The fatty acid compositions are still similar and therefore the biodegradation rate and intermediate non-volatile biodegradation product levels are close despite the chemical alteration (Lapinskiené & Martinkus 2007).

This means that the effects on the ecosystems must be studied thoroughly so that in cases of fuel spillages adequate responses can be taken. The re-newable liquid fuels (biodiesel and bio-oils) are said to be biodegradable (Sendzikiene et al. 2007) but nevertheless these still have an impact on the ecosystem (plant growth etc.) if an oil or fuel spill happens (Lauhanen et al. 2000, Lapinskiene et al. 2006, Peterson and Möller 2004, Gong et al. 2008).

Biodiesel is reported to biodegrade more rap-idly and to be less toxic than normal crude oil based diesel fuels (Lapinskiené et al. 2006, Mariano et al. 2008, Zhang et al. 1998). In those reported tests the biodiesel has filled the EN 14214 standard. Also the biodegradation rate of vegetable oils is faster in comparison to mineral based oils (Aluyor et al. 2009, Kuokkanen et al. 2004). It has been shown that both mineral and bio-oil contamination in for-ests have a negative effect on wood growth; when the foliage has been contaminated (Lauhanen et al. 2000). Vegetable oil processing residues such as olive processing waste is also found to act as an herbicide to certain types of weeds (Boz et al. 2010).

In this work we used rapeseed oil and rape-seed methyl ester that were stored in storage tanks for over a year. Both of these were still usable in fuel production and as a fuel for tractors and combine harvesters. The RSO and RME studied didn’t have any detectable properties which would have led us to believe that they had gone rancid (appearance, smell, viscosity). However, the stor-age life for vegetable oils and biodiesels is lim-ited: because they all contain fatty acids that tend to oxidize (Vauhkonen et al. 2009). As a result of oxidation the chemical and physical properties, such as: acid value, viscosity and peroxide value start to change (Knothe 2007, Thompson et al. 1996). Oxidation also causes formation of formic acid and other organic acids (e.g. acetic acid) in the oils and biodiesels. This means that the toxicity of these substances changes when compared to fresh oil and biodiesel. These oxidation products might therefore change the biodegradation rates of these studied substances.

The main aims of the current study were to determine 1) the phytotoxicity of RSO and RME



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on barley growth when typical fuel-leakage type contamination takes place, 2) the biodegradability of stored RSO and RME in OECD 301F conditions and in ground water.

Material and methods

The RSO used in this study was cold pressed in Ilmajoki at Seinäjoki University of Applied Sciences, School of Agriculture and Forestry, using a screw press. The rapeseeds were harvested in the autumn of 2006 and the oil was pressed in the summer of 2007. The rapeseed oil was therefore two years old at the beginning of these experiments in 2009.

The RME used in this study was taken directly from its storage tank at the school of agriculture and forestry in Ilmajoki and was being used as a fuel for tractors during the spring and summer of 2009.

Rapeseed oil methyl ester was produced using a commercial biodiesel processor manufactured by Preseco Ltd, Espoo, Finland. The processor uses a transesterification process where the oil is allowed to react with an alkali based catalyst and methanol to remove glycerol from the fatty acid chains.

The fuel properties of RSO and RME were measured at the Biofuel Laboratory of Vaasa En-ergy Institute and at the Department of Chemistry, University of Oulu. These measurements were made to evaluate the quality of the liquids used in this study.

Test sites and experimental design

Two different test sites were chosen for this study. The test sites were cultivated with two different barley (Hordeum vulgare) cultivars during the summer of 2009. The “Koulutila” test site (62°43’N 22°31’E) was cultivated using spring barley cultivar Saana (two-rowed) and the “Rahkakorpi” test site (62°45’N 22°39’E) with Vilde (multi-rowed), also a spring barley cultivar.

The Koulutila soil was classified as very fine sand and Rahkakorpi as mould when the nutrient analyses of these test fields were carried out in the year 2007. The pH value and nutrient contents of the soils at the test sites are presented in Table 1. The amounts of minerals, besides potassium, were higher in the Rahkakorpi soil than in the Koulutila soil. Both of these sites were cultivated in 2008 and therefore the nutrient levels in these soils had changed from those determined in 2007.

Cultivation of test fields

The field where the Koulutila test site was located was harrowed using a disk harrow at the end of May 2009. Sowing was made two days after the harrowing. The field had been fertilized with dry manure in the autumn of 2008 (10 tonnes ha-1) and again in the spring of 2009 (18 tonnes ha-1). The last fertilization was done during sowing using 40.5 kg N ha-1 (Suomensalpietari, N-P-K: 27-0-1, Yara International ASA). The total fertilization of the test field was 79 kg N ha-1.

The Rahkakorpi test site was rotary harrowed in May 2009 and was fertilized with 60 kg N ha-1 (YaraMila Pellon Y1, N-P-K: 26-2-3, Yara Interna-tional ASA) at the same time as the sowing of the field took place. Pesticide treatment was made for both test sites using K-MCPA (active substance: MCPA, 1.00 l ha-1, BASF, Ludwigshafen, Ger-many) and Express 50T (active substance: Triben-uronmetyl, 7.50 g ha-1, E. I. du Pont Nemours & Co, (Inc.), USA) at the end of June.

Table 1. Results of the soil analyses from the year 2007.

Koulutila Rahkakorpi

pH 6.3 5.5

Calcium, mg l-1 1090 2600

Phosphorus, mg l-1 6.9 16.0

Potassium, mg l-1 125 88

Magnesium, mg l-1 135 310

Sulphur, mg l-1 17.0 21.0



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The total yields from the Koulutila and Rahka-korpi test fields were 2800 kg ha-1 and 3350 kg ha-1, respectively.

Founding the test sites

The test matrices were established 3 weeks after cultivation. The barley sprouts were approximately 10–15 centimeters long and the overall growth had been normal up to that point.

At both of the test sites the test matrices had 15 test plots (50 cm × 50 cm). The test matrices had three reference plots with zero contamination and three plots for each of the two contamination levels: 0.5 and 2.0 l m-2 for both contamination substances RSO and RME. The test plots for the different contamination levels were chosen at ran-dom within the matrix.

The RSO and RME were poured on the barley sprouts. This method was chosen to resemble an oil or fuel leakage from a tractor or combine harvester. However, the spreading of the contaminants was still made as evenly as possible on the test plots.

After the spreading there was a distinct film of RSO and RME on the leaves that had been in direct contact with the substances used. Most of the contaminants settled on the soil surface under the barley sprouts. At the Koulutila test site both of the contaminants left a puddle on the surface of the soil. At the Rahkakorpi test site both of the contaminants were absorbed into the soil.

Climate conditions during the study

The climatic condition (temperature and rainfall) was followed at two different locations (Saari’s Weather Station at Jouppila, Seinäjoki and at the School of Agriculture and Forestry) that were close to the test sites Rahkakorpi and Koulutila, respectively. The effective heat sum was 1090 °C during the growing season (109 days) for the test matrices. The total rainfall during this time was 110.8 mm at Jouppila weather station and 175 mm at the Koulutila test

site. The difference in the rainfalls according to the measurement data can be explained by harder local rainfall at Koulutila test site on 3 to 4 days during the total growing season.

Phytomass measurements

The influence of the contaminants with different contamination levels at different test sites were de-termined by measuring the total phytomass growth of the test plots. In these tests the measured values were total wet mass, dry mass and the weight of 1000 kernels that were harvested from each test plot. The effect of the contaminants was also determined using visual inspection (for withering).

The barley crop from the tests plots were har-vested and collected into sheaves at the start of September 2009. The weight of the sheaves (total wet mass) from the test plots was measured. The crop was then dried using a cold air dryer for a period of 3 weeks. After the sheaves were dried the dry masses of the sheaves were measured and the kernels were separated from the straw by hand and stored in air tight containers.

The harvested kernels were weighed at the Bio-fuel Laboratory of Vaasa Energy Institute in Vaasa. To determine the weight of 1000 kernels 50 kernels were counted from each test plot and weighed. Af-ter each measurement the kernels were mixed back into the kernels harvested from the same test plot. These measurements were repeated three times for each batch of kernels harvested from the test plots. The official measurement for the mass of 1000 kernels should be made by weighing a mass of 500 kernels. This was impossible to do due to the small test plots and therefore small kernel yield. The moisture contents of the kernels were deter-mined using the ISO 712 oven method (ISO 712).

Statistical analysis

Statistical analyses were performed by using the IBM SPSS Statistics 18.0 statistical software. The



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influence of the contamination level on growth was conducted by using one-way ANOVA for both con-taminants RSO and RME separately. The influence of the test site and contamination level was analyzed by using 2-way ANOVA where the contamination level and the test site were used as factors. This was done for both contaminants separately. The effect of the contamination level on the weight of 1000 kernels was also conducted using one-way ANOVA. The effect of the test place wasn’t evaluated due to the fact that the test sites had different types of barley cultivars.

Biodegradation tests

Biodegradation of RSO and RME were measured both in OECD 301F tests conditions (OECD 1992) and in the ground water provided by the water treat-ment plant Paavolan Vesi Ltd in Ruukki.

The biodegradation in OECD 301F conditions is used to measure the biodegradation in optimal conditions. The biodegradation in ground water was measured for ground water protection pur-poses to have information on how these substances behave if contaminants leak and transfer though the soil into the ground water.

The biodegradation tests were carried out us-ing the manometric respirometric BOD (Biological Oxygen Demand) Oxitop® method. This method is based on very accurate automatic pressure mea-surements in closed bottles under constant temper-ature (here 20.0 ± 0.2 °C). When organic matter degrades, it requires a certain amount of oxygen, Equation (1):

Corg + O2(g) → CO2(g) (1)CO2(g) + 2NaOH(s) → Na2CO3(s) + H2O(l) (2)

When oxygen is consumed from the gas phase the pressure falls and at the same time carbon dioxide gas is produced; but in this method the carbon dioxide is absorbed onto solid sodium hydroxide pellets and therefore it does not affect the measured pressure, Equation (2). The measurement time of 28 days was used in our biodegradation tests. The

measurement is fully automated and for measure-ments in solutions the instrument calculates the BOD value in the unit [mg L-1] using Equation (3)

(3)

where M (O2) is the molecular weight of oxygen (32000 mg mol-1), R is a gas constant (83.144 l hPa mol−1 K−1), Tm is the measuring temperature (K), T0 is 273.15 K, Vtot is the bottle volume (ml), Vl is the liquid phase volume (ml), α is a Bunsen absorption coefficient (0.03103) and Δp (O2) is the difference in partial oxygen pressure (hPa) as given by WTW. The degree of biodegradation of the substance in percentages can be calculated from the Equation (4):

(4)

where ThOD is the theoretical oxygen demand and is calculated here from the carbon content of the oil [mg mg-1]. BOD in Equation (4) is also in the unit [mg mg-1] and is calculated from the BOD [mg L-1] value by Equation (5):

(5)

where Vsample is the volume of sample [L] and msample is the mass of the sample [mg].

Results

Fuel specifications of RSO and RME Some physical properties of RSO and RME were measured to evaluate the quality of these liquids. The density, surface tension, water content and oxidation stability were measured at the Biofuel Laboratory of Vaasa Energy Institute. The higher heat value, viscosity and acid number were measured at the Department of Chemistry, University of Oulu. Measured values are presented in Table 2.

7

Corg + O2(g) → CO2(g) (1) 179

CO2(g) + 2NaOH(s) → Na2CO3(s) + H2O(l) (2) 180

181

When oxygen is consumed from the gas phase the pressure falls and at the same time carbon 182

dioxide gas is produced; but in this method the carbon dioxide is absorbed onto solid sodium hydroxide 183

pellets and therefore it does not affect the measured pressure, Equation (2). The measurement time of 184

28 days was used in our biodegradation tests. The measurement is fully automated and for 185

measurements in solutions the instrument calculates the BOD value in the unit [mg L-1] using Equation 186

(3) 187

188

��

� ��

�� (3) 189

190

where M (O2) is the molecular weight of oxygen (32000 mg mol-1), R is a gas constant (83.144 l hPa 191

mol−1 K−1), Tm is the measuring temperature (K), T0 is 273.15 K, Vtot is the bottle volume (ml), Vl is the 192

liquid phase volume (ml), α is a Bunsen absorption coefficient (0.03103) and Δp (O2) is the difference 193

in partial oxygen pressure (hPa) as given by WTW. The degree of biodegradation of the substance in 194

percentages can be calculated from the Equation (4): 195

196

��%� � �� % (4) 197

198

,where ThOD is the theoretical oxygen demand and is calculated here from the carbon content of the oil 199

[mg mg-1]. BOD in Equation (4) is also in the unit [mg mg-1] and is calculated from the BOD [mg L-1] 200

value by Equation (5): 201

202

��

(5) 203

204

where Vsample is the volume of sample [L] and msample is the mass of the sample [mg]. 205

Results 206

7

Corg + O2(g) → CO2(g) (1) 179


181






(3) 187

188

��

� ��

�� (3) 189

190






196

��%� � �� % (4) 197

198




202

��

(5) 203

204


Results 206

7

Corg + O2(g) → CO2(g) (1) 179


181






(3) 187

188

��

� ��

�� (3) 189

190






196

��%� � �� % (4) 197

198




202

��

(5) 203

204


Results 206



137136

Table 2. Physical and chemical properties and elemental analyses of the rapeseed oil (RSO) and rapeseed oil methyl es-ter (RME).

Property RSO RME Standard

Density at 20°C, kg m-3 918.0 880.0 ISO 649Surface tension at 20°C, dyn cm-1 32.9 31.4 ISO 6889Water content, mg kg-1 414.0 462.5 ISO 12937

Oxidation stability, h 1.97 1.64 EN 14112

Higher Heat Value, MJ kg-1 39.4 40.0 ASTM D240

Viscosity at 20°C, mm2 s-1 58.0 6.4 ASTM D2983

Acid Number, mg KOH g-1 5.45 0.86 EN 12634

Carbon, % 77.8 74.8 EN 14538

Hydrogen, % 12.1 11.6 EN 14538

Phosphorus, mg kg-1 16.0 0.1 EN 14538

Sodium, mg kg-1 0.8 0.5 EN 14538

Iron, mg kg-1 <0.1 0.3 EN 14538

Potassium, mg kg-1 13.5 0.2 EN 14538

Magnesium, mg kg-1 2.7 <0.1 EN 14538

Calcium, mg kg-1 35.4 0.4 EN 14538

Copper, mg kg-1 <0.1 <0.1 EN 14538Zinc, mg kg-1 0.2 0.1 EN 14538

Fig. 1. The withering effect of rapeseed oil (on left) and rapeseed methyl ester (on right) with contamination level of 2.0 liters per square meter at the Koulutila test site.

Effects on the vegetation

The barley sprouts showed signs of stress and started to wither straight away at the beginning of

the experiment. After one week the barley vegetation inside the test plots with the RME contamination started to wither from where the RME had been in direct contact with the leaves.



137136

This effect was not detected at the same time on test plots with RSO contamination. However, later on the effect of the RSO increased and the sprouts in the test plots with RSO contamination started to wither rapidly (Fig. 1).

When the harvesting of the barley vegetation from the test plots was started, the vegetation with the highest contamination level of both contami-nants was almost totally withered.

Three months after the actual test ended there were noticeable differences in the test matrices. The test plots with the largest concentration of contaminants RSO and RME, had only a small amount of undergrowth compared with those with zero contamination. The soil surface inside the plots which had had 2.0 liters of RSO contamina-tion had also turned dark (Fig. 2).

Effects on phytomass

The contamination with both RSO and RME had phytotoxic effect on the barley growth at both test sites. Both RME and RSO caused significant losses of vegetation by the end of the test period (Table 3 and Fig. 3).

The RSO contamination at the Koulutila test site reduced (F = 13.13, p = 0.01) the harvested wet mass by 29% with a contamination level of

Fig. 2. The effect of rapeseed oil (RSO) in the Koulutila test matrix 3 months after the test ended.

Table 3. The relative reduction of phytomasses caused by the contaminants rapeseed oil (RSO) and rapeseed oil methyl ester (RME).

Rahkakorpi Koulutila

Contamination level RSO RME RSO RME

0.0 l m-2 0% 0% 0% 0%

0.5 l m-2 -29% -41% -41% -46%

2.0 l m-2 -84% -55% -77% -71%

Fig. 3. The measured phytomasses (±SD) of barley crops with different contamination levels of rapeseed oil (RSO) and rapeseed oil methyl ester (RME) at the Koulutila and Rahkakorpi test sites.

0

100

200

300

400

500

600

700

RSO (Koulutila)

RME(Koulutila)

RSO(Rahkakorpi)

RME(Rahkakorpi)

Wet mass, grams

0.0, l m⁻² 0.5, l m⁻² 2.0, l m⁻²

0.5 liters per square meter and by 84% with a con-tamination level of 2.0 liters per square meter. At the same test site the RME reduced (F = 18.68, p < 0.001) the recovered wet mass by 41% with a contamination level of 0.5 liters per square meter and by 55% with a contamination level of 2.0 liters per square meter.

RSO contamination levels of 0.5 and 2.0 liters per square meter decreased (F = 24.67, p < 0.001) the amount of harvested wet masses at the Rahka-korpi test site by 41% and 77%, respectively. The wet mass reduction (F = 167.51, p < 0.001) in the test squares, with RME as a contaminant, was 46%



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with 0.5 liters per square meter and 71% with 2.0 liters per square meter.

There was considerable variations between some of the test plots (RSO 0.5 liters per square meter at Koulutila and RSO 0.5 and 2.0 liters per square meter at Rahkakorpi). This might be the re-sult of the uneven spread of the contaminant.

The mass reduction was similar at both of the test sites despite different cultivars and the fact that the Koulutila test site was partly contaminated with oat growth. The oat was transferred to the test site along with the dry manure.

The cold air drying reduced the total mass of the sheaf’s by 34.80 ± 1.06% (mean value ± SD) and because of the consistent reduction of mass, after the drying, the effects on the phytomass was only evaluated using harvested wet masses. The variance analyses using dry masses showed simi-lar dependency between contamination levels vs. harvested dry masses.

The weight of 1000 kernels

The measured kernel masses from the plots with-out contamination were higher compared to those reported with the Saana and Vilde cultivars; 46.1 g and 41.9 g, respectively (Kangas, et al. 2009). The moisture content of the kernels was determined for kernels harvested from the test plots. The barley harvested from the Koulutila test site had a moisture content of 9.2 ± 0.2% and at Rahkakorpi it was 10.4 ± 0.2% (mean value ± mean error).

The weight of 1000 kernels was also influenced by both of the contaminants (see Table 4). The

changes in the kernel masses were influenced by the contamination level introduced to the test plots. The RSO (F = 13.59, p < 0.001) and RME (F = 10.87, p < 0.001) showed a small increase in kernel masses with the 0.5 liter contamination level at the Rahkakorpi test site and the 2.0 liter contamination level decreased the kernel masses.

The effects of RSO at the Koulutila test site was similar to other measurements taken but they were still insignificant (F = 0.47, p = 0.63). RME contamination at the Koulutila test site slightly in-creased (F = 3.69, p = 0.04) the kernel masses with contamination level of 0.5 liters per square meter and the 2.0 liter contamination level decreased the kernel masses.

Biodegradation

The biodegradations of RSO and RME in OECD 301F conditions are presented in Fig. 4. The bio-degradation levels (BOD/ThOD) after a time period of 28 days for RSO and RME, were 65.8% and 60.6%, respectively. The small difference between the biodegradation levels can be explained by the differences in the chemical compositions of RSO and RME, and therefore their different biodegrad-ing components. The studied liquids had been aged which can be seen with the increase in their acid value. The differences in chemical compositions mean differences in the oxidation components in the RSO and RME which can inhibit the biodegrada-tion of these substances. The RME should actually biodegrade more rapidly than the RSO because the RSO was more stable in the oxidation stability test.

Table 4. The effect of rapeseed oil (RSO) and rapeseed oil methyl ester (RM) to the measured dry masses (g) of 1000 kernels harvested from the test plots. Data are mean ± SD

Rahkakorpi Koulutila

Contamination level RSO RME RSO RME

0.0 l m-2 45.1±1.6 45.1±1.6 48.0±2.0 48.0±2.0

0.5 l m-2 47.1±1.5*** 45.3±2.3*** 48.7±3.4ns 49.9±1.7**

2.0 l m-2 40.1±4.6*** 41.6±1.6*** 47.4±1.4ns 47.9±1.5**Statistically significant differences are indicated as follows: ns = not significant, *, p<0.05; **, p<0.01; ***, p<0.001



139138

The biodegradation results determined in the ground water differed from those measured in OECD 301F optimal conditions (Fig. 5). RSO and RME both started to degrade in an almost similar way when compared to each other. The biodegra-dation level after the 28 day period was 9.9% for RSO and 10.0% for RME. Both substances still started to degrade during this measurement period and therefore both are fairly biodegradable also in the ground water.

Discussion

Fuel properties

The physical parameters measured for RSO and RME are similar to the ones measured previously in our own studies (Niemi et al. 2009). The only properties that could indicate that these substances might have been stored for a period of time are the oxidation stability and acid number which doesn’t meet the limits set by the EN 14112 standard (6 h and less than 0.5 mg KOH g-1, respectively).

Oxidation stability doesn’t have a straight cor-relation to the time that the RME is stored. The reasons for these changes in oxidation stability are related to: the fatty acid composition of the oil or biodiesel, the concentration of certain metals (e.g. Cu and Fe) and water content (Vauhkonen et al. 2009). The oxidation stability doesn’t therefore have a straight correlation to the age and/or the quality of the vegetable oil or biodiesel as a fuel.

As a result of oxidation, formic and other or-ganic acids are formed in vegetable oils and biodie-sel. These substances increase the acid value. The RSO and RME investigated had high acid values 5.45 and 0.86 mg KOH g-1, respectively. This in-dicates that both RSO and RME have been partly oxidized.

The RSO had large amounts of phosphorus (16.0 mg kg-1), potassium (13.5 mg kg-1), magne-sium (2.7 mg kg-1) and calcium (35.4 mg kg-1) com-pared to those of RME. This might be a result of a high pressing temperature and a lack of adequate filtering of the oil.

Phosphorus is normally fixed in the plant fib-ers and a high pressing temperature can transfer phosphorus into the pressed oil. If the rapeseed oil has a large phosphorus content it is dangerous to the engine because it is abrasive. Large amounts of phosphorus can also cause blocking of the fuel filters and deposits on valves and pistons (Thuneke 2006). Studies have also shown that phosphorus can have a poisoning effect to the catalyst materials in exhaust gas catalyzers (Kröger 2007, Rokosz et al. 2001). Magnesium and calcium can cause po-lymerization, similar to making soap with rapeseed

Fig. 4. The biodegradation of rapeseed oil (RSO) and rapeseed oil methyl ester (RME) in OECD 301F conditions.

Fig. 5. The biodegradation of rapeseed oil (RSO) and rapeseed oil methyl ester (RME) in ground water.

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Biodegradation level, %

Time (d)

RSO (OECD) RME (OECD)

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Biodegradation level, %

Time (d)

RSO (Ground water) RME (Ground water)



141140

oil (Baquero et al. 2010). High concentrations of these elements have also been related to the ash content of the oils and biodiesels in our previous measurements. Kröger (2007) has concluded that calcium also can decrease the catalytic activity of components used in automotive catalysts. The RSO used in these measurements therefore could not be used in diesel engines, as such.

The alkali metals sodium and potassium are both measurable values in the EN 14214 standard for biodiesels. For RME these values are under the maximum limit (less than 5.0 mg kg-1) set for biodiesels. The concentrations of hazardous heavy metals copper and zinc were also measured in this work. The concentrations of these metals in both RSO and RME were lower than 0.2 mg kg-1, and therefore these metals shouldn’t cause problems in the soil or to the vegetation.

The concentration of iron in RME was 0.3 mg kg-1 which is high enough concentration to have a negative impact to the oxidation stability. Sarin et al. (2009) reported that an iron concentration of 0.5 mg kg-1 decreased the oxidation stability of Jatropha oil methyl ester by an hour.

Phytotoxicity tests

The results of this study indicate that both RSO and RME have a negative impact on barley growth. The phytomass growth (wet mass) was highly influenced by the amount of contaminant introduced to the test sites. Despite the contaminants not being spread evenly on the sprouts some of the test plots showed almost total loss of vegetation.

Similar types of contamination measurements have been earlier carried out with pine seedlings (Lauhanen et al. 2000, Flykt et al. 2003) and in these studies there have been noticeable disorders in the plant’s physiology. In our study we used barley sprouts that are much more sensitive to contaminants than pine seedlings due to the larger respiration surfaces of the barley sprouts compared to those of pine seedlings.

Gong et al. (2008) found that sunflower oil had an inhibitory effect on Brassica rapa L. bio-

mass growth, the effect increased with increasing amounts of sunflower oil. Similar effects, such as reduction of biomass, was also shown in this work with both of the studied contaminants RSO and RME. Boz et al. (2010) showed that the olive processing residues can also inhibit the growth of certain weeds.

The contaminants left a noticeable film on the barley sprouts. The rapeseed oil and also rapeseed oil methyl ester, both being in contact with air, start to form polymers as a result of oxidation. This means that contaminants in the case of polymeriza-tion formation will suffocate the plant. Therefore stored rapeseed oil and rapeseed oil methyl esters can be said to act as herbicides.

The dry weight of kernels was also influenced by both of the contaminants. Small contamination levels showed a small increase in kernel masses and higher contamination levels reduced the ker-nel masses. There could be several reasons for this effect. The studies with pine seedlings (Flykt et al. 2003) showed that the bio-oil contamination increased the amount of dissolving nitrogen in the soil at low oil contamination levels. This might partly explain the small increase of the measured kernel masses with small contamination levels in this study. The reduction of kernel masses might be explained by the uneven spread of the rapeseed oil and rapeseed oil methyl ester. The leaves that were contaminated by the substances used withered and therefore the natural growth of the barley was disturbed. The contaminants were absorbed into the soil more efficiently at the Rahkakorpi test site because the soil type was more porous than the soil at the Koulutila site. This means that the contami-nants might have reached the root zone and dam-aged the root system. This could explain the reduc-tion of kernel masses at the Rahkakorpi test site. Both the rapeseed oil and rapeseed oil methyl ester will also change the pH level of the soil and there-fore also have an effect on the micro-organisms and their interrelationships (Lapinskiene et al. 2006). The contaminants used might have also therefore affected the normal metabolism of the microbes in the soil. Microbes have been reported to be able to consume small amounts of bio-oils and the amount of microbes has increased and larger contamination



141140

levels have decreased the amount of microbes in the soil (Flykt et al. 2003). Al-Darbi et al. (2005) showed that the different types of oils had different kind of effect to the amount of microbes in seawa-ter and waste water contaminations.

Biodegradability tests

The studied rapeseed oil and rapeseed oil methyl ester were effectively biodegradable in OECD 301F test conditions but also at a slower rate in ground water. The large difference in biodegrada-tion levels between these two conditions is caused by the fact that the OECD 301F condition is the optimal condition for biodegradation and the ground water test is closer to the natural environment for biodegradation. The biodegradation rates of RSO and RME measured in these test vary from those reported in various studies in both; optimal and also in aquatic conditions (Peterson and Möller 2004, Zhang et al. 1997). The reason for this might be that the substances used in this study had been stored over a period of time and the fuel charac-teristics had changed. The acid values were higher due to the increased amounts of organic acids (e.g. formic acid) in these fuels as a result of oxidation. The organic acids can also be formed as a result of microbial activity (Schricher et al. 2009). It has also been suggested that the viscosity, structure and compostion of the oils and fats have an effect on the biodegradation rates and also oxidation products can either accelerate or inhibit the biodegradation (Al-Darbi et al. 2005). Lapinskiené and Martinkus (2007) studied the intermediate biodegradation products of biodiesel, sunflower oil, diesel and beef grease. These measurements showed that the biodegradation products that are formed consist of acetic acids and propionic acids that both are antibacterial substances. There is also evidence that the autoxidation as a part of the deterioration process can accelerate the biodegradation process by producing smaller and easier compounds to be biodegraded or inhibits it by producing antibacterial products (e.g. formic acid) (Al-Darbi et al. 2005). Therefore it can be concluded that the oxidation

products could have affected the biodegradation tests in this study.

Conclusions

This study concentrated on phytotoxic effects of stored rapeseed oil and rapeseed oil methyl ester and the biodegradability of these substances in OECD 301F conditions and ground water. Some of the fuel properties (acid value and oxidation stability) had been affected by the storing and did not meet the values set by the standards. The results indicate that the stored rapeseed oil and rapeseed oil methyl ester used in this study both had a phytotoxic effect when they were in direct contact with barley sprouts. Both substances biodegraded in the laboratory tests and therefore it can be concluded that, in small spill-like contaminations, they should not cause long term environmental problems to the soil or ground water. There were also indications that the seed masses could be affected by the contamination.

Acknowledgements. The authors are grateful to the Oiva Kuusisto Foundation for the financial support of this study. The staff of Biofuel Laboratory of Vaasa Energy Institute are thanked for making part of the measurements for this study.

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Sendzikiene, E., Makareviciene, V., Janulis, P. & Makarev-iciute D. 2007. Biodegradability of biodiesel fuel of ani-mal and vegetable origin. European Journal of Lipid Sci-ence and Technology 109: 493-497

Scheicher, T., Werkmeister, R., Russ, W. & Meyer-Pittroff, R. 2009. Microbial stability of biodiesel-diesel-mixtures. Bioresource Technology 100: 724-730

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© Agricultural and Food Science Manuscript received August 2010

Comparison of different DNA extraction methods from hair root follicles to genotype Finnish Landrace

boars with the Illumina PorcineSNP60 BeadChipAnu Sironen*, Pekka Uimari, Johanna Vilkki

MTT Agrifood Research Finland, Biotechnology and Food Research, FI-36100 Jokioinen, Finland

*email: [email protected]

Recent developments in sequencing methods have enabled whole genome sequencing of several species and the available sequence information has allowed the development of high throughput genotyping chips. However, these genotyping methods require high quality DNA. The possibility to genotype samples based on DNA from non-invasive sources would permit retrospective genotyping of previously collected samples and also facilitate the analysis of large populations e.g. for genomic selection. In this study we have developed and evaluated different DNA preparation methods from porcine hair root follicles for high throughput geno-typing with the PorcineSNP60 Genotyping BeadChip (Illumina). We describe a method for DNA extraction from porcine hair root samples, which produces results from high throughput genotyping with the same high degree of accuracy as previously reported for DNA extracted from sperm, blood or tissue samples. This method was used for the genotyping of 273 hair follicle samples. When the DNA concentration was > 30 ng/µl all samples had the same high call rate ( > 99%) as sperm samples confirming the robustness of this DNA extraction method for high throughput genotyping. Our data also establishes the suitability of the PorcineSNP60 BeadChip for genotyping the Finnish Landrace population.

Key-words: DNA extraction, Finnish Landrace, genotyping, hair follicles, pig, SNP


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Introduction

High throughput single-nucleotide polymorphism (SNP) genotyping methods are becoming increas-ingly important in population (Decker et al. 2009, Vonholdt et al. 2010) and association studies (Goddard and Hayes 2009) and also for genomic selection (Meuwissen et al. 2001). High throughput genotyping is now feasible due to the large number of SNP discovered by genome sequencing of vari-ous species and the development of new methods to efficiently genotype a large numbers of SNPs (Hayes et al. 2009). Genomic selection is based on a large reference population, in which animals are both phenotyped and genotyped. Previously collected samples from a reference population with reliable breeding values are required to predict genomic breeding values (GEBV) in subsequent generations. Similarly, existing samples can be used for the analysis of population diversity and identi-fication of disease associated genomic regions by high throughput genotyping. DNA extracted from sperm, blood or tissue samples can be used for high quality genotyping using chip technology (Li et al. 2008, Jiang et al. 2010), but invasive procedures are required to collect such samples causing un-necessary pain and distress for sampled animals. Furthermore, these samples are also expensive and time consuming to collect.

The hair root is known to contain DNA and therefore represents a non-invasive source of DNA. The collection, transportation and storage of hair samples do not require any special procedures and as a result offers a painless and inexpensive alternative to the sampling of other tissues. Sev-eral methods have been described for polymerase chain reaction (PCR) amplification of single mark-ers from hair samples (Amendola-Pimenta et al. 2009, Hayashida et al. 2009, Sironen et al. 2010). However, the low quality of DNA obtained has pre-vented the use of these samples for high throughput genotyping. In this study, we have assessed several possible methods for DNA preparation from por-cine (Sus scrofa) hair roots for genotyping with the chip technology. Based on our analysis we have developed a reliable method for the preparation of

DNA from the hair root for genotyping with the PorcineSNP60 Genotyping BeadChip (Illumina).

Material and Methods

DNA samplesGenomic DNA was prepared from porcine (Finn-ish Landrace) hair roots collected during years 2000–2006 (samples of 273 boars were used for this study) and stored at room temperature. Control DNA was extracted from sperm samples using a phenol/chloroform extraction protocol. For the assessment of various DNA preparation methods replicate of samples (n > 4) including the hair follicle of 5–15 hairs were used:

1. The hair roots were lysed in lysis buffer [proteinase K 0.5 mg/ml and 2 µl Mg-free PCR Buffer (Dynazyme DNA polymerase, Finnzymes) in dH2O] at 55 ºC for 60 min following Proteinase K inactivation at 98 ºC for 10 min.

2. DNA purification from the lysed samples (first preparation method) by ethanol (EtOH) pre-cipitation with 100% ethanol and sodium acetate (0.3M, pH 5.2) at -20 °C for 30 min following cen-trifugation at 13000 rpm for 10 min. Any residual salt was washed with 70 % ethanol and centrifuged at 13000rpm for 10min. Precipitated DNA was air dried and dissolved in 20 µl of distilled H2O.

3. DNA extraction from the lysed samples (first preparation method) by phenol/chloroform.

4. DNA purification from the lysed samples (first preparation method) by InstaGene matrix (BioRad) following the protocol supplied by the manufacture.

5. Hair roots were lysed in ATL lysis buffer (Qiagen) with proteinase K (10 mg/ml) at 55 ºC for 60 min following Proteinase K inactivation at 98 ºC for 10 min. Thereafter the DNA was purified with ethanol precipitation.

6. Hair roots were lysed in lysis buffer (10mM Tris HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% SDS) with proteinase K (10 mg/ml) at 55 ºC for 60 min following Proteinase K inactivation at



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98 ºC for 10 min. Thereafter, DNA was purified by precipitation with ethanol.

7. Extraction of DNA with DNeasy Blood & Tissue Kit (Qiagen) according to the instructions supplied by the manufacturer.

For the lysis protocols (1, 5–6) the effect of ad-dition of MgCl2 (2mM) and DTT (100µM) and dis-solution of the hair root by mechanical force (with a rod or FastPrep homogenization system) were also tested. The concentration of extracted DNA was measured with a Nanodrop spectrophotometer (NanoDrop Technologies) and a Qubit Quantitation Platform (Invitrogen). Nanodrop was also used for analysing the purity of extracted DNA. Absorbance at 260 nm quantified the amount of DNA and pro-tein contamination was detected at 280 nm. The ratio of absorbance at A260/280 and A230/260 was used to determine the purity of DNA samples. The Qubit platform uses fluorescence-based Quant-iT™ assays, where fluorescence label binds specifi-cally to DNA. Although the specificity to dsDNA increases the accuracy of the DNA concentration, this detection system does not give any information about the purity of the sample. The accuracy of these concentration measurements were also con-firmed by quantitative PCR (qPCR).

The qPCR was performed with an ABI 7000 Sequence Detection System in 96-well microtiter plates using Absolute qPCR SYBR Green ROX Mix (VWR). DNA was amplified using primers for the microsatellite marker SW2411 (Forward CCT-GGACTCATTCTTGCTTTG, reverse TTCCTAT-TCTGTCCTGCCTTG). Amplification by qPCR contained 12.5 μl of Absolute qPCR SYBR Green Mix, 20 ng of DNA, and 70 nM of each primer in a final volume of 25 μl. Amplifications were initiated with 15 min enzyme activation at 95 °C followed by 40 cycles of denaturation at 95 °C for 15 s, prim-er annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. All samples were amplified in duplicate and a water sample was used as a negative control. A standard curve was produced by serial dilutions of DNA extracted from boar sperm (quantified with Nanodrop spectrophotometer). Quantities of DNA in the sample were estimated from the standard curve. Raw data were analyzed with the sequence detection software (Applied Biosystems).

Genotyping

For high throughput genotyping selected samples were analyzed on the PorcineSNP60 Genotyping BeadChip (Illumina Ltd) in the Institute for Molecu-lar Medicine Finland (FIMM). The PorcineSNP60 BeadChip has recently been developed as an out-come from the porcine whole genome sequencing project (Ramos et al. 2009). The concentration of samples analyzed by the BeadChip was estimated by Qubit measurements and the purity was confirmed by Nanodrop. The concentration varied between 2–100 ng/µl.

DNA samples of three boars with three differ-ent DNA preparation methods (1, 2 and 7) from hair roots and a reference sample extracted from sperm were selected for genotyping with the Porci-neSNP60 BeadChip. Furthermore, a duplicate sam-ple of DNA from boar sperm was included on the chip in order to compare the differences amongst identical sperm samples and variation between DNA preparation methods and sperm samples. Based on these analyses the most reliable DNA extraction method for hair samples was selected and an additional 280 samples were analyzed with the PorcineSNP60 BeadChip.


The effect of DNA concentration and genotyping batch on call rate was evaluated by multivariate ANOVA-method (SAS Enterprise Guide 4.3, SAS Institute Inc.).

Results and discussion

Evaluation of DNA preparation methodsA wide range of DNA preparation methods were tested including a basic lysis protocol with different lysis buffers. The hair root lysis has been success-fully implemented in single marker analysis (Sironen



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et al. 2010), however the purity is extremely low in these samples. No clear differences in DNA yield or purity were detected between lysis buffers (methods 2, 5 and 6) or following modification of the lysis protocol (addition of DTT/MgCl2 or dissolution, Table 1). The most consistent results were produced using 15 hair roots, even though 10 or even 5 hair roots yielded adequate amounts of DNA depend-ing on the sample (data not shown). Therefore the lysis buffer (method 1) used in our previous studies (Sironen et al. 2010) with 15 hair roots was selected for further purification.

DNA extraction with the InstaGene matrix (method 4) and by chloroform/phenol (method 3) resulted in low DNA concentrations (Table 1). Ethanol precipitation (method 2) and a commercial extraction kit (method 7) yielded relatively high amounts of good quality DNA (Table 1). Thus, for high throughput genotyping, DNA extracted from samples using methods 2 and 7 were selected. In addition, samples prepared by lysis (method 1) were also genotyped, since the concentration of DNA was assumed to be highest in these samples although the purity was low and interfered with

the determination of DNA concentration by Qubit and Nanodrop.

Comparison of different concentration assays

The concentration of DNA in various preparations was evaluated with the Nanodrop spectrophotometer and Qubit platform. Concentrations of DNA were found to be approximately 10 times higher based on the Nanodrop than Qubit analysis (Table 1). Therefore, the determination of DNA concentration was evaluated further by qPCR and a standard curve prepared from DNA extracted from boar sperm (quantified with Nanodrop spectrophotometer). The same amount (20 ng) of DNA based on each concentration assay was added to the qPCR am-plification and the concentration was compared to the standard curve. Qubit measurements appeared to be consistent with the results from qPCR and were therefore used to evaluate the concentration of DNA for chip genotyping.

Table 1. Protocols tested for the preparation of total DNA from porcine hair roots. Results from two different concen-tration measurement methods are presented. Sample purity (260/280 and 260/230 ratios) was assessed by Nanodrop.

Protocol (n) Nanodrop ng/µl (mean, SD)

260/280 ratio 260/230 ratio Qubit ng/µl

(mean, SD)

1. Lysis (10) 58–500 (202, 157) <1 <0.5 0.9–29 (12, 11.7)

2.Lysis+EtOH precipitation (36) 15–500 (168, 137) 1.8–2 0.9–1.5 1.5–100 (21, 17)

3.Lysis+phenol/chloroform (3) 22–47 (36, 12.5) 1.8–1.9 2 –

4. Lysis+BioRad matrix (5) 35–83 (58, 24) 1.3–1.7 0.8 0.5–3 (1.8, 1.2)

5. ATL Qiagen lysis (5) 2.5–21.7 (11, 8) <1 <0.6 0.2–0.5 (0.35, 0.12)

6. SDS+EDTA lysis (5) 2–22 (12, 8.5) <1 <0.5 0.14–0.5 (0.3, 0.15)

7. Qiagen kit extraction (28) 32–450 (144, 139) 1.8–2 0.5–1.8 2–100 (38, 26)

n = number of samples



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SNP quality measures of the Porcine-SNP60 Genotyping BeadChip

The total number of SNPs in the PorcineSNP60 BeadChip is 62163. However, 2815 SNPs did not work for any of the genotyped samples and 9216 SNPs were monomorphic in the data set of Finnish Landrace pigs. Excluding those SNPs, the average minor allele frequency was 0.25 (SD = 0.14). Fur-thermore, the observed distribution of P-values of the Hardy-Weinberg equilibrium test statistic did not differ from expectations; 183 SNPs (exclud-ing the SNPs on the X-chromosome) had p-values lower than 1.0E-06 which is much less than could be expected by chance. Thus, the PorcineSNP60 BeadChip appears to be a robust and reliable method for genotyping of Finnish Landrace pigs.

Quality of the PorcineSNP60 BeadChip genotyping results with hair root sam-

plesThe comparison of the genotyping results from three different DNA preparation methods illustrated the importance of the quality of analysed DNA samples. The lowest call rate (CR, 90–93 %, Table 2) was detected with the unpurified lysis samples (method 1). When compared with the sperm sample, the percentage of missing alleles was 2–5 % and the percentage of different allele calls (e.g. A in one sample and G in the other) was 0.09–0.31 % (Table 3). These differences substantially reduce the reli-ability of genotypes produced from lysed hair root samples. Samples of DNA extracted by the ethanol precipitation (method 2) and Qiagen purification (method 7) methods showed high call rates; 94–95 % and 95% for methods 2 and 7, respectively (Table 2) and very low percentage of differences when compared with sperm samples (Table 3). These values are consistent with the differences seen in duplicate DNA samples extracted from sperm (Table 3). Excluding the 2815 SNPs, that did not work for any of the genotyped samples, the average CR

Table 2. The overall call rate (CR) for three samples (1-3) prepared by different DNA extraction methods and a control sample (4a and 4b) after Illumina Beadchip genotyping. The corrected CR indicates the CR after excluding SNPs that did not work for any of the sam-ples analyzed. The number or letter listed in the method column indicates the DNA preparation method: S=DNA extracted from sperm, 1=lysis, 2=lysis+EtOH precipita-tion and 7=Qiagen kit.

Sample Method CR CR corrected1 S 0.950 0.9951 1 0.901 0.9441 7 0.950 0.9951 2 0.940 0.9852 S 0.950 0.9952 1 0.931 0.9752 7 0.950 0.9952 2 0.950 0.9943 S 0.950 0.9953 1 0.913 0.9563 7 0.950 0.9953 2 0.937 0.9814a S 0.950 0.9944b S 0.950 0.994

for all Qiagen and EtOH precipitated samples was 0.9982 (SD = 0.007).

Additional extractions of DNA were performed using the Qiagen Blood and Tissue kit (method 7), since the overall call rates were slightly higher in these samples compared with EtOH precipitated samples. However, the EtOH precipitation from hair root lyses appears to be a feasible method for DNA preparation for high throughput genotyping. The CR for 231/270 (86 %) additional hair root samples was > 99 % (after exclusion of SNPs that did not work in any samples). For 28 samples the call rate was > 90 %, for 18 samples > 72 % and three samples did not work. The purity (260/280 ratio) was > 1.7 for all genotyped samples and did not explain the lower call rates, but the DNA con-centration of the samples varied between 2–100 ng/µl. Samples with a lower DNA concentration were also genotyped, because for some hair root samples it was extremely difficult or even impos-sible to obtain a sufficiently high DNA concentra-



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Table 3. Differences in allele calls after Beadchip genotyping of hair root samples prepared by various DNA extraction methods. Missing genotypes: number of genotypes that are missing in the comparison between hair and sperm samples. Different genotypes: number of genotypes that are different in the comparison between hair and sperm samples. The letter or number after the iden-tification number indicates the DNA preparation method: S=DNA extracted from sperm, 1=lysis, 2=lysis+EtOH precipitation and 7=Qiagen kit.

Missing genotypes

% Different genotypes

%

PIG.1S PIG.1_1 3068 4.94 193 0.31PIG.1S PIG.1_7 48 0.08 32 0.05PIG.1S PIG.1_2 618 0.99 38 0.06PIG.1_7 PIG.1_2 604 0.97 71 0.11



PIG.4aS PIG.4bS 64 0.10 52 0.08

tion (> 50 ng/µl). Thus, with the aim of genotyping a large population of animals with minimal effort the range in DNA concentration was considered acceptable. When the call rates were analysed in groups based on the DNA concentration, a clear as-sociation between concentration and CR was iden-tified (Fig. 1). With low DNA concentrations (< 30 ng/µl) the proportion of samples with adequate call rates was lower than when the concentration exceeded 30 ng/µl. Based on pair-wise compari-sons using the Tukey’s test there was a significant difference between sperm samples and hair sam-ples when DNA concentration fell below 30 ng/µl, but no difference when the concentration was higher than 30 ng/µl (Table 4). Significant differ-ences were also found between batch number four and batch number three (Table 4). Part of this may be due to the age and storage conditions of the hair samples, but some variation may also arise from handling of the sample during DNA extraction protocol. The acceptable limit for DNA concen-

Fig. 1. Distribution plot of call rates against DNA con-centration of hair root samples. At low concentrations the proportion of samples with an adequate call rate is lower than with samples containing DNA concentration above 30 ng/µl.

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100

Call rate

Qubit ng/µl



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tration appeared to be 30 ng/µl producing CR > 99 % corresponding to the CR with DNA extracted from sperm or blood. However, samples with lower concentration of DNA also exhibited high call rates indicating that some deviation can be tolerated for successful genotyping. Therefore, depending on the experimental design, it may be necessary to increase sample numbers by decreasing the DNA concentration streshold.

Conclusion

Our results show that DNA from porcine hair roots using an appropriate extraction method gener-ates reliable genotyping results with the Illumina PorcineSNP60 Genotyping BeadChip. These data allow a straightforward and inexpensive means of genotyping previously collected samples and/or

large animal populations. The concentration of DNA appears to be the limiting factor in genotyping DNA from hair roots and can be used to confirm the high CRs. However, if the genotyping of large popula-tions is required analysis of samples containing lower DNA concentrations may remain viable. The Qubit platform appeared to be a valid method for the measurement of DNA concentration. Furthermore, the SNPs on PorcineSNP60 BeadChip were highly polymorphic in the Finnish Landrace population highlighting the feasibility of this technology for genotyping of Finnish Landrace pigs.

Acknowledgements. Funding for this study was provided by the Finnish Ministry of Agriculture and Forestry (Makera). The assistance of Tiina Jaakkola and Tarja Hovivuori in DNA extraction and Päivi Lahermo (Institute for Molecular Medicine Finland, FIMM) in genotyping with PorcineSNP60 Geno-typing BeadChip (Illumina) is greatly appreciated.

Table 4. Descriptive statistics of call rates (CR) for hair samples containing different DNA concen-trations (ng/µl Qubit) and between different extraction batches. The CR for samples extracted from hair root follicles with a low DNA concentration (< 30 ng/µl) was significantly different from CR for DNA samples extracted from sperm. The LS mean corresponds to multivariate ANOVA least square mean estimate. P–value refers to the significance of differences between sperm samples and different DNA concentrations extracted from hair roots and between batch 4 and other batches based on Tukey’s test statistics.

DNA source Class n Mean SD LS mean p–valueHair root <10 24 0.885 0.084 0.900 <.0001Hair root 10 – 30 80 0.953 0.052 0.969 0.005Hair root 30 – 50 71 0.979 0.004 0.994 1Hair root 50 – 100 98 0.980 0.005 0.994 1Sperm 130 0.993 0.007 0.995

DNA batch 1 21 1.000 0.0002 0.975 0.0992 87 0.995 0.0003 0.970 0.1033 16 0.990 0.0139 0.981 0.0034 279 0.963 0.0453 0.954

Overall 403 0.973 0.041



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Manninen, M. et al. Silage digestibility and concentrate protein in feeding of bulls Vol. 20(2011): 151–168.

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Effects of grass-red clover silage digestibility and concentrate protein concentration on performance,

carcass value, eating quality and economy of finishing Hereford bulls reared in cold conditions

Merja Manninen1*, Markku Honkavaara2, Lauri Jauhiainen3, Arja Nykänen4 and Anna-Maija Heikkilä1

1MTT Agrifood Research Finland, Economic Research, FI-00410 Helsinki, Finland2Finnish Meat Research Institute, FI-13110 Hämeenlinna, Finland

3MTT Agrifood Research Finland, Services Unit, FI-31600 Jokioinen, Finland4MTT Agrifood Research Finland, Plant Production Research, FI-50100 Mikkeli, Finland

*Present address: Evira, Finnish Food Safety Authority, Control department, FI-32200 Loimaa, Finland, email: [email protected]

The aim of the present experiment was to study the effects of (1) digestibility of grass-red clover silage (GCS) and (2) concentrate protein concentration on the performance, eating quality and economy of Hereford bulls during a six months pre-slaughter period, and reared in cold indoor facilities. Thirty-one bulls with an initial live weight (LW) of 289 kg were selected for a 2 × 2 factorial design experiment consisting of two primary growth GCSs harvested at different maturities (in vitro digestible organic matter (OM) in dry matter (DM), D value: Early-cut, E, 750 g kg-1 DM; Late-cut, L, 699 g kg-1 DM) and two concentrate crude protein concentrations (Medium, M, 170 g kg-1 DM; High, H, 210 g kg-1 DM). The concentrate comprised milled barley and pelleted commercial protein compound and was offered daily on average 3.2 kg DM, including 0.45 and 1.13 kg of rapeseed cake in M and H, respectively. Grass-red clover silage was offered ad libitum. The target cold carcass weight was 330 kg.The proportion of concentrate of the total daily DM intake averaged 0.337 during the entire experiment. Treatments had no effect on the daily intake of GCS, total intake of DM, DM intake kg-1 LW0.75 and metabolizable energy averaging 6.0 and 9.4 kg DM, 97.4 g and 109.4 MJ, respectively. The digestibility of dietary OM and neutral detergent fibre was lower (p < 0.05, 0.733 vs. 0.769 and 0.625 vs. 0.665) on diet L than on diet E. The animals on diet E tended to consume daily on average 1.29 kg less (p < 0.10) DM kg-1 net weight gain than those on diet L. The time to achieve the target carcass weight was on average 18 days longer (p < 0.01) on diet L than on diet E. During the entire experiment the LW gain averaged 1795 and 1609 g d-1 (p < 0.01) on diets E and L, respectively. The concentrate protein concentration did not affect animal performance. Treatments had no significant effect on the kill-out proportion, EUROP carcass conformation and carcass fat classifica-tion which averaged 537 g kg-1, 6.5 and 3.6, respectively. The eating quality of the tested loins was good. Treatments had only a minor effect on the yield of valuable cuts. It is concluded that the digestibility of silage is important since the early-cut silage improved the growth rate and shortened the finishing period of bulls significantly compared with those fed late-cut silage. The lower yield and, thus, higher unit cost of early-cut silage may, however, invalidate its superiority compared with the late-cut silage. There was no benefit from using concentrate of high protein concentration.

Key-words: Beef production, concentrate protein concentration, economy, eating quality, silage digestibility


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compounds available are based on rapeseed meal or rapeseed cake. In earlier studies with light (final LW between approximately 390 and 510 kg) dairy-breed bulls (Aronen 1990, Aronen et al. 1992), rapeseed meal supplementation increased the ani-mal growth rate, particularly at the beginning of the finishing period, partly due to an increased silage and energy intake. In several studies, protein sup-plementation has not increased the growth rate (e.g. Steen 1996a, Huuskonen et al. 2008, Huuskonen 2009a), but there is evidence that finishing cattle may respond to supplementary protein in barley-based concentrates when the grass silage digest-ibility is moderate or low (Waterhouse et al. 1985) and in situations where the animals have very high growth potential (Steen 1996b). In some studies, excess protein supplementation has increased car-cass fat classification (Steen and Moore 1988, 1989, Steen 1996a) but Berge et al. (1993) reported that steers which were given protein supplementation had leaner carcasses than steers not given protein supplementation. Meat tenderness is the most im-portant property of beef meat for consumers and it has been studied widely (e.g. Berge et al. 1993, Manninen et al. 2010) but the effects of protein sup-plementation on high digestible grass silage-based diet on eating quality are scarce. Furthermore, over-feeding of protein may cause extra costs to beef producers and load to the animals’ metabolism and environment.

No studies, made in Nordic conditions having timothy-meadow fescue grasses, are available on the effects of silage digestibility and protein supple-mentation with a low amount of concentrate on the performance of animals having a high growth po-tential. Therefore, the aim of the present experiment was to study the effects of grass-red clover silage (GCS) digestibility and concentrate protein con-centration during a six months pre-slaughter period with Hereford (Hf) bulls, and reared in cold indoor facilities. However, the best economic performance is not necessarily reached with the feeding strategy that gives the best biological results. The effects of treatments on feed intake, diet digestibility, animal growth rate, feed conversion, carcass and eating quality, yield of valuable cuts and, finally, on the economy are discussed in this paper.

Introduction

Beef production from suckled beef-breed calves is increasing in Finland. The feeding of finishing cattle is largely based on grass silage. The nutritive value of this depends on the stage of growth at harvesting and the changes in the chemical composition dur-ing ensiling (e.g. Beever et al. 2000, Huhtanen et al. 2007). During the primary growth of grass, the daily decline in D value (digestible organic matter (OM) in dry matter (DM), g kg-1 DM) in Finnish conditions with timothy-meadow fescue grasses has typically been 5 g kg-1 DM (Rinne et al. 1999, Rinne et al. 2002) but lower values have been reported for the harvest of primary growth perennial ryegrass herbage (Keady et al. 2000). If the silage contains red clover, the decline is slower (Rinne and Nykänen 2000). According to Kuoppala et al. (2008), postpon-ing the harvest of primary growth grass decreased silage DM intake (DMI) of dairy cows by 0.48 kg and the energy-corrected milk yield by 0.61 kg 10 g-1 decrease in silage D value. On the other hand, postponing the silage harvest increases the DM yield ha-1 and thus decreases the unit cost of silage DM. With growing cattle, several studies have confirmed that harvesting primary growth grass at an earlier stage of maturity improved the animal growth rate (e.g. Scollan et al. 2001, Nadeau et al. 2002, Steen et al. 2002, Keady et al. 2008). According to Steen (1988a), increasing the digestibility of grass silage by harvesting the grass at an earlier stage of growth may be the most effective method of increasing animal performance from silage.

In Finland, most producers use varying amounts of protein supplements with grain on grass silage-based diets. Protein supplementation for finishing cattle has been studied extensively, but the re-sponses to supplementation have been inconsist-ent. The discrepancies may be due to the differ-ences in animal live weight (LW) and the nutritive value of roughage as well as the amount and type of supplement offered. Additionally, the response to protein supplementation may depend on total diet crude protein concentration, stage of maturity of the beef animal and gender. At present, rapeseed is the most important protein supplement used for cat-tle in Finland and most of the commercial protein



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Animals, experimental design and housing facilities

The experiment was carried out at Tohmajärvi Re-search Station, MTT Agrifood Research Finland, located in eastern Finland (62°20’N, 30°13’E) where the average vegetation growth period is 155 days. Thirty-two Hf bulls were taken for the experiment but one animal was removed from the experiment on 26 January due to acute lameness, possibly due to muscle injury. All data for this animal is deleted. Therefore, thirty-one Hf bulls (the dams were Hf cows) with an initial LW of 289 kg (standard devia-tion (SD) 37.7 kg) and age of 225 d (SD 22.6 d) on 18 November were selected for the experiment. The bulls were born at the Research Station between 13 March and 10 June (17 in March, nine in April, three in May and two in June). The birth weight averaged 42.0 kg (SD 4.81 kg). At pasture, dam milk and grass were the sole feeds for the bulls. From weaning on 17 September to the onset of the experiment on 19 November, the bulls were kept in an uninsulated barn and had free access to grass silage. During the last two weeks pre-experiment, milled barley, at most 2.0 kg DM d-1, was given to facilitate adaptation to the experimental diet. The daily live weight gain (LWG) from birth to the onset of the experiment averaged 1097 g (SD 128.7 g).

In the present experiment, four treatments in a 2 × 2 factorially arranged design consisted of two primary growth digestibilities, GCSs (Early-cut, E; Late-cut, L) and two concentrate crude protein (CP) concentrations (Medium, M; High, H). Initial LW, age and sire were used to allocate the animals to four groups. Thereafter, the treatments were randomly assigned to groups. The animals were group-fed, four animals per pen and two pens per treatment. The two pens for each treatment were allotted in the barn so that pens having same treat-ment were not alongside.

During the experiment the animals were kept in an uninsulated barn in eight pens. Each pen was 37 m2, including 26.5 m2 of bedding area and 10.5 m2 of passage. Straw and peat were used as bed-

ding materials. The bulls had access to an asphalted outdoor exercise area of 53 m2 for one to two hours two to three times weekly while bedding material was added. During the experiment the temperature in the barn was measured daily at 8:00 and 14:00 hours. The coldest temperature (-20.7 °C) was measured on 12 February at 8:00 hours and the highest temperature (+23.3 °C) on 7 May at 14:00 hours. The mean temperature during the experi-mental months was +2.0 °C.

Feeds and feeding

The aim was to have silages with D values of 700 and 650 g kg-1 DM, but the target was not met due to weather conditions. Wilted silages were harvested from two fields at the Research Station, 17–18 June (E) and 31 June–1 July (L), with a mower conditioner and a precision chopper. The herbage was ensiled in bunker silos using a formic acid-based additive (AIV 2 Plus: formic acid 760 g kg-1, ammonium formate 55 g kg-1, Kemira Oyj, Oulu, Finland) applied at 5 l t-1 fresh weight. The sward for silage was a second-year timothy (Phleum pratense L.), meadow fescue (Festuca pratensis Huds.), red clover (Trifolium pratense L.) mixture sown in the proportions 650, 300 and 50 seeds m-2, respectively. The sward was fertilized in spring using nitrogen (N) 33.5 kg ha-1. The energy value of the GCS was evaluated prior to the experiment using in vitro OM digestibility (OMD, Friedel 1990).

The concentrate comprised milled barley and pelleted commercial protein compound (CPC), having a CP concentration of either 170 (M) or 210 (H) g kg-1 DM. The energy content in M and H was 12.88 and 12.55 MJ metabolizable energy (ME) kg-1 DM, respectively. Thus, the difference between M and H was 0.33 MJ ME kg-1 DM. Bar-ley was milled using a 7 mm riddle. The CPC (the diameter of pills was 5 mm, Futura-Maituri 140 L, Raisio Feed Ltd, Raisio, Finland) included rape-seed cake (680 g kg-1), wheat bran (105 g kg-1), molassed sugarbeet pulp (70 g kg-1), mixed mo-lasses (50 g kg-1), wheat middlings (42 g kg-1), oat bran (30 g kg-1), calcium carbonate (11 g kg-1),



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sodium chloride (6 g kg-1) and premix (6 g kg-1). The amount of barley and CPC given was calcu-lated on the basis of the pre-evaluated feed analysis of both feeds. The proportion of CPC in the con-centrate was either 0.185 or 0.459 on an air-dry basis in diets M and H, respectively. During the experiment the animals received 280 g DM d-1 of mineral mixture (Luonnon Viher-Minera: Ca 84, P 34, Na 60 and Mg 70 g kg-1, Suomen Rehu Oy, Vaasa, Finland). No vitamin mixture was given to the animals.

The concentrate was offered at 2.0, 3.0 and 4.0 kg DM d-1 during the periods P1 (56 d), P2 (57 d) and P3 (min 27 d, max 104 d, mean 74 d, SD 22.2 d, until slaughter), respectively. The animals were fed at 7:30 hours. Barley and CPC were spread on the feeding table evenly and, thereafter, the animals were tied up for maximum 2 h so that each animal could eat its own portion. Silage was offered ad libitum (at least 22 hours per day) during the entire experiment at an excess level of 1.05 of the daily intake. The amount of feeds offered and refused was recorded daily. Water was offered ad libitum via heated water-pipes and -cups.

Feed and faecal sampling and analysis

The swards were pre-sampled on 11, 18 and 23 June, i.e. once before the harvest of E and three times before the harvest of L. The D value and CP concentration of the sward were determined in order to monitor the change in sward digestibility. The pre-samples were cut by scissors from the swards from four 0.25 m2 areas of the two fields, weighed, dried and analysed for DM content, as well as for D value and CP concentration by FOSS NIR Systems 5000 (Near Infra-Red Spectroscopy, FOSS, Eden Prairie, MN 55344, USA). At the time of harvest, similar samples were taken for botanical analyses to determine the red clover content of the swards. During the experiment, samples of GCS were taken for P1 and P2 and two samples for P3. One representative feed sample, pooled over twelve sub-samples, of barley, CPC and mineral mixture was taken at the onset of the experiment. The CPC

and mineral mixture originated from one production batch and the barley from one harvest.

The total-tract apparent OM and protein as well as neutral detergent fibre (NDF) dietary digestibil-ity were estimated using acid insoluble ash as an internal marker (European Commission 1971). Spot faecal samples were collected from each bull on two occasions, and on each occasion they were taken once daily for three consecutive days. Sam-ples were taken on 3–5 February during P2 and 29 –31 March during P3. The samples were pooled on a pen basis (on a wet basis, equal amount per ani-mal), thoroughly mixed, sub-sampled and stored at -20 oC. Totally 16 faecal samples were collected and analysed, i.e. one sample per pen per sampling period. Extra feed samples (four GCS samples, two barley samples and two CPC samples) were col-lected on the faecal collection days.

The GCS, barley, CPC and mineral mixture DM contents were determined by oven drying at 105 °C for 16 hours and GCS corrected for volatile losses according to Huida et al. (1986). Feed and faecal samples were analysed for ash (AOAC 1990, method No. 942.05), ether extract (AOAC 1990, method No. 920.39A) and crude fibre according to the EEC standard (92/89, ASN 3802) using the FiberCap 2021/2023 system (Foss Tecator AB, Höganäs, Sweden), total N of mineral mixture by the Dumas method using a Leco FP 428 nitrogen analyser (AOAC 1990, method No. 968.06, Leco Corp., St Joseph, MI 49085, USA) and total N of GCS, barley and CPC using a Foss Kjeltec 2300 Analyzer Unit (Foss Tecator AB, Höganäs, Swe-den) and for NDF according to Van Soest et al. (1991). In vitro OMD was measured using a cellu-lase enzyme complex according to Friedel (1990). Fresh GCS samples were analysed for pH and wa-ter-soluble carbohydrates (WSC) by the method of Somogyi (1945), lactic acid (Haacker et al. 1983), volatile fatty acids (Huhtanen et al. 1998), ammo-nia N (McCullough 1967) and ethanol with an en-zymatic kit (Cat No. 981680, KONE Instruments Corporation, Espoo, Finland) and soluble N by the Kjeldahl method using Cu as a digestion catalyst (AOAC 1990, method No. 984.13).

The ME value for GCS was calculated assum-ing a ME content of 16 MJ kg-1 digestible OM



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(DOM, MTT 2006). The D value was based on in vitro measurement. The AAT values were cal-culated using the measured D value and the CP concentration (MTT 2006). The intake index (IN) for GCS was calculated according to Huhtanen et al. (2002).

The energy value for barley was calculated us-ing the determined chemical composition and av-erage digestibility coefficients reported by MTT (2006). The energy value for CPC was calculated using the average chemical composition and av-erage digestibility coefficients of each component (MTT 2006).

Live weight, slaughter procedures and eating quality

The animals were weighed on two consecutive days at the onset of the experiment and at the end of P1, P2 and P3 before feeding. Additionally, the animals were weighed once every 28 days.

The target cold carcass weight was 330 kg. The animals were selected for slaughter based on LW, LWG pre-slaughter and an assumed dressing pro-portion (0.550) which was assessed based on ear-lier studies (unpublished data) in Finland with Hf bulls. The animals were slaughtered in 11 slaughter batches, i.e. three batches in April (seven animals in all), four batches in May (ten animals in all) and four batches in June (14 animals in all). Feed was not offered on the morning of slaughter, but there was still silage available for all animals. The ani-mals were slaughtered in the Atria Oyj slaughter-house in Kuopio, 190 km away from the Research Station. The time interval from the departure to the slaughter was approximately six hours.

The carcasses were classified for conformation (12 classes: S, E, U, R+, R, R-, O+, O, O- and P+, P, P-) and fat cover (5 classes: 1, 2, 3, 4 and 5) us-ing the EUROP quality classification scale (Com-mission of the European Communities 1991). The kill-out proportion was calculated as the propor-tion of cold carcass weight (hot carcass weight × 0.98; Ministry of Agriculture and Forestry 1995) to final LW and expressed as the proportion of kg

cold carcass weight to kg final LW. The carcass temperature was chilled below 7 °C for 24 hours, after which the pH value of the loin was measured on the 11th rib of a half carcass. The right side of each carcass was then quartered at the 5th rib into a pistola hind quarter without the flank (Swatland 2000). The pistola was cut into valuable cuttings and tallow (subcutaneous fat). It is well known that the most valuable cuts come from the back and whole round of a half carcass. Thus, loin and tenderloin were cut from the back, whereas the whole round cuttings were outside round, inside round, corner round and roast beef. All cuttings and the tallow were weighed and their yields were expressed as percentages of the carcass cold weight (0.98 × carcass hot weight 50 min post mortem).

During cutting, a loin sample of 2 kg was taken from between the 5th and 8th ribs and vacuum pack-aged. After that, samples were sent to the Finnish Meat Research Institute (LTK) for further analyses. At LTK, the loin samples were aged for 17 days at 4 °C, making a total ageing time of 19 days, and thereafter frozen (-20 °C) for four months before sensory evaluation and shear force value measure-ment. After thawing, the organoleptic quality and shear force value of the loins were analysed. For the organoleptic evaluation, four 1.5 cm slices from each loin were heated to 68 °C in a rolling grill (Palux Rotimat, Germany) and evaluated by six trained sensory panellists for tenderness, juiciness and taste. These traits were scored on a 7-point scale (4=satisfactory, 5=good, 6=desirable and 7=most desirable). In addition, the panellists re-corded off-flavours, if any, during the organoleptic evaluation.

For shear force value measurements loin sam-ples were heated in a water bath of 85 °C until the core temperature of meat was 70 °C. After chilling for 24 hours (4 °C), loin samples of about 6 cm long (parallel to the myofibres), 1 cm high and 1 cm wide (square probe of 1 cm x 1 cm surface area) were placed in a Warner-Bratzler shear blade to be sheared perpendicularly to the muscle fibre longi-tudinal axis in Instron testing machine. Maximum force was recorded and results were expressed as kg cm-2 (Honkavaara et al. 2003).



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Economic evaluation

The economic performance was determined by calculating the return on fixed inputs, or on inputs which were constant per day regardless of feeding strategy. In this examination, feeding cost and calf cost were considered as variable costs. The feeding cost varied because of diverse feed rations and feed prices. The treatment affected also the length of the finishing period and, thus, the calf cost per day. Market revenue and subsidies for beef production formed the gross return of the calculation. Since some premium payments are coupled to carcass weight, some to growing time (Niemi and Ahlsted 2008), the subsidies had to be included in the evalu-ation. The results are presented per day to allow for the varying length of the finishing period.

The unit prices and subsidy rates are shown in Table 1. The meat price expresses the price for meat in EUROP conformation R- and EUROP fat classification from 1 to 3. One step downwards or upwards in the conformation results in a 0.10 € kg-1 decrease or increase of the price, respectively. Fat classification 4 or 5 causes a 0.30 and 0.60 € kg-1 decrease of the price, respectively. The meat price is graded by carcass weight and calf price increases

with LW (M. Ilola, Atria, Seinäjoki, Finland; per-sonal communication). The subsidy rates represent the subsidy level in Central Finland (subsidy area C2).

Barley, CPC and mineral mixture were priced at the market prices for May 2009. A milling cost of 0.016 € kg-1 was added to the market price of barley (ProAgria 2009a). The price of silage was set ac-cording to its production cost, 1.123 € ha-1 when the subsidies paid ha-1 were subtracted from the total cost (ProAgria 2009a). Storage losses were estimated to be 15% (McDonald et al. 1991) and feeding losses 5% referring to the intended excess level of 1.05 in the feeding of silage.

A correlation between the harvesting time and yield of silage was considered while calculating the unit cost of E and L. Experiments undertaken in Central Finland in summer 2008 were used as a basic guideline in estimating the yield differences of varying cutting times (Vanhanen 2009). Using 100 kg N ha-1 as a reference fertilizing level, the later harvesting of silage produced a 24% higher DM yield ha-1 than the earlier harvest. As those experimental yields were fairly high compared to yields in farm conditions, the observed relative yield difference was transferred to two lower yield

Table 1. Input prices, meat prices and subsidy rates for beef production in 2009.Weight limits Unit Euro

Silage, early-cut € kg-1 DM 0.144Silage, late-cut € kg-1 DM 0.116Barley € kg-1 DM 0.134Commercial protein compound € kg-1 DM 0.295Mineral mixture € kg-1 DM 0.567Calf live weight 140 kg € calf-1 365.00Calf live weight 141–300 kg + € kg-1 2.20Calf live weight > 300 kg + € kg-1 1.30Meat carcass weight 290–319 kg € kg-1 2.83Meat carcass weight 320–349 kg € kg-1 2.89Meat carcass weight ≥350 kg € kg-1 2.91Special beef premium € bull-1 157.50Slaughtered bull premium carcass weight ≥ 330 kg € / slaughtered bull 30.30National aid € / livestock unit a / year 422.00a bull older than 6 months and younger than 2 years equals 0.6 livestock unit



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levels. A yield level of 8 700 kg DM ha-1, which the best quarter of farmers are able to produce (ProAgria 2009b), was assumed in the basic solu-tion. The other solution was based on an average yield, 6 000 kg DM ha-1 (ProAgria 2009b). The two levels were used to test the sensitivity of eco-nomic results with respect to silage price. Based on the results of Kuoppala et al. (2008), the effects of a change in the price difference between E and L were also analysed. Moreover, the sensitivity analysis concerned changes in the price of grain and meat and a reduction in subsidies.


Diet digestibility, daily intake, feed conversion (ratio of DM, ME, CP and AAT intake and kg net weight gain) and the ratio of DMI and kg-1 LW0.75

were measured at the group level only and one-way analysis of variance was used to analyse the data. The rest of the variables were measured individu-ally. Because treatments were assigned to groups, group was used as an error term when treatments were compared. The statistical model used in these analyses was: yijkl = µ + φl +αi + βijl + εijkl

where yijkl is the observed value of the response variable for the kth animal in the ith treatment in the jth group, µ is the overall mean, αi is the effect of the ith treatment, βijl is the effect of group, ϕl is the blocking effect for groups and gijkl is the residual error. Animals were divided to groups according to animals’ age at the start of the study (young-est - oldest) and blocking effect takes into account this. The effect of αi was divided into three parts: main effect of silage digestibility, main effect of concentrate protein concentration and their interac-tion. Statistical analyses were carried out using the SAS/GLM software (SAS 2004).

SAS/MIXED software was used only for eco-nomic variables. In the SAS/MIXED analyses the effect of group was used as a normally distributed random effect. MIXED analysis was rejected for the analysis of the non-economic variables, be-

cause the estimated variance for group effect was not positive for most variables.

Results

FeedsThe DM, CP and D values of the sward pre-samples were 191, 228 and 212 g kg-1, 182, 162 and 137 g kg-1 DM and 763, 755 and 740 g kg-1 DM, respectively. The red clover content of the sward varied from 15 to 20% in DM with no difference between E and L. The D values for silages E and L averaged 750 and 699 g kg-1 DM, respectively. The contents of DM, ash, NDF, WSC as well as pH and ammonia and soluble N in total N were higher in L than in E (Table 2).

Diet digestibility and feed intake

The digestibility of diet OM was lower (p < 0.05, 0.733 vs. 0.769) and the digestibility of diet protein tended to be lower (p < 0.10, 0.667 vs. 0.697) on diet L than on diet E (Table 3). The digestibility of diet NDF was lower (p < 0.05, 0.625 vs. 0.665) on diet L than on diet E and tended to be lower (p < 0.10, 0.631 vs. 0.660) on diet M than on diet H.

During the entire experiment the daily intake of concentrate was almost identical on all diets as expected due to the fixed feeding regimens averag-ing 3.17 kg DM and including 0.452 and 1.129 kg of rapeseed cake in M and H, respectively (Table 4). No refusals of milled barley or pelleted CPC were observed due to the moderate amounts of both feeds offered, maximum 4.0 kg DM daily pre-slaughter (P3). Treatments had no effect on the daily intake of GCS, the total intake of DM, OM, DMI kg-1 LW0.75 and ME averaging 6.0, 9.4 and 8.6 kg DM, 97.4 g and 109.4 MJ, respectively. The daily intake of CP was 65 g lower (p < 0.05) on diet L compared with diet E and 158 g lower (p < 0.01) on diet M compared with diet H. The daily



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Table 2. Harvests of grass-red clover silages and mean chemical compositions and feed values of experimental feeds.

Silage, Early-cut

Silage, Late-cut

Barley Commercial protein

Mineral mixture

Mean SDa Mean SD compoundHarvest date 17-18 June 31 June-1 JulyGrowing timeb, (d) 42 55Degree daysb, (°C) 256 354Number of samples 4 4 1 1 1Dry matter (DM, g kg-1) 251 44 307 22.4 879 881 968In DM (g kg-1) Ash 76 2 85 3.8 29 88 550 Crude protein 162 19.9 151 7.4 143 289 67 Ether extract ndc nd 20 84 nd Crude fibre nd nd 55 121 nd Neutral detergent fibre 418 4.3 483 11.6 226 313 97 Lactic acid 73 4.8 45 10 Acetic acid 28 2.5 18 3.4 Butyric acid 0.4 0.388 0.38 0.573 Ethanol 8 4.21 4.4 0.94 Water-soluble carbohydrates 26 11.1 52 25.2pH 3.78 0.116 4.03 0.075In total nitrogen (g kg-1) Ammonia N 40 5.7 45 3.1 Soluble N 511 69 547 43.9D valued, g kg-1 DM 750 4.4 699 10.2Intake index 108 1.4 103 1.8Feed value, kg-1 DM Metabolizable energy, MJ 12 0.07 11.2 0.16 13.1 11.9 AATe, g 91 1.1 86 0.5 107 137a Standard deviation.b Calculated from the beginning of growing season on 6 May 2003.c Not determined.d Digestible organic matter in dry matter.e Amino acids absorbed in the small intestine.

Table 3. Mean treatment effects on in vivo dietary digestibility coefficients.Silage digestibility (S) Early-cut Late-cut Significanceb

Concentrate protein concentration (P) Medium High Medium High SEMa S P S×PNumber of groups 2 2 2 2Organic matter 0.766 0.773 0.734 0.732 0.0067 *Protein 0.691 0.703 0.656 0.677 0.0104 oNeutral detergent fibre 0.642 0.688 0.619 0.632 0.0112 * oa SEM = standard error of mean.b o p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.



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intake of AAT was 43 g higher (p < 0.05) on diet H than on diet M. The daily intake of NDF was 429 g higher (p < 0.01) on diet L than on diet E.

The proportion of concentrate in the total daily DMI averaged 0.337 during the entire experiment. The proportion of CP and NDF in the total daily DMI on diets EM, EH, LM and LH were 0.163, 0.177, 0.155 and 0.168, and 0.350, 0.358, 0.389 and 0.400, respectively.

Animal growth and feed conversion

The health of the animals was good and no signs of diseases were observed. The concentrate protein concentration had no effects on animal performance (Table 5). The duration of the entire experiment was on average 18 days longer (p < 0.01) on diet L compared with diet E. The final LW was as planned equal for all animals, averaging 606 kg. During the entire experiment the LWG and the net weight gain were on average 187 (p < 0.01) and 116 g d-1 (p < 0.05) higher on diet E compared with diet L. The animals on diet E tended to consume daily on

average 1.29 kg less DM (p < 0.10) kg-1 net weight gain than those on diet L. The daily consumption of CP, ME and AAT kg-1 net weight gain was on average 1614 g, 113.3 MJ and 930 g, respectively.

Carcass and meat evaluation

The treatments had no significant effect on the kill-out proportion, carcass conformation and carcass fat classification which averaged 537 g kg-1, 6.5 and 3.6, respectively (Table 5), or on the juiciness and shear force value of the meat which averaged 4.5 and 4.3 kg cm-2, respectively (Table 6). The taste of meat L tended to be better than that of meat E (p < 0.10, 4.9 vs. 4.4). In pH and sensory evaluation of tenderness there were significant interactions (p < 0.05) between silage digestibility and concentrate protein concentration. The tenderness was better and the pH lower in meat EM compared with meat EH with the opposite being true in meat L. Normal pH values of beef are from 5.50 to 5.90 a day after slaughter. On diet EM, three meat samples had a very low pH value (≤ 5.50). Correspondingly, on

Table 4. Mean daily intakes on experimental diets.Silage digestibility (S) Early-cut Late-cut Significanceb

Concentrate protein concentration (P) Medium High Medium High SEMa S P S×PNumber of groups 2 2 2 2Dry matter, kg Grass-red clover silage 5.88 5.98 5.86 6.13 0.157 Concentratec 3.13 3.16 3.2 3.2 0.014 ntd nt nt Mineral mixture 0.28 0.29 0.28 0.28 0.001 nt nt nt Total 9.30 9.43 9.34 9.61 0.149Dry matter, g kg-1 LW0.75 95.8 96.2 98.0 99.8 1.39Organic matter, kg 8.58 8.64 8.57 8.76 0.137Metabolizable energy, MJ 110.9 111.4 106.7 108.7 1.69Crude protein, g 1518 1666 1443 1611 19.8 * **AATe, g 890 928 862 911 12.6 *Neutral detergent fibre, g 3250 3372 3637 3842 72.1 **aSEM = standard error of mean.bo p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.cConcentrate: Including barley and commercial protein compound.d Not tested.eAmino acids absorbed in the small intestine.



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Table 5. Age of animals, duration of the experiment, live weights, live and net weight gains, slaughter data and feed conversion.Silage digestibility (S) Early-cut Late-cut Significanceb

Concentrate protein concentration (P) Medium High Medium High SEMa S P S×PNumber of animals 8 8 7c 8Age, d Initial 229 215 232 225 6.5 At end of experiment 407 395 430 420 7.5 oDuration of the experiment, d 179 179 198 195 1.8 **Live weight, kg Initial 288 288 287 288 0.4 Final 604 610 601 606 3.8Live weight gain, g d-1 1782 1809 1588 1630 22.1 **Net weight gaind, g d-1 1009 1048 902 923 20.2 *Slaughter data Carcass weight, kg 324 331 321 324 4.1 Kill-oute, g kg-1 0.536 0.543 0.535 0.535 0.0038 EUROP conformationf 6.5 6.6 6 6.6 0.34 EUROP fat classificationg 3.5 3.6 3.3 3.9 0.17Feed conversion, kg-1 net weight gainh

Dry matter, kg 9.24 9.01 10.36 10.46 0.576 o Metabolizable energy, MJ 110.2 106.5 118.3 118.3 6.57 Crude protein, g 1508 1592 1600 1754 95.8 AATi, g 884 886 956 992 52.3a SEM = standard error of mean.b o p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.c The SEM given should be multiplied by 1.0801 when making comparisons with other means except for feed conversiond Kill-out proportion of 50 used for calculation of net weight gain.e Ratio of cold carcass weight to final live weight.f Conformation: O- = 4, O = 5, O+ = 6, R- = 7, R = 8, R+ = 9.g Fat cover: 1 = leanest,…,5 = fattest.h Two groups per treatment.i AAT = Amino acids absorbed in the small intestine.

Table 6. Loin sensory evaluation, shear force value and pH.Silage digestibility (S) Early-cut Late-cut Significanceb

Concentrate protein concentration (P) Medium High Medium High SEMa S P S×PNumber of samples 8 8c 7d 8Sensory evaluatione

Tenderness 5.6 5.1 5.2 5.9 0.13 * Juiciness 4.5 4.3 4.7 4.8 0.23 Taste 4.8 4.1 4.9 5.0 0.17 oShear force value, kg cm-2 4.4 4.6 4.2 4.1 0.23pH 5.52 5.59 5.57 5.54 0.011 *a SEM = standard error of mean.b o p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.c N = 7 for pH.d The S.E.M. given should be multiplied by 1.0801 when making comparisons with other means.e Sensory evaluation: Scale from 1 to 7.



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diets EH, LM and LH, two, one and one samples had a very low pH value. On diet EM, the minimum and maximum pH values were 5.40 and 5.62, re-spectively. The corresponding values on diets EH, LM and LH were 5.48 and 5.75, 5.42 and 5.73 and 5.38 and 5.62, respectively. Thus, all pH values were below 6.00.

On diet EM, two meat samples were recorded as being slightly dry and one dry with slight off-flavour. Two samples had liver flavour, one was tasteless and another dry on diet EH. On diet LM, one sample was slightly dry and one had liver fla-vour. Two samples had off-flavour and one was dry on diet LH.

The treatments had no significant effect on the amount (kg) and yield (%) of inside round, roast beef, loin, tenderloin and tallow in a carcass, av-eraging 11.5, 5.6, 10.3, 4.0 and 34.5 kg and 3.5, 1.7, 3.2, 1.2 and 10.6%, respectively (Table 7). The

amount and yield of outside round tended to be higher (p < 0.10, 18.2 vs. 17.5 kg and 5.6 vs. 5.4%) on carcasses fed diet E than diet L. The amount of corner round was higher (p < 0.05, 10.6 vs. 10.1 kg) on carcasses fed diet E than diet L. The amount of valuable cuttings tended to be higher (p < 0.10, 100.9 vs. 98.6 kg) on carcasses fed diet E than diet L.

Economic performance

The results of the economic analysis (Table 8) indicated that the diet EH caused the highest feed cost per day (p < 0.001). A reason for this was the highest unit cost of feed since the daily DMI was unaffected by the diet (Table 4). Carcass weights, carcass conformation and carcass fat classification

Table 7. Valuable cuts of the animals.

Silage digestibility (S) Early-cut Late-cut Significanceb

Concentrate protein concentration (P) Medium High Medium High SEMa S P S×P

Number of animals 8 8 7c 8Outside round, kg 18.1 18.3 17.4 17.7 0.24 o From yield, % 5.6 5.5 5.3 5.5 0.06 oInside round, kg 11.8 11.6 11.1 11.4 0.40 From yield, % 3.6 3.5 3.4 3.5 0.07Corner round, kg 10.7 10.5 10.1 10.2 0.09 * From yield, % 3.3 3.2 3.1 3.1 0.04 o oRoast beef, kg 5.5 5.9 5.5 5.4 0.21 From yield, % 1.7 1.8 1.7 1.7 0.06Loin, kg 10.5 10.0 10.3 10.4 0.38 From yield, % 3.2 3.0 3.2 3.2 0.06Tenderloin, kg 4.1 4.0 3.9 3.9 0.11 From yield, % 1.3 1.2 1.2 1.2 0.02Tallow, kg 35.2 33.7 33.4 35.4 0.83 From yield, % 10.9 10.1 10.2 10.9 0.40Yield from carcass weight, kg 101.5 100.2 97.2 100.0 0.88 oYield from carcass weight, % 31.4 30.2 29.8 30.9 0.37 oa SEM = standard error of mean.b o p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.c The SEM given should be multiplied by 1.0801 when making comparisons with other means.



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caused some variation in the meat price, but the differences between the diets were not statistically significant. However, the diet E resulted in slightly higher market revenue per day than the diet L (p < 0.10) because of a higher LWG per day and, thus, a shorter finishing period to reach the targeted carcass weight (Table 6). The premiums paid per animal decreased with the length of the finishing period and, therefore, the subsidies per day were slightly higher on diet E than on diet L (p < 0.10). Also the calf cost per day decreased, as expected, with the length of the finishing period, but not statistically significantly.

As the fluctuations in costs and returns can-celled each other out, the average return on the fixed inputs was nearly the same on all the diets. No statistically significant differences were found although the average return of diet LH was some lower than the returns of other diets. Sensitivity analysis revealed the stability of the results. Most of the analysed price changes did not change the ranking of the treatments; they just affected the lev-el of the return on fixed inputs. Increase of 0.05 € kg-1 DM (L) and 0.06 € kg-1 DM (E) in the unit cost of silage would decrease the return on fixed inputs by 0.46 € d-1 (E) and 0.37 € d-1 (L). A smaller price difference (19%) between E and L would result in about 0.02 € d-1 higher (E) or lower (L) return on

fixed inputs compared to the basic solution where the difference was 24%. A rise of 30% in the price of grain would cause a reduction in the return on fixed inputs ranging between 0.05 € d-1 (LM) and 0.10 € d-1 (EM). Only LM with the cheapest feed cost would result in a positive return to fixed inputs if the meat price fell off by 20%. Equal cutting of subsidies would not cause as dramatic drop in the return. The reduction would vary from 0.33 € d-1 (LH) to 0.36 € d-1 (EM and EH). An increase of 20% in the basic price of meat would raise the return considerably, more than double on E. Such a price change would also mean that E would give a better economic result than L (p < 0.10).

Discussion

FeedsIn the present study, a cold onset to the growing season delayed the decrease in the sward D value. In addition, the development of red clover is slower than that of grasses (Rinne and Nykänen 2000), which probably slightly affected the decrease in digestibility. In the present study, the daily decline

Table 8. Unit price of meat and feed, gross return, expenses and return on fixed inputs.

Silage digestibility (S) Early-cut Late-cut SEMa Significanceb

Concentrate protein concentration (P) Medium High Medium High S P S*P

Number of groups 2 2 2 2Meat price € kg-1 2.690 2.665 2.700 2.575 0.0678–0.0703Average feed price € kg-1 DM 0.173 0.180 0.159 0.166 0.0053 * oGross returnMarket revenue € d-1 4.82 4.98 4.32 4.20 0.321–0.331 ◦Subsidies € d-1 1.82 1.81 1.69 1.67 0.125–0.127 ◦

ExpensesCalf cost € d-1 3.83 3.88 3.38 3.46 0.620–0.625Feed cost € d-1 1.81 1.91 1.67 1.80 0.006 *** *** o

Return on fixed inputs € d-1 1.00 1.00 0.96 0.61 0.229–0.233aSEM = standard error of mean.b o p < 0.10; * p < 0.05; ** p < 0.01; *** p < 0.001.



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from pre sampling to early cut was on average 1.9 g kg-1 DM, and from early cut to late cut 3.6 g kg-1 DM. Because the swards were needed for grazing studies, the harvest had to be done at an earlier stage of maturity than planned, resulting in on average 50 g kg-1 DM higher D values in the silages than originally planned.

The fermentation quality was good in both si-lages, while the DM content in L was higher than in E. The E silage was slightly more fermented in-cluding less WSC than the L silage. In both silages, the CP concentration was fairly typical for primary growth silage, but the content of NDF was quite low, probably due to the early stage of maturity and the inclusion of red clover in the silage (MTT 2006). The wilted silages did not freeze and were therefore suitable for this type of feeding in cold conditions. The last group of animals was slaugh-tered on 23 June, but the quality of silage remained acceptable in May and June.

Effects of silage digestibility on animal performance

The digestibility of the silage did not affect the DMI of the silage, corresponding to the results reported by Nadeau et al. (2002) and Cummins et al. (2007). The similar intake of both silages may be due to the high digestibility and good fermenta-tion quality of both silages and, partly, due to the higher DM content of L compared with E. However, several studies with growing cattle (Steen 1988b, Martinsson 1990, Steen 1992, Scollan et al. 2001, Steen et al. 2002, Keady et al. 2008) have confirmed an increased intake of silage in response to higher digestibility. Aronen et al. (1992) observed early-cut grass silage to improve the LWG of light (initial LW 123 kg) dairy-breed bulls during the first six months of growth but the bulls offered late-cut silage compensated the difference during the following six months pre-slaughter. This was mainly due to the larger grass silage intake of the early-cut bulls compared to the late-cut bulls. However, the dif-ference between the harvest times of those silages was one week. Furthermore, Aronen et al. (1992)

concluded that the bulls were not able to take full advantage of the high protein concentration (160 g kg-1 DM) of the early-cut silage. In the present study, the CP concentration of E and L averaged 162 and 151 g kg-1 DM. Thus, the difference in the CP concentration of silages was small compared with a big difference in D value.

As in the present study, in several studies with growing cattle and sheep, postponing the harvest of silage has reduced the digestibility of silage as a sole feed (Drennan and Keane 1987, Dawson et al. 2002, Keady et al. 2008) or the digestibility of diets (Steen 1988b, Martinsson 1990, Steen 1992, Scollan et al. 2001). Harvest date is the major fac-tor effecting silage digestibility.

In the present study, the calculated ME intake was similar on all the diets and thus did not ex-plain the difference in the growth rate. The growth rate can be considered as very high for all animals. Steen et al. (2002) concluded that high-digestibility (0.743 DOM in DM) silage had potential relative to high-concentrate diet. It can be assumed that the main reason for the very high growth rate in the present study was the effect of high silage digest-ibility leading to high energy intake and optimal conditions for microbial protein synthesis in the rumen. According to Schroeder and Titgemeyer (2008), energy supply affects the efficiency of pro-tein utilization. The improved LWG on early-cut silage or on silage of high digestibility has earlier been confirmed in several studies (e.g. Scollan et al. 2001, Nadeau et al. 2002, Steen et al. 2002, Keady et al. 2008), but also opposing results exist (Steen 1988b, Cummins et al. 2007). In a review of literature, Steen (1988a) summarized that in 11 comparisons of unsupplemented silages, increasing digestibility increased daily LWG and carcass gain of finishing cattle by 45 and 33 g, respectively, per 10 g kg-1 increase in digestibility. In eight compari-sons in which the silages were supplemented with concentrates (20 to 37% of total DMI), increasing digestibility increased daily LW and carcass gains by 37 and 28 g, respectively, per 10 g kg-1 increase in digestibility. In the present study, daily LWG and carcass gain were increased by 37 and 23 g per 10 g kg-1 increase in silage digestibility, well in agreement with Steen (1988a).



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The silage digestibility had no effect on the kill-out proportion and the carcass fat and conforma-tion scores were in accordance with earlier stud-ies (Steen 1988b, Cummins et al. 2007). In some experiments, high digestible silage has increased carcass fatness (Drennan and Keane 1987, Steen et al. 2002, Keady et al. 2008). The varying responses to silage digestibility may be due to differences in silage digestibility, final LW, breed, breed maturity, gender and proportion of concentrate in the diet.

When Hf bulls were offered concentrate, ei-ther restricted or ad libitum, and grass silage (D value 700 g kg-1 DM) ad libitum for three months pre-slaughter, 35% of the carcasses had a fat score of 5, without treatment effects (Manninen et al. 2010). Those carcasses were approximately 30 kg heavier than the carcasses in the present study. The results of the present study and those observed by Manninen et al. (2010) agree with Steen and Kil-patrick (2000) who concluded that for cattle reared on high-forage diets, reducing slaughter weight is likely to be a more effective approach to reduce carcass fat content than reducing energy intake dur-ing the finishing period.

Effects of concentrate protein concentra-tion on animal performance

The concentrate protein concentration did not affect the intake of silage and total DMI kg-1 metabolic LW, which was consistent with the results observed with heavy dairy-breed bulls (Huuskonen 2009a). Additionally, in earlier studies with suckled conti-nental-cross bulls (Drennan et al. 1994) or heavy steers (Steen 1988b, Steen 1996a), silage intake was unaffected by protein supplement. On the contrary, rapeseed meal supplementation increased the silage (Aronen 1990, Aronen et al. 1992) intake of light dairy-breed bulls. The positive response of intake to protein supplement may be more evident in animals of lower LW than in the bulls in the present study, as also Aronen (1990) suggested.

The digestibility of dietary OM was unaffected by concentrate protein concentration, in accord-ance with Huuskonen et al. (2007, 2008) and Hu-

uskonen (2009a). In numerous studies (e.g. Steen 1988b, Huuskonen et al. 2007, 2008, Huuskonen 2009a), protein supplement increased the digest-ibility of dietary protein, which was not observed in this study.

The growth response to protein supplementa-tion depends generally on animal LW, silage di-gestibility, proportion of concentrate in the diet and breed. If the supply of energy is good, the mi-crobial protein synthesis is generally sufficient to sustain a high LWG in animals of a LW over 250 kg (Huuskonen 2009b). Titgemeyer and Löest (2001) presented that, while amino acids were the limiting factor with lighter calves offered grass silage, ener-gy availability was the limiting factor with heavier animals. Later, Schroeder and Titgemeyer (2008) concluded that energy supply affects the efficiency of protein utilization but the effects may be differ-ent, depending on which amino acid is the most limiting. In the present study, the supply of energy was sufficient with a diet with a moderate amount of concentrate and high-digestibility silage. Wa-terhouse et al. (1985) reported that Friesian steers were most likely to respond to supplementary pro-tein in barley-based concentrate when the grass silage in vitro digestibility was below 0.65. In the present study, there was no benefit from the higher concentrate protein concentration, suggesting that the amino acid supply from feed protein and micro-bial protein synthesis in the rumen was sufficient on diet L for a high daily LWG. Correspondingly, in recent studies (Huuskonen et al. 2007, Huusko-nen et al. 2008, Huuskonen 2009a) with heavy dairy-breed bulls, rapeseed meal supplementation did not improve the growth rate, as also observed with heavy steers (Steen 1988b, Steen 1996a) and suckled beef bulls (Drennan et al. 1994) fed fish meal/soybean meal supplementation.

According to Lowman and Lewis (1991), the performance of bulls is not very sensitive to a range of protein concentrations between 130 and 180 g CP kg-1 DM. In the present study, the diet CP con-centrations were 163, 177, 155 and 167 g CP kg-1 DM on diets EM, EH, LM and LH, respectively. However, the diet CP concentrations do not take into account the quality of protein.



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The carcass traits were unaffected by the con-centrate protein concentration being consistent with recent studies (Huuskonen et al. 2007, Huuskonen et al. 2008, Huuskonen 2009a). On the contrary, increased protein intake tended to increase the car-cass fatness of steers and heifers (Steen and Robson 1995, Steen 1996a). Although not observed in this study, protein supplementation of high (D value about 710 g kg-1 DM) digestibility silage tended to reduce carcass fatness but had no effect with me-dium (D value about 650 g kg-1 DM) digestibility silage (Steen 1988b). It seems that the opportuni-ties to affect carcass fatness by protein supplement may be limited since the carcass fatness on grass silage-based feeding is also dependent on the qual-ity of the silage.

Eating quality

The results of the present study showed that the average sensory quality of the loins was assessed good or acceptable by the sensory panel. In fact, the analysed beef samples were evaluated as tender, quite juicy and quite tasty.

Beef from the bulls on diet LH had the best sensory quality and lowest shear force values, whereas EH beef had the lowest sensory quality and the highest shear force values. In this study, the measured average Warner-Bratzler shear force value of 4.3 kg cm-2 for the beef was lower than the average value of 5.5 kg cm-2 achieved in a pre-vious study in Finland (Honkavaara et al. 2003). The difference in these shear force values can be explained by prolongation of ageing time, which was 19 days (followed by freezing) in the former and ageing time of eight days (without freezing) in the latter. The former were Hf bulls, whereas most of the latter were milk breed bulls. In both cases, carcasses were suspended from the achilles tendon overnight. The method for shear force value measurement was also the same in both studies. Post mortem hanging of carcasses also affects meat shear force value. Keady et al. (2008) improved meat shear force value by aitch bone hanging in-stead of achilles tendon suspension. According to

Meisinger et al. (2006), liver-like off-flavours are specific to individual animals and pH and heme iron are not strongly related to off-flavour notes.

Economy

Price estimates have an important role in the eco-nomic comparison of feeding strategies. In this study, especially the price relation of E and L was an es-sential factor in the evaluation of different strategies.

Determination of the unit cost of silage requires information on both the first and the second cut of the sward. In this experiment, the re-growth was utilized as a pasture and the price setting had to be based on previous experiments. The quality of silage was not similar in the referred and in the present study but, as the quality effect was taken into consideration in the growth rates of the bulls, the subject of our interest was only the relative yield difference between E and L. For the determination of this difference, the earlier experiments were ap-plicable.

Berthiaume et al. (1996) concluded that in a steer’s diet it is technically possible to compensate for a lower forage digestibility by an addition of grain, but it is not necessarily economical. They based their statement on the average daily gain without including any price information in their analysis. Giving economic values to the inputs and outputs may change the result considerably as in-dicated in our study. Price relations are highly de-pendent on the economic environment where the beef producers operate and, therefore, the results of this study cannot be generalized. The study does, however, show that it is important to pay attention to the economic analysis, not only to growth rates, while seeking a profitable feeding strategy for fin-ishing bulls.

All the tested feeding strategies gave nearly the same return for a beef producer. Moreover, the sensitivity analysis proved that this result is very stable; the price changes affect more the level of the return than the ranking of the treatments. Thus, the beef producer can adjust the harvesting time and make the feeding decisions according to farm-



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specific resources and production conditions. The most important thing is to know the feeding value of the silage and, thus, be able to give a proper amount of protein supplementation to reach the intended growth rates.

Conclusions

Health was good and production performance high in the uninsulated housing conditions used. The results confirmed the importance of silage digestibility in the feeding of finishing beef bulls since early-cut silage improved the growth rate and shortened the finishing period significantly compared with later-cut silage. Animal performance was unaffected by the concentrate protein concentration and, thus, use of concentrate of higher protein concentration was not beneficial. All carcass traits were unaffected by the treatments. The eating quality of the tested loins was good and treatments had only a minor effect on the yield of valuable cuts. The same economic perform-ance was achieved with different feeding strategies, which allows the producers to adjust the feeding flexibly to the prevailing production conditions.

Acknowledgements. The authors are indebted to Mrs. Ulla Eronen and her staff for technical assistance during the ex-periment. The personnel of the Slaughterhouse of Lihakunta in Kuopio and the staff of the Finnish Meat Research Insti-tute in Hämeenlinna are thanked for their help in slaughter procedures and meat evaluation. Doctor Seija Jaakkola is warmly thanked for her comments on the manuscript. The commercial protein compound was supplied by Raisio Feed Ltd, which is gratefully acknowledged.

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Wiander, B. and Palva, A. Impact of mineral salt, garlic and algae on sauerkraut fermentation Vol. 20(2011): 169–175.

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© Agricultural and Food Science Manuscript received July 2009

Sauerkraut and sauerkraut juice fermented spontaneously using mineral salt, garlic and algae

Britta Wiander1* and Airi Palva2

1Finnish Food Safety Authority Evira, PO Box 111, FI-32201 Loimaa, Finland, 2University of Helsinki, Faculty of Veterinary Medicine, PO Box 66, FI-00014 Helsingin yliopisto, Finland

*e-mail: [email protected]

The use of mineral salt in natural fermentation of white cabbage into sauerkraut and sauerkraut juice, in order to evaluate whether the amount of NaCl could be lowered, was studied. Mineral salt differs from ordinary salt because NaCl is partially replaced by KCl. In the fermentations mineral salt was used in vari-ous amounts (0.8–1.5%) and in combination with garlic and algae. The final NaCl concentrations in these fermentation trials were 0.5–0.9%. Fermentations were also carried out with cabbage sliced to different degrees. The sauerkraut juice fermented by using 0.8% mineral salt (0.5% NaCl) was found to have the best sensory quality. The yield of sauerkraut juice increased as the coarseness of the cabbage mix decreased.

Key-words: sauerkraut, sauerkraut juice, salt, garlic, algae and seaweed

Introduction

Fermented sauerkraut has a long history and is gen-erally considered to be a health promoting product. Traditional sauerkraut fermentations have usually been carried out in the presence of rather high NaCl amounts. However, consumers who want to eat health promoting products prefer products with low sodium content.

The fermentation of white cabbage into sau-erkraut traditionally proceeds in the presence of NaCl. There are reports on using different salt concentrations (Delanoe and Emard 1971, Gango-padhyay and Mukherjee 1971, Mayer et al. 1973, Niven 1980). Low percentages of NaCl have been used in natural sauerkraut fermentations by Tolo-nen et al. 2002 and Viander et al. 2003. Sauerkraut fermentations with lowered salt percentages have also been studied by Pederson 1940, Fleming and


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McFeeters 1985, Trail et al. 1996, Johanningsmeier et al. 2007, Martinez-Villaluenga et al. 2009, Penas et al. 2010). Sauerkraut has been prepared utilising hydrolysed protein and a salt content of 1.0 - 4.5% (Hsu et al. 1984, Wedral et al. 1985). A patent has been worked out for making sauerkraut where part of the normally added salt is replaced by an alco-hol/acid mixture (Owades 1991). Delclos (Delclos 1992) has studied the use of a reduced NaCl con-centration in combination with lactic acid bacteria starter cultures, the NaCl concentration being 1%. Kimchi is traditionally produced by using NaCl, but there have been studies on replacing part of the NaCl with KCl (Choi et al. 1994).

In this study we have further optimised the use of mineral salt (containing 28% KCl and 57% NaCl) in natural fermentation of white cabbage. Various amounts of mineral salt (0.8–1.5%) were used with final NaCl concentrations of 0.5–0.9%. Fresh garlic was used in combination with 0.8% mineral salt. Garlic extract is known to inhibit the growth of moulds (Sutabhaha et al. 1992). Vacame algae in combination with 0.8% mineral salt were also used. It has been reported that fresh edible sea-weeds can be preserved by lactic acid fermentation and that the juice may be drained off at the end of the fermentation and replaced by sauerkraut juice to improve the sensory quality (Oltz and Hubert 1990). In this study white cabbage was also cut into slices of different size and fermented using 0.8% mineral salt to study the impact of the coarseness of the cabbage on the resulting yield of sauerkraut juice.


Fermentation trialsIn the first fermentation trial the amounts of mineral salt used were 0.8%, 1.2% and 1.5%. The treatments were carried out in duplicate. The sliced white cabbage (cultivar Nosomi) was fermented in steel vessels. Mineral salt was mixed with the sliced

cabbage, the slices were pressed tightly together and covered with a plastic film, on which water was poured to inhibit air from entering the cabbage mixture and CO2 from escaping from the mixture. The amount of sliced cabbage per vessel was 2.5 kg. Fermentations were carried out at 20 °C.

In the second fermentation trial the amount of mineral salt was 0.8%. Fresh domestic garlic (0.2%) or Vacame algae from Japan (1%) was add-ed to the sliced cabbage (cultivar Nosomi). The treatments were carried out in duplicate. One dupli-cate treatment was used as a control and contained neither garlic nor algae. Mixing of the salt, pressing of the cabbage mix (2.5 kg) and other fermentation conditions were as described above.

In the third fermentation trial 0.8% mineral salt was used and white cabbage (cultivar Erdeno) was cut into slices of various sizes. The treatments were carried out in duplicate. The sizes of the slices were approximately: 1 mm × 1 mm, 2 mm × 10 mm and 3 mm × 40 mm. The sliced cabbage (1 kg) was fermented in glass vessels and the fermentation conditions were as described above.

Mineral salt containing 28% KCl and 57% NaCl was used in all fermentation trials. The min-eral salt (Pansuola®) contains 57% sodium chlo-ride, 28% potassium chloride, 12% magnesium sul-phate, 2% lycine hydrochloride, 1% silicon dioxide and 0,0036% potassium iodide. The mineral salt has been produced for and marketed by Oriola Oy (Espoo, Finland).

Sampling

Samples were taken regularly during the fermen-tation processes by using sterile pipettes. To get representative samples, equal volumes of cabbage juice were taken from three different places in the fermentation vessels from a depth of approximately 5 cm. The three samples were mixed into one sam-ple. When the samples were taken the plastic film covering the cabbage mixture was partly carefully removed avoiding water from entering the cabbage mixture.



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Microbiological analyses

Lactic acid bacteria were enumerated by cultivation on M.R.S nutrition medium (Biokar Diagnostics, France or Difco Labs, USA) containing 0.02% sodium azide and 1.5% agar for 2–3 days at 30 °C. Yeasts and moulds were grown on Yeast extract glucose chloramphenicol agar (Difco Labs, USA) for 7 days at 25 °C. All microbiological analyses were carried out either in duplicate or triplicate.

Chemical analyses

The pH of the cabbage juice was measured by us-ing a pH-meter (RadiometerPHM93, Radiometer Analytical, Denmark) during the fermentation. Total acidity, given as total lactic acid, was measured by titration using 0.1 N NaOH with phenolphtalein as indicator. All chemical analyses were carried out either in duplicate or triplicate.

Sensory evaluation

The sensory quality of the sauerkraut juices was evaluated by a taste panel consisting of 5 trained persons. A scale of 1-5, where number 1 refers to not acceptable and number 5 to excellent taste and qual-ity, was used in the evaluation. The quality scale of Karlsruhe was used (Tuorila and Hellemann 1993).

Results

Optimisation of mineral salt concentra-tion in sauerkraut fermentation

The decrease in pH was more rapid in the treatments in which 1.5% mineral salt was used compared to the pH drop in the treatments with 0.8% mineral salt. The titratable acidity increased more rapidly during the fermentation process when using 1.5% mineral salt (Fig. 1). In the fermentations with

Fig. 1. Change of pH (squares) and amount of lactic acid (tri-angles) during spontaneous fer-mentation of sauerkraut at 20 °C using mineral salt. The symbols refer to the used mineral salt concentrations: 0.8% mineral salt (□ ▲), 1.5% mineral salt ( ■ ▲). Asteriks (*) refers to pressed sauerkraut juice. Mean values of two parallel samples are shown.

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0.8% and 1.2% mineral salt pH was similar in all pressed sauerkraut juices. In the treatments with 1.5% mineral salt the pH of the pressed juices was somewhat lower compared to the juices produced by fermentation with 0.8% and 1.2% mineral salt.

The number of lactic acid bacteria was highest and the number of yeasts and moulds was lowest in

Fig. 2. Number of lactic acid bacteria in sauerkraut juice enu-merated immediately after the juice was pressed after spon-taneous fermentation at 20 °C for 20 days using mineral salt. The bars refer to the mean val-ues and standard deviations of bacterial colony forming units (cfu) in three parallel juice sam-ples of juices with three differ-ent mineral salt concentrations: 0.8% mineral salt (□), 1.2% mineral salt (■) and 1.5% min-eral salt (■).

Fig. 3. Number of yeasts and moulds in sauerkraut juice pressed after spontaneous fermentation at 20 °C using mineral salt. The bars refer to the mean values and stand-ard deviations of colony forming units (cfu) of yeasts and moulds in three parallel juice samples of juices with three different mineral salt concentrations: 0.8% mineral salt (□), 1.2% mineral salt (■) and 1.5% mineral salt (■).

the fresh sauerkraut juices resulting from the treat-ments with 1.5% mineral salt (Fig. 2, 3).

The sensory evaluation of the sauerkraut juic-es showed that the best sensory quality was ob-tained by fermentation with 0.8% mineral salt (4-5 scores). This treatment resulted in a very smooth-tasting juice.

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The number of yeasts and moulds was lowest in the treatments with fresh garlic and highest in the control fermentations (Fig. 5).

The sensory evaluation of the sauerkraut juices showed that the sauerkraut juices having the best taste were the juices obtained from the treatments with added garlic (3–4 scores) and the control (4–5 scores) sauerkraut juices. The taste panel did not find the juices produced from the fermentations with algae (2 scores) very appealing, even though they were considered acceptable.

Fig. 4. Change of pH (squares) and amount of lactic acid (tri-angles) during spontaneous fer-mentation of sauerkraut at 20 °C using mineral salt with addition of garlic or Vacame algae. The symbols refer to the used min-eral salt concentration in com-bination with garlic or Vacame algae: 0.8% mineral salt + 0.2% garlic ( □▲ ), 0.8% mineral salt + 1% Vacame algae ( ■▲ ) and 0.8% mineral salt with no add-ed garlic nor Vacame algae ( ■▲ , control fermentation). Mean values of two parallel samples are shown.

Fig. 5. Number of yeasts and moulds in sauerkraut juice pressed after spontaneous fer-mentation at 20 °C using min-eral salt. The bars refer to the mean values and standard devi-ations of colony forming units (cfu) of yeasts and moulds in three parallel juice samples of three different juices with 0.8% mineral salt in combination with garlic or Vacame algae: 0.8% mineral salt + 0.2% garlic (□), 0.8% mineral salt + 1% Vacame algae (■) and 0.8% mineral salt with no added garlic nor Vacame algae (■ , control fermentation).

Garlic and algae supplements

When 0.8% mineral salt was used in combination with 0.2% fresh garlic pH decreased somewhat more rapidly compared to the treatments with 1% Vacame algae. The pH decreased most rapidly at the beginning of the control fermentations with no addition of either garlic or algae (Fig. 4).

The titratable acidity increased more slowly in the treatments with garlic and algae compared to the control fermentations. At the end of the fer-mentations the titratable acidity was highest in the treatments with garlic (Fig. 4).

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Cabbage slicing

The pH decreased rather similarly in all treatments, except for a somewhat slower decrease at the begin-ning of the treatments in which the cabbage was sliced into a very fine mix (approximately 1 mm × 1 mm). The titratable acidity increased most in the treatments with the cabbage shredded into a very fine mix (Fig. 6).

The highest yield of pressed sauerkraut juice was obtained from the treatments with the cabbage cut into a very fine mix (1 mm × 1 mm) being nearly 80%. When the size of the cabbage slices was 2 mm × 10 mm and 3 mm × 40 mm the yield of pressed sauerkraut juice was 70% and 60% re-spectively.

Discussion

The preliminary results of this study show that it is possible to produce sauerkraut and sauerkraut juice by natural fermentation using mineral salt (0.8%,

Fig. 6. Change of pH (squares) and amount of lactic acid (tri-angles) during spontaneous fermentation of sauerkraut at 20 °C using 0.8% mineral salt. The symbols refer to the differ-ent sizes of the fermented cab-bage slices: 1 mm x 1 mm ( □▲ ), 2 mm x 10 mm ( ■▲ ) and 3 mm x 40 mm ( ■▲ ). Mean values of two parallel samples are shown.

1.2% and 1.5%) with final NaCl concentrations of 0.5%, 0.7% and 0.9%. All the sauerkraut juices were found to have a smoother taste compared to sauerkraut juices produced by using ordinary salt. However, the preliminary sensory evaluation of the sauerkraut juices showed that the best sensory quality was obtained by fermentation using 0.8% mineral salt resulting in a very smooth-tasting juice. On the other hand the number of lactic acid bacteria was highest when 1.5% mineral salt was used in the fermentation trials and the number of yeast and moulds was lowest in these trials. Although the used mineral salt amounts were low the fermentation process proceeded well and the pH decreased to the desired level, pH 3.8 in a time of 20–25 days. The highest yield of pressed sauerkraut juice was obtained from the treatments where the cabbage was cut into a very fine mix, the cabbage slices being approximately 1 mm × 1 mm.

The preliminary sensory evaluation results show that it is possible to produce sauerkraut and sauerkraut juice with low sodium content with gar-lic and algae supplements. Consumers who want to consume fermented vegetable products with low sodium content would perhaps find these kinds of products interesting.

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Acknowledgements. The authors are grateful to Dr. Ilkka Palva for participating in this study. We also wish to thank Mrs. Pirjo Satka for technical assis-tance and Mrs. Heli Vähä-Touru for assistance in preparing the graphics. Furthermore, we are thankful to Arktinen Bio-Lacto Oy for delivering the cabbage needed in the fermentation trials.

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Delanoe, R. & Emard, L.O. 1971. Experimental manufac-ture of sauerkraut in Quebec. Quebec Laitier et Alimen-taire 30: 11–14.

Delclos, M. 1992. Vegetable preservation by a mixed or-ganic acid fermentation. Dissertation Abstracts Inter-national -B 52: 4537.

Fleming, H.P. & McFeeters, R.F. 1985. Residual sugars and fermentation products in raw and finished commer-cial sauerkraut. New York State Agricultural Experiment Station Special Report 56: 25–29.

Gangopadhyay, H. & Mukherjee, S. 1971. Effect of differ-ent salt concentrations on the microflora and physico-chemical changes in sauerkraut fermentation. Journal of Food Science and Technology 8: 127–131.

Hsu, J.Y., Wedral, E.R. & Klinker, W.J. 1984. Preparation of sauerkraut utilizing hydrolysed protein. United States Patent US4428968.

Johanningsmeier, S.D., McFeeters, R.F., Fleming, H.P. & Thompson, R.L. 2007. Effects of Leuconostoc me-senteroides starter culture on fermentation of cabbage with reduced salt concentrations. Journal of Food Sci-ence 72: M166–M172.

Martinez-Villaluenga, C., Penas, E., Frias, J., Ciska, E., Honke, J., Piskula, M.K., Kozlowska, H. & Vidal-Val-verde, C. 2009. Influence of fermentation conditions on glucosinolates, ascorbigen, and ascorbic acid content in white cabbage (Brassica oleracea var. capitata cv. Tal-er) cultivated in different seasons. Journal of Food Sci-ence 74: C62–C67.

Mayer, K., Pause, G. & Vetsch, U. 1973. Bildung biogener Amine während der Sauerkrautgärung. Industrielle Obst und Gemüseverwertung 58: 307–309.

Niven, C.F. 1980. Technology of sodium in processed foods: general bacteriological principles, with empha-sis on canned fruits and vegetables, and dairy foods. In: Sodium and potassium in foods and drugs, Na & K Sym-posium. American Medical Association, USA. p. 45–48.

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Penas, E., Frias, J., Sidro, B. & Vidal-Valverde C. 2010. Chemical evaluation and sensory quality of sauerkrauts obtained by natural and induced fermentations at differ-ent NaCl levels from Brassica oleracea var. capitata cv. Bronco grown in eastern Spain. Effect of storage. Jour-nal of Agricultural and Food Chemistry 58: 3549–3557.

Pederson, C.S. 1940. The relation between quality and chemical composition of canned sauerkraut. New York State Agricultural Experiment Station Bulletin 693: 1-15.

Sutabhaha, S., Suttajit, M. & Niyomca, P. 1992. Studies of aflatoxins in Chiang Mai, Thailand. Kitasato Archives of Experimental Medicine 65: 45–52.

Tolonen, M., Taipale, M., Viander, B., Pihlava, J.-M., Kor-honen, H. & Ryhänen, E.-L. 2002. Plant-derived biomol-ecules in fermented cabbage. Journal of Agricultural and Food Chemistry 50: 6798–6803.

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Viander, B., Mäki, M. & Palva, A. 2003. Impact of low salt concentration, salt quality on natural large-scale sau-erkraut fermentation. Food Microbiology, 20: 391–395.

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© Agricultural and Food Science Manuscript received February 2010

Preliminary studies on using LAB strains isolated from spontaneous sauerkraut fermentation in

combination with mineral salt, herbs and spices in sauerkraut and sauerkraut juice fermentations

Britta Wiander1* and Hannu J.T. Korhonen2

1Finnish Food Safety Authority Evira, P.O. Box 111, FI-32201 Loimaa, Finland, 2MTT Agrifood Research Finland, Food Research, ET-talo, FI-31600 Jokioinen, Finland

*e-mail: [email protected]

The use of mineral salt, herbs and spices in combination with isolated lactic acid bacteria strains in sauer-kraut fermentation was studied. Mineral salt differs from ordinary salt because NaCl is partially replaced by KCl. The mineral salt contains 28% KCl and 57% NaCl. The final NaCl content in the sliced white cabbage mixture was 0.5%. In approximately 20 hours the pH dropped to the desired level. All the pressed sauerkraut juices had a good microbiological quality. The sensory quality of all pressed juices was found to be either good or acceptable.

Key-words: sauerkraut, sauerkraut juice, salt, aniseed, fennel seeds, caraway, dill, garlic, mint

Introduction

Even though sauerkraut is mostly produced by spontaneous fermentation starters are also used. The selection criteria for lactic acid bacteria used in vegetable and vegetable juice fermentations

has been summarised in 1993 by Buckenhüskes (Buckenhüskes 1993, Daeschel et al. 1987, Lücke et al. 1990, Buckenhüskes 1992). Daeschel and Fleming (1984) have also discussed the selec-tion of lactic acid bacteria to be used in vegetable fermentations and the use of starters has further been studied in several works (Frank 1973, Flem-


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ing et al. 1985, Buckenhüskes et al. 1986, Delclos 1992, Harris et al. 1992, Breidt et al. 1993, Breidt et al. 1995, Halasz et al. 1999, Savard et al. 2000, Gardner et al. 2001, Tolonen et al. 2002, Wiander and Ryhänen 2005, Johanningsmeier et al. 2007, Wiander and Ryhänen 2007, Wiander and Ryhänen 2008, Martinez-Villaluenga et al. 2009, Penas et al. 2010). Commercial starters are available on the mar-ket, but they are not widely used in sauerkraut and sauerkraut juice fermentations. The most common reasons have been discussed by Lücke et al. (1990) and Hammes (1991). In spite of the advantages the use of starters also increases fermentation costs. One of the benefits of commercial starters according to Lücke et al. (1990) is that the starters ensure the accurate proceeding of the fermentation process.

There are studies in which lactic acid bacteria strains have been isolated from spontaneous sau-erkraut fermentations (Stetter and Stetter 1980, Valdez de et al. 1990, Harris et al. 1992, Wiander and Ryhänen 2008) and further studies in which such isolated lactic acid bacteria strains have been used as starters in the fermentation of sauerkraut (Breidt et al. 1993, Tolonen et al. 2002, Wiander and Ryhänen 2008).

The fermentation of white cabbage into sau-erkraut traditionally proceeds in the presence of NaCl. There are reports on using different salt concentrations (Delanoe and Emard 1971, Gango-padhyay and Mukherjee 1971, Mayer et al. 1973, Niven, 1980). Low concentrations of NaCl (0.3% and 0.5%) have been used in sauerkraut fermenta-tions (Viander et al. 2003, Wiander and Ryhänen 2005, Tolonen et al. 2002) and NaCl concentra-tions of 0.6% have also been used (Pederson 1940, Fleming and McFeeters 1985, Trail et al. 1996). Sauerkraut has been prepared utilising hydrolysed protein and a salt content of 1.0 – 4.5% (Hsu et al. 1984, Wedral et al. 1985). A patent has been worked out for making sauerkraut where part of the normally added salt is replaced by an alcohol/acid mixture (Owades 1991). Delclos (1992) has studied the use of a reduced NaCl concentration in combination with lactic acid bacteria starter cul-tures, the NaCl concentration being 1%. Kimchi is traditionally produced by using NaCl, but there

have been studies on replacing part of the NaCl with KCl (Choi et al. 1994).

In this work we studied the impact of low concentration of mineral salt, different herbs and spices in combination with isolated lactic acid bac-teria strains on the fermentation of white cabbage into sauerkraut and sauerkraut juice. The herbs and spices used in this study were aniseed, fennel seeds, caraway, dill, garlic and mint. Mineral salt with low sodium content was used because this is in line with the general trend in industrialized coun-tries of reducing the salt level of foods to prevent cardiovascular diseases.


Fermentation trialsIn the first trial 0.9% mineral salt was used in com-bination with isolated Leuconostoc mesenteroides (105 cfu/g) and isolated Pediococcus spp. Nr. 4 (103 cfu/g). Fresh mint (2%) was used as a supplement. The treatments were carried out in triplicate and one triplicate treatment without mint was used as a control. Hungarian cabbage was used in the treat-ments and the amount of sliced cabbage per vessel was 6 kg. The fermentation vessels were made of steel. Mineral salt was mixed with the sliced cab-bage, the slices were pressed tightly together and covered with a plastic film, on which water was poured to inhibit air from entering the cabbage mixture and CO2 from escaping from the mixture. Fermentations were carried out at 20 °C.

In the second trial 0.9% mineral salt was used and the sliced cabbage was inoculated with isolated Leuconostoc mesenteroides (1.36 x 106 cfu/g) and isolated Pediococcus dextrinicus (1.02 x 106 cfu/g). Domestic aniseed, fennel seeds, caraway, dill and garlic were used as supplements (1%). Domes-tic cabbage was used in these treatments and the amount of sliced cabbage per vessel was 1.6 kg. Each treatment was carried out once. The fermenta-tion conditions were otherwise as described above, but the fermentation temperature was 21 °C.


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Mineral salt containing 28% KCl and 57% NaCl was used in all treatments. The mineral salt (Pansuola®) contains 57% sodium chloride, 28% potassium chloride, 12% magnesium sulphate, 2% lycine hydrochloride, 1% silicon dioxide and 0,0036% potassium iodide. The mineral salt has been produced for and marketed by Oriola Oy (Es-poo, Finland).

Isolation, identification and enrichment of LAB strains

The Leuconostoc mesenteroides and Pediococcus dextrinicus strains were isolated from spontaneous sauerkraut fermentations and identified in a previous study (Wiander and Ryhänen 2008).

Pediococcus spp. Nr. 4 was isolated from spon-taneous sauerkraut fermentation and identified by using the API test consisting of API 50 CH strips and API 50 CHL Medium (bioMérieux sa, France).

The isolated lactic acid bacteria strains used in the fermentation trials were enriched in autoclaved juice pressed from sliced raw white cabbage for 2 – 3 days.

Inoculation of cabbage and sampling

The sliced white cabbage mixed with herbs or spices was inoculated either with isolated Leuconostoc mesenteroides and isolated Pediococcus dextrinicus or isolated Leuconostoc mesenteroides and isolated Pediococcus spp. Nr. 4.

Samples were taken regularly during the fer-mentation processes by using sterile pipettes. To get representative samples, equal volumes of cab-bage juice were taken from three different places in the fermentation vessels from a depth of approxi-mately 5 cm. The three samples were mixed into one sample.

Microbiological, chemical and sensory analyses

Lactic acid bacteria were enumerated by cultivation on M.R.S nutrition medium (Biokar Diagnostics, France or Difco Labs, USA) containing 0.02% sodium azide and 1.5% agar for 2–3 days at 30 °C. Yeasts and moulds were grown on Yeast extract glucose chloramphenicol agar (Difco Labs, USA) for 7 days at 25 °C. Enterobacteria were cultivated on Violet red bile agar (Biokar Diagnostics or Difco Laboratories) supplemented with 0.1% glucose and grown for 2-3 days at 37 °C. All microbiological analyses were carried out either in duplicate or triplicate.

The pH of the cabbage juice was measured by using a pH-meter (RadiometerPHM93, Radiometer Analytical, Denmark) during the fermentation. To-tal acidity, given as total lactic acid, was measured by titration using 0.1 N NaOH with phenolphtalein as indicator. All chemical analyses were carried out either in duplicate or triplicate.

The sensory quality of the sauerkraut juices was evaluated by a taste panel consisting of 3 trained persons.

Results

Isolated lactic acid bacteria strains in combination with mineral salt and mint

The number of lactic acid bacteria in the raw mate-rial was enumerated before the fermentation started and was found to be 10 cfu/g in the cabbage and 15 cfu/g in the mint. The number of yeasts and moulds was 2 250 cfu/g in the cabbage and 13 500 cfu/g in the mint and the number of enterobacteria in the cabbage and mint was 148 cfu/g and 440 cfu/g respectively.

The pH was measured during the fermentation process and was found to decline slightly slower in the mint treatments compared to the control treat-



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ments. However, in approximately 25 hours the pH in all treatments had decreased to 4.0 (Fig. 1).

The number of lactic acid bacteria was enu-merated during the fermentation and the number was higher in the pressed sauerkraut juice from the fermentations with mint (0.23 x 108 cfu/ml) compared to the juices produced without mint (0.17 x 106 cfu/ml).

The number of yeasts and moulds decreased radically as the fermentation process proceeded and was in the pressed sauerkraut juice produced from the fermentation with mint 12 cfu/ml and in the sauerkraut juice produced without mint 22 cfu/ml.

No enterobacteria were detected in any of the pressed sauerkraut juices.

Isolated lactic acid bacteria strains in combination with mineral salt and sup-

plements

In the treatments with aniseed, fennel seeds, caraway, dill and garlic supplements pH was measured and the decrease in pH was quite similar in all treat-ments, except for the treatment with garlic. The reduction in pH was slower in the treatment with garlic compared to the other treatments. However, in 24 hours the pH in all treatments had decreased to 4.0 (Fig. 2).

The growth of lactic acid bacteria was slowest in the treatment with garlic as supplement (Fig. 3).

The number of yeasts and moulds decreased rapidly during the fermentation and no yeasts and moulds or enterobacteria were detected in any of the pressed sauerkraut juices.

Fig. 1. Change of pH during fermentation of sauerkraut at 20°C by using a mixture of Pediococcus spp. Nr. 4 and Leuconostoc mesenteroides with (○) or without (□) added mint (mean values and stand-ard deviations).

Fig. 2. Change of pH dur-ing fermentation of sauer-kraut at 21°C by using a mix-ture of Leuconostoc mesenter-oides and Pediococcus dextrin-icus with added herbs or spic-es. ( ○ ) aniseed, ( □ ) fennel seeds, (▲) caraway, (○) dill, (□) garlic and (▲) control with-out added herbs or spices.

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Sensory evaluationThe preliminary results of the sensory evaluation suggest that the kind of sauerkraut juices produced in this study could be accepted by the consumers. The taste of the pressed sauerkraut juices produced from the treatments with mint, aniseed, fennel seeds, and dill was considered the best (5 scores of 5), garlic was considered good (4 scores of 5), whereas the taste of the juice from the treatment with caraway was not considered very appealing although it was acceptable (2 scores of 5). The taste of the sauerkraut juices produced from the control fermentations was considered good (4 scores of 5).

Discussion

The results of this preliminary study show that it is possible to produce sauerkraut and sauerkraut juice of good quality and with a pH of approximately 3.8 by using mineral salt with a final concentration of 0.5% NaCl. The sauerkraut juices were considered to have a smoother taste compared to sauerkraut juices produced by using ordinary NaCl which is in line with earlier studies (Tolonen et al. 2002, Viander

et al. 2003, Wiander and Ryhänen 2005, Wiander and Ryhänen 2008). By using herbs and spices in combination with isolated lactic acid bacteria strains as starters and mineral salt with a low NaCl content it was possible to produce sauerkraut and sauerkraut juices with a good microbiological and sensory quality. The pH dropped in all treatments below 4 in 20 - 25 hours which is a considerably shorter fermentation time compared to what is needed in natural sauerkraut fermentations. This study also showed that isolated Pediococcus spp. Nr. 4 can be used in sauerkraut fermentations with good results.

These products with low sodium content are in accordance with the general trend in trying to lower the salt content in foods and promote the consumption of healthier foods. The products are alternatives to consumers who want to eat ferment-ed products with low sodium content.

Acknowledgements.The authors are grateful to Dr. Ilkka Palva for participating in this study. We also wish to thank Mrs. Pirjo Satka for technical assist-ance and Mrs. Heli Vähä-Touru for assistance in preparing the graphics. Furthermore, we are thankful to Arktinen Bio-Lacto Oy for delivering the cabbage needed in the fermentation trials.

Fig. 3. Number of lactic acid bac-teria during fermentation of sau-erkraut at 21°C by using a mix-ture of Leuconostoc mesenter-oides and Pediococcus dextrin-icus. ( ○ ) aniseed, ( □ ) fennel seeds, (▲) caraway, (○) dill, (□) garlic and (▲) control with-out added herbs or spices.

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© Agricultural and Food Science Manuscript received November 2010

Impact of temperature and germination time on the success of a C4 weed in a C3 crop: Amaranthus

retroflexus and spring barley Terho Hyvönen

MTT Agrifood Research Finland, Plant Production Research, FI-31600 Jokioinen, Finland

e-mail: [email protected]

Elevation in temperatures due to climate change could promote the invasion by C4 weed species of arable fields in the boreal region, which are dominated by C3 crops. The success of Amaranthus retroflexus L. (a C4 weed) in spring barley (a C3 crop) was studied at current and elevated temperatures (3 °C difference) in a greenhouse experiment in southern Finland. The competition treatments included no competition and four levels of competition with barley, differing in terms of germination time. The success of A. retroflexus was measured as growth (height and biomass) and seed production (number and biomass). Elevation in temperature enhanced seed production of A. retroflexus, but the impact on growth was minor (only dif-ference in plant height in one treatment). The growth and seed production of A. retroflexus in competition with barley was minimal although the growth of barley decreased with the rise in temperature. The results indicate that climate change could improve growth of a C4 weed such as A. retroflexus, but it is unlikely to succeed in spring barley.

Key-words: arable weed, climate change, seed production, species invasion

Introduction

Climate change has been predicted to broaden the distribution areas of arable weed species (Patter-son 1995). A successful range expansion of arable weeds requires not only adaptation to the climatic

conditions of a new region but also colonisation of cultivated fields. Therefore, crop-weed interac-tions must be considered in connection with range expansion of arable weeds as a consequence of global warming.

The key issue to understanding crop-weed in-teractions with respect to climate change lies in the


Hyvönen, T. Amaranthus retroflexus and spring barley

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different responses of C3 and C4 species to eleva-tion in temperature and CO2 concentration (Pat-terson and Flint 1980, Ziska 2001, 2003). C3 plants benefit from the elevated CO2 levels, whereas C4 plants benefit from the elevated temperature (Pear-cy et al. 1981, Sage and Kubien 2007). As a result climate change will affect crop-weed interactions disproportionately depending on the photosynthet-ic pathway of the crop and weed species (Ziska 2001, 2003). C3 and C4 species occupy a temporal niche in natural vegetation that is determined by temperature (see Sage and Pearcy 2000). This is also evident in arable weed communities since C4 weeds usually occur in C4 crops (e.g. Schroeder et al. 1993, Bunce and Ziska 2000), which are alike with respect to temperature requirements for germination and growth. Since the majority of the “world’s worst weeds” are C4 plants (Holm et al. 1977) and most crops are C3 plants (Pat-terson 1995), global warming can be assumed to strengthen the competition from these weeds in the future (Ziska 2003, Tungate et al. 2007). Regard-ing range expansion, the pressure on C4 weeds to invade northern regions, where they are currently rare, can be assumed to increase (Holm et al. 1997). This will require, however, the colonisation of C3 crops by C4 weeds.

One of the most widely distributed C4 weeds in the world is Amaranthus retroflexus L., which is found in 60 crops in 70 countries (Holm et al. 1997, Costea et al. 2004). The main distribution area of A. retroflexus in Europe is in southern and central Europe, the northern border of the distribu-tion area being in the Nordic and Baltic countries. At the margins of the distribution area, including Finland and Estonia, A. retroflexus is regularly found as a casual alien (Hämet-Ahti et al. 1998, Kukk and Kull 2005), i.e. a species that cannot es-tablish permanent populations and is dependent on the regular supply of new seeds (Richardson et al. 2000). Based on its current distribution, A. retro-flexus can be regarded as one of the most probable C4 weed species to extend its distribution into the boreal region as a consequence of climate change. Previous studies showed that elevation in tem-perature increases growth of A. retroflexus (Guo and Al-Khatib 2003) but decreases seed produc-

tion (Weaver 1984), indicating opposing respons-es regarding success as a consequence of climate change. Seed production is lowered in competition with a crop (McLachlan et al. 1995). The success of A. retroflexus as a competitor with C3 cereals has not been established experimentally, but field observations suggest it is poorer than with C4 crops (Costea et al. 2004).

The aim of the present study was to explore the growth response and the seed production of A. retroflexus at an elevated temperature with and without competition from a C3 crop (spring bar-ley). The study was conducted in Finland, where the dominant C3 crop species is spring barley and which represents the northern distribution limit of A. retroflexus. Since the timing of germination is a critical factor affecting crop-weed competition (Knezevic et al. 1997) and the optimum germina-tion temperature for A. retroflexus is high compared with that for barley (Ghorbani et al. 1999, Guo and Al-Khatib 2003), which indicates later germination and emergence times, several germination dates for A. retroflexus were included in the study. I expected the elevation in temperature to enhance growth but decrease seed production of A. retroflexus.

Material and Methods

Plant materialThe seeds of Amaranthus retroflexus were collected from southern Sweden (55°45´ N; 13°19´ E) in Sep-tember 2004. In summer 2008, the seeds were sown in pots and grown both outdoors and in a greenhouse in Jokioinen, southern Finland (60°49’ N; 23°29’ E) in order to produce seeds for the experiment. The seeds were stored at +4 °C but kept at -18 °C for two days in October 2008. The seeds of spring barley, Hordeum vulgare L., cultivar Rolfi, were obtained from a local agricultural market.

The plants for each treatment (see below) were grown in 3.5 l pots (diameter 18 cm) in a com-mercial peat-sand mixture (‘Kylvöseos’ by Kek-kilä Oyj, Mellilä, Finland; pH 6.0, N: 70 mg l-1, P:



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21 mg l-1, K: 140 mg l-1) and fine sand (grain size 0.2-1 mm) at a ratio of 2:1. The pots were watered regularly and fertilised (a mixture of 2.5 kg ferti-lization [N-P-K content 14-5-21 %, respectively] with 25 litres water mixed with irrigation water in a proportion of 1:50) 7 times during the experi-ment (total amount of fertiliser-water mixture per pot was 2.7 l).

The sowing density of barley was 12 seeds per pot sown in two rows (row width 7 cm), the same sowing density as in field cropping, and one seed or seedling of A. retroflexus placed in the middle of the pot. The seeds of A. retroflexus were either sown in the pot (treatments 1 and 2) or planted as 1–2 cm long seedlings (treatments 3–5). The seed-lings were germinated on Petri dishes in a growth chamber at a temperature regime of 35/10 °C (16/8 h) with full light. If a seedling of A. retroflexus died within two weeks after the sowing date it was replaced by a new seedling of the same age grown in the same greenhouse. Seventy-seven (32.1% of all plants in the experiment) such replacements were made during the experiment. At the end of the experiment, 12 A. retroflexus individuals were dead of which 9 were replacement seedlings. Two dead barley plants were also replaced with new seedlings after 10 days from the beginning of the experiment.

Experimental design

The greenhouse experiment was conducted in Jokio-inen between 10th February and 8th May 2009. The experiment was conducted in four greenhouses of which two had a temperature regime of 20/10 °C (18/6 h) (hereafter, current) and the other two 23/13 °C (18/6 h) (hereafter, elevated). The temperature regimes were randomized among greenhouses. The temperature regime with lower values was in line with the long-term (1971–2000) average minimum and maximum temperatures for the summer months (June–August) recorded at Jokioinen meteorological station (Drebs et al. 2002). The 3 degree elevation in temperature conforms to the predicted median temperature value for summer months (June-August)

for the time period 2040–2069 according to four climate scenarios (Jylhä et al. 2004). The light/dark regime was 19/5 h for both temperature regimes, equivalent to the day length for Jokioinen in June.

The experiment included the following treat-ments: 1) A. retroflexus seedling planted on the 1st day of the experiment, no barley, 2) A. retroflexus seedling planted and barley sown on the 1st day of the experiment, 3) A. retroflexus seedling planted on the 7th and barley sown on the 1st day of the ex-periment, 4) A. retroflexus seedling planted on the 14th and barley sown on the 1st day of the experi-ment, 5) A. retroflexus seedling planted on the 21st and barley sown on the 1st day of the experiment and 6) Barley sown on the 1st day of the experiment, no A. retroflexus. Treatments 1–5 were placed on the same table as a row column design with three replicates (10 cm between pots). Four such tables were placed in each greenhouse (n = 12 for each treatment per greenhouse). The distance between the tables was about 1 metre. Barley control (treat-ment 6) consisted of two pots placed in close prox-imity to each table (n = 8 per greenhouse).

Measurements and data analyses

At the end of the experiment, plant height, above ground biomass (air-flow drying at 40 °C) and number and weight of seeds of A. retroflexus were recorded. The height of each barley plant and the above ground biomass of 12 barley plants per pot were also measured. Prior to analyses, the heights of barley plants were averaged and the data were pooled for treatments 1–5. The averages for barley controls were calculated for above ground biomass and height.

The data were analysed using linear mixed models. The treatment and greenhouse were fixed factors and the interaction of greenhouse and table a random factor in the models used for the analyses of all barley data sets and the height of A. retroflex-us. However, for the data sets of seed number and weight, as well as for the biomass of A. retroflexus, comparisons within treatment 1 only were possi-ble due to missing data (seeds) or highly skewed



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data distributions (biomass). In these analyses, the greenhouse was a fixed factor and the interaction of greenhouse and table a random factor. In both anal-yses, the paired comparisons between temperatures within treatment or between treatments within tem-perature were made using two-sided t-tests. All the A. retroflexus data were square root-transformed and the height and biomass data for barley (treat-ments 1–5) log (x+1) transformed prior to analyses. The statistical analyses were performed using the MIXED procedure in SAS (Littell et al. 2006).

Results

Seed production of A. retroflexus was affected both by competition with barley and temperature. The impact of competition was so strong that only 9 A. retroflexus plants in treatment 2, 2 plants in treatment 3 and a single plant in treatments 4 and 5 produced seeds. The majority of these cases were in the greenhouses at an elevated temperature: only one plant produced seeds in competition with barley at the current temperature. Due to the scattered data for seed production, the impact of competition and

Fig. 1. The number and biomass of Amaranthus retroflexus seeds per plant (mean and SD) at the two temperature regimes.

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the interaction of competition and temperature could not been verified using statistical testing. In the comparison of treatment 1 (i.e. no competition with barley) at different temperatures, a greater number (1655 ± 896 vs. 5465 ± 2711 seeds per plant, t = 4.3, df = 12, p < 0 .001) and weight (0.7 ± 0.4 vs. 2.1 ± 1 g per plant, t = 4.2, df = 12, p < 0.01) of seeds were associated with an elevated rather than a current temperature (Fig. 1).

The biomass of A. retroflexus did not differ be-tween temperature treatments (treatment 1: t = 1.8, df = 12, p < 0.05). The impact of temperature on the height of A. retroflexus was dependent on the competition treatment (treatment and temperature interaction F4, 48 = 4.8, p < 0.01), the height be-ing greater (t = -5.61, df = 59.3, p < 0.001) only for treatment 2 (Fig. 2). For barley, the elevated temperature resulted in both lower biomass and re-duced height for all treatments (biomass: t = 3.6-4.1, df = 46.2, P < 0.001; height: t = 5.7-6.9, df = 17.7, p < 0.001). The same pattern was detected in control barleys, where both height (63.3 ± 3.6 vs. 55.6 ± 2.8 cm) and biomass (34.8 ± 2.3 vs. 30.8 ± 1.8 g) were higher at current rather than at an elevated temperature (height: t = -5.2, p < 0.001; biomass: t = -4.8, P < 0.001).



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Fig. 2. The height and biomass of Amaranthus retroflexus and barley individuals (mean and SD). In T1, no barley included. The treatments T2-T5 differed in terms of sowing date of A. retroflexus.

Discussion

Contrary to expectations, elevation in the tem-perature enhanced seed production and had a minor impact on the growth of the C4 weed A. retroflexus.

The growth and seed production of A. retroflexus in competition with a C3 crop, spring barley, was minimal although barley growth was decreased by the elevation in temperature. The results indicate that climate change could improve the success of A. retroflexus populations but success in spring barley is unlikely.



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Previous studies showed C4 weeds to respond positively to elevation in temperature (Guo and Al-Khatib 2003). However, the temperature optimum for photosynthesis of C4 plants is typically high (Sage and Kubien 2007); for A. retroflexus over 35 °C (Pearcy et al. 1981). This suggests that the maximum temperature used in the present study (23 °C) was too low to give an advantage to A. retroflexus in terms of photosynthetic rate. Interest-ingly, the 3 degree elevation in temperature used in the present study was high enough to limit the growth of barley, in accordance with earlier find-ings (Peltonen-Sainio et al. 2010, 2011). This sug-gests a decline in the competitive ability of spring cereals against weeds, in a future climate. For C4 weeds this would mean potential improvement in the success in the crop rotations typical of boreal regions. The results of the present study, however, suggest that even these changes will not promote the success of A. retroflexus in competition with spring cereals in the boreal region.

Previous competition experiments (e.g. Kneze-vic et al. 1997) showed considerable yield losses due to competition by A. retroflexus. In the present study, yield loss was not evident but A. retroflexus was unable to effect any decline in the biomass of barley either. Previous studies included crops other than spring cereals as study species. Under field conditions A. retroflexus is more common in such crops than in cereals (e.g. Costea et al. 2004), which suggests the growth patterns to be more alike with these species. One of the factors related to growth patterns is the germination tem-perature which determines the emergence patterns of seedlings. In the present study, the germination temperature (35 °C) was high compared with the day temperatures used during growth (20 or 23 °C). Under field conditions, A. retroflexus would have germinated later than barley. Therefore, the treatment used in the study benefited A. retroflexus. However, the seedlings of A. retroflexus were ger-minated on Petri dishes and the seedlings planted in the pots to ensure the same germination date for each individual within treatments. This procedure appeared not to be successful since one third of the seedlings had to be replaced with new seedlings during the experiment. This likely contributed to

the poor success of A. retroflexus in competition with barley.

Even though A. retroflexus did not affect de-crease in barley biomass, seed production showed a positive response to elevation in temperature. The finding was contradictory to previous findings by Weaver (1984), who reported an increase in seed production when the temperature regime was changed from 28/22 °C to 22/14 °C. The latter tem-perature regime is comparable with the elevated temperature regime used in this study. Therefore, the results are not entirely contradictory. The seed production level recorded in the present study was lower compared with those reported earlier (Weav-er 1984, Costea et al. 2004). The increase in seed production at the elevated temperature suggests an improvement in the success of establishment of permanent populations. The finding suggests that some individual plants could produce seeds even in competition with barley. However, the building up of the populations, which would effect consid-erable yield losses, would require a crop rotation including a crop species less competitive against A. retroflexus (e.g. sugar beet). Even though the present study focused only on seed production, neglecting many components of the population demography (e.g. Morse and Bazzaz 1994), the suggestion is that A. retroflexus could survive un-der these conditions.

Range expansion of C4 weeds into the boreal re-gion of Europe as a consequence of global warming is of concern. Several C4 weed species comprise an important part of the weed community in central Europe, but C4 weed species typically occur in C4 crops (Schroeder et al. 1993). The results of the present study suggest that A. retroflexus is unlikely to take hold in spring cereals. This is confirmed by the field observations: none of the 188 weed species found in a Finnish weed survey (Salonen et al., 2001) was a C4 species. In addition, none of the C4 species belong to the most important weeds of C3 crops in Europe (Schroeder et al. 1993). The results suggest that even though the 3 degree eleva-tion in average temperature decreased the growth of a C3 crop and improved the seed production of a C4 weed, C3 crop species could act as a barrier to invasion by the “world’s worst weeds” into the



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boreal region. Furthermore, climate warming will advance the sowing times of C3 crops (Kaukoranta and Hakala 2008), thus reinforcing the competitive benefit of C3 crops in spring time. The invasion of C4 weeds cannot be expected to take place before the introduction of C4 crops (e.g. maize), which has been predicted to occur not before the end of this century in Finland (Peltonen-Sainio et al. 2009).

Acknowledgements. I am indebted to Lars Andersson for providing Amaranthus seeds for the experiment, Timo Hurme and Lauri Jauhiainen for their help in planning the experimental design and conducting the statistical analyses, and to Auli Kedonperä and Tuula Viljanen for taking care of the greenhouse experiment. The study was financed by the Academy of Finland (project number 122477).

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AGRICULTURAL AND FOOD SCIENCE

A G R I C U L T U R A L A N D F O O D S C I E N C EVol. 20, No. 2, 2011

ContentsSalputra, G., Chantreuil, F., Hanrahan, K., Donnellan, T., van Leeuwen, M. and Erjavec, E.Policy harmonized approach for the EU agricultural sector modelling

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Vauhkonen, V., Lauhanen, R., Ventelä, S., Suojaranta, J., Pasila, A., Kuokkanen, T., Prokkola, H. and Syväjärvi, S.The phytotoxic effects and biodegradability of stored rapeseed oil and rapeseed oil methyl ester

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Sironen, A., Uimari, P. and Vilkki, J. Comparison of different DNA extraction methods from hair root follicles to genotype Finnish Landrace boars with the Illumina PorcineSNP60 BeadChip.

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Manninen, M., Honkavaara, M., Jauhiainen, L., Nykänen, A. and Heikkilä, A.-M. Effects of grass-red clover silage digestibility and concentrate protein concentration on performance, carcass value, eating quality and economy of finishing Hereford bulls reared in cold conditions

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Wiander, B. and Palva, A. Sauerkraut and sauerkraut juice fermented spontaneously using mineral salt, garlic and algae.

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Wiander, B. and Korhonen, H.J.T. Preliminary studies on using LAB strains isolated from spontaneous sauerkraut fermentation in combination with mineral salt, herbs and spices in sauerkraut and sauerkraut juice fermentations.

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Hyvönen, T. Impact of temperature and germination time on the success of a C4 weed in a C3 crop: Amaranthus retro-flexus and spring barley.

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