Sans Pierre

Lassaut Bernard
email: blassaut@wanadoo.fr

Čandek-Potokar Marjeta
email: meta.candek-potokar@kis.si

EUROPEAN PROTECTION OF AGRICULTURAL PRODUCTS AND FOODSTUFFS IN THE NEW EU MEMBER COUNTRIES. THE EXAMPLE OF COLLABORATION BETWEEN FRANCE AND SLOVENIA EVROPSKA ZAŠČITA POIMENOVANJ ZA POSEBNE KMETIJSKE PROIZVODE IN ŽIVILA V NOVIH ČLANICAH EU. PRIMER SODELOVANJA MED FRANCIJO IN SLOVENIJO

Keywords: European quality policy, geographical denomination, registration, certifi cation, cooperation, Slovenia IZVLEČEK evropska politika kakovosti, geografsko poimenovanje, registracija, certifi ciranje, sodelovanje, Slovenija

The objective of this article is to present the main results of an advising and assisting program, which took place in years 2001 until 2003, and which involved the Slovenian Ministry of Agriculture, Forestry and Food and two French institutions, French Agency for Standardization and National Institute for Designations of Origin. Authors report their experience in implementation of a system for the registration and certifi cation of special agricultural products and foodstuffs in the Republic of Slovenia, in accordance with the quality policy and legislation of the European union (Council Regulations 2081/92 and 2082/92). They highlight the key success factors for an effi cient implementation of the registration system for the special agricultural products and foodstuffs in the new EU members.

SANS Pierre, LASSAUT Bernard and ČANDEK-POTOKAR Marjeta

POVZETEK (SUMMARY)

V prispevku predstavljamo rezultate sodelovanja med Francijo in Slovenijo pri vzpostavitvi sistema zaščite poimenovanj posebnih kmetijskih proizvodov in živil v Sloveniji, skladno z zakonodajo EU. Slovenija je majhna a geografsko in klimatsko zelo raznolika dežela. Posledica te naravne raznolikosti ter kulturnega vpliva sosedskih dežel je tudi kulinarično bogastvo dežele. V želji po zaščiti teh proizvodov in možnostmi, ki jih takšna zaščita nudi v sklopu skupnega trga v Evropski uniji (EU), je ministrstvo pristojno za kmetijstvo želelo vzpostaviti način zaščite, ki bi bil skladen z zakonodajo EU (Council regulation 2081/92 in 2082/92). V ta namen je v letih 2001 in 2003 med slovenskim ministrstvom za kmetijstvo in francoskim inštitutom INAO ter francosko Agencijo za standardizacijo potekal program Phare, tekom katerega je bil ob pomoči francoskih strokovnjakov vzpostavljen sistem registracije (zaščite) poimenovanj ter certifi ciranja posebnih kmetijskih proizvodov in živil. Program je potekal na dveh ravneh in sicer na organizacijski ravni, kjer je šlo za pomoč pri vzpostavitvi zakonskih in administrativnih okvirjev za izpeljavo zaščite ter na operativni ravni, kar je pomenilo prikaz delovanja sistema na praktičnem primeru skozi vse faze vzpostavitve zaščite. Pred začetkom programa je Slovenija, ob pomoči francoskih ekspertov, pripravila spremembe zakonodaje in v Zakon o kmetijstvu (UL RS 54/2000) vpeljala sistem zaščite poimenovanj posebnih kmetijskih proizvodov in živil po vzoru EU. Ta poleg same zaščite imena predvideva tudi certifi ciranje oziroma sistem neodvisnega nadzora, ki ga je bilo ravno tako potrebno vzpostaviti. Ključna težava pri vzpostavljanju sistema zaščite poimenovanj je bilo pripraviti vse, od proizvajalcev, profesionalnih združenj, uradnikov in drugih, ki jih zaščita kakorkoli zadeva, na razumevanje fi lozofi je zaščite. Zaščita poimenovanj posebnih kmetijskih izdelkov in živil pomeni izjemo pri prostem pretoku blaga znotraj EU in zahteva prostovoljen, odprt in kolektiven pristop. Kot rezultat tega sodelovanja je Slovenija konec leta 2004 v Bruselj poslala trinajst prošenj za zaščito poimenovanj za posebne kmetijske proizvode oziroma živila (Preglednica 2), kar je veliko za tako majhno deželo. V prispevku smo na primerih dveh proizvodov, "kraškega pršuta" in "prekmurske gibanice", ponazorili ključne dejavnike uspeha pri postopku zaščite, ki so motivacija (interes) za zaščito, težave pri usklajevanju protagonistov ter možna ali realna nasprotovanja zaščiti. V primeru "kraškega pršuta" je bil v začetku pristop protagonistov defenziven, saj je bila država tista, ki je spodbujala zaščito. Obstajala so tudi precejšnja razhajanja, na eni strani med prašičerejci, ki so želeli da se zaščiti tudi poreklo, ter pršutarji, ki so se temu upirali, ker so surovino v glavnem uvažali; razhajanja so obstajala na drugi strani tudi med samimi pršutarji, ki so želeli različno geografsko razmejitev področja. Za nameček je bilo potrebno protagoniste podučiti, da takšen sistem ne more biti zaprt in da vanj lahko kadarkoli vstopi kdorkoli, ki sprejme pogoje. Na koncu je bil sprejet kompromis, ustanovil se je konzorcij "kraški pršut", ki se je odločil za zaščito geografske označbe. Prvi pršuti s certifi katom so na trg prišli jeseni 2005. Pri "prekmurski gibanici" pa je bilo nasprotno opaziti ofenziven pristop k zaščiti, saj je Društvo za zaščito prekmurskih dobrot s tem ukrepom želelo zagotoviti, da se pri izdelavi te cenjene slaščice spoštuje tradicionalni recept. Razhajanja, ki so se pojavila med protagonisti v tem primeru so bila predvsem posledica razlik v načinu izdelave (sestavine, oblika, število in zaporedje plasti) pri različnih proizvajalcih, ki ga je bilo potrebno uskladiti. Hkrati je bilo potrebno sprejeti, da v primeru zaščite tradicionalnega poimenovanja ne morejo izdelave geografsko omejiti samo na Prekmurje. Na primeru Slovenije se je ponovno potrdilo, da je uspešnost zaščite v največji meri odvisna od motivacije in sposobnosti ljudi, ki projekte zaščite spravijo v življenje. Vendar pa za tržni uspeh samo zaščiteno poimenovanje ni dovolj; potrebno je zagotoviti dobro in standardno kakovost ter prepoznavnost takšnega izdelka na trgu. Naloga države pa je, da zagotovi učinkovit in verodostojen sistem zaščite s skrbno presojo vlog, spoštovanjem nadzora ter stalno podporo preko svoje kmetijske politike.

INTRODUCTION

In order to give a reader a brief impression of Slovenia and its agriculture we present some basic statistical indicators (Table 1) assembled from offi cial statistics [1,2]. The natural conditions of Slovenia situate more than three quarters of its territory to the zone where agriculture is limited by climatic, pedological or topographic (mountains) factors. A half of the Slovenian territory is covered by forests, a quarter of its territory represents utilised agricultural land of which more than a half is under permanent grassland. Contrary to the other Central European countries, Slovenian agriculture was relatively little collectivized before its independence in 1991. However even today, Slovenian agriculture remains characterized by small parcels. According to the last available data [1] 77,000 agricultural holdings perform agricultural activity in the Republic of Slovenia. An average agricultural holding uses 6.3 ha of agricultural area and breeds 6.6 livestock units, while only 15 % of agricultural holdings uses more than 10 ha and only 101 (0.1%) agricultural holdings uses more than 100 [START_REF] Phare | Project management of programmes[END_REF]. The aim of this program was to implement the system of protection and certifi cation of special agricultural products and foodstuffs in the Republic of Slovenia which would be compatible with European quality policy and legislation [5,6].

MATERIAL AND METHODS

Preparing Slovenia for her accession to the European Union (EU)

Within the framework of the accession to the EU, it meant assisting the Slovenian Ministry of Agriculture, Forestry and Food in the drafting of new laws but also in the construction of a system for registration of products and their certifi cation. Three major goals were pursued, (i) to determine the optimal structures of the new organizations which would be in charge of these functions, by presenting various options and by proposing a solution, (ii) to succeed in fully involving all the operators and institutions (Chamber of Agriculture, Chamber of Commerce) concerned by the protection of special agricultural products and foodstuffs, (iii) to ensure the diffusion of the results of the technical support so that all the concerned parties would have clear and reliable information in case they decided to present an application for the protection of particular product.

Methodology

In order to achieve previously mentioned goals, we decided to work simultaneously on two levels; at fi rst level, the organizational (or "system") level, the objective was to ensure that the legislative and administrative frame in Slovenia would be compatible with the requirements of the EU as regards the protection (registration) of special agricultural products. After the review of the Slovenian legislation in force, we participated to the preparation of new legislative texts and proposed the scheme of operational system for the registration and certifi cation of products. The second level, on which we exerted the support, was the operational (or "product") level, within which the formerly taken decisions and installed tools were applied on the practical examples of Slovenian products. The decision to make a "demonstration through example" came from the wish to actually evaluate the range of the European texts concerning the protection of the denominations and to identify the dangers or obstacles to be encountered at the time of the drafting or implementing the complex reference frames. It was also a question of supporting the immediate involvement of the actors concerned by the product certifi cation, while contributing to their formation and their information.

RESULTS

A complete legislative frame and the assistance to the carriers of project

A few months before the beginning of our mission, the Slovenian Ministry of Agriculture, Forestry and Food decided to create an Offi ce for the recognition of denominations of the agricultural products and foodstuffs. This offi ce, in collaboration with the competent sector (food quality and safety) of the same ministry, was charged to draft the basic legal act 1 defi ning fi ve offi cial quality signs for the recognition of special agricultural products and foodstuffs: the protected designation of origin, the protected geographical indication, the traditional specialty, a sign of higher quality, a sign for organic agricultural products and a sign for integrated agricultural products. We took part in the drafting of the three legal texts (rules) 2 of application of the law which respectively specifi ed the defi nitions of each sign, characteristics of the petitioning groups and the procedure to be followed to register a product. The implementation of this new legal frame assumed the distribution of the tasks; the registration of denominations was entrusted to the Offi ce for the recognition of denominations of the agricultural products and foodstuffs while the external control and certifi cation was to be trusted to the various offi ces, existing organizations, or ones to be created. At that point, entrusting the external control to the offi cial services was necessary, as no private structure was able to assume this task at that time. However the obligation was clear to distinguish between the offi cial control which would be binding to all the operators (veterinary services, inspection of the frauds...) and that carried out within the voluntary approach in certifi cation 3 . The certifi cation was foreseen for the organization of certifi cation created at the later stage (in the respect of the standard EN 45011).

The validation of the reference frames as suggested by the applicants was to be performed by the Offi ce for the recognition of denominations of the agricultural products and foodstuffs after acquiring the opinion of the one of the six sector commissions4 created at the later stage. These commissions engage experts of various fi elds (pedology, climatology, technology of the product...) coming from the scientifi c and professional circles. In order to help the petitioning groups in their actions to register the product, practical guides were written: these represent the entire procedure (and the requirements related to each stage) from the preliminary strategic analysis (why to protect the product?) until the fi nal registration. Census on the products having been the subject of an applications and/or a subject of a request for registration The products being subject of the exemplary applications were proposed by the Slovenian Ministry of Agriculture, Forestry and Food (Table 2 and Figure 1). The initial choices considered by the Slovenian Ministry of Agriculture, Forestry and Food (Table 2; foreseen protection) refl ect the diffi culties encountered to set up a legislative frame corroborating the requirements of the European legislation: -in case of the "Slovenian honey", neither the Article 2 of the Council Regulation 2081/92, narrowly limiting the protection of the names of the state, had been taken into account, nor the diversity of the characteristics of the honeys produced in different Slovenian regions.

-in case of the "Piran salt", the absence of salt in the list of the products concerned by the Council regulation 2081/92 had been ignored; -in case of the cheese "Tolminc", the name had already been registered as the trade mark by one industrial operator; what's more for a cheese that was not in conformity with the specifi cations. The coherence with the article 14 of the Council regulation 2081/92 [5] had not been validated. However, there had been no opposition formulated by the holder of the trade mark during the public survey carried out to justify this request for protection. As a result of the program, thirteen requests were transmitted to the European Commission in 2004 by the Republic of Slovenia (Table 2); seven demands for protected designation of origin, fi ve demands for protected geographical indication and one demand for the sign traditional specialty. However a demand for protected designation of origin for Slovenian honey ("Slovenski med") was replaced by the demand for protected designation of origin for Honey of Kočevje forest ("Kočevski med"), the demand for protected designation of origin for Pumpkin seed oil of Štajerska and Prekmurje ("Štajersko prekmursko bučno olje") was sent as a request for protected geographical indication, while the demand for Piran salt ("Piranska sol") remains waiting the decision of the European Commission for the eligibility of salts for protection, like other European salts. Compared to the eight requests for protection having been the subject of an application, six more were transmitted to the EU Commission by the Slovenian Ministry of Agriculture, Forestry and Food (Table 2). It was important to understand that the same person can not perform the tasks of the offi cial control and the certifi cation for the same producer.

SANS Pierre, LASSAUT Bernard and ČANDEK-POTOKAR Marjeta

DISCUSSION

In order to synthesize the major lessons of the program, we will present the case on two products: "Prekmurska gibanica" or in translation Layer-pie of Prekmurje region and "Kraški pršut" or Dry-ham of Kras (Frames 1 and 2). These two products make it possible to cover a broad pallet of the problems emerging when preparing a demand for protection of product and its name. We will elucidate the common points and the differences observed between Slovenian situation and the approach observed in the European Union (and in particular in France).

Strong similarities (i) Motivations of the applicants for protection. The strategic approach in protection can be either defensive or offensive [START_REF] Čandek-Potokar | Perspectives de marché pour le Jambon de Prekmurje en relation avec les caractéristiques de consommation des produits carnés secs en Slovénie. 5th Int. Symp. Mediterranean Pig[END_REF]. The defensive strategy can be illustrated by the case of "Kraški pršut", the highly recognised product of the Slovenian meat industry. The Slovenian Ministry of Agriculture, Forestry and Food, in dialogue with the professionals, justifi ed the request for the protection by the fear that, after the accession to the EU, similar products from other countries would come to the market, using the identity of Kras. Some tradesmen indeed placed on the Slovenian market cheaper dry hams (usually of lower quality), imported from Italy, using a denomination "Kraški pršut" 5 . In the case of "Prekmurska gibanica", on the contrary, an offensive strategy was observed: the regional source of "Prekmurska gibanica" was seen as an asset differentiating by itself and indeed supported by the use of collective trade mark "Diši po Prekmurju" for Source of the map: Geografski inštitut Antona Melika, ZRC SAZU, Ljubljana, Slovenia [10]

Figure 1. Localization of the Slovenian products involved in a registration procedure. 5 The term "pršut", is close to Italian word "prosciutto", and is used only in this region bordering Italy ; this term however became in a way a generic name for this type of product (non-smoked dry ham) and itself associates good quality in the mind of Slovenian consumers [START_REF] Lassaut | Les motivations des groupements demandeurs d'IGP et leurs interprétations du règlement CEE 2081/92 : quelles sont les causes de confl its entre entreprises et les risques de concurrence déloyale[END_REF]. In other parts of Slovenia, where dry hams (smoked) are produced, a term "šunka" is used (close to german "schinken") …however the old genuine Slovenian word for dry ham is "gnjat " (which is not used and can be found mainly in older Slovenian texts). "Prekmurska gibanica" (Figure 2) is a traditional cake of the Prekmurje region (North-eastern part of Slovenia). This product was the subject of a demand for protection carried out by the Association for the promotion and the protection of the culinary specialties of Prekmurje (in Slovene language "Društvo za zaščito prekmurskih dobrot") founded in 1999 and whose objective is to defend the products with reputation such as the dry ham of Prekmurje [START_REF] Lassaut | Les motivations des groupements demandeurs d'IGP et leurs interprétations du règlement CEE 2081/92 : quelles sont les causes de confl its entre entreprises et les risques de concurrence déloyale[END_REF], horse radish and pumpkin seed oil. "Prekmurska gibanica" is a pie composed of eight layers of different ingredients (in order: poppy grains, curd cheese, ground walnuts and grated apples) all repeated twice and separated by a layer of fl aky pastry and posed on a "base" made up of a pie crust pastry. It is produced three industrial enterprises (50% of production) and by many restaurants and independent confectioners. It belongs to a broader family of layer pies resembling the "Prekmurska gibanica" but not exactly identical (differences in number of layers, order of layers, shape, size, appearance, ingredients) and whose reputation is less eminent. "Prekmurska gibanica" was traditionally prepared for festive occasions. "Prekmurska gibanica" was protected at the Slovenian level in 2004 as a traditional specialty and the demand for registration was transmitted to Brussels. The fi rst certifi ed products came to the market at the end of 2005.

SANS Pierre, LASSAUT Bernard and ČANDEK-POTOKAR Marjeta

Frame 2. "Kraški pršut" (or Dry-ham of Kras)

"Kraški pršut" is a non-smoked dried ham, contrary to the majority of dry hams produced on the Slovenian territory, a product well known since more than one hundred years and enjoying a reputation of top-level product by the consumers of this country (Figure 3). It is traditionally associated to festive meals. Presently it is manufactured and marketed by two big meat industry fi rms (MIP and KRAS), medium-sized specialised fi rm (Pršutarna Lokev) and many small producers, these latest with a circuit of sale mainly limited to the local market. In a strict sense, the term Kras (which is the origin of internationally acknowledged term for karstic landscape and landforms) defi nes grounds made up of limestone and red soil rich with iron (in italian "terra rosa"). From a geographical point of view, the area of production of "Kraški pršut" corresponds to a zone of plateau (average altitude of 500 meters) limited by fringes of lower altitude (100-200 m). The major part of the production of "Kraški pršut" is sold on the Slovenian territory in the circuit of the great distribution at relatively high prices taking into account the standard of living (11 € a kg of a entire and 35 € a kg of pre-sliced ham packed under modifi ed atmosphere). "Kraški pršut" was protected at Slovenian level as a protected geographical indication in april 2004 and the demand for protection on the EU level was transmitted to Brussels in october 2004. The fi rst certifi ed products were put on the market in the autumn 2005.

which the translation would be "Savours of Prekmurje". The choice to protect the name with the traditional specialty sign corresponds to main goal of applicants which was to guarantee that all the Slovenian producers respect the traditional recipe and that differentiation from other similar layer-pies is assured.

(ii) Diffi culties of coordination between actors. The confl icts observed relate to the choice of the sign of protection between protected designation of origin and protected geographical indication, on the specifi cation standards and on the delimitation of the geographical areas concerned. They often rise from the very great heterogeneity of the actors [START_REF] Barjolle | Facteurs de succès des produits d'origine certifi ée dans les fi lières agro-alimentaires en Europe : marché, ressources et institutions[END_REF]. In the case of "Kraški pršut", the Slovenian pig producers affi rm their will to obtain a protected designation of origin which would be founded on a raw material coming from Slovenia and a zone of ham transformation limited to Kras, whereas 80% of hams manufactured by meat industry were imported. They criticized the practice of imports of hams which induces a heterogeneous quality and harms seriously the image of "Kraški pršut" by the consumers. On the contrary, the meat industry focuses on the protection sign protected geographical indication and underlines the defi cit and the qualitative inadequacy of the local pig production (pig breeding oriented towards the fresh meat marketing leading to light hams, absence of appropriate genetics) causing diffi culties in sorting and high price. In addition, two biggest producers, industrials MIP and KRAS were opposed on the delimitation of the geographical area of Kras, each one having its own defi nition. The confl ict relates primarily to the inclusion or not of the town of Nova Gorica, located at the North-West of Kras (outside the zone of the plateau Kras) (Figure 1). Indeed, the enterprise MIP carries out the salting of hams in its factory located in this town and later transfers hams into their drying unit located on Kras. On the contrary, company KRAS carries out all the stages of the process in the zone defi ned previously. In Figure 3. "Kraški pršut" -a traditional non-smoked dry ham of Slovenian region Kras (Photo: "GIZ Kraški pršut")

SANS Pierre, LASSAUT Bernard and ČANDEK-POTOKAR Marjeta the case of "Prekmurska gibanica", the confl icts related to the certifi cate of the conformity of the specifi cation standards to the specifi c elements of the traditional recipe. The divergences between operators were primarily due to the fact that each one of them evolved its manufacturing mode according to its own constraints, as well technical as commercial. These divergences, as for the manufacturing process, relate mainly to the following elements: possible addition or not of aromatizing substances such as lemon or cinnamon, use of vegetable fats or butter in the place of grease, possibilities of freezing in the course of process and of the fi nished product.

(iii) Potential or actual oppositions. The oppositions could originate in the delimitation of the geographical areas of protection. In the case of "Kraški pršut", this delimitation collides with the unknown factor on the localization (and the exact number) of the small (on farm) producers, in the absence exhaustive preliminary survey. Another potential opposition could come from KRAS company, for its' products, the company registered a trade mark KRAS at the Slovenian offi ce for protection of the industrial property and was reticent about protection of denomination using the same geographical name, proposing the anteriority of its trade mark. Specifi cities related to the Slovenian history and the apprenticeship regarding EU and its institutions One of the major tasks of the advising and assisting program was to answer the need for new legislative texts, in agreement with the European regulations and with other elements of agricultural policy and the trade-mark law. Moreover, it was necessary to assist the Slovenian Ministry of Agriculture, Forestry and Food in the creation of the adequate institutions, and decisions needed to be taken by the last, with no previous experience. Besides, the success of implementation of the EU quality policy in terms of protection of denominations was dependent on fi nding solution to two obstacles (i) to overcome the linguistic barriers, in particular in case of operators who seldom use other languages, and which were observed during the workshops that we organised, (ii) to understand the nature of exceptions to the rule of free circulation of goods within EU market payment and its implications, in particular the nature of voluntary and collective approach. The voluntary approach resides on the initiative of the operators. It was an important message to pass and diffi cult task to achieve, bringing all the concerned operators to this state of mind, while the majority of the projects, following the example of "Kraški pršut", resulted from the suggestions of the national authorities, with no preliminary strategic and market analysis to measure the viability of the project, interest for differentiation and the capacity to segment the market. The collective approach led to identify the operators able to ask, obtain and maintain the protection signs while remaining opened to all newcomers respecting the specifi cations for product fabrication: in case of "Kraški pršut" we advised strongly the creation of consortium charged to prepare the rules and specifi cations for fabrication and we insisted on the necessity to integrate also small (on farm) producers.

The Consortium for dry ham of Kras was actually created in November 2003, comprising two industrials, one medium size producer and one small (on-farm) producer of "Kraški pršut". The compromise regarding the specifi cations for fabrication was reached among operators: "Kraški pršut" is a dry ham aged at least 12 months and made of properly shaped hams of at least 9 kg; the provenance of hams is unrestrained, only salt and no other additives can be added, the entire process of fabrication must be done according to the specifi cation in the geographically defi ned area of Kras. However, on a purely derogatory basis and for a transitional period of 8 years, the company MIP preserves the right to carry out the salting phase in its factory in Nova Gorica 6 . In case of "Prekmurska gibanica", the capacity to structure the sector in accordance with the specifi cations of European legislation shall also be a key success factor in the protection of this traditional pastry. Namely, in the initial step, the applicant association meant to exclude all the operators outside the region Prekmurje, even if they respected the manufacturing specifi cations (three principal industrial fabricants commercialise the product under the name "Prekmurska gibanica").

Particularities related to the size of the country

The number of fi led demands for the protection of special agricultural products and foodstuffs is relatively important for such a small country which has a size of an average EU region. The natural diversity of this country together with cultural infl uence of neighbouring regions provided a heritage of many special products which merit protection. However, it is diffi cult for such a small country to mobilise fi nancial and human resources 7 in terms of competence and availability necessary to counsel, examine the fi les, register the name, and manage the control over the product. It is noteworthy to mention that the company MIP, in June 2005, launched a new dry-ham product on the market named "Vipavski pršut" in translation Dry-ham of Vipava, with a very similar product and fabrication characteristics but with a Slovenian origin of the raw material. It remains to see if this action could mean a turning away from the initiative of the Consortium.

The Offi ce for the recognition of denominations of the agricultural products and foodstuffs no longer exists and was integrated to the sector of food quality and safety of the Ministry for agriculture

CONCLUSIONS

In order to ensure the economic success of the protected products, the Slovenian professionals must profi t from the assets of this collaboration. It appears essential to guarantee and facilitate the access of all the operators concerned with the protection, in particular small farm and artisan producers in order to avoid situations observed elsewhere (for example in France for protected geographical indication for goose-liver of south-west "Foie gras du Sud-ouest"). Moreover, the operators need to ensure the collective approach to the development of protected designations and to make the control over the product or certifi cation by an independent organization; it is a question of accepting a control as a guarantee of protection, as an approach to the progress in quality, and not as an act of repression. Finally, the actors will have to adapt their marketing policies and to engage in communication policies with the support of the EU. These efforts shall not be crowned with success without a constant support of the public institutions which have the role of guaranteeing the system credibility and effectiveness while taking care of consistent and thorough examination of the application fi les and the respect of the control. Moreover, they must assure that the system of protection is supported and promoted in all the actions concerning agricultural policy of the country. The effectiveness and the viability of the system rest above all on the competence and motivation of people which brings up the importance of the education of experts suited to perform tasks of preparing and managing a project of protection. It is a true challenge for a country as small as Slovenia: by way of example, our interlocutors in charge of mission during the program were often very young people without or not having yet fi nished their university course (on probation) or detached offi cers from other administrations. One year after the end of the program, more than half of them were no longer at that post. Let us bet that the Slovenians as well professionals as offi cials, with enthusiasm which they expressed, will be able to take up this challenge!

1

  Law on agriculture published in Offi cial Gazette of the Republic of Slovenia (UL RS 54/2000). 2 Acts (rules) UL RS 58/2001, UL RS 33/2005, UL RS 76/2003 as amended by the act (rule) UL RS 47/2005 (in the order of citation).

3

 3

Figure 2 .

 2 Figure 2. "Prekmurska gibanica" -a traditional pastry of Slovenian region Prekmurje (Photo: "Društvo za zaščito prekmurskih dobrot")

6

 6

Table 1 .

 1 Some statistical indicators on Slovenia and its agriculture

	Total area

SANS Pierre, LASSAUT Bernard and ČANDEK-POTOKAR Marjeta

	ha. There are about 67,000 stockbreeders in Slovenia
	(86% of the agricultural holdings) of which only 15%
	breed more than 10 livestock units. The relative share
	of the vegetable (42%) and animal productions (56%)
	in the agricultural production evolves little in a context
	dominated by mixed breeding farms of small dimension.
	Despite its weak economic importance (≈2 % of gross
	domestic product (GDP) and 4.6 % of active population),
	agriculture preserves an important role in maintaining
	social and territorial equilibrium. The sector of food
	industries represents about 3.2 % of gross domestic
	product (GDP) and includes approximately 370 fi rms
	with 19,000 employees [3]. The share of the production
	intended for food industries is relatively weak; the
	on-farm sale of agricultural products or sale on local
	markets is widespread. Besides, the sale by way of on-
	farm tourism is also increasing. The statistically recorded
	sale of agricultural products in relation to the agricultural
	production is above 50% only for meat (poultry, beef
	and pork) and milk. A signifi cant part of milk production
	is transformed on the farm or by smaller scale artisan
	dairies.
	In this context, characterized by the diversity of the
	food productions and the coexistence of industrial and
	artisan way of production, the Slovenian authorities
	would like to develop a market for special agricultural
	products and foodstuffs, recognized as such, towards the
	larger European market. Moreover, Slovenian authorities
	expressed a keen interest for the protection system
	according to European legislation (Council regulations
	2081/92 and 2082/92). Before that, Slovenia knew and
	practiced only the protection of geographical designation
	under the law on intellectual properties, which considered
	the protection of names but without quality certifi cation.

The program, for which the methodological support was offered by France and its experts, was carried on during the years 2001 to 2003. It was performed within the framework of a convention binding AFNOR (French Agency for Standardization) and the National Institute for Designations of Origin (INAO) to the Slovenian Ministry of Agriculture, Forestry and Food

Table 2 .

 2 List of studied agricultural products and requests for the product registration sent to the European Commission by the Republic of Slovenia

	Slovenian name of product	English translation of	Group	Foreseen	Demanded
		product's name		protection	protection
	A. Products having made the object of an application
	"Nanoški sir"	Nanos cheese	Cheese	PDO	PDO
	" Tolminc"	Cheese Tolminc	Cheese	PDO	PDO
	"Štajersko prekmursko	Oil of pumpkin seed of	Oils and fats	PDO	PGI
	bu�no olje"	regions Štajerska and
		Prekmurje
	"Slovenski med"	Slovenian honey	Other products	PDO	not transmitted
	"Ko�evski gozdni med"	Honey of Ko�evje forest	of animal origin		PDO
	"Piranska sol"	Piran salt	-	PDO	not transmitted
	"Kraški pršut"	Dry ham of Kras region	Meat products PDO or	PGI
				PGI
	"Prleška tünka"	Cured and smoked pork	Meat products PGI	PGI
		plunged into fat of Prlekija
		region
	"Prekmurska gibanica"	Layer-pie of the region	Pastry and	PGI or	TSG
		Prekmurje	bakery	TSG
			products
	B. Additional requests initiated by the Slovenian authorities
	"Mohant"	Cheese Mohant	Cheese	-	PDO
	"Bovški sir"	Bovec Cheese	Cheese	-	PDO
	"Prekmurska šunka"	Dry ham (smoked) of	Meat products	-	PDO
		Prekmurje region
	"Ekstra deviško olj�no olje	Extra virgin olive of	Oils and fats	-	PDO
	Slovenske Istre"	Slovenian Istria
	"Zgornjesavinjski	Stomach of Upper Savinja	Meat products	-	PGI
	želodec"	valley
	"Šebreljski želodec" 9	Stomach of Šebrelje	Meat products	-	PGI
	PDO: protected designation of origin; PGI: protected geographical indication; TSG: traditional specialty guaranteed.

Milk and dairy products; meat and meat products; honey; pastry and bakery products; oils; butter and fats; salt.

ACKNOWLEDGEMENT

Authors are indebted to the associations "GIZ Kraški pršut" and "Društvo za zaščito prekmurskih dobrot" for providing the photos of dry ham "Kraški pršut" and traditional pastry "Prekmurska gibanica".


Philippe Bich
email: bich@univ-paris1.fr

An answer to a question of Herings et al

Keywords:

teaching and research institutions in France or abroad, or from public or private research centers.

The result

In [START_REF] Herings | A fixed point theorem for discontinuous functions[END_REF], Herings et al. prove the following new fixed point theorem for possibly discontinuous mappings: Theorem 1.1 Let P a non empty polytope, i.e. the convex hull of a finite subset of R n ; let f : P → P which is "locally gross direction preserving" in the following sense: for every x ∈ P such that f (x) = x, there exists V x , an open neighborhood of x in P such that for every u and

v in V x , f (u) -u, f (v) -v ≥ 0.
Then f admits a fixed-point, i.e. there exists

x ∈ P such that f (x) = x.
This theorem is a generalization of Brouwer fixed point theorem (see [START_REF] Brouwer | Über Abbildung von Mannigfaltigkeiten[END_REF]) which says that every continuous mapping from the unit closed ball of R n to itself admits a fixed point. Yet, there is a restriction in Theorem 1.1: the set P must be a polytope. But typical strategy sets in game theory are rather compact and convex sets. Thus, an important question in practice is to know if Theorem 1.1 holds true for such subset of R n . In [START_REF] Herings | A fixed point theorem for discontinuous functions[END_REF], one can read: "whether locally gross direction preserving is sufficient to guarantee the existence of a fixed point on an arbitrary non empy convex and compact set is still an open question".

1

In this note, we prove that Herings et al. result holds true when P is a nonempty convex and compact subset of R n . This proof rests on a fixed point theorem of S. Toussaint ([4]) we describe at the end of this paper.

Here, and throughout this paper, for every x ∈ R n and y ∈ R n , x, y denotes the eulidean scalar product of x and y. The set P is a nonempty convex and compact subset of R n . Lemma 1.2 If f : P → P is locally gross direction preserving and has no fixed points, then for every x ∈ P such that x = f (x), there exists p x ∈ R n and V x , an open neighborhood of x in P , such that for every x ∈ V x , one has

p x , f (x ) -x > 0.
Proof. Suppose that f : P → P is locally gross direction preserving and has no fixed points. Let x ∈ P , and let V x be an open neighborhood of x in P such that for every u and

v in V x , f (u) -u, f (v) -v ≥ 0. Let {f (x 1 ) - x 1 , ..., f (x k ) -x k } be a basis of the vector space F :=span{f (y) -y, y ∈ V x },
where k ∈ N * , and x 1 , ...,x k are in V x . Then define

p x = k i=1 (f (x i ) -x i ). Let x ∈ V x . One clearly have p x , f (x ) -x ≥ 0 (1)
from "locally gross direction preserving" property and from the definition of p x . Besides, since one has f (x i )x i , f (x )x ≥ 0 for every i = 1, .., k, Inequation 1 is an equality if and only if for every i = 1, ..., k, one has f (x )x , f (x i )x i = 0. This last property would imply f (x )x ∈ F ⊥ ∩ F = {0}, a contradiction with the assumption that f (x ) = x . Thus, Inequality 1 is strict, which ends the proof of the lemma. Now, for every x ∈ P , one introduces the (possibly empty) multivalued mapping T x defined for every y ∈ P by

T x (y) = {z ∈ P, z -y, p x > 0}.
Clearly, for every x ∈ P , T x has convex values. Besides, for every y ∈ P , one has y / ∈ T x (y). Lastly, for every z ∈ P , the set

T -1 x (z) = {y ∈ P | z ∈ T x (y)
} is an open subset of P . A multivalued mapping T x satisfying theses assumptions is called KF in [START_REF] Toussaint | On the existence of equilibria in economies with infinitely many commodities and without ordered preferences[END_REF].

Besides, from the definition of p x , there exists a neighborhood V x of x such that for every x ∈ V x , one has f (x ) ∈ T x (x ): one says that T x is a KF -majorant of f at x (see [START_REF] Toussaint | On the existence of equilibria in economies with infinitely many commodities and without ordered preferences[END_REF]).

To summarize, we have proved that a locally gross direction preserving mapping f : P → P without fixed point is KF -majorized in the sense that for every x ∈ P , there exists a KF -majorant T x of f at x. Now, state Theorem 2.2. in [START_REF] Toussaint | On the existence of equilibria in economies with infinitely many commodities and without ordered preferences[END_REF]: it says that given any non empty convex and compact subset X of a topological space, given any multivalued mapping F : X → X which is KF-majorized, there exists x ∈ X such that F (x) = ∅. Apply this theorem to F = f and X = P . Thus, there should exists x ∈ P such that f (x) = ∅, a contradiction. This proves that any locally gross direction preserving mapping f : P → P must admit a fixed point.

Acknowledgements

I wish to thank an anonymous referee for valuable comments, and in particular for suggesting me to simplify the main result of my paper by using a fixed point theorem by Sabine Toussaint.


Jean-Marc Lelièvre
email: jean-marc.lelievre@jouy.inra.fr

Daniel Le Bourhis

Amandine Breton

Hélène Hayes

Jean-Luc Servely

Xavier Vignon

Heat-induced and spontaneous expression of Hsp70.1Luciferase transgene copies localized on Xp22 in female bovine cells

Background: Expression of several copies of the heat-inducible Hsp70.1Luciferase (LUC) transgene inserted at a single X chromosome locus of a bull (Bos taurus) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell.

Findings: Transgene copies were mapped to bovine Xp22. In XX LUC female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active X LUC chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic (female) embryos obtained after IVF and in male and female embryos obtained after SCNT. Conclusions: The X LUC chromosome is normally inactivated but at least part of the inactivated X-linked Hsp70.1Luciferase transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the X LUC chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.

Background

Menck and colleagues have reported a luminescent screening system based on the integration of a transgene composed of scaffold attachment regions flanking the murine HSP70.1 gene promoter linked to firefly luciferase cDNA [START_REF] Menck | Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos[END_REF]. Among the transgenic fetuses obtained, one male carried a cluster of 20 to 30 copies of the transgene [START_REF] Menck | Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos[END_REF]. Later, somatic cell nuclear transfer (SCNT) cloning with cells from this fetus generated a healthy and fertile bull for which we have localized the transgenic cluster on the X chromosome (this report). Thus an interesting animal model was available to investigate the inactivation/activation status of transgenes in bovine female fetuses from this bull. Indeed, dosage compensation between male and females is achieved after X-chromosome inactivation (XCI) in mammalian female cells, i.e. one of the two X, the inactive X (Xi) chromosome, is in great part transcriptionally silent [START_REF] Payer | X chromosome dosage compensation: how mammals keep the balance[END_REF][START_REF] Heard | Dosage compensation in mammals: fine-tuning the expression of the X chromosome[END_REF]. At least in domestic mouse, XCI occurs in two waves early during development (reviewed in [START_REF] Payer | X chromosome dosage compensation: how mammals keep the balance[END_REF][START_REF] Heard | Dosage compensation in mammals: fine-tuning the expression of the X chromosome[END_REF]). First, both X chromosomes are transcriptionally active during a short developmental window of the cleavage phase. Then, most studies agree that the paternally inherited X P chromosome becomes inactivated by the blastocyst stage [START_REF] Payer | X chromosome dosage compensation: how mammals keep the balance[END_REF][START_REF] Heard | Dosage compensation in mammals: fine-tuning the expression of the X chromosome[END_REF] and also in most placental cells. In the epiblast cells, both X chromosomes are again transiently active, before random XCI during gastrulation [START_REF] Mak | Reactivation of the paternal X chromosome in early mouse embryos[END_REF]. This results in a mosaic of two somatic cell types expressing X-linked genes inherited either from the mother or the father. Some X-linked genes maintain bi-allelic expression in female cells [START_REF] Payer | X chromosome dosage compensation: how mammals keep the balance[END_REF][START_REF] Heard | Dosage compensation in mammals: fine-tuning the expression of the X chromosome[END_REF]. On the human submetacentric X chromosome, 5% [START_REF] Johnston | Large-scale population study of human cell lines indicates that dosage compensation is virtually complete[END_REF] to 15% [START_REF] Carrel | X-inactivation profile reveals extensive variability in X-linked gene expression in females[END_REF] of the genes escape inactivation; they are preferentially found in clusters and more frequently on the short arm than on the long arm [START_REF] Carrel | X-inactivation profile reveals extensive variability in X-linked gene expression in females[END_REF][START_REF] Tsuchiya | Chromosomal domains and escape from X inactivation: comparative X inactivation analysis in mouse and human[END_REF]. On the mouse acrocentric X chromosome, homologs of the human genes escaping inactivation are mostly inactivated and only two non-clustered genes with no homolog on the Y have been shown to escape inactivation [START_REF] Chow | Silencing of the mammalian X chromosome[END_REF]. Thus, the phenomenon of inactivation escape may depend on genomic context, including either the absence of sequence elements necessary for silencing spreading [START_REF] Tsuchiya | Emergent genome-wide control in wildtype and genetically mutated lipopolysaccarides-stimulated macrophages[END_REF][START_REF] Carrel | Genomic environment predicts expression patterns on the human inactive X chromosome[END_REF] or the presence of insulators/ barriers that prevent XCI-coupled silencing [START_REF] Tsuchiya | Emergent genome-wide control in wildtype and genetically mutated lipopolysaccarides-stimulated macrophages[END_REF][START_REF] Li | Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus[END_REF].

Similarly, XCI-related silencing of X-linked transgenes may depend on the insertion site, the transgene's intrinsic properties or other unknown factors. Furthermore, it may vary between cell lineages or during development ( [START_REF] Li | Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus[END_REF][START_REF] Hadjantonakis | An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta[END_REF][START_REF] Ciavatta | A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation[END_REF][START_REF] Goldman | Comparative methylation analysis of murine transgenes that undergo or escape X-chromosome inactivation[END_REF][START_REF] Tam | Expression of an X-linked HMG-lacZ transgene in mouse embryos: implication of chromosomal imprinting and lineage-specific X-chromosome activity[END_REF] and references therein), as reported for 10% of the human X-linked genes [START_REF] Carrel | X-inactivation profile reveals extensive variability in X-linked gene expression in females[END_REF][START_REF] Carrel | Heterogeneous gene expression from the inactive X chromosome: an X-linked gene that escapes X inactivation in some human cell lines but is inactivated in others[END_REF] and one mouse Xlinked gene [START_REF] Sheardown | The mouse Smcx gene exhibits developmental and tissue specific variation in degree of escape from X inactivation[END_REF].

Expression of an X-linked transgene has rarely been observed during early development and only in the domestic mouse [START_REF] Tam | Expression of an X-linked HMG-lacZ transgene in mouse embryos: implication of chromosomal imprinting and lineage-specific X-chromosome activity[END_REF][START_REF] Eggan | X-Chromosome inactivation in cloned mouse embryos[END_REF] in which surprisingly, one Xlinked transgene has been shown to display delayed expression when paternally inherited [START_REF] Tam | Expression of an X-linked HMG-lacZ transgene in mouse embryos: implication of chromosomal imprinting and lineage-specific X-chromosome activity[END_REF].

Analysis of SCNT cloned embryos can provide further insight on how XCI influences gene expression. For an Xi-associated transgene, silencing reversion has been reported in SCNT cloned early mouse embryos [START_REF] Eggan | X-Chromosome inactivation in cloned mouse embryos[END_REF] but for an autosomal insertion, transgene-related and/or position effect-related silencing was found unchanged in SCNT cloned cattle [START_REF] Bordignon | Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells[END_REF].

Indirect evidence suggests that bovine and mouse XCI profiles are quite similar. De La Fuente and colleagues [START_REF] La Fuente | X inactive-specific transcript (Xist) expression and X chromosome inactivation in the preattachment bovine embryo[END_REF] have shown that in some cells from bovine embryos the two X chromosomes replicate asynchronously, one early and one late in S phase, thus XCI is established at the blastocyst stage 7 days after in vitro fertilization (IVF) in cattle. Indeed, late replicating regions including the Xi are generally transcriptionally inactive while early-replicating regions including the Xa are generally transcriptionally active (except in the mouse immediately after imprinted XCI; [START_REF] Heard | Dosage compensation in mammals: fine-tuning the expression of the X chromosome[END_REF]). Furthermore, two reports clearly indicate that the paternally-inherited X is preferentially inactivated in the placenta [START_REF] Xue | Aberrant patterns of X chromosome inactivation in bovine clones[END_REF] or in the chorion only [START_REF] Dindot | Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses[END_REF], suggesting that imprinted XCI takes place earlier in the associated cell lineage(s), i.e., at least in the trophoblast, at the blastocyst stage. In somatic bovine cells, both X are inactivated, [START_REF] Xue | Aberrant patterns of X chromosome inactivation in bovine clones[END_REF][START_REF] Dindot | Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses[END_REF], suggesting that XCI occurs randomly in the bovine epiblast during gastrulation as in the mouse. To date, it has not been established whether some genes escape inactivation on the bovine X.

Several studies have shown that the expression of the Hsp70.1Luciferase transgene mimics that of the murine Hsp70.1 gene, i.e. the level of luciferase activity increases after heat-shock (HS) induction in both mouse and bovine transgenic embryonic and somatic cells and also spontaneously during embryonic genome activation (EGA) in early mouse transgenic embryos ( [START_REF] Menck | Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos[END_REF] and references therein). To investigate whether some copies of the X-linked transgene remained inducible, we first analyzed the HS-induced luciferase activity and/or protein level in transgenic female somatic cells and blastocyst embryos. Second, we took advantage of the spontaneous activity of the transgene in early embryos before XCI and the relative success of SCNT cloning in cattle [START_REF] Yang | Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning[END_REF] to measure the influence on gene expression of the chromosomal environment inherited from spermatozoa or from male and female cells in early IVF embryos and SCNT embryos respectively.

Materials and methods

Results and discussion

Transgenes are located on the X chromosome of the transgenic bull After hybridization with a probe specific for the whole transgene, a unique strong signal was observed on the short arm of the bovine submetacentric X in chromosome preparations from the transgenic bull, precisely in the early-replicating R band Xp22 (Figure 1A). The bovine Xp region is conserved with part of the human Xq [START_REF] Rubes | Assignment of chromosome rearrangements between X chromosomes of human and cattle by laser microdissection and Zoo-FISH[END_REF][START_REF] Goldammer | Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle[END_REF] while the human Xp22 region, in which about 30% of the genes may escape inactivation [START_REF] Carrel | X-inactivation profile reveals extensive variability in X-linked gene expression in females[END_REF], is conserved with part of the bovine Xq [START_REF] Rubes | Assignment of chromosome rearrangements between X chromosomes of human and cattle by laser microdissection and Zoo-FISH[END_REF].

In XX LUC somatic female cells inactivation frequencies of both X are similar Since XCI occurs randomly in the somatic bovine lineages [START_REF] Xue | Aberrant patterns of X chromosome inactivation in bovine clones[END_REF][START_REF] Dindot | Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses[END_REF], about half of the transgenic female somatic cells are expected to have an active X LUC chromosome inherited from the bull. Presence of a strong bias would indicate preferential inactivation of one of the X or preferential survival of the cells that inactivate one X. Although normal random XCI is reported for mouse and bovine clones [START_REF] Eggan | X-Chromosome inactivation in cloned mouse embryos[END_REF][START_REF] Xue | Aberrant patterns of X chromosome inactivation in bovine clones[END_REF][START_REF] Dindot | Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses[END_REF][START_REF] Nolen | X chromosome reactivation and regulation in cloned embryos[END_REF], it was important to check whether this was the case in IVF females carrying the X of the transgenic bull since the bull was obtained by SCNT cloning, a technique which can result in developmental anomalies generally associated with abnormal epigenetic processes [START_REF] Yang | Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning[END_REF].

Analysis of metaphase chromosome spreads prepared from synchronized female XX LUC fibroblast cells cultured in the presence of BrdU during late S phase showed no bias. An equivalent number of BrdU-labeled X chromosomes or inactive Xi (N = 22) and partly BrdU-labeled X chromosomes or active Xa (N = 21) carried copies of the transgene. Furthermore, the transgenes' presence had no visible influence on Xp22 inactivation/activation since the X LUC p22 region replicates late on the Xi (Figure 1B) and on the normal X [START_REF] Coppola | Spatial distribution of histone isoforms on the bovine active and inactive X chromosomes[END_REF]. Thus, inactivation of the X carried by the bull's sperm is normal, which indicates that the presence of multiple copies of the transgene on either Xi or Xa is not toxic (counter-selected) to cell physiology and does not interfere with random XCI.

Proportions of cells expressing the luciferase protein after heat-shock induction are similar in X LUC Y male and XX LUC female population of cultured fibroblasts

We compared the level of luciferase activity and protein after heat-shock (HS) induction in somatic female cells derived from two fetuses generated by IVF with the sperm of the transgenic original bull and referred to as F616 and BSF731 cells, and in male cells from the original transgenic bull (referred to as OV7060 cells). HS-induced luciferase activity ranged from 0.99 × 10 6 RLU.μg protein -1 .min -1 in female BSF731 fibroblast cells to about 2.60 × 10 6 RLU.μg protein -1 . min -1 in both male OV7060 and female F616 fibroblast cells (Table 1), corresponding to a many-fold increase in the three cell populations. Since in the BSF731 cells the numbers of Xi LUC and Xa LUC are similar after random XCI (see above), we compared the proportions of luciferase-positive cells after HS induction and immunostaining in this female cell population and the male OV7060 cell population (Table 1; Figure 2) They were very similar (Figure 2 and [additional File 2 Additional data]). It indicated that the X-linked luciferase transgene was expressed in most cells, strongly suggesting that at least some transgene copies remain active on the Xi in BSF731 cells. In tissue biopsies of three fetal organs tested from the F616 female fetus, a strong increase in the level of luciferase activity after HS-induction was observed (Table 2). Overall, these data strongly suggest that the presence of 20 to 30 transgene copies [START_REF] Menck | Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos[END_REF] does not prevent the HS-induced expression of at least some of them in the different cells and tissues tested, whether they are on the Xi or Xa.

The pattern of spontaneous Hsp70.1Luciferase transgene activity during early development was conserved in all bovine embryos

During normal development, between the one-cell and the 4-8-cell stages, the level of luciferase activity was null in at least 80% of the IVF and SCNT transgenic bovine embryos and very low in the remaining 20%

(Figure 3; Table 3). Since the sperm used here was obtained from a bull hemizygous at the X-linked transgenes, statistically half of the IVF embryos, all female, should be transgenic. As expected, about 50% of the "8-16-cell" IVF embryos displayed a high level of luciferase activity at days 3 and 4, suggesting that the transgenes are expressed in most female IVF embryos. At the morula stage, 32% of the IVF embryos remained luciferasepositive but the level of luciferase activity per embryo had already decreased significantly (P < 0.05). At the blastocyst stage, less than 10% of the IVF embryos displayed luciferase activity, and all at a low level. Moreover, the percentages of luciferase-positive embryos differed significantly between the 8-16-cell stage and the 4-8-cell or blastocyst stages in the three embryo types (Table 3). Before nuclear transfer, the BSF731 female donor cells carried the transgenes either on the Xa or the Xi with a similar probability (see above). However, in female BSF731-derived SCNT embryos, we found no evidence for two sub-populations displaying two different levels and patterns of luciferase activity and the standard error to the mean was similar to that observed in the two Lung 2930

a the level of specific luciferase activity (RLU.μg protein -1 .min -1 ) was measured in explants of the transgenic female F616 fetus after HS induction and compared with the level measured in non-induced explants. b all biopsies were recovered from the transgenic female F616 fetus. c average value based on the measurement of luciferase activity on two independent extracts for each tissue and each condition. other embryo types (Table 3). This indicates that after cloning, the number of active genes was similar in these female SCNT embryos as expected if their XCI-dependent silencing in the donor cell was not achieved (as suggested from Table 1; Figure 2) or was reverted after cloning [START_REF] Eggan | X-Chromosome inactivation in cloned mouse embryos[END_REF]. In turn, the variation in HS response of BSF731 fibroblast cells may result either from a variable number of inactivated transgenes on the Xi or from a variable HS response in the BSF731 cell population. Since the Hsp70.1Luciferase transgene can be similarly active at the 8-16-cell stage in both IVF and cloned embryos, this further suggests that neither the paternal origin of the transgenic X LUC in the case of IVF embryos, nor the origin, male or female, of the somatic cells, in the case of SCNT embryos, prevented spontaneous, oocytedriven, expression of the X-linked transgenes.

We detected an increased level of luciferase activity after heat-shock induction in IVF female blastocysts [additional File 3 Additional Table S1] and placental tissue (Table 2) in which imprinted XCI, i.e. inactivation of the paternallyinherited X LUC , is expected. However, we cannot yet conclude whether the transgenes escaped inactivation after imprinted XCI since the presence of cells in which XCI had not occurred or had occurred randomly is likely.

In conclusion, using a species other than mouse and different approaches we have investigated the expression of an X-linked transgene to determine its innocuousness as well as that of the transgene insertion site, and to test its sensitivity to XCI-dependent or -independent silencing. The results indicate that the transgenic X inherited from the cloned bull is normally inactivated/activated in somatic female cells and that at least some of the transgene copies at this locus escape XCI-coupled silencing in these cells. Whether this is due to HS-dependent [START_REF] Brown | Disruption of downstream chromatin directed by a transcriptional activator[END_REF][START_REF] Inouye | Heat Shock Transcription Factor 1 Opens Chromatin Structure of Interleukin-6 Promoter to Facilitate Binding of an Activator or a Repressor[END_REF] or HS-independent [START_REF] Thompson | Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues[END_REF] properties of the transgene, to the insertion site and/or to the creation of a new genomic environment remains to be determined.

  All samples were generated according to the International Guiding Principles for Biomedical Research involving animals of experimental farms. The research work on cloned animals was approved by COMEPRA (Ethical and Precaution Committee for Agronomic Research Application) in December 1999. The remaining of this section is found in [additional file 1: Material and methods].

Figure 1

 1 Figure1The luciferase transgenes are located on bovine Xp22 and do not alter random X-chromosome inactivation. (A) A whole chromosome metaphase spread from the transgenic bull. Late-replicating DNA of synchronized fibroblasts cells was labeled by BrdU. Metaphase spreads were prepared on slides, fixed with methanol/acetic acid (3:1) and then used for fluorescent in situ hybridization (FISH) using the biotin-labeled probe corresponding to the plasmid containing the whole 8 kb-long Hsp70.1Luciferase transgene. After DNA counterstaining with propidium iodide in alkaline conditions, BrdU-rich bands appear as dark chromosomal bands while early replicated R bands fluoresce red. In these conditions the transgenic locus was detected on the Xp22 band by immunolabeling of the biotin probe and immunodetection with FITCconjugated secondary antibodies. No other signal was detected on complete metaphase spreads. (B) (B') Partial metaphase spread of a female BSF731 fibroblast cell. Metaphases were prepared as above before (B) immunodetection or (B') DNA counterstaining. (B) After DNA denaturation and FISH with the DIG-labeled transgene probe, late incorporation of BrdU was immunolabeled with anti-BrdU antibodies and immunodetected with FITC-conjugated secondary antibodies; the DNA probe was immunolabeled with anti-DIG antibodies and detected with TRITC-conjugated secondary antibodies; in the case shown, the transgene was localized on the late-replicating X chromosome. Arrows indicate the position of the transgene.

Figure 2

 2 Figure2Heat-induced immunostained luciferase proteins in bovine cells. Male X LUC Y (OV7060) and female XX LUC (BSF731) transgenic fibroblast cells were fixed in cold methanol after continuous culture at 39°C or after 20 min at 45°C and 7 hours at 39°C. Immunostaining was performed with monoclonal mouse antibodies directed against the firefly luciferase and FITC-conjugated anti-mouse secondary antibodies (bright signal in left panels and green signal in right panels). Nuclear DNA was counterstained with Hoechst 3342 (blue signal on right panel). In these photographs (X400), 30 (97%) out of 33 OV7060 cells and 24 (82%) out of 29 BSF731 cells were counted positive after HS induction since they displayed a level of immunostaining two standard deviations above the mean value observed in untreated cells (see [additional File 2 Additional data]).

Figure 3

 3 Figure3Evolution of the percentage of luciferase-positive embryos in early IVF and SCNT transgenic bovine embryos. In vitro matured bovine oocytes were used to obtain IVF embryos (black bars) after in vitro fertilization with the semen of the transgenic OV7060 X LUC Y bull, and male X LUC Y OV7060 (open bars) and female X LUC X (grey bars) SCNT embryos after nuclear transfer of fibroblast cells. Embryos were harvested individually at the correct stage and their spontaneous (in the absence of heat-shock induction) luciferase activity measured. Embryos were considered luciferase-positive when the level of luciferase activity was above the background level. The percentage of positive embryos increased at the 8-16-cell stage in all embryo types.

Table 1

 1 Heat-shock response vs. sex in bovine fibroblast cells carrying the Hsp70.1Luciferase transgenes

	Origin of the fibroblast cultures Sex	Mean luciferase specific activity a RLU.μg protein -	Immunostained luciferase-positive
			1 .min -1 ± SD		cells after heat shock b
			Control	After heat shock
	Transgenic adult bull "OV7060"	Male (X LUC Y)	205 ± 76	2.58 × 10 6 ± 0.32 × 10 6	92%
	Transgenic fetus "BSF731"	Female (X LUC X)	315 ± 79	0.99 × 10 6 ± 0.13 × 10 6	80%
	Transgenic fetus "F616"	Female (X LUC X)	875 ±160	2.62 × 10 6 ± 0.35 × 10 6	nd

a RLU = relative light unit. b percentage of cells, which after HS induction displayed cytoplasmic immunostaining values two standard deviations above the mean value observed in noninduced cells.

Table 2

 2 Rate of increase in luciferase activity in fetal and placental female tissues after heat shock a

	Origin of biopsy b	Rate of increase after HS c
	Placenta	38
	Heart	141
	Muscle	282

Table 3

 3 Changes in luciferase activity and in the percentage of luciferase-positive embryos during early development different letters indicate that values on the line are significantly different (P < 0.05). * SEM; SEM could not be calculated when 0 or only one embryo displayed a level of luciferase activity above the background level.

	Embryo type

abc

Acknowledgements

We are indebted to two anonymous referees and to the editor for helpful comments. We thank Yvette Lavergne, Rose-Marie Placide, Matthieu Chamand and Etienne Laloy for their technical help and kindness in the initial phase of this project and Dr Gilles Charpigny for his help on the SYSTAT software. The expertise of Dr Michel Guillomot and Dr Yvan Heyman, and of all the staff of the UCEA experimental farm in Bressonvilliers is greatly acknowledged for recovering the transgenic female fetuses and the semen.

Authors' contributions DLB and XV generated the bovine bull; XV and JLS generated cell cultures, JML, AB, MC, HH and JLS generated IVF embryos, performed FISH and immuno-detection, and measured luciferase activity and protein concentrations; DLB generated SCNT embryos, JML designed the experiments, performed statistical tests and wrote the draft. HH and XV edited the draft and all authors agreed on the final version of this report.

Competing interests

The authors declare that they have no competing interests.