ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 46 Slov Vet Res • Ljubljana • 2009 • Volume 46 • Number 2 • 43-79 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 46 Slov Vet Res • Ljubljana • 2009 • Volume 46 • Number 2 • 43-79 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Previously: RESEARCH REPORTS OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA Prej: ZBORNIK VETERINARSKE FAKULTETE UNIVERZA V LJUBLJANI 4 issues per year / izhaja štirikrat letno Editor in Chief / glavni in odgovorni urednik: Gregor Majdič Technical Editor / tehnični urednik: Matjaž Uršič Assistant to Editor / pomočnica urednika: Valentina Kubale Dvojmoč Editorial Board / Uredniški Odbor: Vojteh Cestnik, Polona Juntes, Matjaž Ocepek, Zlatko Pavlica, Modest Vengušt, Milka Vrecl, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani Editorial Advisers / svetovalca uredniškega odbora: Gita Grecs-Smole for Bibliography (bibliotekarka), Leon Ščuka for Statistics (za statistiko) Reviewing Editorial Board / ocenjevalni uredniški odbor: Ivor D. Bowen, Cardiff School of Biosciences, Cardiff, Wales, UK; Antonio Cruz, Departement of Clinical Studies, Ontario Veterinary College, Guelph, Ontario, Kanada; Gerry M. Dorrestein, Duch Research Institute for Birds and Exotic Animals, Veldhoven, The Netherlands; Wolfgang Henninger, Veterinärmedizinische Universität Wien, Austria; Simon Horvat, Biotehniška fakulteta, Univerza v Ljubljani, Slovenia; Nevenka Kožuh Eržen, Veterinarska fakulteta, Univerza v Ljubljani, Slovenia; Louis Lefaucheur, INRA, Rennes, France; Bela Nagy, Veterinary Medical Research Institute Budapest, Hungary; Peter O'Shaughnessy, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Scotland, UK; Milan Pogačnik, Veterinarska fakulteta, Univerza v Ljubljani, Slovenia; Peter Popelka, University of Veterinary Medicine, Košice, Slovakia; Detlef Rath, Institut für Tierzucht, Forschungsbericht Biotechnologie, Bundesforschungsanstalt für Landwirtschaft (FAL), Neustadt, Germany; Hans-Peter Sallmann, Tierärtzliche Hochschule Hannover, Germany; Marko Tadic, Veterinarski fakultet, Sveučilište u Zagrebu, Croatia; Frank J. M. Verstraete, University of California Davis, Davis, California, US Slovenian Language Revision / lektor za slovenski jezik: Viktor Majdič Address: Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov: Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, 47 79 129, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by / tisk: Birografika Bori d.o.o., Ljubljana Indexed in / indeksirano v: Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich's International Periodicals Directory, Science Citation Index Expanded, Journal Citation Reports/Science Edition http://www.vf.uni-lj.si/vf/index.php/Slovenski-veterinarski-zbornik.html SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2009; 46 (2) Review Papers Ornik D, Cadonic-Spelic V. Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states within 15-years period...............................................47 Fazarinc G. Enzyme-immunohistochemical aspects of muscle fiber type classification in mammals...............61 Original Research Paper Jakovac-Strajn B, Pestevsek U, Knafelc T. Influence of gradual change in feed use ,of acidifier and prebiotic on rabbits in the period of weaning..........................................................71 Slov Vet Res 2009; 46 (2): 47-60 UDC 636.025:34-179.3(497.12)(4) Review Paper RECORDS ON THE USE OF ANIMALS IN EXPERIMENTS IN THE REPUBLIC OF SLOVENIA AND IN OTHER EU MEMBER STATES WITHIN A 15-YEAR PERIOD Dragica Ornik*, Vida Cadonic-Spelic Veterinary Administration of the Republic of Slovenia, Parmova 53, 1000 Ljubljana, Slovenia Corresponding author, E-mail: dragica.ornik@gov.si Summary: Scope of this paper is to present data on the number and species of animals used in experiments and on the purposes of use of animals in the Republic of Slovenia and in the European Union within a 15-year period. According to data collected in the particular years of the period from 1992 to 2006 on the use of animals in experiments in the Republic of Slovenia, the trend of the use of animals in experiments has been found to be on the decline. The total number of experimental animals in 1992 amounted to 37,212, and in 2006 to 13,181 only. In the Republic of Slovenia, the use of experimental animals in applied research shows a downward trend on account of validated alternative methods in use, which do not require animals. In the most recent years in particular, the Slovenian legislative activity in the field of protection of experimental animals has been most productive with the scope of harmonising the Slovenian legislation with the EU law. The European Commission published five reports on the use of animals in experiments. The total number of experimental animals as reported by the EU Member States amounted to 11.79 million in 1991, to 11.64 million in 1996, to 9.81 million in 1999, to 10.73 million in 2002, and to 12.11 million in 2005. The first two reports provided a limited scope of analysis due to the absence of a consistent system of reporting the data on the use of experimental animals in the Member States. The third and the fourth reports were based on agreed harmonized tables. This facilitated a more extensive interpretation of results on the use of experimental animals in the EU, despite some inconsistencies in the data submitted by the Member States. The second report covered for the first time the data collected by the 3 new Member States, and the fifth report by the 10 new Member States. However, it is not possible to draw conclusions on the evolution of use of animals for experimental purposes in the EU by comparing these data with those of the previous reports. The total number includes different animal species, from cold-blooded vertebrates on the one side, to mammals on the other, including farmed animals or anthropoid primates as used in some Member States. Comparison between the national reports is rendered impossible on account of the non-aligned methods of reporting by the EU Member States. These reports give a general survey only of the use of animals in experiments at Community level. Key words: experimental animals; legislation; report Background In Europe and in other developed countries, methods are sought which would decrease the use of animals in experiments. Significant international development in regulating the protection of experimental animals was perceived at adoption of the European Convention (1) and Directive (2, 3). By Decision of the Received: 24 December 2008 Accepted for publication: 13 May 2009 Council of Europe 1999/575/EC (4), the Community was signatory to the Convention of the Council of Europe, ETS 123 (1), increasing thereby its commitment to endeavours of replacing experimental animals and protecting those still used in experiments. The objective of Directive (2) and European Convention (1) was to provide for the harmonisation of provisions on the protection of experimental animals in the national legislations of the Member States. By complying with the proposed standards, the disparities would be abolished, and the measures for 48 D. Ornik, V. Cadonic-Spelic the protection of experimental animals harmonised in providing for the adequate conditions of rearing, care and use and, in particular, to avoid the unnecessary duplications of experiments on animals, by complying with the uniform standards and mutual recognition of test results obtained by experiments already conducted on animals. The requirement that experiments on animals shall not be conducted if another acceptable, feasible and scientifically satisfactory method is available, which does not require the use of experimental animals, has contributed to a decreased use of experimental animals and stimulated the development of alternative methods for experiments on animals. In 1991, the European Centre for the Validation of Alternative Methods - ECVAM was set up in Italy, which directly contributes to the protection of animals, using the 3 R concept (Replacement, Reduction, Refinement) and providing for the validation of alternative methods (5). Greater significance is awarded to the implementation and monitoring of policies in the different spheres of consumer protection and safety testing of chemicals, cosmetic products, biocides, foodstuffs, biological substances and medical devices. At implementation of relevant legislation on safety testing, the alternative methods, which exclude the use of experimental animals, are becoming more and more important. One of the seven main objectives of the action plan of the future policy on chemicals, set out in the White Paper of2001 (6), is to support the developing of tests requiring no use of animals. Alternative methods can produce_reliable information in the most up-to-date and proven tests, which are more rapid and cost-effective than the existing experiments on animals. It has been estimated that the requirements (expenses and animals) for experiments within the REACH Programme (Registration, Evaluation and Authorisation of Chemicals) - including the registration, valuation, authorisation and restriction of chemicals, could decrease by 70 % if using the intelligent strategies for testing (7). A final goal is to replace_experiments on animals with methods that do not require the use of animals. At experiments which still do require the use of live animals, the goal is to decrease the number of animals used, and to improve the methods so as to cause less pain, suffering and harm. Activities towards the full implementation of the 3 R concept need to proceed in all the spheres of use of animals, the harmonisation of Directive 86/609/ EEC (2) and legislation requiring experiments on animals shall be fully implemented, and agreements on the mutual acceptability and recognition of data shall be subjected to scrutiny. Setting up a Community Reference Laboratory for the validation of alternative methods shall additionally improve the quality of alternative methods for testing, and speed up the validation procedure (6, 7). Legal basis for data collection on experimental animals In Slovenia, the Veterinary Administration of the Republic of Slovenia keeps records based on annual reports by the user organisations, in compliance with Article 24 of the Animal Protection Act (8). In 2004, the methods of collecting data on experimental animals were laid down in the Rules (9, 10). Pending the entry into force of these Rules, the data were collected from the reports submitted by scientists performing a particular experiment. Using a questionnaire prepared to this end and partly summing up the European Convention, Appendix B (1), and including the visits to user organisations, data were gathered for the period from 1992 to 1996, and presented to the public for the first time in 1999 (11, 12). Data collected on entry into force of the Animal Protection Act (8) were made available to the public and to all the applicants as public information (13). Based on Article 13 of Council Directive 86/609/ EEC (2), the competent authorities of the EU Member States collect and make available to the public the statistical data on the use of animals in experiments. In Article 26, the said Directive is laying down that the European Commission shall prepare regular reports for the Council and the European Parliament, using the data presented by the EU Member States. As the Directive does not lay down the form and scope of reporting, a common form was unanimously adopted to this end after the multi-annual discussions of the national competent authorities. The European Union requires the reporting to be performed using eight tables, called the EU Tables. The European Convention (1) requires the member states of the Council of Europe to provide reports in five tables, called the Convention Tables. The reservation of the European Community as to the reporting remained unchanged in Council Decision 2003/584/ EC (14) that is laying down the simplified procedure of amending the Appendices to the European Convention. The Republic of Slovenia, having ratified the European Convention in 2006 (15), provided as well for a reservation in accordance with Article 34 to the effect that it did not consider itself bound by the requirement of reporting statistical data. Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 49 Data on experimental animals used in the Republic of Slovenia in a 15-year period Table 1 shows data on experimental animals used in the Republic of Slovenia in a 15-year period, collected by the Veterinary Administration of the Republic of Slovenia. In 1992, the number of experimental animals totalled 37,212, as compared to 2006 with 13,181 animals only, which is by 65 % less. Most used were the laboratory mice, followed by laboratory rats and rabbits. Within the first five years of data collection on experimental animals there stands out a high number of poultry. The numbers include also the poultry used in nutritional experiments under the normal rearing conditions and without the invasive treatment of animals. Such use of poultry was excluded from the subsequent reports. Table 1 includes a column on the protected animal species that are protected in the Republic of Slovenia in accordance with the Decree (16, 17). Of the protected animal species, mostly the amphibians (frogs) were used in experiments. Most experiments are conducted on laboratory rodents in pharmaceutical industry for substance testing, and are carried out in accordance with the applicable legislation, the rules of pharmacopoeias applied, and international laws, regulations and administrative provisions. This information is evident from Table 2. Institutes and laboratories of university faculties of the medical, veterinary, biological and zootechnical programmes use animals in the baseline biological research, and in scientific and research studies. To a lesser extent, animals are used for diagnosing diseases, training and education, and for other purposes that are not particularly specified. Table 1: Number and species of animals used in experiments in the Republic of Slovenia in the period from 1992 to 2006 Species 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Mice (Mus musculus) 19,754 16,475 15,555 16,274 15,163 15,233 11,796 12,900 10,394 9,773 9,024 8,388 7,560 8,556 7,590 Rats (Rattus norvegicus) 7,458 6,472 5,974 5,659 5,066 4,011 4,387 3,261 4,284 3,504 3,201 3,056 4,429 2,732 4,767 Guinea Pigs (Cavia porcellus) 532 553 450 567 482 400 468 139 79 50 112 120 121 38 26 Hamsters (Mesocricetus) 0 0 0 0 0 10 3 0 0 0 0 0 0 0 0 Other Rodents (other Rodentia) 6 86 0 0 0 10 0 0 177 35 18 0 Rabbits (Oryctolagus cuniculus) 1,251 1,207 1,387 909 1,451 1,107 1,439 781 744 712 795 597 582 533 472 Cats (Felis catus) 43 50 50 57 29 60 50 83 55 44 38 0 1 0 0 Dogs (Canis familiaris) 19 52 44 18 19 14 17 21 3 12 14 34 7 15 6 Horses, donkeys, crossbreeds (Equidae) 10 0 1 0 0 1 1 4 26 1 0 Pigs (Sus) 22 22 40 26 27 69 239 82 246 29 106 6 11 16 0 Goats (Capra) 0 0 0 10 60 0 0 0 0 0 Sheep (Ovis) 19 21 21 19 17 35 22 21 36 47 47 43 21 57 50 Cattle (Bos) 22 22 22 22 22 22 2 1 36 20 0 0 0 0 0 Primates (Prosimia) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Poultry (Poultry) 8,004 7,394 8,524 8,575 7,625 361 520 334 60 438 225 0 0 0 0 Other birds (pigeon) 0 11 3,000 150 150 0 0 270 22 265 Fish (Pisces) 20 10 100 522 180 245 146 25 270 813 120 137 326 0 0 Other animals 706 606 756 172 287 236 81 4 0 4 Protected animals* 68 53 65 59 45 305 268 227 270 146 26 39 145 3 1 Total 37,212 32,337 32,318 32,707 30,136 22,578 19,975 21,631 16,819 16,086 13,945 12,682 13,538 11,991 13,181 Legend to Table 1: * Protected or endangered animal species in accordance with the Decree (16, 17) applicable at the time 50 D. Ornik, V. Cadonic-Spelic Table 2: Number of animals used in experiments as to the purpose of use in the Republic of Slovenia in the period from 1992 to 2006 Research type 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Baseline biological research (1) 13,107 12,711 14,526 13,863 13,656 2,668 4,276 4,001 2,688 3,688 3,975 3,700 4,391 1,888 1,457 Applied research (2) 23,282 18,701 16,649 16,018 15,545 17,974 14,048 13,018 12,071 10,081 8,685 8,202 8,182 9,420 10,564 Research of substances for protecting the public, animals and environment (3) 130 50 140 539 200 477 305 273 859 1,467 120 0 0 * * Education and practice (4) 177 85 87 182 112 832 812 745 475 468 336 239 454 283 214 Forensics (5) 4 11 2 1 0 0 0 0 0 0 0 0 0 0 0 Diagnosing diseases (6) 512 779 914 2,104 623 539 331 565 368 267 339 446 414 378 683 Other (7) 88 203 3,029 358 115 490 95 97 22 263 Total 37,212 32,337 32,318 32,707 30,136 22,578 19,975 21,631 16,819 16,086 13,945 12,682 13,538 11,991 13,181 (1) Baseline biological research: research of the anatomy or operation of living organisms, organs, tissues and cells (2) Applied research: research, development, quality control, pharmacological and toxicological analysing and testing of efficiency of medicinal products and medicinal substances in human medicine and dentistry, and in veterinary medicine. (3) Research of substances for protecting the public, animals and environment with toxicological and other testing (excluding medicinal products and medicinal substances) (4) Education and practice (5) Forensics: human or veterinary (6) Diagnosing diseases: tests for the specific determination of pathogens or for the production of diagnostic reagents (7) Other: other purposes of use of animals in experiments. * Number of animals used in the research of substances for protecting the public, animals and environment was in the period 2005 - 2006 included in the number of animals used in the applied research. Data on experimental animals used in the European Union in a 15-year period European Commission published five reports, which include data on experimental animals in the EU Member States. Table 3 shows the total number of animals used in experiments in the European Union in 1991, 1996, 1999, 2002 and 2005 (18, 19, 20, 21, 22). The first report on animals used in experiments in the EU Member States (18) includes data on the total number of animals in 1991 only, and was published in 1994. France and Portugal reported the data of 1992, and Belgium and Luxemburg did not present any relevant reports. Total number of experimental animals used in 1991, as reported by the EU Member States, amounted to 11.79 million. Data are shown in column 2 of Table 3. The second report on animals used in experiments in the EU Member States (19) provides data of 1996, and was published in 1999. The report includes the data of three new Member States, Austria, Finland and Sweden. France and Sweden presented their respective reports in the EU Tables, and the other Member States in the Convention Tables. France reported the data of 1997. This report and the subsequent reports include animals used for the education and training purposes. Such a purpose of use of animals is not laid down in Directive (2), but in Council Resolution 86/C331/02 (23). Some Member States included in their reports the animals which are neither indicated in the Directive nor in the Resolution, as for instance, the production of harmful mutants and transgenic animals. The number of animals used in experiments in the European Union in 1996 totalled 11.64 million. Table 4 shows relevant data. The second report includes also the data on the purpose of use of animals in experiments, as shown in Table 5. Thirteen Member States reported on the purposes of use of animals in experiments. In case of some Member States, the data on the total number of animals used in experiments did not tally with the data on the purpose of use of animals in experiments. Per purpose of use of animals in experiments, the number of animals used in experiments in 1996 in the thirteen Member States totalled 8.81 million. The third report on animals used in experiments in the EU Member States (20) provides the data of 1999, and was published in 2003. Member States presented their respective reports in the EU Tables, excluding one Member State that presented its re- Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 51 Table 3: Total number of animals used for experimental purposes in the EU Member States in 1991, 1996, 1999, 2002 and 2005 Abbreviation Member State 1991 1996 1999 2002 2005 AT Austria 204,825 130,295 192,062 167,312 BE Belgium 1,515,867 790,089 695,091 718,976 CY Cyprus 967 C Z Czech Republic 330,933 DE Germany 2,402,710 1,509,619 1,591,394 2,071,568 1,822,424 DK Denmark 304,370 350,226 323,444 371,072 365,940 EL Greece 25,439 19,280 9,686 515,423 926,092 ES Spain 558,823 506,837 475,726 262,042 595,597 EE Estonia 4,900 FR France 3,645,708* 2,609,322** 2,309,597 2,212,294*** 2,325,398 HU Hungary 297,290 IE Ireland 25,199 77,107 73,929 52,203 37,940 IT Italy 683,293 1,094,185 987,771 924,889 896,966 LV Latvia 13,319 LT Lithuania 5,767 LU Luxembourg 1,003 3,060 5,320 4,120 MT Malta 0 NL The Netherlands 876,058 652,300 621,466 640,930 531,199 PL Poland 358,829 FT Portugal 87,117* 49,520 39,851 44,577 41,621 FI Finland 110,659 228,334 644,880 256,826 SI Slovenia 11,991 SK Slovakia 23,369 SE Sweden 286,012 324,067 281,184 505,681 UK United Kingdom 3,181,768 2,659,368 1,905,462 1,817,485 1,874,207 Total 11,790,485 11,646,130 9,814,171 10,731,020 12,117,583 * data of 1992 ** data of 1997 *** data of 2001 port in the Convention Tables. The number of animals used in experiments in the European Union in 1999 totalled 9.81 million. Table 6 shows relevant data. The third report made by 14 Member States included also the data on the purpose of use of animals in experiments, as shown in Table 7. Data on the total number of animals used in experiments do not tally with the number of animals per purpose of use of animals in experiments. The fourth report on animals used in experiments in the EU Member States (21), as shown in Table 8, provides the data of 2002, and was published in 2005. All the fifteen Member States presented their respective reports in the EU Tables, with the exception of France that reported the data of 2001. The number of animals used in experiments in the European Union in 2002 totalled 10.73 million. Part 2 of the report conveys the data of the particular Member States, including clarifications. Purposes of use of animals are described in detail, including the required conditions and types of testing. The fourth report includes the data on the purpose of use of animals in experiments, as shown in Table 9. All the Member States reported on the purposes of use of animals in experiments. Data on the total number of animals used in experiments do tally with the data on the purposes of use. 52 D. Ornik, V. Cadonic-Spelic Table 4: Total number of animals used for experimental purposes in 1996 in the EU Member States in 1996 Species AT BE DE DK EL ES FR* IE IT LU NL PT n SE UK Total Rodents and rabbits 200,640 711,748 1,258,110 307,513 17,091 481,950 2,411,358 54,925 1,071,856 1,003 500,720 46,567 76,759 266,922 2,348,758 9,755,920 Cold - blooded vertebrates (1) 2,158 736,165 134,952 24,604 1,930 1,090 103,024 19,021 9,193 0 44,787 118 29,608 11,489 146,924 1,265,063 Birds (2) 0 54,982 94,793 9,347 129 17,736 67,652 94 9,218 0 86,071 329 1,912 3,178 113,691 459,132 Artio + Perissodactyla (3) 0 9,073 14,026 7,028 126 5,126 18,054 2,554 1,868 0 17,865 2,457 2,097 3,070 32,413 115,757 Carnivores(4) 274 2,899 5,887 1,710 2 812 6,545 513 1,254 0 1,763 44 248 1,266 12,980 36,197 Prosimians + Monkeys + apes 164 600 1,519 18 2 53 2,622 0 772 0 1,082 0 17 46 3,786 10,681 Other animals 1,589 400 332 6 0 70 67 0 24 0 12 5 18 41 816 3,380 Total 204,825 1,515,867 1,509,619 350,226 19,280 506,837 2,609,322 77,107 1,094,185 1,003 652,300 49,520 110,659 286,012 2,659,368 11,646,130 Species % AT BE DE DK EL ES FR* IE IT LU NL PT n SE UK Mean Rodents and rabbits 97.96 46.95 83.34 87.80 88.65 95.09 92.41 71.23 97.96 100 76.76 94.04 69.37 93.33 88.32 83.77 Cold - blooded vertebrates (1) 1.05 48.56 8.94 7.03 10.01 0.22 3.95 24.67 0.84 0.00 6.87 0.24 26.76 4.02 5.52 10.86 Birds (2) 0.00 3.63 6.28 2.67 0.67 3.50 2.59 0.12 0.84 0.00 13.20 0.66 1.73 1.11 4.28 3.94 Artio + Perissodactyla (3) 0.00 0.60 0.93 2.01 0.65 1.01 0.69 3.31 0.17 0.00 2.74 4.96 1.90 1.07 1.22 0.99 Carnivores (4) 0.13 0.19 0.39 0.49 0.01 0.16 0.25 0.67 0.11 0.00 0.27 0.09 0.22 0.44 0.49 0.31 Prosimians + Monkeys + apes 0.08 0.04 0.10 0.01 0.01 0.01 0.10 0.00 0.07 0.00 0.17 0.00 0.02 0.02 0.14 0.09 Other animals 0.78 0.03 0.02 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.01 0.03 0.03 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 * = France reporting for 1997 For abbreviations see Table 3 (1) = reptiles + amphibians + fish (2) = quails and other birds (3) = horses, donkeys and crossbreeds + pigs + goats and sheep + cattle (4) = cats + dogs + ferrets + other carnivores Table 5: Number of animals used for selected purposes versus species in the EU Member States in 1996 Species Baseline biological research Research, development and quality control of products and devices for human medicine and dentistry and for veterinary medicine Toxicological and other safety valuations (including valuation of products) Diagnosis of disease Education and training Other Total Rodents and rabbits 1,820,483 3,644,125 536,527 317,145 75,090 751,776 7,145,146 Cold - blooded vertebrates(1) 190,605 23,458 59,750 8,257 5,699 661,885 949,654 Birds (2) 64,592 68,256 6,226 5,406 936 45,423 190,839 Artio + Perissodactyla (3) 22,963 17,753 739 5,427 2,476 8,855 58,213 Carnivores (4) 8,381 14,982 3,403 814 693 211 28,484 Prosimians + Monkeys + apes 1,187 5,737 631 254 41 242 8,092 Other animals 98,561 129,136 152,167 51,064 7,019 1,337 439,284 Total 2,206,772 3,903,447 759,443 388,367 91,954 1,469,729 8,819,712 Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 53 Table 6: Total number of animals used for experimental purposes in 1999 in the EU Member States Species AT BE DE DK EL ES FR IE IT LU NL PT FI SE UK Total Mice 91,194 446,677 775,932 163,680 3,566 261,301 1,552,330 31,251 410,788 3,000 277,774 23,669 89,959 184,230 990,162 5,305,513 Eats 12,699 169,662 403,227 96,864 1,900 134,070 460,407 14,484 500,625 20 159,768 9,836 32,519 84,374 526,904 2,607,349 Guinea - Pigs 7,367 37,397 42,891 10,431 240 13,892 77,021 1,041 18,474 20 10,246 1,452 1,737 9,355 61,308 292,872 Other Rodents 396 19,641 18,020 1,310 0 1,227 25,605 133 6,023 0 5,267 1,211 1,763 550 18,848 99,994 Rabbits 15,056 20,968 50,623 6,543 632 19,496 49,836 915 19,030 20 9,222 730 1,686 5,031 27,578 227,366 Cold - blooded vertebrates(1) 1,447 65,097 179,869 29,018 1,840 20,605 29,042 20,052 7,995 0 47,428 539 89,094 28,249 130,595 650,870 Birds (2) 1,367 19,726 92,792 5,225 80 19,027 86,610 1,229 20,157 0 92,823 267 5,228 6,920 105,931 457,382 Artio+ Perisso-dactyla (3) 670 8,874 17,765 9,004 1,426 4,181 18,735 4,370 3,295 0 17,430 1,752 2,347 4,165 29,376 123,390 Carnivores(4) 92 1,557 7,531 1,358 2 1,831 7,417 441 847 0 1,153 94 1,844 774 10,632 35,573 Prosimians+ Monkeys+apes 7 490 2,084 0 0 96 2,322 0 512 0 320 0 9 66 3,191 9,097 Other Mammals 0 0 660 11 0 272 13 25 0 45 301 2,148 353 937 4,765 Total 130,295 790,089 1,591,394 323,444 9,686 475,726 2,309,597 73,929 987,771 3,060 621,466 39,851 228,334 324,067 1,905,462 9,814,171 Species % AT BE DE DK EL ES FR IE IT LU NL PT FI SE UK Mean Mice 69.99 56.54 48.76 50.61 36.82 54.93 67.21 42.27 41.59 98.04 44.70 59.39 39.40 56.85 51.96 54.06 Rats 9.75 21.47 25.34 29.95 19.62 28.18 19.93 19.59 50.68 0.65 25.71 24.68 14.24 26.04 27.65 26.57 Guinea - pigs 5.65 4.73 2.70 3.22 2.48 2.92 3.33 1.41 1.87 0.65 1.65 3.64 0.76 2.89 3.22 2.98 Other rodents 0.30 2.49 1.13 0.41 0.00 0.26 1.11 0.18 0.61 0.00 0.85 3.04 0.77 0.17 0.99 1.02 Rabbits 11.56 2.65 3.18 2.02 6.52 4.10 2.16 1.24 1.93 0.65 1.48 1.83 0.74 1.55 1.45 2.32 Cold - blooded vertebrates(1) 1.11 8.24 11.30 8.97 19.00 4.33 1.26 27.12 0.81 0.00 7.63 1.35 39.02 8.72 6.85 6.63 Birds (2) 1.05 2.50 5.83 1.62 0.83 4.00 3.75 1.66 2.04 0.00 14.94 0.67 2.29 2.14 5.56 4.66 Artio +Perisso-dactyla (3) 0.51 1.12 1.12 2.78 14.72 0.88 0.81 5.91 0.33 0.00 2.80 4.40 1.03 1.29 1.54 1.26 Carnivores (4) 0.07 0.20 0.47 0.42 0.02 0.38 0.32 0.60 0.09 0.00 0.19 0.24 0.81 0.24 0.56 0.36 Prosimians+ Monkeys+apes 0.01 0.06 0.13 0.00 0.00 0.02 0.10 0.00 0.05 0.00 0.05 0.00 0.00 0.02 0.17 0.09 Other Mammals 0.00 0.00 0.04 0.00 0.00 0.00 0.01 0.02 0.00 0.00 0.01 0.76 0.94 0.11 0.05 0.05 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 For abbreviations see Table 3 Table 7: Number of animals used for selected purposes versus species in the EU Member States in 1999 Species Baseline biological research Research, development and quality control of products and devices for human medicine and dentistry and for veterinary medicine Toxicological and other safety valuations (including safety valuation of products) Diagnosis of disease Education and training Other Total Mice 1,452,583 2,347,842 285,132 93,218 27,719 219,937 4,426,431 Rats 567,904 1,265,125 284,940 4,837 36,157 24,959 2,183,922 Other Rodents 40,631 215,796 51,397 3,618 1,571 11,897 324,910 Rabbits 22,701 84,159 30,104 9,108 3,316 9,850 159,238 Cold - blooded vertebrates (1) 215,412 56,186 82,113 21,317 11,300 82,470 468,798 Birds (2) 101,487 165,879 18,571 4,107 1,707 71,472 363,223 Artio + Perissodactyla (3) 45,687 34,135 3,584 3,573 4,824 13,129 104,932 Carnivores (4) 6,930 8,963 9,190 221 594 1,995 27,893 Prosimians + Monkeys + apes 1,279 1,796 3,687 22 4 206 6,994 Other Mammals 3,430 312 274 0 0 89 4,105 Total 2,458,044 4,180,193 768,992 140,021 87,192 436,004 8,070,446 54 D. Ornik, V. Cadonic-Spelic Table 8: Total number of animals used for experimental purposes in the EU Member States in 2002 Species AT BE DE DK EL ES FR* IE IT LU NL PT FI SE UK Total Mice 153,034 460,487 1,071,282 221,557 3,589 200,821 1,370,293 16,790 466,640 3,000 288,706 27,616 98,078 163,041 914,795 5,459,729 Rats 13,175 116,340 483,470 80,518 4,021 38,544 471,234 8,282 377,573 2,200 128,975 12,302 27,563 73,862 473,285 2,311,344 Guinea - Pigs 7,566 34,305 39,913 7,613 310 1,932 59,184 35 18,722 100 8,752 633 757 2,738 43,779 226,339 Other Rodents 132 19,315 24,057 6,966 135 587 24,099 6 9,106 0 7,788 93 3,822 1,283 13,820 111,209 Rabbits 15,560 10,805 132,833 5,542 1,492 2,292 53,545 130 12,481 20 8,093 908 1,235 2,165 20,574 267,675 Cold - blooded vertebrates (1) 1,176 26,235 208,805 36,171 502,360 14,888 109,831 21,046 6,202 0 32,426 2,399 502,400 19,383 165,938 1,649,260 Birds (2) 417 20,352 78,882 5,275 340 1,625 94,932 0 28,892 0 143,100 198 6,872 14,053 140,029 534,967 Artio + Perisso dactyla (3) 536 5,486 22,867 6,621 3,141 1,138 17,770 5,520 3,771 0 20,761 394 2,969 3,422 32,009 126,405 Carnivores (4) 388 1,191 6,468 794 35 141 7,518 262 1,071 0 1,968 34 494 1,049 8,699 30,112 Prosimians+ Monkeys+apes 78 567 1,844 5 0 74 3,840 0 420 0 270 0 0 91 3,173 10,362 Other Mammals 0 8 1,147 10 48 132 11 91 690 97 1,384 3,618 Total 192,062 695,091 2,071,568 371,072 515,423 262,042 2,212,294 52,203 924,889 5,320 640,930 44,577 644,880 281,184 1,817,485 1,0731,020 Species % AT BE DE DK EL ES FR* IE IT LU NL PT FI SE UK Mean Mice 79.68 66.25 51.71 59.71 0.70 76.64 61.94 32.16 50.45 56.39 45.04 61.95 15.21 57.98 50.33 50.88 Rats 6.86 16.74 23.34 21.70 0.78 14.71 21.30 15.86 40.82 41.35 20.12 27.60 4.27 26.27 26.04 21.54 Guinea - Pigs 3.94 4.94 1.93 2.05 0.06 0.74 2.68 0.07 2.02 1.88 1.37 1.42 0.12 0.97 2.41 2.11 Other Rodents 0.07 2.78 1.16 1.88 0.03 0.22 1.09 0.01 0.98 0.00 1.22 0.21 0.59 0.46 0.76 1.04 Rabbits 8.10 1.55 6.41 1.49 0.29 0.87 2.42 0.25 1.35 0.38 1.26 2.04 0.19 0.77 1.13 2.49 Cold - blooded vertebrates (1) 0.61 3.77 10.08 9.75 97.47 5.68 4.96 40.32 0.67 0.00 5.06 5.38 77.91 6.89 9.13 15.37 Birds (2) 0.22 2.93 3.81 1.42 0.07 0.62 4.29 0.00 3.12 0.00 22.33 0.44 1.07 5.00 7.70 4.99 Artio + Perisso dactyla (3) 0.28 0.79 1.10 1.78 0.61 0.43 0.80 10.57 0.41 0.00 3.24 0.88 0.46 1.22 1.76 1.18 Carnivores (4) 0.20 0.17 0.31 0.21 0.01 0.05 0.34 0.50 0.12 0.00 0.31 0.08 0.08 0.37 0.48 0.28 Prosimians + Monkeys +apes 0.04 0.08 0.09 0.00 0.00 0.03 0.17 0.00 0.05 0.00 0.04 0.00 0.00 0.03 0.17 0.10 Other Mammals 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.25 0.00 0.00 0.01 0.00 0.11 0.03 0.08 0.03 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 * = France reporting for 2001 For abbreviations see Table 3 Table 9: Number of animals used for selected purposes versus species in the EU Member States in 2002 Species Baseline biological research Research, development and quality control of products and devices for human medicine and dentistry and for veterinary medicine Toxicological and other safety valuations (including safety valuation of products) Diagnosis of disease Education and training Other Total Mice 2,125,001 2,473,444 358,090 187,231 54,716 261,297 5,459,779 Rats 638,337 1,196,783 375,656 8,548 52,062 39,908 2,311,294 Other Rodents 47,140 201,372 69,792 2,931 2,435 13,878 337,548 Rabbits 19,621 178,776 45,067 8,232 2,095 13,884 267,675 Cold - blooded vertebrates(1) 690,261 472,495 175,220 2,486 218,566 90,232 1,649,260 Birds (2) 141,623 197,706 18,975 6,631 4,934 165,098 534,967 Artio + Perissodactyla (3) 56,065 37,871 3,227 10,528 6,741 11,973 126,405 Carnivores (4) 5,754 9,202 13,188 712 408 848 30,112 Prosimians + Monkeys + apes 1,738 1,580 6,832 34 7 171 10,362 Other Mammals 2,886 58 0 0 3 671 3,618 Total 3,728,426 4,769,287 1,066,047 227,333 341,967 597,960 1,0731,020 * = France reporting for 2004 Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 55 The fifth report on animals used in experiments in the EU Member States (22) provides the data of 2005, and was published in 2007. All the twenty-five Member States presented their respective reports in the EU Tables. France presented its report of 2004. This report includes the data presented by the ten new EU Member States, i.e. Cyprus, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Malta, Poland, Slovakia and Slovenia. The number of animals used in experiments in the European Union in 2005 totalled 12.11 million. Data by the old Member States are presented in Table 10, and data by the new Member States in Table 11. The number of animals used in the ten new Member States represents 8.6 % of the total number of animals used in the twenty-five Member States. Part 2 of the report conveys data presented by the particular Member States, including clarifications. Purposes of use of animals are described in detail, including the required conditions and types of testing. Data on the total number of animals used in experiments do tally with the data on the purposes of use. Table 12 shows relevant data. Table 10: Total number of animals used for experimental purposes in the 15 EU Member States reporting up to 2005 Species AT BE DE DK EL ES FR* IE IT LU NL PT FI SE UK Total Mice 128,634 488,125 1,084,358 208,375 15,340 393,217 1,510,334 17,776 534,614 3,280 240,048 28,318 120,636 213,727 1,052,064 6,038,846 Rats 11,920 106,483 435,417 85,664 6,024 125,754 424,387 7,722 279,774 720 116,608 6,793 28,358 83,321 411,501 2,130,446 Guinea - Pigs 3,149 39,530 37,761 5,046 574 16,780 79,350 4 11,533 100 7,479 379 563 2,014 28,918 233,180 Other Rodents 224 4,134 15,538 6,783 40 1,202 21,374 0 3,840 0 8,411 129 3,313 1,436 11,962 78,386 Rabbits 18,439 21,159 103,329 5,805 1,255 11,878 93,282 379 9,916 20 8,251 594 1,214 2,112 15,523 293,156 Cold - blooded vertebrates (1) 2,104 40,286 74,905 36,852 902,275 31,013 66,072 6,420 19,598 0 18,076 4,799 93,240 188,545 203,173 1,687,358 Birds (2) 1,025 13,691 41,607 7,784 21 8,425 106,263 2,024 31,697 0 111,233 112 5,773 7,838 115,000 452,493 Artio + Perissodactyla (3) 1,664 3,530 20,622 8,603 548 6,094 13,540 3,281 4,420 0 18,963 460 2,569 4,378 22,787 111,459 Carnivores (4) 97 1,530 6,686 843 14 1,090 7,007 286 1,094 0 1,790 36 188 1,596 7,623 29,880 Prosimians+ Monkeys+apes 56 449 2,086 0 1 84 3,789 0 412 0 327 0 0 75 3,115 10,394 Other Mammals 0 59 115 185 0 60 0 48 68 0 13 1 972 639 2,541 4,701 Total 167,312 718,976 1,822,424 365,940 926,092 595,597 2,325,398 37,940 896,966 4,120 531,199 41,621 256,826 505,681 1,874,207 1,1070,299 Species % AT BE DE DK EL ES FR IE IT LU NL PT FI SE UK Mean Mice 76.88 67.89 59.50 56.94 1.66 66.02 64.95 46.85 59.60 79.61 45.19 68.04 46.97 42.27 56.13 53.07 Rats 7.12 14.81 23.89 23.41 0.65 21.11 18.25 20.35 31.19 17.48 21.95 16.32 11.04 16.48 21.96 19.28 Guinea - Pigs 1.88 5.50 2.07 1.38 0.06 2.82 3.41 0.01 1.29 2.43 1.41 0.91 0.22 0.40 1.54 2.12 Other Rodents 0.13 0.57 0.85 1.85 0.00 0.20 0.92 0.00 0.43 0.00 1.58 0.31 1.29 0.28 0.64 0.79 Rabbits 11.02 2.94 5.67 1.59 0.14 1.99 4.01 1.00 1.11 0.49 1.55 1.43 0.47 0.42 0.83 2.58 Cold - blooded vertebrates (1) 1.26 5.60 4.11 10.07 97.43 5.21 2.84 16.92 2.18 0.00 3.40 11.53 36.30 37.29 10.84 15.07 Birds (2) 0.61 1.90 2.28 2.13 0.00 1.41 4.57 5.33 3.53 0.00 20.94 0.27 2.25 1.55 6.14 5.44 Artio + Perissodactyla (3) 0.99 0.49 1.13 2.35 0.06 1.02 0.58 8.65 0.49 0.00 3.57 1.11 1.00 0.87 1.22 1.16 Carnivores (4) 0.06 0.21 0.37 0.23 0.00 0.18 0.30 0.75 0.12 0.00 0.34 0.09 0.07 0.32 0.41 0.33 Prosimians + Monkeys + apes 0.03 0.06 0.11 0.00 0.00 0.01 0.16 0.00 0.05 0.00 0.06 0.00 0.00 0.01 0.17 0.09 Other Mammals 0.00 0.01 0.01 0.05 0.00 0.01 0.00 0.13 0.01 0.00 0.00 0.00 0.38 0.13 0.14 0.08 Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 * = France reporting for 2004 For abbreviations see Table 3 56 D. Ornik, V. Cadonic-Spelic Table 11: Total number of animals used for experimental purposes in the 10 New Member States in 2005 Species % CY CZ EE HU LV LT MT PL SI SK Total Mice 967 82,252 4,350 138,312 10,480 5,116 0 126,492 8,556 14,975 391,500 Rats 0 31,703 484 109,479 2,376 493 0 51,558 2,732 6,761 205,586 Guinea - Pigs 0 4,075 0 8,360 297 0 0 10,763 38 594 24,127 Other Rodents 0 6,018 0 518 0 0 0 11,069 18 0 17,623 Rabbits 0 5,567 66 9,152 166 158 0 3,101 533 782 19,525 Cold - blooded vertebrates(1) 0 71,186 0 11,315 0 0 0 56,413 3 0 138,917 Birds (2) 0 126,241 0 17,434 0 0 0 62,618 22 251 206,566 Artio + Perissodactyla (3) 0 3,193 0 1,303 0 0 0 24,026 74 0 28,596 Carnivores (4) 0 459 0 1,330 0 0 0 7,728 15 6 9,538 Prosimians + Monkeys + apes 0 51 0 6 0 0 0 0 0 0 57 Other Mammals 0 188 0 0 0 0 0 5,061 0 0 5,249 Total 967 330,933 4,900 297,209 13,319 5,767 0 358,829 11,991 23,369 1,047,284 Species % CY CZ EE HU LV LT MT PL SI SK Mean Mice 100 24.85 88.78 46.54 78.68 88.71 0 35.25 71.35 64.08 37.38 Rats 0.00 9.58 9.88 36.84 17.84 8.55 0 14.37 22.78 28.93 19.63 Guinea - Pigs 0.00 1.23 0.00 2.81 2.23 0.00 0 3.00 0.32 2.54 2.30 Other Rodents 0.00 1.82 0.00 0.17 0.00 0.00 0 3.08 0.15 0.00 1.68 Rabbits 0.00 1.68 1.35 3.08 1.25 2.74 0 0.86 4.45 3.35 1.86 Cold - blooded vertebrates (1) 0.00 21.51 0.00 3.81 0.00 0.00 0 15.72 0.03 0.00 13.26 Birds (2) 0.00 38.15 0.00 5.87 0.00 0.00 0 17.45 0.18 1.07 19.72 Artio + Perissodactyla (3) 0.00 0.96 0.00 0.44 0.00 0.00 0 6.70 0.62 0.00 2.73 Carnivores (4) 0.00 0.14 0.00 0.45 0.00 0.00 0 2.15 0.13 0.03 0.91 Prosimians + Monkeys + apes 0.00 0.02 0.00 0.00 0.00 0.00 0 0.00 0.00 0.00 0.01 Other Mammals 0.00 0.06 0.00 0.00 0.00 0.00 0 1.41 0.00 0.00 0.5 Total 100 100 100 100 100 100 0 100 100 100 100 Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 57 Table 12: Number of animals used for selected purposes versus species in the all EU Member States* in 2005 Species Baseline biological research Research, development and quality control of products and devices for human medicine and dentistry and for veterinary medicine Toxicological and other safety valuations (including safety valuation ofproducts Diagnosis of disease Education and training Other Total Mice (Mus musculus) 2,465,474 2,727,254 384,741 225,524 86,597 551,356 6,440,946 Rats (Rattus norvegicus) 677,533 1,161,517 350,275 13,564 50,048 72,876 2,325,813 Other Rodents (other Rodentia) 53,241 230,403 56,006 4,512 2,606 6,548 353,316 Rabbits (Oryctolagus cuniculus) 15,463 237,411 38,761 8,322 3,856 8,829 312,642 Cold - blooded vertebrates(1 ) 485,858 942,973 116,123 5,905 40,236 235,180 1,826,275 Birds (Aves) (2) 251,443 249,024 53,935 9,723 5,440 89,494 659,059 Artio + Perissodactyla (3) 64,419 41,079 4,542 4,100 9,491 16,341 139,972 Carnivores (Carnivore) (4) 11,605 9,309 14,884 348 674 2,339 39,159 Prosimians + Monkeys + apes 1,456 1,397 7,004 16 42 536 10,451 Other Mammals (other Mammalia) 8,978 214 15 0 4 739 9,950 Total 4,035,470 5,600,581 1,026,286 272,014 198,994 984,238 12,117,583 * = France reporting for 2004 Table 13 shows the comparison between proportions of groups of animals used in the EU in experiments in 1996, 1999, 2002 and 2005 (22). As seen above, the most used group of animals represent the rodents and rabbits with around 80 %, with the highest use in 1999 and the lowest use in 2005. The second most used group of animals Discussion In the Republic of Slovenia, the data on animals used in experiments have been known since 1992; however, the data collection method was defined in 2004 only (in a specific regulation). Data collected in represent cold-blooded vertebrates, and their use ranges between 10 and 15 %, with the rather low use in 1999. Birds represent a third most used group of animals, which ranges between 4.7 and 5.4 %. A fourth most used group represent the equi-dae and ungulates with around 1 %. the Republic of Slovenia show a downward trend in animal use in experiments. In the beginning of data collection it was believed that the number of animals used in experiments would increase from year to year owing to the more comprehensive methods of data collection, though the real number of animals Table 13: Comparison between proportions of classes of animals used in EU in 1996, 1999, 2002 and 2005 Class of species 1996* 1999 2002** 2005*** % Rodents - rabbits (1) 81.3 86.9 78.0 77.5 % Cold - blooded vertebrates (2) 12.9 6.6 15.4 15 % Birds (3) 4.7 5 5.4 % Artio + Perissodactyla (4) 1.2 1.2 1.1 * = 14 Member States reporting for 1996; 1 for 1997 ** = 4 Member States reporting for 2002; 1 for 2001 *** = 24 Member States reporting for 2005; 1 for 2004 58 D. Ornik, V. Cadonic-Spelic used in experiments would be smaller. However, the presentation of the total number of animals used in experiments, by species and in a longer period of time, clearly demonstrates the opposite. In the Republic of Slovenia, the use of experimental animals in applied research shows a downward trend on account of validated alternative methods in use, which do not require animals, even if authorised for use in experiments by the law. In the most recent years in particular, the Slovenian legislative activity in the field of protection of experimental animals has been most productive with the scope of harmonising the Slovenian legislation with the EU law. Current activities are focused on improving the minimum accommodation standards and conditions of care of particular animal species, including those not covered by the applicable legislation. This is resulting from the Protocol of Amendment (ETS 170) to Convention (ETS 123) of the Council of Europe (23), which was fully transposed as recommendation by the European Commission (24) in Directive 86/609/EEC (2). A more extensive amendment of the applicable legislation is envisaged to take place, including the European Commission's proposal for a directive amending Directive 86/609/EEC. The proposed draft Directive (25) shall take into account the most recent developments in animal welfare and ethical concerns of animal use in experiments. The proposed draft Directive shall harmonise disparities between the national laws of the EU Member States so as to harmonise actions of protecting experimental animals, decreasing the number of animals used in experiments, and avoiding the unnecessary duplication of experiments. The Republic of Slovenia supports the strategy of protecting experimental animals, by urgently requiring the numbers of animals used in experiments to decrease, by introducing alternative methods and providing for the utmost protection and, at the same time, by providing for the welfare of animals which are still used in experiments on the reasonable and justifiable grounds. Reviewing the number of animals used in experiments in the particular EU Member States through all the five years of reporting, it may be established that the number of animals used in experiments has been oscillating in many Member States. Most animals were used in experiments in France, United Kingdom and in Germany. Data presented by the Member States show a general survey only of the use of animals in experiments in their respective countries. Data cannot be compared on account of the differing reporting methods. For this very reason it is important that the EU Member States present the data in a standardised way so as to facilitate comparison. It may be envisaged with certainty that the number of animals used in experiments will decrease in future, in the Republic of Slovenia as well as in the European Union. This fact will be influenced by the more rigorous legislation, more severe inspection and control, replacement of animals by alternative methods, authorisation granting procedures for the implementation of experiments which will take into account the opinions stated by the ethical commissions, staff qualification, higher responsibility of the researchers and their improved attitude towards experimental animals that shows in preparing the precise experimental protocols, in selecting the methods and in the implementation of experiments as such. In decreasing the numbers of experimental animals, the mutual cooperation of institutions, researchers at state and interstate levels, as well as active approach of animal protection societies will be of key importance. In decreasing the numbers of experimental animals in pharmaceutical industry in particular, in addition to the validated alternative methods, the interstate recognition of results obtained by experiments already conducted on animals, the improved biometric methods, improved baseline research stages of new substances, and the use of cell cultures, tissues or smaller groups of animals play a significant role. References 1. Council of Europe. European convention for the protection of vertebrate animals used for experimental and other scientific purposes. ETS No. 123. Strasbourg: Council of Europe, 1986: 51 str. 2. EEC. Council Directive 86/609/EEC on the approximation of laws, regulations and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes. OJ EEC 1986; L 358 (29): 1-28. 3. EEC. Directive 2003/65/EC of the European Parliament and of the Council of 22 July 2003 amending Council Directive 86/609/EEC on the approximation of laws, regulations and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes. OJ EEC 2003; L 230 (46): 32-3. 4. EEC. Council Decision of 23 March 1998 concerning the conclusion by the Community of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes. OJ EEC 1999; L 222 (42): 29-30. Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states... 59 5. Commission of the European Communities: establishment of a European Centre for the Validation of Alternative Methods (ECVAM). Communication from the Commission to the Council and the European Parliament. SEC (1991) 1794 final. Brussels: CEC, 1991: 6 str. 6. Commission of the European Communities. White paper: Strategy for a future chemicals policy. COM (2001) 88 final. Brussels: CEC, 2001: 1-32. 7. Commission of the European Communities. Commission working document on a Community action plan on the protection and welfare of animals 2006-2010. Strategic basis for the proposed actions. SEC (2006) 65. COM (2006) 14 final. Brussels: CEC, 2006: 1-25. 8. Animal Protection Act (ZZZiv-UPB2). UL RS 2007; 17 (43): 5943-56. 9. Rules on the professional, staffing and technical conditions for the carrying out of experiments on animals. UL RS 2004; 14 (36): 4330-2, corrigendum 14 (40): 4779-810. 10. Rules on conditions for experiments on animals. UL RS 2006; 16 (88): 9477-510. 11. Ornik D. Use of animals in experiments and of the system of control: doctoral thesis. Ljubljana: Veterinary Faculty, 1999: 141-2. 12. Ornik D, Pogacnik M, Lazar P, Skubic V, Tadic M. Use of animals in experiments in the Republic of Slovenia. Slov Vet Res 2000; 37 (1/2): 21-30. 13. Access to Public Information Act (official consolidated text). UL RS 2006; 16 (51): 5585-94. 14. EEC. Council Decision 2003/584/EC of 22 July 2003 concerning the conclusion of the Protocol of Amendment to the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes. OJ EEC 2003; L 198 (46): 10. 15. Act ratifying the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (MEKZVU). UL RS-MP 2006; 16 (20): 1558-67. 16. Decree on the protection of endangered animal species. UL RS 1993; 3 (57): 2851-5, 2000; 10 (69): 8610. 17. Decree on protected wild animal species. UL RS 2004; 14 (46): 5963-6016; 14 (109): 12972; 2007; 17 (115): 16486-7. 18. Commission of the European Communities. 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Commission of the European Communities. Proposal for a directive of the European parliament and of the Council on the protection of animals used for scientific purposes. COM, 2008 543/5. Brussels: CEC: 2008: 89str. 60 D. Ornik, V. Cadonic-Spelic POROČILA O UPORABI ŽIVALI V POSKUSIH V REPUBLIKI SLOVENIJI IN DRUGIH DRŽAVAH, ČLANICAH EVROPSKE UNIJE V 15-LETNEM OBDOBJU D. Ornik, V. Čadonič-Špelič Povzetek: Namen prispevka je prikazati podatke o številu in vrsti uporabljenih živali v poskusih ter o namenih uporabe živali v Republiki Sloveniji in Evropski uniji v obdobju 15 let. V Republiki Sloveniji na podlagi zbranih podatkov v letih od 1992 do 2006 ugotavljamo, da trend uporabe poskusnih živali pada. V letu 1992 jih je bilo 37.212, v letu 2006 pa le 13.181. V Sloveniji upada uporaba poskusnih živali zaradi uporabe validiranih alternativnih metod, ki ne zahtevajo živali. Zlasti v zadnjih nekaj letih poteka v Sloveniji aktivna zakonodajna dejavnost na področju zaščite poskusnih živali, da bi harmonizirali slovensko zakonodajo z evropsko. Evropska komisija je objavila pet poročil o uporabi živali v poskusih. Kot so poročale države članice, je bilo v letu 1991 11,79 milijona poskusnih živali, v letu 1996 11,64 milijona, v letu 1999 9,81 milijona, v letu 2002 10,73 milijona in v letu 2005 12,11 milijona. Prvi dve poročili dajeta nepopolno analizo zaradi neenotnega navajanja podatkov o uporabi poskusnih živali v državah članicah. Tretje in četrto poročilo temeljita na dogovorjenih enotnih tabelah. To omogoča razširjeno obrazložitev zbranih podatkov o uporabi poskusnih živali v Evropski uniji, kljub določenim neskladnostim pri poročanju držav članic. Drugo poročilo prvič vsebuje podatke, zbrane v treh novih državah članicah, peto poročilo pa v desetih novih državah članicah. Podatkov o uporabi živali v poskusne namene ni mogoče primerjati s tistimi iz prejšnjih poročil. Skupno število vključuje različne živalske vrste, od hladnokrvnih vretenčarjev na eni strani do sesalcev, kot so rejne živali ali človeku podobni primati v nekaterih državah članicah na drugi strani. Zaradi neenotnega poročanja držav članic primerjava med nacionalnimi poročili ni mogoča. Poročila dajejo le splošen pregled nad uporabo poskusnih živali na nivoju skupnosti. Ključne besede: poskusne živali; zakonodaja; poročilo Slov Vet Res 2009; 46 (2): 61-70 UDC 611.018.62:612.015.1:57.083.3 Review Paper ENZYME-IMMUNOHISTOCHEMICAL ASPECTS OF MUSCLE FIBER TYPE CLASSIFICATION IN MAMMALS Gregor Fazarinc Institute of Anatomy, Histology and Embryology, Veterinary faculty, University of Ljubljana, Gerbiceva 60, SI-1000 Ljubljana, Slovenia 'Corresponding author, E-mail: gregor.fazarinc@vf.uni-lj.si Summary: Skeletal muscles are the most abundant and adaptable tissue in mammalians. They are composed of heterogeneous muscle fibers, in which distinct sets of structural proteins and metabolic enzymes are expressed. The percentages of different muscle fibers in the muscle define its morphological and functional characteristics. In this review, we summarize enzyme-immunohistochemical techniques to present muscle fiber type characteristics and their diversity in somatic skeletal muscles of various animal species. The principal methods to define myofiber properties on the tissue sections are based on the immunohistochemical determination of myosin heavy chain (MHC) isoforms and the myosin ATPase and metabolic enzyme histochemistry Four MHC isoforms (-I, -Ila, -IIx and -lib) have been detected in somatic skeletal muscles of small mammals. Fibers that co-express more than one MHC isoform simultaneously are labeled as hybrid myofibers and are indicators of muscle fiber transition. The maximal shortening velocity of MHC fibers increases in the following order: -I < -IIa < -IIx < -IIb. On the basis of the myosin ATPase activity myofibers have been classified as types I, IIA, IIB and IIC. Type IIC fibers represent an intermediate type between MHC-I and type MHC-IIa fibers. Most large mammals do not possess fastest MHC-IIb isoform, although some recent studies in pigs and llamas have shown the existence of all three fast MHC isoforms in their skeletal muscles. Additional MHC isoforms are present transitorily during development, and in some highly functionally specialized muscles such as extraocular, laryngeal and masticatory muscles (MHC-extraocular, MHC-m). Embryonic and neonatal MHC isoforms are expressed during muscle development and regeneration. Slow MHC-I myofibers show high oxidative capacity, whereas fast MHC-II myofibers revealed entire spectrum of metabolic enzymes activity with large overlaps between contractile fiber types. Combining the contractile classification with metabolic enzymes activity, myofibers can be basically defined as slow-twitch oxidative (SO), fast-twitch oxido-glycolytic (FOG) and fast-twitch glycolytic (FG). In most cases enzyme and immunohistochemical techniques are not fully interchangeable, which makes combination of different techniques necessary to get a reliable classification of myofibers. Key words: skeletal muscle; myosin heavy chain; muscle fiber type; histochemistry; mammals Introduction The principal muscle functional properties, such as contraction speed and fatigue resistance are mostly related to the proportions of myofiber types. Therefore, defining muscle fiber type composition became an essential step of any functional and applicative research in clinical and sports medicine as well as in animal muscle development and meat quality studies. Received: 7 March 2009 Accepted for publication: 1 June 2009 In most mammals, skeletal muscle tissue represents about 55% of individual body mass and plays vital roles in locomotion, heat production and overall metabolism. In 19th century the French anatomist Louis Ranvier Antoine already observed that some muscles were darker and contracted more slowly during longer periods than paler muscles. This early observation was the basis for the distinction of red and white muscles, which was later found to be related to myoglobin content, an iron-containing oxygen transport protein in the muscle fibers (1). In the sixties and seventies of the last century, new histochemical procedures enabled to 62 G. Fazarinc distinguish muscle fibers on the basis of their contractile and metabolic properties. Furthermore, it was established that mammalian skeletal muscles were composed of different proportions of muscle fiber types, which define the properties of muscles as functional units. The proportions of the various myofiber types vary between muscles and between individuals for a given muscle (2). It is well known that endurance athletes have a greater proportion of slow-twitch oxidative fibers, whereas sprinters and weightlifters have more fast-twitch glycolytic fibers (3). Diverse myofiber type composition between individuals has been also reported in horses and dogs exhibiting different athletic abilities, as well as in different breeds of domestic pigs (4, 5). On the cryosections the contractile properties of the myofibers are usually established either through immunohistochemical detection of the myosin heavy chain (MHC) isoforms or enzymehisto-chemical determination of myosin ATPase activity, while the energy metabolism is estimated on the basis of the histochemically demonstrated metabolic enzymes activity in the myofibers. Although all three techniques provide valuable data about the myofiber properties, the results could be sometimes erroneously interpreted, above all due to a lack of correspondence between myofiber classification systems within species and between species and because of antibodies immunoreactivity, which show certain diversity among various species (6). Thus the main goal of this paper is to describe the cellular basis for the myofiber typing and present some particularities in the enzyme-immunohisto-chemical myofiber classification in different animal species. Myosin heavy chain fiber type classification The heterogeneity of mammalian skeletal muscle fibers is related to the diversity of myofibrillar proteins, predominantly the myosin heavy chain (MHC). Myosin is a large molecule composed of two myosin heavy chains: (200,000 kDa each) and four myosin light chains (MLC, app. 20 kDa) (7). MLC are divided into two alkali (essential) light chains and two regulatory light chains. The exact role of MLC in contraction is not fully established; however, it is assumed that they are involved in the regulation of shortening velocity of muscle fibers (8). The role of MHC is better established. It is both, a structural protein and an enzyme, which hydro- lyses ATP and is therefore essential in determining excitation -contracting coupling and movement (9). It is well documented that MHC composition determines the force-velocity characteristics, making MHC composition a good tool to type myofibers functionally. In mammalian skeletal muscles up to 9 MHC isoforms have been identified: -I, -IIa, -IIb, -IIx, -IIm, -neonatal, -embryonic, -extraocular and two cardiac. Each of them is encoded by a distinct gene and has its own myosin ATPase activity (10). They are grouped in clusters located in different chromosomes and forming distinct subfamilies. The subfamily of fast isoforms comprises genes coding for three fast isoforms (MHC-IIa, -Ilx and -lib) expressed in adult fast fibers of limb and trunk muscles and genes coding for extraocular, embryonic and neonatal isoforms. The subfamiliy od cardiac isoforms is composed of two genes, coding for slow (also p-cardiac) MHC expressed in cardiac muscle and in slow (type I) myofibers of skeletal muscles and for a-cardiac expressed in cardiomyocytes and in specialized skeletal muscles (masticatory, extraocular, laryngeal). Only the gene coding for masticatory (-IIm) MHC belongs to the third subfamily. This gene represents a autonomous subfamily because of the distinct chromosomal localization and also because sequence analysis carried out in cat, dog, and human shows a large diversity compared with all other MHC genes. (11). The main MHC isoforms in adult locomotory skeletal muscle are -I, -IIa, -IIx and -IIb. MHC-I is a slow contracting isoform, while the three MHC-II isoforms are fast contracting; however, with different shortening speed. The polymorphism among adult MHC isoforms is functionally relevant as they determine not only myosin ATPase activity and fati-gability, but the maximum shortening velocity of myofibers as well. Therefore, the existence of several MHC isoforms enables the skeletal muscles to fulfill different physiological demands. Studies on the isolated myfibers in rodents showed that the maximal shortening velocity increased in the following order: -I < -IIa < -IIx < -IIb (8, 12). Muscle fibers are capable of altering their phenotype under various conditions, such as altered neuromuscular activity, mechanical loading, hormonal profiles and aging. The changes in MHC isoforms follow a general scheme of reversible transitions from fast to slow and slow to fast in a order: MHC-I « MHC-IIa « MHC-IIx « MHC-IIb (13, 14). The consequence of the MHC isoform tran- Enzyme-immunohistochemical aspects of muscle fiber type classification in mammals 63 sition scheme is that expression of two adjacent MHC isoforms in the same myofiber is possible. Such myofibers are designated as hybrid ones in contrast to so called pure myofibers, which contain only one MHC isoform. Recently, it has been established that different developmental and fast MHC isoforms could be co-expressed in the same muscle fiber during development, muscle regeneration and electrical stimulation, as well as in some highly specialized muscles such as extraocular, laryngeal and masticatory muscles (15). Embryonic and neonatal MHC isoforms are typically expressed during muscle development and regeneration, yet they are also found in the intrafusal fibres (14). Masticatory (-IIm) MHC is phylogenetical-ly ancient and confers high maximal muscle force and power. It is highly jaw-specific and is expressed in reptiles and fish. It is also found in several orders of mammals including carnivores, primates, chirop-terans and diprotodonts.. In some species among listed mammals, masticatory myosin is replaced by some other isoform. It is postulated that during mammalian evolution, mastication of food became important, and in some yaw-closing muscles the masticatory myosin is replaced with a-cardiac, developmental, slow or fast limb isoforms to adapt to variety of diet (16, 17) Extraocular MHC isoform has been shown in ex-traocular and some laryngeal muscles. The shortening speed of these muscles has proved to be even faster than that of masticatory ones (18, 19). Finally, a-cardiac MHC is fast contracting MHC isoform contained in cardiac muscle. It has been identified in adult human and rabbit masticatory muscles and is also temporally expressed during postnatal muscle development in pig and horse skeletal myofibers (20, 21) and in skeletal myofibers after chronic low frequency stimulation in rabbit (22). Currently, the shortening speed of adult MHC isoforms decrease in order -extraocular > -IIm > -lib > -IIx > -Ila > a-cardiac > -I (23). The expression of distinct MHC isoforms in my-ofibers, their function and the comparison of fiber type characteristics between different skeletal muscles and species has been studied using different procedures. Immunohistochemical detection with sets of monoclonal antibodies raised against different MHC is the most frequently used method to type myofibers. However, specifity of antibodies is still a problem in distinguishing between fast MHC isofoms in different species. Finally, in situ hybridization using nucleic acid probes for MHC isoforms and RT-PCR are used to analyze the expression of MHC at the mRNA level (24). Figure 1: MHC isoforms expression in serial cross sections of canine triceps brachii muscle. MHC-I fibers were demonstrated using MHC-slow antibody (Fig. 1a). All myofibers that remained unstained were uniformly labeled with A4.74 antibody (fig 1b). The BF-35 antibody that recognizes all the MHC isoforms except -IIx, weakly stained a subpopulation of fast myofibers implying that they express the MHC-IIx isoform. Since these myofibers reacted positively with the A4.74 antibody, which is specific for MHC-IIa of rat, these fibers were first erroneously designated as hybrid co-expressing MHC-IIa and -IIx isoforms (MHC-IIa/x) (25). Further investigation showed that A4.74 antibody in canine muscles recognized both MHC-IIa and -IIx isoforms (26). Thus the BF-35 weakly stained myofibers were probably pure MHC-x myofibers, which were previously misclassified due to the nonspecific reaction of A,4.74 antibody 64 G. Fazarinc Myosin ATPase histochemistry Barany (9) demonstrated that if myosin was extracted from skeletal muscle and activated in the presence of actin, the acto-myosin ATPase activity was directly proportional to the speed of shortening of the muscle from which the myosin was extracted. Since ATPase activity is ubiquitous in living organism, specific techniques to reveal myosin ATPase activity have been developed. They are all based on the precipitation of inorganic phosphate coming from the hydrolysis of ATP by myosin ATPase in the presence of Ca2+. The staining procedure is performed on frozen unfixed sections, since fixation destroys enzyme activity. Subsequently, differences in the pH stability of myosin ATPase formed the basis for distinction of type I (slow) and type II (fast) myofibers (27). This method distinguished both fiber types at pH 9.4, because the fast type II fibers exhibited a much higher myosin ATPase activity at this pH than slow type I fibers. Further histochemical techniques based on propoperties of myosin ATPase activity revealed the presence of fast subtypes II fibers (28). Pre-incubation of serial cryosections in acid or alkali buffers before myosin ATPase staining could distinguish between type II fibers. Thus, three fast and one slow type could be demonstrated in small mammals (29, 30). The fast subtypes were shown to contain MHC-IIa, -IIx and -IIb, whereas the slow type contain MHC-I. These four types represent so called pure fibers containing only one MHC isoform. Alkali and acid stable type IIC fibers correspond to a hybrid my-ofiber population, containing both slow MHC-I and -IIa isoforms. Otherwise other hybrid fibers remain difficult to detect when using only myosin ATPase histochemisty. Their detection must be confirmed with complementary techniques such as immuno-histochemistry. It should be stressed that interspe-cies differences exist for the pH stability/lability of myosin ATPase which makes identification of fiber types by myosin ATPase slightly different among species. (31). Metabolic enzyme histochemistry Another widely used method for determining the muscle fiber properties is histochemistry for selected enzymes of energy metabolism. Several metabolic enzymes have been chosen to represent metabolic pathways involve in either oxidative or glycolytic fuel utilization. Thus, different mitochondrial enzymes are markers of the potential oxidation of diverse substrates including fatty acids, carbohydrates and amino acids. Different enzymes of the glycolysis are used to determine the potential anaerobic catabolism of glycogen and glucose to lactate. In practice, succinate dehydrogenase (SDH) and a-glycerophosphate dehydrogenase (a-GPDH) are most frequently used to characterize oxidative and glycolytic potential capacities of myofibers, respectively (32, 33). The combination of myosin ATPase with metabolic enzyme activities distinguishes, three basic muscle fiber types in mammalian muscles, i.e. slow-twitch oxidative (SO), fast-twitch oxido-glyc-olytic (FOG) and fast twich glycolytic (FG) (34). SO fibers are slow-contracting and are fatigue resistant. Structurally, they exhibit a small fiber diameter; possess a high mitochondrial and capillary density and a high myoglobin content. Energetically, these myofibers are rich in triglyceride droplets but have low level of glycogen and high energy creatine phosphate, which is usually used for explosive movements. Functionally, these fibers are used for aerobic activities like walking and maintaining posture. FOG fibers are fast contracting and resistant to fatigue. They have a high levels of mitochondria, capillary and myoglobine. They are rich in creatine phosphate and glycogen, moderately rich in triglycerides and exhibit an oxido-gly-colytic metabolism. These myofibers are capable of prolonged anaerobic activity with a relatively high force output. FG fibers are fast contracting and very sensitive to fatigue. They have a low mi-tochondrial, capillary myoglobin and trigliceryde content (35) but exhibit high creatine phosphate and glycogen concentrations. FG fibers have large diameters and are used for short anaerobic activity with high force production such as galloping or jumping. Slow type I myofibers are mostly oxidative and exhibit a rather uniform metabolic properties, whereas subtypes II fibers can be either oxido-glycolytic or glycolytic with large overlaps between subtypes. Moreover, MHC-IIb and -IIa fibers do not always correspond to FG and FOG fibers, respectively, and the discrepancy between myofiber classification becomes even more important when considering MHC-IIx myofibers. Therefore, the mixing of different classification systems can be misleading (36). Enzyme-immunohistochemical aspects of muscle fiber type classification in mammals 65 Figure 2: Pig longissimus dorsi muscle. Determination of slow-twitch oxidative (SO), fast-twitch oxido-glycolytic (FOG) and fast-twitch glycolytic (FG) fibers on a single cry-osection using combine SDH histochemistry and immu-nolabeling with an anti-MHC-I (slow) isoform (41). A highly organized pattern and unique distribution of fibers composed of clusters of SO fibers, which are surrounded by FOG fibers and more external FG fibers can be observed Because of its simplicity, muscle fiber classification into SO, FOG and FG is still widely used above all in studies in which basic information about contractile and metabolic properties of the muscles are required. To make the conventional enzyme-histochemical fiber typing more friendly to use, combined histochemical methods based on the successive staining of myosine ATPase and different metabolic enzymes such as SDH or NADH-TR on a single cryostat section have been developed (37, 38, 39). However, these techniques lead sometimes to unreliable fiber staining because of some incompatibilities in enzyme optimal conditions (40). More reliable results are usually obtained when combining successively metabolic enzyme staining and immu-noistochemnical labeling of fibers (Fig. 2). Patterns of muscle fiber type distribution in mammals The distribution and proportions of fiber types vary between species and muscles. In most mammalian species, skeletal muscles exhibit a random spatial distribution of different fiber types. Fibers belonging to the same motor unit i.e. innervated by the same motorneurone, exhibit similar contractile and metabolic characteristics and are interspersed between fibers of other motor units. In small mammals, like rodents and lagomorphs, three fast MHC isoforms, -Ila, -IIx and -lib are expressed in fast fibers (42, 43). On the contrary, MHC-IIa and -IIx isoforms are present in skeletal muscles of humans (44, 45), cats (46), dogs (25), cattle (47), goats (48), horses (49, 50) and brown bear (51). The muscle fiber type composition depends on the specific function of a muscle and, furthermore, extends to species specific differences. From the comparison of fast MHC isoforms concentration between species it seems clear that the relative amount of MHC-IIb isoform decreases as body size increases, whereas that of MHC-IIa and -IIx increases. A possible explanation for such differences in muscle fiber composition between species could be that, that small animals with faster movements need faster twitch myofibers than larger animals which movements are slower. This hypothesis can explain why most large mammal species do not possess the fastest MHC-IIb isoform in their skeletal muscles. Such hypothesis was additionally confirmed in rabbit, where MHC-IIb isoform is more intensively expressed in young than adult animals. Decrease in the relative concentration of MHC-IIb isoform with increasing age possibly relates to the growth of the animal and changes in its locomotion pattern (24). MHC isoforms transformation associated with the process of growth was described also in large mammals, although they usually do not contain MHC-IIb isoform. In these species, the proportions of MHC-I and MHC-IIa myofibers increase, while that of MHC-IIx myofibers decreases during growth. In the early postnatal period the increased expression of MHC-I and -IIa isoforms is the consequence of a transition from developmental to adult MHC profile (52, 53). However, during later periods of growth some MHC-IIx myofibers obviously transform into MHC-IIa myofibers via hybrid MHC-IIa/x myofibers and into MHC I via hybrid MHC-I/IIa myofibers. Such transformations were observed in adolescent bears (51) and up to six years of age in different horse breeds (54, 55). With increasing age the percentage of hybrid fibers decreases, which supports their transitional role in muscle maturation. Taken together, the slower and more fatigue resistant characteristics of skeletal muscles with increasing age likely relate to a progressive adaptation to increasing body weight. The lack of MHC-IIb isoform expression in most adult large mammals has been hypothesized to be related to body size and muscle fiber length. In large mammals the shortening of the fastest MHC-IIb isoform would produce such a high force that muscle could be injured (45). However, recent stud- 66 G. Fazarinc ies have shown the existence of all three fast MHC isoforms , including MHC-IIb, in adult pig longis-simus (56) and llamas semitendinosus (57), which did not support hypothesis suggesting no expression of MHC-IIb in large mammals. In fact, gene coding for MHC-IIb isoform has even been discovered in humans; however, its expression in skeletal muscles remains to be confirmed (58). The reason why this isoform would be expressed only in pigs and llamas among large mammals is not known. Both species exhibit a so called type grouping distribution of the muscle fiber types with central clusters of MHC-I myofibers surrounded by MHC-IIa, then MHC-IIx and finally more external MHC-IIb myofibers (5, 56). In other species such fiber type grouping can be observed in relation to some neuromuscular disorders, whereas it is a normal spatial distribution in porcine skeletal muscle (59). Highly organized focal arrangement is supposed to be functionally relevant. Thus, central clusters of MHC-I myofibers would be most easily mobilized first for weak long-lasting contraction, whereas MHC-IIb fibers would be mobilized last for short-lasting forceful contraction. However, such a muscle fiber type distribution is not prerequisite, since most mammals exhibit random mosaic fiber distribution and are nevertheless fully functional. As well, it could be speculated that MHC-IIb isoform is expressed in some large glycolytic pig skeletal muscles as a result of intense selection for high muscularity and growth efficiency; however, further research is needed to test this hypothesis (60). Correspondence between myofiber classification systems It is well documented that maximal shortening velocity of muscle fibers expressing homologous MHC isoforms greatly decreases with increasing body size (61). Such functional diversity of the homologous fast MHC isoforms between species is likely related to different structural characteristics. This is probably one reason why MHC antibodies, which are usually raised against rat isoforms, can have diverse reactivity with homologous MHC in large mammals and why myosin ATPase histochemistry protocols must be adjusted to each animal species. Because of some important differences in the reactivity of MHC antibodies between species (Table 1), a set of different antibodies is usually used to avoid myofiber misclassification. Table 1: Reactivity of commonly used antibodies raised against MHC isoforms in different species according to Smer-du at al. (6, 26, 51), Lefaucheur et al. (60) and Rivero et al. (49) ^^ Antibody MHC\. isoforms MHC-slow A4.74 SC-71 F113, 15f4 BF-35 BF-F3 MHC-I Rat + - - - + - Human + - - - + - Dog + - - - + - Bear + - - - + - Horse + - - - + - Pig + - - - + - MHC-IIa Rat - + + + + - Human - + + + + - Dog - + + + + - Bear - - - + + - Horse - + + + + - Pig - + + + + - MHC-IIx Rat - - - + - - Human - ± ± + + or - - Dog - + + + - - Bear - + + + - - Horse - - - + - - Pig - - ± + - - MHC-IIb Rat - - - - + + Human - - - + + - Dog - - - + + - Bear - - - + + - Horse - - - + + - Pig - - - + + + (- = negative reaction, +/- = weak reaction, + = positive reaction) The specificity of MHC-slow antibodies is unambiguous because they revealed type I myofib-ers in skeletal muscles of all species (25, 48, 49, 60), suggesting that the slow MHC-I isoform is highly conserved among species. On the opposite, fast MHC isoforms can show different antigenic properties between species. Thus, both A4.74 and SC-71 antibodies are specific to MHC-IIa isoform of rat, but cross-react with MHC-IIx in human, dog, pig, goat (26, 48, 60, 62, 63). In bear skeletal muscles both antibodies actually recognize MHC-IIx and not MHC-IIa isoform (51). This was confirmed with antibody BF-35, which reveals all MHC exept IIx in rat (42). Similar problems were Enzyme-immunohistochemical aspects of muscle fiber type classification in mammals 67 observed with the antibody F113.15F4, which recognizes MHC-IIa, -IIx and -IIb in rat, and only MHC-IIa and -IIx in dog and bear (25, 51). Some misclassification of myofibers between species also occurs using myosin ATPase histochemistry. The staining pattern of fibers depends upon the lability of myosin ATPase to pH preincubation and is related to the MHC isoforms content within a single myofiber. When two isoforms are expressed in the same myofiber, the staining pattern of the myosin ATPase is ambiguous and can lead to mis-classification of the muscle fiber type, especially of fast type II fibers. In the past myosin ATPase based classification led to some contradictory reports on fast fiber sub-types in large mammals. In some studies of canine muscles only type IIA and IIC myofibers were found (64, 65, 66, 67). On the contrary, other authors claimed that type IIB myofibers are present even in dogs, although they were slightly less acid-labile than type IIB in other species (68). The immunohistochemical labeling of MHC isoforms demonstrated that strongly acid- stable subclass of canine fast fibers, which were dark after preincubation at pH 4.6 and would thus correspond to type IIB myofibers of other species, actually expressed MHC-IIa isoform, and the more acid labile sub-class, which were named as IIDog fibers (69, 70) actually corresponded to MHC-x fibers (26). Such integrated use of both myosin AT-Pase and immunohistochemical labeling of MHC isoforms demonstrated that type IIB fibers have been misclassified in numerous previous studies based upon traditional myosin ATPase histochemistry in other large mammals as well (71). Myosin ATPase can also lead to fiber misclassification because of partial denaturation of the enzyme. Thus, the rapid postmortem acidification combined with increased muscle temperature encountered in some glycolytic muscles of stress susceptible pigs can lead to irregular and altered myosin ATPase staining these PSE (pale, soft and exudative) muscles (Figure 3). In such case, the use of antibodies against MHC isoforms is a far more reliable technique to type myofibers (72). 3c 3d III» IB 0 1IB v ' 1 1 17' Figure 3: Alkali stable myosin ATPase activity (a, b) (73) and myosin ATPase activity pH 4.3 preincubation (c, d) of a normal (a, c) and PSE (b, d) pig longissimus dorsi muscle. Three different fiber types are distinctly recognized in normal muscle (I, IIA, IIB); whereas, the staining pattern of the alkali stable myosin ATPase is altered in PSE muscle. The PSE condition mostly inactivated the alkali stable myosin ATPase activity in peripheral fast-twich glycolytic IIB fibers Conclusion Big differences in muscle fiber type composition exist between muscles and species, and between individuals within species. It is well documented that skeletal muscles is a highly adaptable tissue which can be influenced by many intrinsic and extrinsic factors, such as age, altered neuromuscular activity and mechanical loading. The principal methods to type myofibers on the tissue cryosection are the immunohistochemical detection of MHC isoforms and the myosin ATPase and metabolic enzyme his-tochemistry. 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Arch Neurol 1977; 34: 171-3. ENCIMSKO-IMUNOHISTOKEMIČNI VIDIKI RAZVRšČANJA TIPOV MIšIČNIH VLAKEN PRI SESALCIH G. Fazarinc Povzetek: Skeletne mišice so pri sesalcih tkivo, ki ga je v telesu največ in je tudi najbolj prilagodljivo. Sestojijo iz mišičnih vlaken, ki se razlikujejo po vsebnosti strukturnih proteinov kot tudi po aktivnosti presnovnih encimov. Zato številčni deleži tipov vlaken v mišici določajo njene morfološke in funkcionalne značilnosti. V članku so predstavljene osnovne encim-sko-imunohistokemične tehnike, na osnovi katerih prepoznavamo značilnosti posameznih tipov mišičnih vlaken kot tudi njihovo raznovrstnost v skeletnih mišicah različnih živalskih vrst. Na tkivnih rezinah razvrščamo mišična vlakna na osnovi imunohistokemičnega določanja vsebnosti izoform težkih miozinskih verig (MHC), aktivnosti miozinske ATP-aze in aktivnosti presnovnih encimov. V somatskih skeletnih mišicah manjših sesalcev so dokazane štiri različne izoforme težkih miozinskih verig (MHC-I, -Ila, -IIx in — Ilb). Glede na vsebnost izoforme raste hitrost krčenja mišičnih vlaken v naslednjem zaporedju MHC: -I < -IIa < -IIx < -IIb. V t. i. hibridnih vlaknih sta izraženi dve izoformi MHC in sta pokazatelj preobrazbe mišičnih vlaken. Na osnovi aktivnosti miozinske ATP-aze vlakna razvrščamo v tipe I, IIA, IIB in IIC. Vlakna tipa IIC predstavljajo prehodni tip med vlakni MHC-I in MHC-IIa. Mišice pri večini velikih sesalcev ne vsebujejo najhitrejše MHC-IIb izoforme, vendar pa so zadnje študije pokazale prisotnost vseh treh hitrih MHC izoform tudi pri domačem prašiču in lami. Zrkelne, grlne in žvekalne mišice, ki se tako funkcijsko kot razvojno razlikujejo od somatskih, vsebujejo tudi t. i. ekstraokularno (MHC-ekstraokularna) oz. mastikatorno (MHC-m) izoformo, med razvojem in regeneracijo pa so v mišicah prisotne še razvojne izoforme (MHC-embrionalna, MHC-neonatalna). Počasna MHC-I vlakna kažejo veliko oksidativno presnovno zmožnost, v hitrih MHC-II vlaknih pa je aktivnost presnovnih encimov zelo različna. Na osnovi krčljivostnih lastnosti mišičnih vlaken in ob upoštevanju njihove presnovne aktivnosti jih lahko razvrstimo v tri osnovne tipe: počasi krčljiva oksidativna (SO), hitro krčljiva oksidativ-no-glikolitična (FOG) in hitro krčljiva glikolitična (FG). Razvrščanje vlaken na osnovi samo ene od opisanih metod je večkrat problematično, zato je za natančno in zanesljivo določitev lastnosti mišičnih vlaken potrebno sočasno uporabiti različne encimsko-imunohistokemične tehnike. Ključne besede: skeletna mišica; težka miozinska veriga; tip mišičnega vlakna; histokemija Slov Vet Res 2009; 46 (2): 71-79 UDC 636.92.083.14:636.082.4.084/.087 Original Research Paper influence of gradual change in feed, use of acidifier and prebiotic on rabbits in the period of weaning Breda Jakovac-Strajn1*, Uroš Pestevšek1, Tomaž Knafelc2 11nstitute of Hygiene and Pathology of Animal Nutrition, Veterinary Faculty, Gerbičeva 60, 2Jata Emona d.o.o., Agrokombinatska 84, 1000 Ljubljana, Slovenia Corresponding author, E-mail: breda.jakovac-strajn@vf.uni-lj.si Summary: To establish the possibility of improvement of rabbit production results, the use of feed acidifier, supplements to feed and gradual change from feed for does to feed for weanlings was investigated. Ten days before parturition 3 groups of 15 pregnant does each were formed. They were fed feed containing coccidiostatic drug Robenidin (1 mg/ kg). To the first two groups acidifier Acid Pac 4 Way (2 g/l) was administered in drinking watter. Between day 22 and 30 of weanlings age gradual change from feed for does to feed for weanling rabbits, that contained prebiotic Bio Mos (2 g/ kg), was carried out in the first group. The second group got the same feed but there was no gradual change of feed. In the third group gradual change of feed was performed, but for weaned rabbits only basic feed without supplements was used. The results of parturitions, feed consumption, number and weight of weaned rabbits to the 40th day of age and losses of does and their youngs were registered. Statistically significant difference (x ±SE) in the number of equalized and weaned rabbits were stated between 2nd (7.3±0.3, 6.7±1.91) and 3rd (8.7±0.21, 9.5±1.24) group (P<0.05). At weaning the greatest average weight of youngs was found in the 1st group (640.7±19.29 g) and at the age of 40 days in the 2nd group (934.1±10.41 g). The results showed that feed supplements can contribute to better results in intensive rabbit production. Key words: animals, feeding; feed, additives; oligosaharides; animals, suckling; rabbits Introduction In the last decades, the performance of intensive rabbit production improved a lot, due to the development of specialised strains (hybrids), increasing use of artificial insemination, adapted diets and management rationalisation. However, mortality of youngs before and after weaning is still very high and mounts in total to nearly 25%. The major part of these losses is due to diarrhoea, whose etiology is multifactorial (1). The nutrition requirements of a rabbit derive from physiological processes of digestion. Being herbivorous a rabbit needs crude fibres, which assure normal physiological activity Received: 10 January 2009 Accepted for publication: 15 May 2009 of digestion and reduce the appearance of metabolic disorders. Selective separation of particles inside ileo-cecal region and the ability of repeated utilization of soft faeces by cecotrophy represent two particularities of rabbit digestion with specific influence on digestive processes (1, 2). For economical reasons in the intensive breeding of rabbits pelleted feeds are used (3). Due to small number of rabbit farms in Slovenia and correspondingly small demand for rabbit feeds, their production still represents a lateral program of feed industry. Customary three types of feed are used (for does, for weanling rabbits and for animals in fattening), despite the known fact that different categories of rabbits cannot be fed the same feed by regulating its amount in correspondence to the category (4). 72 B. Jakovac-Strajn, U. Pestevsek, T. Knafelc Actually the nutrition requirements of young rabbits after weaning and their mothers are antagonistic. Does have great demands for energy while the use of feed with low starch and high fibres content before weaning benefits the health status of weaned youngs. By classical technology of feeding young rabbits are fed together with their mothers from the same vessel and get special feed for weanling animals only after weaning. Frequently transition from milk to solid pelleted feed represents greater stress than weaning itself (1,5). Main problem of growing rabbits nutrition is thus securing of physiological balance between adequate provision of nutritional substances and avoiding of metabolic disturbances related to disharmonies of this provision. Symptoms of such digressions are in principle diarrhoea and higher mortality, especially in the period of weaning, causing great economic losses (6). Besides the improvement of technological procedure of young rabbits transition from suckling period to the period after weaning, in intensive rabbit production different supplements (enzymes, nutritive antibiotics, flavours, probiotics, acidifiers etc.), helping in diminishing the stress, are used in feed or water. Because of illness complexity and lack of special products for rabbits the influence on microbial population of guts is in principle less successful. Anyhow some researches show positive effect of probiotics and acidifiers supplementation on rabbit production, feed conversion and on reduction of enteritis frequency (5,7). It is also considered that young rabbits with higher body weight by weaning show lower sensibility to weaning stress (1). The presented research was performed to study the possibilities for diminishing of digestion disturbances in young rabbits by corresponding mode of feeding with the aim of lowering their mortality and achieving higher body weight at weaning. Material and methods Animals The experiment was carried out in facilities for parent stock and for fattening rabbits of a bigger rabbit farm. All the technological procedures except feeding, where daily consumption of feed was controlled, were standard for the farm. The experiment included 45 pregnant, clinically healthy females of New Zealand - Californian crossbreed, from 38 do 90 weeks old, weighing between 3700 g and 5230 g. The experiment started ten days before parturition when the does were divided into three groups of 15 animals each. At the day 13 after delivery the does were inseminated and 18 days later the fertility control was carried out. After delivery the offsprings were left in the groups of their mothers and were controlled until the day 10 after weaning - to the average age of 40 days. Two days after the last delivery in the group, the litters inside the group were equalized by size and number of kits, considering the principle that the litter must be composed from young rabbits with the same outliving ability and that weak litters belong to the does with higher milk production. In the case of doe's loss, the kits were divided among other litters of the same group. Weanling rabbits were transferred to fattening stall at the average age of 30 days. For the first time they were weighed on the day of equalization and later at average age of 10, 17, 24, 31, and 40 days. Health status and mortality of animals were controlled during the whole experiment. Feed andfeeding The pelleted feed was prepared in the farm owned feed factory following the standard prescription used in the farm. The consumption was not limited and the composition of basic feed was as follows: - feed for does contained: 37% alfalfa, 20% wheat bran, 15% oats, 11% sunflower meal, 8% dried beet slices, 4.25% barley, 1.5% vegetable oil, 1% pinotan (lignin sulphate as pellet binder), 0.7% dicalcium phosphate, 0.25% salt, 0.2% lisin, 0.1% alimet (liquid metionin - 88%), 1% premix for does and 1.4g/kg of natural tannin extract (extract from chestnut wood), - feed for weanling rabbits: 37% alfalfa, 25% wheat bran, 23.7% oats, 6% dried beet slices, 5% soybean meal, 1% vegetable oil, 1% pinotan, 0.2% salt, 0.1% alimet, 1% premix for weanling rabbits and 2.0g/kg of natural tannin extract (extract from chestnut wood). Regarding the program of the trial, the basic feeds were supplemented with Robenidin (coccidi-astatic) or Bio Mos (Alltech Inc.) - beer leaven from yeast Sacharomyces cerevisae containing mannoo-ligosaccharides, structural parts of yeast cell wall. The samples of feeds were analysed at The Institute for Hygiene and Pathology of Animal Nutrition Influence of gradual change in feed, use of acidifier and prebiotic on rabbits in the period of weaning 73 of Veterinary Faculty in Ljubljana. Chemical composition was determinated by Weende analyses (8), whereas macro- and microelements: calcium, magnesium, sodium, potassium (9), manganese, zinc, copper and iron (10) were determined by atomic absorption spectrometry and phosphorus spectro-photometricaly (11). The microbiological quality of feeds was established in accordance with the procedure by Schmidt et al. (12). The number of grown colonies of mesofilic aerobic bacteria, moulds and yeasts was ascertained and expressed in colony forming units per gram feed. For the gradual change from feed for does to feed for weanling rabbits, carried out between 22nd and 30th day of young rabb^ age, mixtures of both feeds were prepared and fed in appointed proportions. At the day 22nd and 23rd of the young rabbits age mothers and their offsprings were fed 80% of feed for does and 20% of feed for weanling rabbits. Next 3 days both feeds were mixed in proportion 50:50. Afterwards, up to the 29th day the mixture contained 20% of feed for does and 80% of feed for weanling rabbits. At the day of weaning (30th day of age) they got only feed for weanling rabbits. The does returned to their own feed after three days. At the first day they got 80% of feed for weanling rabbits and 20% of feed for does, at the second day 50% of feed for weanling rabbits and 50% of feed for does and at the third day only 20% of feed for weanling rabbits and 80% of feed for does. At the fourth day they were fed only feed for does. During gradual change of feed does got feed for weanling rabbits with no coccidiostatic drug. Therefore their faeces were coprologically controlled 3 times: at delivery, 3 weeks after delivery and 3 days after weaning. Collective faeces sample of each group was taken from ten places of the floor under cages. Acidifier in the drinking water In the 1st and the 2nd group of animals acidifier Acid Pac 4 Way (Alltech Inc.), in concentration of 2 g/l, was supplemented in fresh drinking water, which was permanently available to does and their kits until weaning. Afterwards, weaned rabbits got water with no acidifier. Basic feeds, supplements and feeding technology used in the experiment are presented in table 1. Table 1: Scheme of feeds, supplements and feeding technology used in the experiment group feed supplements Acid Pac 4 Way in water (only for does) gradual change from feed for does to feed for weanling rabbits feed for does feed for weanling rabbits 1 feed A feed 1 YES YES 2 feed A feed 1 YES NO 3 feed A feed 2 NO YES Legend: feed A: Basic feed for does + coccidiostatic Robenidin (1mg/kg) feed 1 : Basic feed for weanling rabbits + prebiotic Bio Mos (2 g/kg) feed 2 : Basic feed for weanling rabbits Statistical methods The results were statistically evaluated by one way ANOVA, followed by posthoc Scheffe test. All data were analysed using SPSS (Statistical Package for Social Sciences - Version 12, November 2003) software package. Results Feed analyses The results of chemical analyses of feeds (table 2) were estimated regarding producer's declaration. Considering maximum permitted deviations and measurement uncertainty of used methods no digression from declared values was found. Consumption of feed In the first fifteen days after delivery feed was available only to does and afterwards also to their offsprings. No statistical significant difference between groups was observed regarding average consumption of feed (does + litter) from delivery to weaning. The lowest average consumption was observed in the second group (440.6 ± 185.1 g feed per day) and the highest in the first group (486.9 ± 199.3 g feed per day). The highest average day consumption of feed from weaning to the 40th day of age, calculated on single weaned rabbit, was found in the 1st group (table 3). 74 B. Jakovac-Strajn, U. Pestevsek, T. Knafelc Table 2: Analyzed composition and microbial content of diets for does and weanling rabbits analysis feed for does feed for weanling rabbits Bio Mos no supplement dry matter (g/kg) 892.1 904.3 895.2 humidity (g/kg) 107.9 95.7 104.8 crude proteins (g/kg) 159.2 150.6 151.1 crude fibres (g/kg) 163.5 150.7 149.6 crude fat (g/kg) 30.0 28.0 30.0 ash (g/kg) 72.8 71.0 70.7 nitrogen free extract NFE (g/kg) 466.6 504.0 493.8 starch (g/kg) 173.9 195.6 163.0 calcium (g/kg) 8.0 7.0 6.8 phosphorus (g/kg) 6.1 4.7 4.6 pothassium (g/kg) 11.3 11.0 10.6 sodium (g/kg) 1.7 2.0 1.8 magnesium (g/kg) 3.0 2.9 2.8 zinc (mg/kg) 157.0 129.7 106.3 copper (mg/kg) 28.7 33.0 22.2 manganesse (mg/kg) 177.4 144.0 125.8 iron (mg/kg) 519.0 570.8 556.6 chlorides (g/kg) 4.4 5.2 5.3 yeasts (v 1000/g) 0 0 0 total number of moulds (v 1000/g) 0 0 1.0 Aspergillus spp. (v 1000/g) 0 0 1.0 Table 3: Average day consumption of feed from weaning to the 40th day of age group 1 group 2 group 3 P n x ±SE n x +SE n x +SE 1 vs.2 1 vs.3 2 vs.3 consumption of feed (g/weanling) 108 105.4 + 9.76 98 95.9 + 11.66 97 84.7 + 6.58 0.732 0.869 0.782 Legend: n = nr. of 40 days old rabbits; x = average day consumption of feed; SE = standard error The highest feed conversion in the first 10 days the 3rd group (2.8). Feed conversion of the 2nd group after weaning was also stated in the 1st group (3.7 was 3.1 kg of feed per 1 kg of weight gain. kg of feed per 1 kg of weight gain) while the lowest in Influence of gradual change in feed, use of acidifier and prebiotic on rabbits in the period of weaning 75 Deliveries The deliveries took place from 30th to 34th day of gestation. Out of 45, 44 does delivered, so the 1st Statistical comparisation between 2nd and 3rd group showed significantly higher number of equalized rabbits (P = 0.034) and weaned rabbits / doe in the 3rd group (P = 0.008). Body weight of young rabbits Average weight of young rabbits by equalization and at the 10th day of age was similar in all three Health status control Mortality observed during the experiment in all groups and categories of animals could be considered as the consequence of technological reasons. Five casualties among does were due to pneumonia and endometritis and the reason for mortality at group was formed only from 14 litters, one less than the other two groups. The greatest number of live born, equalized and weaned rabbits was in the 3rd group (table 4). groups. At the 17th day of age retardation of weight gain was observed in rabbits of the 2nd and especially of the 3rd group (table 5). With exception of rabbits from the 3rd group, the average body weight of rabbits at weaning exceeded 600 g. At the end of experiment (at the age of 40 days) the highest average body weight of rabbits was in the 2nd and the lowest in the 3rd group. young rabbits was mainly diarrhoea. Loss of kits from equalization to weaning was the greatest in the 3rd and the smallest in the 1st group (figure 1). After weaning the mortality in the 3rd group was still increasing while in the 1st and the 2nd group it diminished to the practically equal amount (1.8% and 2%). Table 4: Number of live born, equalized and weaned rabbits in different groups group 1 group 2 group 3 P n x ±SE n x ± SE n x ±SE 1 vs.2 1 vs.3 2 vs.3 live-born 14 8.6 ±0.99 15 8.3 ±0.89 15 9.3 ± 1.01 1.00 0.999 0.991 still-born 14 1.8 ±0.96 15 0.7 ±0.66 15 0.5 ±0.53 0.912 0.865 1.000 equalized 14 8.1 ±0.34 15 7.3 ± 0.30 15 8.7 ±0.21 0.444 0.884 0.034 nr. of weanling 13 7.3 ±3.35 15 6.7 ± 1.91 12 9.5 ± 1.24 0.971 0.093 0.008 Legend: n = number of animals in the group; X = average body weight; SE = standard error Table 5: Average body weight and the number of young rabbits in groups from equalization to weaning body weight of young rabbits (g) group 1 group 2 group 3 P n X ±SE n X ± SE n X ± SE 1 vs.2 1 vs.3 2 vs.3 at equalization 114 93.3 + 5.46 109 91.9 ± 4.60 130 90.5 ± 5.22 1.000 0.999 1.000 10th day of age 111 192.2 ± 8.91 104 187.0 ± 6.49 127 171.1 ±9.26 1.000 0.640 0.813 17th day of age 111 291.1 ± 12.08 102 264.0 ± 10.84 126 254.7 ± 11.89 0.729 0.461 0.997 24th day of age 111 404.3 ± 21.68 100 395.1 ± 11.64 120 351.1 ± 13.77 0.999 0.382 0.583 at weaning 110 640.7 ± 19.29 100 627.0 ± 21.01 114 578.8 ± 28.00 0.999 0.909 0.999 40th day of age 108 922.7 ± 16.36 98 934.1 ± 10.41 97 881.9 ± 27.28 1.000 0.884 0.772 76 B. Jakovac-Strajn, U. Pestevsek, T. Knafelc 1 1 □ group 1 □ group 2 ■ group 3 1 _ Figure 1: Percentage of mortality from equalization to weaning and from weaning to the age of 40 days Coprological examinations of faeces The contamination of does with coccidia ooccysts and eggs of other parasites is presented in table 6. Table 6: Results of coprological examinations of does faeces results of coprological examinations group on the day of delivery 21st day of litter age three days after weaning 1 0 Passalurus ambigus + Passalurus ambigus + 2 Passalurus ambigus + Passalurus ambigus + Passalurus ambigus + 3 single oocysts Passalurus ambigus + Passalurus ambigus + In the facilities for parents stock no clinical case of coccidiosis was stated. The presence of coccidia was confirmed only in the 3rd group, but the low number of stated coccidia prevented their determination. In the groups with gradual change of feed (group 1 and 3) the number of coccidia oocysts was not increased. Discussion Concerning the results of different management of rabbit feeding, there was no significant difference between the three experimental groups regarding body weight of weaned rabbits and average day consumption of feed from weaning to the 40th day of age, representing the essential data of the experiment. The average number of weaned rabbits was statisti- cally significantly higher in 3rd group in comparison with 2nd, but the mortality in this group was during the experiment the highest and the average body weights of rabbits were the lowest. Gradual change from feed for does to feed for weanling rabbits did not improve production results, but the positive effect of prebiotic and acidifier supplementation was clearly obvious from higher body weight and lower mortality in the 1st and 2nd groups where they were used. Feed for does and weanling rabbits that was normally used on the farm was applied in the experiment. Especially the feed for weanling rabbits was problematic due to provision of rough material with higher crude protein and crude fiber content. The content of starch was also too high, which was obvious from the analyses of feed for does and feed for weanling animals where similar values of starch and other nutritive substances were detected (table 2). The comparison of crude fibre with normative values reviewed by Kermauner (13) showed their lack in the feed for weanling rabbits (150.7 and 149.6 vs. > 155 g/kg). Problems of crude fibres, crude proteins and starch content in rabbit feeds are known elsewhere in the world, but the conclusions of different authors involved in that problem are not the same (14, 15, 16, 17, 18). The lowest mortality to weaning (3.5%) in our experiment took place in the 1st group. In comparison to the 2nd group the breeding results in this group over the first ten days were better despite the fact that no other difference was stated until the introduction of gradual change of feed (21st day of age). The rabbits from the 1st group had also the highest body weight at weaning but later, in the period to the 40th day of age, their breeding results were lower from those of the 2nd group. Such results are hard to evaluate. However, great variety of results is obvious also from other experiments on rabbits. Di Meo et al. (19) investigated the effect of two different solid feeds during suckling on productive performance and caeccal content characteristics of rabbits at weaning (28 days). From day 16th, the first group was administered a commercial weaning diet and to the others was given the same feed as to their mothers. After weaning, the rabbits from both groups received ad libidum diet for weanlings, and later a finisher diet. The differences between body weight of mentioned groups were statistically significant only in the 1st week after weaning. The consumption of feed and production rate in the first group was better, which Influence of gradual change in feed, use of acidifier and prebiotic on rabbits in the period of weaning 77 was explained by feeding feed for weaned animals in this group before weaning. Feed intake before weaning has no greater influence on hint gut fermentation but contributes to easier transition from milk to pelleted feed thus diminishing risks of nutritional disturbances. Their opinion was confirmed also by Gidenne and Fortun - Lamothe (20), who investigated different technological procedures of weaning. They emphasize the benefit of feeding young rabbits specifical feed if this feeding is performed between 28th and 35th day of age. In the opposite case suitable technological solutions enabling compromise between nutritional needs of mothers and their offsprings must be taken in consideration (20, 21). However the effect of gradual change of feed performed in our experiment was most probably not clearly obvious because of great composition similarity of feed for does and feed for weaned rabbits. The mortality of young rabbits was the greatest in the 3rd group. Following the results of research including 850 French rabbit farms, Gidenne and Fortun-Lamothe (20) stated that 30% of mortality from birth to slaughtering is not rare. In our experiment the greatest mortality to weaning (12.3%) and 14.9% from weaning to the 40th day of age was found in the 3rd group where only gradual change from feed for does to feed for weanling rabbits was used. In the same group the greatest appearance of digestive disturbances was observed, which resulted in average body weight at weaning lower than 600 g, which was surpassed in both other groups. Comparison of the 3rd group to other two groups also showed that prebiotic (Bio Mos) supplementation can contribute to better results in intensive rabbit production. Probiotics, containing bacteria from Bacillus species or different yeasts, were found to be useful in the nutrition of rabbits also by other authors (22). Use of probiotic Paciflor contributed to significantly better growth rate during fattening and lower mortality in stress situations such as high temperature and low weaning weight (23). Similar results were obtained by Dupperay and Roberton (24) and Szabo-Lacza et al. (25, 26). On the contrary Maertens et al. (27), using the same probiotic, did not report statistically better growth results during fattening. In some researches Bio Mos (Alltech Inc.) was stated to be a perfect supplement to feed for rabbits (28). Supplementation of 2 kg/1000kg of feed lowered mortality caused by enteritis (29). Regarding Tibor et al. (30) in rabbits supplemented with Bio Mos daily weight gain was 9.2% and the body weight at the end of experiment 5.5% higher. Kocher et al. (31) performed the analysis of 20 experiments in which feed for weanling rabbits was supplemented with antibiotics, Bio Mos or contained no supplements. By use of Bio Mos better growth results (P = 0.001) and feed conversion was observed in comparison with control without Bio Mos supplementation. In 19 experiments lower mortality was also stated. Benefits of organic acidifier Acid Pac 4 Way (Alltech Inc.) supplementation in the feed for young rabbits before and after weaning were also described. Its application applenish the production of gastric acids in the critical period of weaning, when a young rabbit has no ability for maintaining of correspondingly low Ph, which represents an important barrier against invasion of pathogenic bacteria (32). Cheeke et al. (33) also stated the beneficial effects of Lacto-sacc and Acid Pac 4 Way supplementation on breeding results, micro-bial digestion in caecum, weight at weaning and diminishing of mortality. In our experiment the supplementation of acidifier in drinking water for does started 10 days before parturitions so the results of deliveries were, due to short period of its use, most probably not influenced. Anyhow the beneficial effect of acidifier supplementation occurred from the 16th day of their age, when they started to take their mothers' feed. After weaning the best results of weight gain was found in the 2nd and the worst in the 1st group. Unfortunately in our experiment following the animal body weight till slaughtering was not possible owing to production technology of the farm. Although no significant difference was observed between the groups, the results lead to conclusion that supplementation of Bio Mos (Alltech Inc.) in feed for weanling rabbits and use of acidifier Acid Pac 4 Way in drinking water effected better production results. Gidenne and Fortun-Lamothe (20) stated that momentary change of feed during lactation reduces feed intake of does and has drastic consequences on milk production. Following their conclusions gradual change from feed for does to the feed for weanling rabbits should improve the technology of rabbit feeding, which was also the reason why it was introduced in our experiment. In our opinion such mode of feeding can be successfully introduced in production technologies where feeding of mothers and their kits cannot be separated. 78 B. Jakovac-Strajn, U. Pestevsek, T. Knafelc References 1. Piattoni F, Maertens L, Mazzoni D. Effect of weaning age and solid feed distribution before weaning on performances and caecal traits of young rabbits. In: 2nd International conference on rabbit production in hot climates. Zaragoza: Mediterranean Agronomic Institute, 1999: 85- 91. 2. Cheeke PR, Grobner MA, Patton NM. Fiber digestion and utilization in rabbits. J Appl Rabbit Res 1986; 9: 25-30. 3. Salcedo-Baca R Ramirez-Luna G, Quinonez-Cruz B, Echegaray-Torres JL. Evaluation of an organic diet for growing rabbits (Oryctolagus cuniculus) based on alfalfa (Medicago sativa) and corn (Zea mays). In: 8th World rabbit congress. Pueblo City: World Rabbit Science Association, 2005: 1507-12. 4. Cheeke PR (1987). Rabbit feeding and nutrition. London: Academic Press, 1987: 302-4. 5. Fortun-Lamothe L, Boullier S. A review on the interactions between gut microflora and digestive mucosal immunity. Possible ways to improve the health of rabbits. Livest Sci 2007; 107: 1-18. 6. Gidenne T. Caeco-colic digestion in the growing rabbit: impact of nutritional factors and related disturbances. Livest Prod Sci 1997; 51: 73-88. 7. Kermauner A, Struklec M, Marinsek-Logar R. Influence of a probiotic added to different feed mixtures on the microbial metabolism in caecum of growing rabbits. Znan Prak Poljopriv Technol 1994; 24: 120-7. 8. AOAC. Official methods of analysis. 16th ed. Washington: Association of Official Analytical Chemists, 1995. 9. ISO. Animal feeding stuffs - determination of the contents of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc - method using atomic absorption spectrometry. Geneva: ISO, 2000. ISO 6869:2000 10. Commission Directive 78/633/EEC of 15 June 1978 Determination of the trace elements iron, cooper, manganese and zinc, Annex (Fe, Cu, Mn, Zn). 11. Commission Directive 71/393/EEC of 18 November 1971c establishing Community methods of analysis for the official control of feedingstuffs (phosphorus) 12. Schmidt HL, Bucher E, Spicher G. Keimgehaltbestimmung von Bakterien, Schimmelpilzen und Hefen in Futtermitteln Nährböden und Methodik. Landwirtschaftl Forsch 1981; 34(4): 242-50. 13. Kermauner A. Fibre in rabbit nutrition: recent re-comendations. Krmiva 2005; 6: 311-19. 14. Gidenne T, Debray L, Fortun-Lamothe L, Le Huërou-Luron. Maturation of the intestinal digestion and microbial activity in the young rabbit: impact of the dietary fibre: starch ratio. Comp Biochem Physiol (A) 2007; 148: 834-44. 15. Carabano R, Badiola I, Chamorro S et al. New trends in rabbit feeding: influence of nutrition on intestinal health. Span J Agric Res 2008; 6: 15-25. 16. Cheeke PR, Patton NM. Carbohydrate overload of the hindgut: a probable cause of enteritis. J Appl Rabbit Res 1980; 3: 20-3. 17. Gutierrez I, Espinosa A, Garcia J, Carabano R De Blas JC. Effect of levels of starch, fiber, and lactose on digestion and growth performance of early-weaned rabbits. J Anim Sci 2002; 80: 1029-37. 18. McNitt JI, Patton NM, Lukefahr SD, Cheeke PR. Rabbit production. Danville: Interstate Publishers, 2000: 182-3. 19. Di Meo C, Bovera F, Piccolo G, Gazaneo MP, Nizza A. Effect of pre-weaning diet on rabbit performance. In: the 8th World rabbit congress. Pueblo City: World Rabbit Science Association, 2005: 792-8. 20. Gidenne T, Fortun-Lamothe L. Feeding strategy for young rabbits around weaning: a review of digestive capacity and nutritional needs. Anim Sci 2002; 75: 169-84. 21. Gidenne T, Lapanouse A. Fortun-Lamonthe L. Feeding strategy for the early weaned rabbit: interest of a high energy and protein starter diet on growth and health status. In: 8th World rabbit congress. Pueblo City: World Rabbit Science Association, 2005: 853-60. 22. Maertens L, Groote G. Effect of a dietary supplementation of live yeast on the zootechnical performance of does and weanling rabbits. J Appl Rabbit Res 1992; 15: 1079-86. 23. Blas C, Garcia J, Alday S. Effect of dietary inclusion of a probiotic Paciflor on performance of growing rabbits. J Appl Rabbit Res 1991; 14: 148-50. 24. Duperray J, Roberton JL. Interet du bioregulateur Paciflor chez le lapin de chair. Cuniculture 1990; 17: 259-62. 25. Szabo-Lacza S, Gippert T, Hullar I, Virag G, Kustos K. Application of Streptococcus faecium M 74 in the feeding of meat rabbit. In: 4th Congress of the World Rabbit Science Association. Budapest: World Rabbit Science Association, 1998: 181-90. 26. Szabo-Lacza S, Gippert T, Hullar I, Virag G. Utilisation de Streptococcus faecium M 74 dans l'alimentation du lapin de chair. Cuniculture 1990; 17: 263-6. 27. Maertens L, Renterghem R, Groote G. Effects of dietary inclusion of Paciflor (Bacillus CIP 5832) on the milk composition and performance of does on caecal and growth parameters of their weanlings. World Rabbit Sci 1994; 2: 67-73. 28. Girard ID, Geliot P, Spring P. Effects of mannano-ligosaccharide on performance of fattening rabbits. In: International Symposium on non-digestible oligosaccha-rides: healthy food for the colon? Wageningen: Wageningen University 1997: 1011. 29. Reed TE. Effect of two levels of Bio Mos (manna-nooligosaccharide) on weight gain and mortality of weanling rabbits. In: 12th Annual Symposium on Biotechnology in the Feed Industry. Lexington: Alltech 1994: 10. 30. Tibor G. Effect of Bio Mos on performance and mortality of growing rabbits. In: 11th Annual Symposium on Biotechnology in the Feed Industry. Lexington: Alltech, 1995: 101. Influence of gradual change in feed, use of acidifier and prebiotic on rabbits in the period of weaning 79 31. Kocher A, Spring P, Hooge DM. Summary analysis of post-weaned rabbit trials with dietary mannan oligosaccharide. In: Animal production in Europe: the way forward in a changing world. Saint-Malo: ZOOPOLE deve-lopement 2004: 261-2. 32. Holister AG, Cheeke PR, Robinson KL, Patton NM. Effects of dietary probiotics and acidifers on performance of weanling rabbits. J Rabbit Res 1990, 13: 6-9. 33. Cheeke PR, Hollister AG, Robinson KL, Lyons TP. Improving feed efficiency and reducing mortality in rabbits: a case study for use in all species. In: Biotechnology in feed industry. Alltech's 6th annual symposium. Nicho-lasville: Alltech, 1989: 224-31. VPLIV POSTOPNEGA PREHODA KRMLJENJA, ZAKISOVALCA IN PREBIOTIKA PRI KUNCIH V OBDOBJU ODSTAVITVE B. Jakovac-Strajn, U. Pestevšek, T. Knafelc Povzetek: V poskusu smo poskušali ugotoviti ali lahko s kombiniranjem zakisovalca, krmnih dodatkov in postopnega prehoda s krme za samice na krmo za odstavljence izboljšamo rejske rezultate pri kuncih. Deset dni pred kotitvami smo oblikovali 3 skupine po 15 brejih samic. Krmili smo jih s krmo, ki je vsebovala kokcidiostatik Robenidin (1 mg/kg). V vodi sta prva in druga skupina samic dobivali zakisovalec Acid Pac 4 Way (2 g/l). V skupini 1 smo od 22. do 30. dne starosti mladičev izvedli postopni prehod s krme za samice na krmo za odstavljence, ki je vsebovala prebiotik Bio Mos (2 g/kg). V skupini 2 postopnega prehoda ni bilo, krma za odstavljence pa je bila enaka kot v skupini 1. V skupini 3 je bil izveden postopen prehod, krma za odstavljence pa ni vsebovala nobenih dodatkov. Spremljali smo rezultate kotitev, porabo krme, število in težo odstavljenih mladičev do starosti 40 dni ter izgube med samicami in mladiči. Statistično značilna razlika ( x±SE) v številu izenačenih in odstavljenih kuncev je bila ugotovljena med drugo (7,3±0.3, 6,7±1,91) in tretjo (8,7±0,21, 9,5±1,24) skupino (P<0,05). Ob odstavitvi so bili najtežji kunci v 1. skupini (640,7 ± 19,29 g), pri starosti 40 dni pa kunci v 2. skupini (934,1 ± 10,41 g). Rezultati kažejo, da krmni dodatki v intenzivni reji kuncev lahko prispevajo k boljšim rejskim rezultatom. Ključne besede: živali, prehrana; hrana, dodatki; oligosaharidi; živali, sesne; kunci INSTRUCTIONS FOR AUTHORS NAVODILA AVTORJEM Slovenian Veterinary Research contains original articles which have not been published or considered for publication elsewhere. All statements in the articles are the responsibility of the authors. The editorial policy is to publish original research papers, review articles, case reports and abstracts of theses, as well as other items such as critical reviews of articles published in Slov Vet Res, shorter scientific contributions, letters to the editor, etc. Authors should send their contributions to the editorial board's address. All articles are subjected to both editorial review and review by an independent referees selected by the editorial board. The editorial board reserves the right to translate titles, summaries and keywords that have not been translated into Slovene by the authors. 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Zaželjena je uporaba elektronske pošte (slovetres@vf.uni-lj.si) in avtorji naj predlagajo tri možne recenzente. Besedilo naj ima dvojni razmik med vrsticami, pri čemer naj bodo vrstice na levi strani oštevilčene. Besedilo naj bo na levi strani od roba oddaljeno 4 cm. Naslovna stran prispevkov se začne z naslovom, sledi ime in priimek avtorja. Kadar je avtorjev več, jih ločimo z vejicami. V naslednjih vrsticah je v rubriki Addresses of authors: za dvopičjem treba navesti polno ime in priimek ter naslov(e) avtorja(ev), tj. ustanovo, ulico s hišno številko, pošto in kraj. Vse navedene podatke ločujejo vejice. Sledi vrstica, kjer je treba navesti ime ter elektronski (E-mail:) in poštni naslov ter telefonsko številko (Phone:) odgovornega avtorja. Sledi besedilo povzetka Summary v obsegu 200 do 300 besed. V naslednji rubriki Key words: se za dvopičjem navedejo ključne besede. Posamezne besede ali sklopi besed morajo biti ločeni s podpičjem. Znanstveni članki in tisti, ki so prikaz lastnih raziskav in dognanj, morajo vsebovati še naslednje obvezne rubrike, s katerimi avtor sam naslovi ustrezne dele besedila v prispevku: Introduction, Material and methods, Results, Discussion in References. Pregledni članki naj vsebujejo uvod, poglavja, ki so glede na vsebino smiselno naslovljena, in literaturo. Podatke o financerjih ali drugih zadevah, pomembnih za prispevek, npr. o tehnični pomoči, avtorji navedejo v rubriki Acknowledgements, ki se uvrsti pred rubriko References. Za rubriko References sledijo spremna besedila k slikam. Priloge, kot so tabele, grafikoni in diagrami naj bodo smiselno vključene v besedilo. Slikovni material naj bo poslan posebej v obliki bmp, jpg, ali tif. Priloge in slike morajo biti poimenovane z besedami, ki jih opredeljujejo, in arabskimi številkami (npr. Table 1:, Figure 1: itn.). Za dvopičjem sledi besedilo oziroma naslov. Vsi navedki (reference), citirani v besedilu, se morajo nanašati na seznam literature. V besedilu jih je treba oštevilčiti po vrstnem redu, po katerem se pojavljajo, z arabskimi številkami v oklepaju. Prvi navedek v besedilu opredeli številko oziroma vrstni red ustreznega vira v seznamu literature. Če se avtor v besedilu ponovno sklicuje na že uporabljeni vir, navede tisto številko, ki jo je vir dobil pri prvem navedku. Citirana so lahko le dela, ki so tiskana ali kako drugače razmnožena in dostopna javnosti. Neobjavljeni podatki, neobjavljena predavanja, osebna sporočila in podobno naj bodo omenjeni v navedkih ali opombah na koncu tiste strani, kjer so navedeni. V seznamu literature so viri urejeni po vrstnem redu. Če je citirani vir napisalo šest ali manj avtorjev, je treba navesti vse; pri sedmih ali več avtorjih se navedejo prvi trije in doda et al. Da bi se morebitni popravki lahko objavili v naslednji številki, jih morajo avtorji pravočasno sporočiti glavnemu uredniku. Načini citiranja Knjiga: Hawkins JD. Gene structure and expression. Cambridge: University Press, 1991: 16. Poglavje ali prispevek v knjigi: Baldessarini RJ. Dopamine receptors and clinical medicine. In: Neve KA, Neve RL, eds. The dopamine receptors. Totowa: Human Press, 1996: 475-98. Članek iz revije ali časopisa: Fuji J, Otsu K, Zorzato F, et al. Identification of mutation in porcine ryanodine receptor asociated with malignant hyperthermia. Science 1991; 253: 448-51. Članek iz zbornika referatov: Schnoebelen CS, Louveau I, Bonneau M. Developmental pattern of GH receptor in pig skeletal muscle. In: the 6th Zavrnik memorial meeting. Lipica: Veterinary Faculty 1995: 83-6. Slov Vet Res 2009; 46 (2) Review Papers Ornik D, Cadonic-Spelic V. Records on the use of animals in experiments in the Republic of Slovenia and in other EU member states within 15-years period...............................................47 Fazarinc G. Enzyme-immunohistochemical aspects of muscle fiber type classification in mammals...............61 Original Research Paper Jakovac-Strajn B, Pestevsek U, Knafelc T. Influence of gradual change in feed use ,of acidifier and prebiotic on rabbits in the period of weaning..........................................................71