ISSN 1580-4003 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 48 Slov Vet Res • Ljubljana • 2011 • Volume 48 • Number 1 • 1-34 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Volume 48 Slov Vet Res • Ljubljana • 2011 • Volume 48 • Number 1 • 1-34 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, Krka, d.d., Novo mesto, 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.slovetres.si/ SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2011; 48 (1) Review Article Rajcevic U. A rodent brain orthotopic model to study human malignant glioma.............................. 5 Original Scientific Articles Prevolnik M, Ocepek M, Candek-Potokar M, Bavec M, Skorjanc D. Growth, carcass and meat quality traits of pigs raised under organic or conventional rearing systems using commercially available feed mixtures........15 Marcincak S, Popelka P, Zdolec N, Martonova M, Simkova J, Marcincakova D. Effect of supplementation of phytogenic feed additives on performance parameters and meat quality of broiler chickens................27 Slov Vet Res 2011; 48 (1): 5-14 UDC 577.2:616.8:616.714.1-006:599.323.4 Review Article A RODENT BRAIN orthotopic MODEL TO STuDY HuMAN MALIGNANT glioma Uroš Rajčevic Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia E-mail: uros.rajcevic@nib.si Summary: Glioblastomas are fatal brain tumors. They have a relatively low incidence, but the fact that regardless of the advances in therapy there hasn't been a major progress in survival over several decades urges the scientists to search for novel diagnostic tools, anti-tumor drug targets and therapies. It appears that our inability to successfully treat brain cancer mostly stems from the lack of understanding of the underlying brain tumor biology. Many rodent orthotopic models have been developed to address issues in drug development as well as biological origins of malignant gliomas. Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. A novel orthotopic rat model of glioblastoma-the most malignant glioma in human-was recently developed, showing some stem-cell properties. The model is based on xenotransplantation of biopsy spheroids from human tumor tissue, into the brain of immunodeficient rats, where they initiated the growth of primary in most cases invasive and angiogenesis-independent glioblastomas. After serial passaging of tumors via spheroids in the subsequent generation of animals, the phenotype of the tumor changed. The most dramatic change was observed in approximately 1/3 of initially invasive tumors that changed into highly angiogenic and very aggressive. Some tumors though, remained invasive even after serial passages, while some were angiogenic from the start. The model thus provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumor progression. In vivo passaging of patient GBM biopsies produced tumors representative of the patient tumors, with high take rates and a reproducible disease course. The model has also been adapted to eGFP expressing immunodeficient mouse in which fluorescently marked tumors can be established in vivo. Key words: malignant brain tumors; glioblastoma; nude rat; e-GFP NOD/SCID mice; xenograft; orthotopic transplantation; biopsy spheroids; translational medicine; molecular neuro-oncology Introduction Gliomas Gliomas are brain tumors with some characteristics of brain support tissue - the glia. Malignant gliomas (anaplastic astrocytoma and glioblastoma multiforme-grade III and IV astrocytomas on WHO scale respectively) are predominant malignant brain tumors in human and have a poor prognosis with an average patient survival under current treatment regimens ranging between 12-14 months. Tumors are characterized by rapid cell growth, extensive neovascularization and diffuse cellular infiltration of normal brain structures (1). They have a relatively low overall incidence, but the fact that in spite of major progress in neurosurgery and oncology (including chemo-, radio- and, more recently biological-therapy) there hasn't been a major progress in malignant gliomas survival over several decades (2-7), urges the scientists to search for novel diagnostic tools, anti-tumor drug targets and therapies. Animal Tumor Models Received: 25 October 2010 Accepted for publication: 17 November 2010 In vivo animal modeling provides essential tumorhost interactions and is a more accurate way of mode- 6 U. Rajcevic ling human cancer than in vitro. We subdivide rodent models into xenograft tumor models - addressed in particular in this review - and models of spontaneous tumor formation in genetically engineered rodents. These models help us address important issues in drug development: toxicity and in vivo antitumor effectiveness (8, 9) (where the models are a matter of highly standardized procedures) as well as other basic phenomena in tumor origin and function such as angiogenesis, invasion and many others (3, 4, 10, 11) in the field of cancer research. In vivo modeling of drug efficacy is a gold standard required by a majority of pharmaceutical companies (12). Xenograft tumors are initiated by implantation or injection of primary tumor cells or cell lines s.c. (under the skin) or orthotopically (into native tumor site) of syngeneic (genetically identical or closely related, so as to allow tissue transplant; immunologically compatible.), xenogeneic (derived or obtained from an organism of a different species, as a tissue graft), allogeneic (genetically different although belonging to or obtained from the same species, as in tissue grafts) or, immunosuppressed, immunodeficient, or newborn immunonaive animals (8, 9, 13, 14). Rodent glioblastoma (GBM) models have been used for over 40 years but the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial (8, 9). The advantages of xenograft glioma models are their highly efficient gliomagenesis, reproducible growth rates, and an accurate knowledge of the location of the tumor (6, 15, 23). However, in xenograft models stepwise genetic changes thought to occur during tumor progression are often missing; injected or implanted cells or cell lines have often been altered by culture or isolation conditions and lack their native tumor stroma. Murine models (e.g. (18) of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies (8). On the other hand, many xenograft tumors lack histologicaly accurate vascularization, and rarely recapitulate tumor-of-origin phenotype (17). Im-munodeficient rodents do not show antitumor immune effects and can produce false positives during drug trials (9). Syngeneic murine models, such as GL26 mouse glioma cells in C57BL6 mice (18) and CNS1 rat glioma cells in Lewis rats (19, 20) are non-immunogenic. Thus, syngeneic glioma models are excellent for studying the response of brain tumors to immunotherapy (19, 20). Establishing clinically relevant animal models of gliomas that would fully reflect the situation in human malignant glioma remains a challenge. Two novel rat and mouse models to study glioblastoma An orthotopic rat xenograft model of glioblastoma was developed (21) to address, among others, the tumor-host interaction issues leading to tumor circumscription, histologically accurate vasculari-zation and to recapitulate accurately the tumor-of-origin phenotype. The model is based on a serial xenotransplantation of glioma biopsy spheroids, generated from glioma tissue (22) into the brain of nude rats. There the glioma spheroids induce the growth of primary, in most cases very invasive glioblastomas in 4-6 months time. These tumors co-opted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers and showed a migratory behavior similar to normal human neural stem cells. When the rats became ill, they were sacrificed and spheroids were generated from their (human) tumors and implanted in the next generation of nude rats. This way, the tumors were passed in a total of 4-6 consecutive generations of nude rats (Figure 1). The most dramatic change was observed in approximately 1/3 of initially invasive tumors that changed into highly angiogenic and very aggressive, less invasive (more circumscribed), but which grew much faster due to good blood supply, killing a rat in 2-3 months. This switch to ang-iogenic phenotype was characterized by a reduction in stem cells markers (21, 23). Some tumors though, remained invasive even after serial passages, while some were angiogenic from the start. At the level of gene expression and immunoblotting proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in invasive tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the invasive tumors (21). Uncoupling of invasion and angiogenesis, represented by the stemlike cancer cells and the cells derived from them respectively, points at two different mechanisms that drive tumor progression. Although the mechanism behind the phenotypic shift is not fully understood, HIF-1 expression seems to be triggered by hypoxia, because it was not constitutively expressed by highgeneration tumor spheroids cultured under nor-moxic conditions (21) . A rodent brain orthotopic model to study human malignant glioma 7 Figure 1: The orthotopic rat glioblastoma model Thus, by serial passaging, this model uncouples and recapitulates for the first time the two major phenotype characteristics of human glioblastoma which makes it one of the best available glioblastoma models and opens the door to multiple applications in basic, translational and pre-clinical research. The major difference and - possibly - the advantage of this model over other described rodent models of glioblastoma (8, 9) arises from the fact that in the biopsy spheroids, which are structures of heterogenic cellular population, at least part of original glioma microenvironment is conserved, a part essential in inducing the tumor upon transplantation. This microenvironment is absent in xenografts generated from immortalized glioma cell lines (8, 9), but may be conserved to some extent in primary glioma cell lines derived from glioma patients and in glioma tissue grafts. The phenotypes, derived from the same tumor sample, may develop through a selection process where most cells in the biopsy specimens die following implantation in the rat brain. The cells that survive show stem cell-like properties, and are able to adapt to the new microenvironment where they divide and produce new tumor clones that show rapid growth and angiogen-esis (21, 24). The concept of the Rodent Glioblastoma model Samples of glioblastoma, resected from the patients are finely minced and grown in cell culture medium. There the cells organize in tumor biopsy spheroids of about 300|m in size. Spheroids are orthotopically implanted into the brain of immu-nodeficient rodents. There, in most cases, they induce the growth of primary glioblastomas of invasive nature, but non-angiogenic in 4-6 months. With serial passaging in the subsequent generations of rodents via spheroids, the tumor pheno-type will change in 1/3 of cases to fully angiogenic in the last generation xenograft with an onset of tumor in 2-3 months. eGFP NOD/Scid mouse model This glioblastoma model was recently adapted to a NOD/Scid mouse expressing enhanced green fluorescent protein (eGFP) (Figure 2). In this model human and mouse tumors marked with red fluorescent protein can be established in vivo, both at subcutaneous and orthotopic locations (25). Using modern microscopy techniques, the intricate co localization of tumor and host cells in situ can be visualized in detail. Technology of fluorescence-activated cell sorting (FACS), allows for complete and reliable separation of the host cells from the tumor cells, thus providing a system for detailed cellular and molecular analysis of tumor-host cell interactions. The fact that tumor and host cells can be reliably identified also allows for detection of double-positive cells, possibly arising from cell fusion events or horizontal gene transfer (two possible origins of tumor initiating - tumor stem cells; (26)). Similarly, the model can be applied for the detection of circulating metastatic cells and for detailed studies on the vascular compartments within tumors, including vasculogenic mimicry (25). Thus, the model should provide significant insight into how tumor cells communicate with their microenvironment. 8 U. Rajcevic J ' V \ A. I > i ' Z / • 'J D Figure 2: Tumor host interaction studies in an eGFP NOD/Scid mouse model U87 dsRed expressing human glioblastoma cells were implanted in the flank of eGFP NOD/Scid mouse. (A-C) Endogenous fluorescence on a frozen histological section from a U87 subcutaneous tumor showing eGFP-expressing host cells within the tumor bed (direct eGFP and dsRed fluorescence). Scale bars = 5 pm (A); 10 pm (B, C). (D) a newborn eGFP NOD/Scid mouse glowing in green fluorescence under the UV light Applications of the rodent glioblastoma model Since its establishment, the rat model of glioblastoma was characterized in detail on several levels and used in multiple assays ranging from basic research to drug testing. MR spectroscopy In this study the metabolic properties of the two experimental phenotypes were analyzed. The early generation (EG) showed an intact blood-brain barrier and normal vascular morphology. In contrast, the high generation (HG) exhibited leaky vessels and necrosis. The rats with HG tumor had raised concentrations of choline and myo-inositol, and decreased concentrations of glutamate and N-acetylaspartate. In the LG tumor group, similar changes in metabolic concentrations were detected, although the alterations were more pronounced. The LG tumors also had higher concentrations of choline, taurine, and lactate. Results of this study showed that metabolic profiles could be used to distinguish between two glioblasto-ma phenotypes. More pronounced anaerobic metabolism was present in the LG stem-cell-like tumors, suggesting a more malignant phenotype (27). A rodent brain orthotopic model to study human malignant glioma 9 Table 1: Applications of the rodent glioblastoma model Applications Model Reference Comments Metabolite analysis Determination of metabolic properties of the xenografts by Magnetic Resonance Spectroscopy (MRS) Nude rat glioma xenograft Thorsen F., et al., 2008 (27) Metabolic profiles produced by MRS could be used to distinguish between two distinct glioblastoma phenotypes Tumor initiation, take and reproducibility Othotopic, stereotactic glioblastoma xenograft model construction and characterization Nude rat glioma xenograft Sakariassen PO et al., 2006 (21) Separation of early /invasive tumor phenotypes and late/angiogenic tumors by serial passaging in nude rats Tumorigenesis; neu-ropathological and radiological features of rat glioblastoma xenograft model Nude rat glioma xenograft Wang J et al., 2009 (23) In vivo passaging of patient GBM biopsies produced tumors representative of the patient tumors, with high take rates and a reproducible disease course. Tumor-host interaction studies eGFP expressing NOD/SCID mouse Niclou SP et al., 2008 (25) Fluorescence-based intricate co localization of tumor and host cells in situ. Anti-tumor drug testing and glioma treatment studies Radio surgery glioblastoma spheroids, nude rat glioma xenograft Thorsen FA et al, 2007 (30) Radio surgery of malignant gliomas might be effective in controlling tumor progression in selected glioblastoma patients. The response of the two phenotypes to doxorubicin Nude rat glioma xenograft Johannessen T-CA et al., 2009, (24) Highly invasive tumors shown to be more chemo resistant than angiogenic tumors derived from the same patients. The effect of hyperoxic treatment on BT4C rat glioma xenografts BT4C rat glioma xenografts Stuhr LEB et al., 2007 (31) Increased pO2-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth, a process characterized by enhanced cell death, reduced vascular density and changes Gene Therapy Adenoviral vector (AAV) transduction Glioma cell lines, glioblastoma spheroids, nude rat glioma xenograft Thorsen FA et al., 2006 (32) AAV4 and AAV5 serotypes may be used to transduce biologically diverse glioma cell lines. They also penetrate and transduce solid human tumor tissue derived from patient biopsies. Lentiviral vector transduction human embryonic kidney cell line 293T, TE671 cell line, nude rat glioma xenograft Huszthy PC et al., 2009 (33) Lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSVG) pseudotyped lentiviral vectors efficiently transduced human glioblastoma cells and cancer stem-like cells. Pseudotyped gamma retroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer. Oncolytic HSV-1 based vector G207 Nude rat glioma xenograft Huszthy PC et al., 2010, (34) Favorable cellular responses to G207 treatment seen from a clinical viewpoint, such as reduced tumor cell proliferation, more frequent events of tumor cell death and a strongly attenuated tumor vascular compartment. Biomarker research The glioma-associated gangliosides 3k-isoLM1, GD3 and GM2 Nude rat glioma xenograft Hedberg KM et al, 2001, (35) Different biological roles for individual gangliosides; antibodies or ligands directed against GD3 and 3k-isoLM1 might be complementary when applied in the treatment of human glioblastomas. Expression of extracellular matrix components in a highly infiltrative glioma model Glioma cell lines, nude rat glioma xenograft Mahesparan R et al., 2003, (16) Possible biological function of tenascin, vitronectin, laminin, fibronec-tin and collagen type IV in highly invasive malignant tumors of glial origin. PDI protein expression in invasive phenotype tumors Nude rat glioma xenograft Goplen D et al., 2006 (36) PDI was shown to be expressed on migrating and invading glioma cells. Tumor initiating cell markers Nude rat glioma xenograft Wang J et al., 2007(29) Cd133- glioma cells are tumorigenic and can produce cd133+ tumors, CD133 expression coincides with the onset of angiogenesis and a shorter survival Global membrane pro-teomics Nude rat glioma xenograft Rajcevic U et al., 2009 (37) Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. Enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. Neural cell adhesion molecule (NCAM) as a glioma marker for the biological aggressiveness Nude rat glioma xenograft Duenisch P et al., 2010, (38) The expression of NCAM-140 inversely correlated with the WHO grade of human gliomas 10 U. Rajcevic Tumor initiation, take and reproducibility The tumor take rates for xenografted GBM biopsies were 96% and close to 100% at subsequent passages in vivo. Only one of four lower grade tumors engrafted. MRI typically revealed changes related to tumor growth, several months prior to the onset of symptoms (23). In another study, CD133 expression was analyzed at various passages. CD 133 is a cell surface marker identified as a putative marker of brain tumor-initiating cells (28). During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis, shorter survival and increased CD 133 expression. CD133 negative cells derived from 6 different patients were tumorogenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In this assay, the authors showed that CD133 negative glioma cells were tumorogenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is up regulated, coinciding with the onset of angiogenesis and a shorter survival. Authors also suggested that CD 133 may not be essential in tumor initiation process (29). Anti-tumor drug testing and glioma treatment studies In a study focused on the radiobiological effects of the Gamma knife (Gamma Knife is currently used to boost treatment of malignant gliomas) the growth and invasiveness of human glioblastoma spheroids xenografted into nude rat brains were assessed after radio surgery. A dose-dependent inhibition of tumor growth and invasion, as well as a dose-dependent increase in animal survival was observed. The results indicated that radio surgery of malignant gliomas might be effective in controlling tumor progression in selected glioblastoma patients (30). In a study aimed at investigating how the two phe-notypes responded in vitro to doxorubicin, a clinically potent cytotoxic drug for solid tumors, highly invasive tumors shown to be more chemo resistant than angiogenic tumors derived from the same patients. It was suggested that treatment resistance in glioblas-tomas could be related to PI3K/AKT activity in stemlike tumor cells, and that targeted interference with the PI3K/AKT pathway might differentiate and sensitize this subpopulation to chemotherapy (24). Another study described the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, ang-iogenesis, apoptosis, general morphology and gene expression parameters. Increased pO2-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process was characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects (31). Gene Therapy In one of the initial studies on the delivery vehicles for gene transfer strategies directed at the central nervous system (CNS), muscle and liver performed on the featuring model the transduction efficacy of AAV serotypes 4 and 5 were compared to AAV2, both in vitro and in intracranial GBM xenografts . While all three AAV serotypes were able to transduce the glioma cell lines when added individually or when they were administered in concert, AAV2 transduced the glioma cells most effectively compared to AAV4 or AAV5. Upon infecting glioblastoma spheroids in vitro, all three AAV serotypes efficiently transduced cells located at the surface as well as within deeper layers of the spheroids. In addition, both AAV4 and AAV5 were able to transduce human glioblastoma xenografts implanted intracranially. Authors suggested that beside AAV2 serotype, AAV4 and AAV5 serotypes may also be used to transduce biologically diverse glioma cell lines and may be used in developing treatment vehicles for human malignant gliomas (32). The rat xenograft model was used to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSVG) pseu-dotyped lentiviral vectors efficiently transduced human glioblastoma cells and cancer stem-like cells. In contrast, pseudotyped gamma retroviral vectors, similar to those evaluated for clinical therapy of glioblas-toma, showed inefficient gene transfer. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. The inefficient gene delivery by gamma retroviral vectors is in line with the results obtained in clinical therapy for GBM and thus con- A rodent brain orthotopic model to study human malignant glioma 11 firms the high reproducibility of the invasive glioma animal model for translational research (33). In another assay the cellular effects of the oncolytic HSV-1 based vector, G207, on the tumor microenvironment were evaluated. The xenografted tumors were quantitatively evaluated 10-30 days after G207 injection for virus-induced changes in proliferation, apoptosis and vascularity. Vector spread and the infiltration pattern of CD68-positive inflammatory cells were assessed. Proliferation indices were lower, whereas apoptotic counts were elevated in plaques as compared with that in non-infected areas of the same lesions, as well as in corresponding control xenografts. A decline in the number of blood vessels was noticed in the plaques and the vascular area fractions were reduced. CD68-positive inflammatory cells accumulated in the plaques. The study highlighted the favorable cellular responses to G207 treatment seen from a clinical viewpoint, such as reduced tumor cell proliferation, more frequent events of tumor cell death and a strongly attenuated tumor vascular compartment (34). Biomarker research A substantial number of biomarker research studies were thus far performed on the model. Initially roles for glioma-associated gangliosides (3k-isoLMl, GD3 and GM2) were assessed in the model, pointing out that different biological roles for individual gangliosides may exist and that the antibodies or ligands directed against GD3 and 3k-isoLM1 might be complementary when applied in the treatment of human glioblastomas (35). In a different study expression of extracellular matrix (ECM) components in this highly infiltrative in vivo glioma model was analyzed. The cellular origin for several of these ECM components was identified using human-specific monoclonal antibodies and polyclonal antibodies detecting epitopes from both species (human and rat). Observed staining patterns clarified the cellular origin and indicated the possible biological function of tenascin, vit-ronectin, laminin, fibronectin and collagen type IV in these highly invasive malignant tumors of glial origin (16). In the initial proteomic assays on biomarkers on the featuring model the researchers showed that PDI, one of the most prominently up-regulated proteins in invasive tumors is strongly expressed on invasive glioma cells, in both xenografts and at the invasive front of human glioblastomas. Using in vitro assays, PDI was shown to be expressed on migrating glioma cells. Functional significance of PDI in cell migration and invasion was tested in vitro, showing an important role of PDI in glioma cell invasion (36). In a global proteomics comparison of the two xe-nograft phenotypes we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phe-notype. The data pointed to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-ang-iogenic phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas (37). In a different assay the relevance of neural cell adhesion molecule (NCAM) as a glioma tissue marker for the biological aggressiveness of these tumors was assessed. The expression of NCAM-140 inversely correlated with the WHO grade of human gliomas. The lost expression of NCAM-140 in human glioblas-tomas and in brain metastases enabled the investigation of the brain- tumor interface and the definition of glioblastoma invasion patterns and showed that brain metastases are more invasive than ever thought (38). Conclusions In vivo passaging of patient GBM biopsies in rats or mice produced tumors representative of the patient tumors, with high take rates and a reproducible disease course. The main advantage of the model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumor progression (Wang J, BMC Cancer 2009) as well as to other in vivo orthotopic rodent GBM models. Thus far, the model has been extensively used in a variety of applications, ranging from basic research of cancer biology and biomark-ers including genomics, transcriptomics, proteomics and metabolomics, through translational research of therapeutics and therapeutic modalities to pre-clinical research of drugs approved for use in other types of cancer. The results of this research provided important novel insights into the mechanisms of tumor initiation, promotion and progression 12 U. Rajcevic through transcriptomic markers, through novel protein biomarkers, validated on clinical and preclinical material. Results also indicated the metabolic changes linked to phenotype switch in the model. 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Rajčevic povzetek: Maligni gliomi so neozdravljivi možganski tumorji. Se relativno redko pojavljajo, vendar ne glede na izboljšave pri terapiji večjega napredka pri preživelosti bolnikov ni bilo že več desetletij. Zaradi tega je pomembno raziskovanje novih diagnostičnih orodij, ugotavljanje novih tarč v tumorskem tkivu in razvijanje novih načinov zdravljenja. Zdi se, da naša nezmožnost uspešno pozdraviti raka na možganih izvira iz nepoznavanja bioloških in biokemičnih lastnosti tumorjev. Z leti je nastalo več glodavskih ortotopičnih modelov, ki so lahko uporabni pri razvoju novih zdravil in pri proučevanju bioloških osnov malignih gliomov. Razvoj klinično pomembnih živalskih modelov za multiformni glioblastom (GBM) ostaja izziv in številni, pogosto uporabljeni modeli na osnovi celičnih linij, ne odražajo vzorcev invazivne rasti GBM pri bolnikih. Nedavno je bil razvit nov ortotopični podganji model glioblastoma - najbolj maligne oblike glioma pri človeku, ki kaže nekatere značilnosti, podobne matičnim celicam. Model je nastal na osnovi medvrstne presaditve biopsijskih sferoidov iz vzorcev človeških tumorjev v možgane podgan z oslabljenim imunskim sistemom, kjer so sferoidi povzročili rast primarnih in večinoma invazivnih, od angiogeneze neodvisnih glioblastomov. Po zaporednem prenosu teh tumorjev preko sferoidov v naslednje generacije živali se je fenotip tumorja spremenil. Najbolj dramatična sprememba je bila ugotovljena pri približno tretjini sprva invazivnih tumorjev, ki so se spremenili v visoko angiogene in zelo agresivne. Nekaj tumorjev je ostalo invazivnih kljub zaporednemu prenosu v naslednje generacije podgan, medtem ko so bili nekateri angiogeni že od začetka. Model torej prinaša kombinacijo angiogenih in invazivnih fenotipov in predstavlja dobro alternativo celičnim linijam, gojenim v pogojih in vitro, za ugotavljanje mehanizmov napredovanja možganskih tumorjev. Zaporedni prenos bolnikovih biopsij GBM in vivo je povzročil nastanek tumorjev, podobnih pacientovim, z visoko ravnijo tumorigeneze in ponovljivim potekom bolezni. Model je bil prilagojen tudi mišim z oslabljenim imunskim odgovorom, ki izražajo zeleno fluorescenčno beljakovino in kjer lahko vzpodbudimo nastanek fluorescenčno označenih tumorjev pri živih živalih. Ključne besede: maligni možganski tumorji; glioblastom, gole podgane; miši e-GFP NOD/SCID; ksenograft; ortotopična transplantacija; biopsijski sferoidis; translacijska medicina; molekularna nevroonkologija Slov Vet Res 2011; 48 (1): 15-26 UDC 636.4.083:631.147:637.51 Original Scientific Article GROWTH, CARCASS AND MEAT QuALITY TRAITS OF pIGS RAISED under organic OR CONVENTIONAL rearing systems using commercially available feed MIXTuRES Maja Prevolnik1, 2, Marko Ocepek1, Marjeta Čandek-Potokar1, 2, Martina Bavec1, Dejan Škorjanc1* 1 Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoče, Slovenia; Agricultural Institute of Slovenia, Hac-quetova 17, 1000 Ljubljana, Slovenia Corresponding author, E-mail: dejan.skorjanc@uni-mb.si Summary: The objective of the study was to evaluate performance from birth until slaughter, as well as final carcass and meat quality, of pigs raised either conventionally (n=32) or respecting organic standards (n=35) using commercially available organic feed mixtures. Lower (22%) feed intake from weaning until week 11 was noted for organic pigs. As a consequence, organic pigs had lower growth rate persisting until week 22. In the last phase (weeks 22 to 26) when food intake was limited, growth rate declined in conventional, and increased in organic, pigs. All pigs were slaughtered at the usual commercial age (26 weeks). Due to the slower growth, organic pigs had lower carcass weight, dressing %, smaller longis-simus dorsi (LD) muscle, and lighter hams, while no differences were observed in fat tissue measurements and carcass leanness (the exception being the area of fat over LD). Analysis for the same slaughter weight indicated that organic pigs would have fatter carcasses as conventional pigs if slaughtered at the same weight. With regard to meat quality, a higher ultimate pH and intramuscular fat content were observed for organic pigs. The results of the present study indicate possible problems (lower feed intake, growth retardation) associated with the use of commercially available organic diets for piglets. On the other hand such diets can increase intramuscular fat content, which is interesting in terms of improved meat quality. Key words: organic farming; growth; carcass quality; meat quality; stress markers; pig Introduction In most European countries, organic pig production has so far played a minor role and the proportion of organically produced pigs is very low (less than 1% (1,2). However, there is a general interest in the European Union (EU) in organic production systems. Consumers often perceive organic food to be healthier, safer, and more palatable and nutritious, owing to the recognition that organic food is wholesome, environmental friendly, traditional and sustainable (3). Received: 9 November 2010 Accepted for publication: 31 January 2011 Owing to the specific standards and requirements, the costs of organic production could be up to 30% higher than those of conventional rearing (4). The legislation associated with organic pig production is aimed at animal welfare, including the provision of environmental conditions that allow livestock to perform their normal behaviour patterns. The animals must be free from hunger and thirst, thermal and physical discomfort, and they must be protected from injuries, diseases, fear and stress. The legislation (5) provides requirements that include specifications for housing conditions, animal nutrition, and animal breeding, as well as animal care, disease prevention and veterinary treatment. 16 M. Prevolnik, M. Ocepek, M. Candek-Potokar, M. Bavec, D. Skorjanc Many studies have been carried out to determine if the consumer perceptions, that quality of organic food is better, are supported scientifically. Growth performance and the carcass and meat quality of pigs from organic and conventional production systems have been compared, and the published results show inconsistencies (1,2,6-11). Discrepancies in the published results can be related to housing, feed, breed or genotype, and their interactions, which are reflected in the health, welfare and productivity of pigs. In view of the inconsistency of the research reports and to test local conditions, an experiment was conducted with the objective to evaluate growth performance and carcass and meat quality traits of pigs reared conventionally or respecting organic standards, but using commercially available organic feedstuffs. Contrary to the majority of the studies, where pigs were put to the test at 20-30 kg live weight, the objective of our experiment was to follow the growth performance of pigs from birth until slaughter and to evaluate the consequences for carcass and meat quality. The differences between organic and conventional rearing system are detailed below and in Table 1 and Table 2. Organic pigs had a larger rearing space, access to an outdoor area, straw bedding, longer lactation, and received a commercially available organic feed mixture. Pigs were weaned after 28 days (conventional) or 42 days (organic) of lactation. After weaning, pigs from the conventional group were allotted to three pens for the whole fattening period. Pigs from the organic group were housed in three pens until week 11 when they were further divided into seven pens in order to fulfil space requirements. Materials and methods Animals, housing and feed The study was conducted at the Pig Research Centre of Faculty of Agriculture and Life Sciences, University of Maribor, Slovenia, and national legislation on animal protection (12) was respected. The experimental pigs were the progeny of landrace* large white dams and landrace*pietrain sires (free of the RYR1 gene). The pigs originated from eight sequential litters born within two months period. The piglets in the first four litters formed a group that was reared according to conventional practices of housing and feeding (n=35; 13 females and 19 castrates). Pigs from the other four litters formed a group of pigs (n=40; 15 females and 20 castrates) that were reared respecting the constraints of EU legislation on organic production (5). Surgical castration of the male pigs was performed at the age of three or four days. Owing to mortality, the final analysis comprised 67 pigs (n=32 conventional, and n=35 organic pigs). The available space (Table 1) was in agreement with relevant legislation (5,12). Pigs were fed with a commercially available feed mixture in pellets (Table 2). The same feeding strategy was used for all pigs, except that the organic pigs received feed mixture of organic origin. From the second week of lactation, in addition to their intake of milk, all the piglets were provided with additional feed mixture (in pellets). During the last four weeks of fattening, only 2.2 kg of feed mixture per pig was provided daily in order to limit fat deposition. Table 1: Available space in conventional and organic pigs according to rearing phase Available space Rearing phase Conventional Organic Lactation (4 or 6 weeks for conventional or organic group, respectively) Pen size 1.8x2.5m2 Pen size 1.8x2.5m2 Growing (weeks 4-11 or 6-11 for conventional or organic group, respectively) Pen size 1.8x2.0m2 0.26-0.36 m2/piglet Pen size 3.8x2.3m2 0.62-0.67 m2/piglet Straw bedding Outdoor area 2.9 x2.3m2 Finishing (weeks 11-26 for both groups) Pen size 3.8x2.1m2 0.66-0.72 m2/piglet Pen size 3.8x2.1m2 1.33 -1.59 m2/piglet Straw bedding Outdoor area 2.9 x2.1m2 Growth, carcass and meat quality traits of pigs raised under organic or conventional rearing systems using commercially. 17 ^Conventional and organic feed mixtures were commercially available; CP - crude proteins; ME - metabolizable energy; Lys - lysine; Supplier Perutnina Ptuj d.d., PC Krmila, DE Proizvodnja krmil, Draženci, SI-2250 Ptuj; 2supplier UNSER LAGERHAUS Warenhandels Ges.m.b.h., Mischfutterwerk, Südring 240, A-9020 Klagenfurt Table 2: Composition of experimental dietsA Conventional Organic Feed mixture Prestarter1 Bek-1 farm1 Bek-2 farm1 Alpenkorn Ferkel2 Alpenkorn Schweine2 Rearing phase Lactation & post-weaning 2-8 weeks Growing 8-16 weeks Finishing 16-26 weeks Lactation & post-weaning 2-9 weeks Growing & Finishing 9-26 weeks Diet ingredients maize 38%, barley 12%, soya meal 17%,milk powder 8%, wheat 8%, fish flour 5%, soya concentrate 2%, minerals, oils & fats, vitamins, Lys, aromatic substances, enzymes, organic acids maize 35%, barley 22%, soya meal 18%, wheat 15%, sunflower meal 3%, fish meal 1%, lucerne 1%, minerals, oils and fats, vitamins, Lys, aromatic substances, enzymes maize 34%, barley 23%, soya meal 15%, wheat 11%, sunflower meal 4%, lucerne 2%, minerals, oils and fats, vitamins, Lys, aromatic substances, enzymes wheat 50%, wheat bran 11%, faba bean 10%, soybean 7%, triticale 6%, barley 5%, rape cake 3%, potato proteins 3%, molasses 2% triticale 25%, wheat 15%, wheat bran 12%, barley 9%, maize 7%, sunflower cake 9%, rape cake 7%, soybean 5%, faba bean 3%, potato proteins 2.5%, molasses 2% CP, g/kg 206 169 158 170 170 ME, MJ/kg 12.3 12.0 12.0 12.8 12.5 Lys, g/kg 12.5 10.0 7.0 8.0 8.0 The pigs were weighed individually one day after birth and at 4, 6, 11, 22 and 26 weeks of age. Daily gains were calculated using the data on body weight and age at weighing for the whole experimental period (0 to 26 weeks; life daily gain) and for the periods between sequential weight measurements. Feed intake was recorded per pen and divided by the number of pigs to give the daily intake per pig. Feed conversion efficiency was calculated per pen as the ratio between feed intake and live weight gain for each period from weaning until slaughter (week 4 or 6 to 26). Transport and slaughter procedures All pigs were slaughtered at the same age (26 weeks) in a commercial abattoir. Pigs from one pen were slaughtered together on the same day. The experimental design required that pigs from the two rearing systems could not be slaughtered on the same day. To avoid confounding of slaughter day and the effect of rearing system, we organised slaugh- ter in six groups (three for conventional and three for organic pigs) with the purpose of randomizing the effect of slaughter day. Pigs were fasted for 12 hours prior to transport to the abattoir. On the day of slaughter, they were loaded between 6 and 8 a.m. and transported for 20 minutes to the local abattoir. The lairage lasted 2-3 hours. During the transport and slaughter procedures there was no mixing of the pigs. Pigs were slaughtered between 8 and 11 a.m. according to the routine abattoir procedure which consisted of CO2 stunning (86 vol. % in air), vertical exsanguination, vapour scalding, dehairing and evisceration, followed by the veterinary inspection and SEUROP carcass classification (13). Plasma stress markers Stress markers were assessed in plasma taken at slaughter in order to monitor for differences in the level of stress between slaughter series. At slaughter, blood samples (approximately 4 ml) were taken into plastic tubes containing EDTA (to prevent blood 18 M. Prevolnik, M. Ocepek, M. Candek-Potokar, M. Bavec, D. Skorjanc coagulation). Immediately after blood collection the tubes were placed on ice and taken to the laboratory where the blood was centrifuged at 1800 rpm for 15 minutes. The supernatants (plasma) were collected and stored at -20°C until analysis of the levels of plasma markers of stress using commercial kits. The tests were based on enzyme immunoassays for the in vitro diagnostic quantitative determination of cortisol, neopterin and Hsp70 according to the manufacturer's instructions (14-16). The intensity of the colour was read at 450 nm using a spectrophotometer (Varioscan Flash) and SkanIt Software Version 2.4.3. RE (Thermo Fisher Scientific Inc. Waltham, MA, USA). The concentrations of cortisol, neopterin and Hsp70 in plasma samples were expressed in |g/ dL, nmol/L and ng/mL, respectively. Carcass quality traits At the end of the slaughter line, the pigs were classified according to SEUROP by the approved classification body (Bureau Veritas), using a method that consists of taking two measurements at the carcass split line: DM fat (minimal fat thickness over the m. gluteus medius) and DM muscle/width (shortest distance between the cranial end of m. gluteus medius and dorsal edge of the vertebral canal). The carcass lean meat percentage was calculated according to the formula (DM meat = 60.81879 - 0.72992xDM fat + 0.12157*DM muscle) approved for Slovenia (17). One day after the slaughter, additional carcass traits were measured. The hind leg (without shank) was cut off the carcass between 6th and 7th lumbar vertebra. It was weighed prior to and after the removal of subcutaneous fat, and the ratio between the weights was calculated. A digital image of the carcass cross section (last rib) was taken with a digital photo camera (Canon PowerShot G3, Canon Inc., Tokyo, Japan). The loin eye area (longissimus dorsi (LD) area), corresponding fat area (fat over LD) and their ratio (LD meat:fat ratio) were determined from the images with LUCIA.NET 1.16.5 software (Laboratory Imaging s.r.o, Prague, Czech Republic). Belly leanness was assessed using a 1-4-7 scale (1 represents only fat, 4 half meat and half fat, and 7 only meat). bach, Switzerland) at one hour (pH1) and 24 hours post mortem (pHU). Duplicate measurements were taken in the LD muscle at the level of the last rib and in the semimembranosus (SM) muscle approximately 4 cm lateral to the os pubis. Measurements of colour CIE L* a* and b* (18) were taken one day after slaughter from a freshly cut surface of LD (at the level of the last rib) using a Minolta Chroma Meter CR-300 (Minolta Co. Ltd, Osaka, Japan) with an 11 mm diameter aperture and D65 illuminant, calibrated against a white tile. At the same time, the colour intensity of LD was also assessed using a 1-6 Japanese colour scale (1 and 2 represent pale, 3 and 4 normal, and 5 and 6 dark meat colour; (19)). A 2.5 cm slice of LD was taken from the loin at the level of the last rib for determination of drip loss (EZ drip loss) according to the method of Christensen (20). Drip loss was determined in duplicate, after 24 and 48 hours of storage at 4°C, and expressed as a percentage of the initial sample weight. Intramuscular fat of the LD was determined with NIR spectroscopy (NIR Systems model 6500, Silver Springs, MD, USA) according to Prevolnik et al. (21). Statistical analysis Analysis of variance (GLM procedure of SAS 9.1, SAS Inc., Cary, NC, USA) was performed in order to evaluate the effect of rearing system. For growth and carcass traits, the model comprised fixed effects of rearing system and sex, random effect of litter nested within rearing system, and interaction between rearing system and sex (which was always non-significant). For meat quality traits and plasma stress markers, an effect of slaughter day within rearing system was added to the model as a random effect. An additional analysis was performed for carcass traits, with warm carcass weight added as a covariate (in the case of meat quality traits it was non-significant). When significant effect of treatment (P<0.05) was detected, least squares means (LS means) were compared (PDIFF option, Tukey adjustment). Results Growth performance Meat quality traits The pH was measured with a MP120 Mettler Toledo pH meter fitted with a combined glass electrode InLab427 (Mettler-Toledo, GmbH; 8603 Schwarzen- Growth performance (body weight and daily gain) differed between conventional and organic pigs (Fig. 1). In Slovenia, the usual slaughter weight of pig fat-teners is about 100 to 110 kg at 6 months of age and growth rate observed for pigs of conventional group Growth, carcass and meat quality traits of pigs raised under organic or conventional rearing systems using commercially. 19 can be considered as an average result in Slovenian situation. In the first six weeks there was no difference in body weight between organic and conventional pigs. At all subsequent stages organic pigs had lower body weight than the conventional pigs. At the end of the experiment the difference in body weight between the organic and conventional pigs was 11 kg (86 vs. 97 kg, respectively), demonstrating lower growth rate of organic pigs when compared with the conventional pigs (462 vs. 523 g/day, respec- tively). Initially (at four weeks of age), when piglets were fed mother's milk, no difference in growth rate was observed (243 vs. 226 g/day, for conventional and organic piglets, respectively). It is only when the feed and feeding regime change that the differences are seen. Lower but non-significant growth rate of organic pigs started in weeks 4-6 when these pigs were still fed milk (the conventional pigs had been already weaned), despite being offered additional concentrate feed. Growth rate decline continued after 105 i 90 : -Conventional Organic *** i -1-r 8 12 16 20 Age (weeks) ns: not significant; ***P<0.001, **P<0.01. 24 28 800 700 3 600 I 500 CD ^ 400 CO <=> 300 200 100 Conventional Organic 0-4 4-6 6-11 11-22 22-26 Fattening period (weeks) Figure 1: Changes in body mass and daily gain (LS means ± s.e.) for pigs raised under conventional and organic rearing system Table 3: Feed intake and feed conversion efficiencyA of pigs raised under conventional and organic rearing system Period Conventional Organic Number of animals 32 35 4th to 6th week Feed intake(kg/day) 0.32 - Feed conversion efficiency 0.781 - 6th to 11th week Feed intake(kg/day) 0.76 0.48 Feed conversion efficiency 0.377 0.274 Weaning to 11th week Feed intake(kg/day) 0.63 0.48 Feed conversion efficiency 0.439 0.274 11th to 22nd week Feed intake(kg/day) 1.85 1.87 Feed conversion efficiency 0.400 0.330 22nd to 26th week Feed intake(kg/day) 2.20 2.20 Feed conversion efficiency 0.306 0.307 Weaning to 26th week Feed intake(kg/day) 1.53 1.59 Feed conversion efficiency 0.383 0.336 AMeasured per pen and calculated per pig. 20 M. Prevolnik, M. Ocepek, M. Candek-Potokar, M. Bavec, D. Skorjanc the weaning (in weeks 6-11). Lower daily gain was recorded for organic pigs until week 22. In the last fattening period (weeks 22-26) organic and conventional pigs had similar daily gain (667 vs. 620 g/day, respectively). During the final period, when feed was rationed to 2.2 kg, a decrease of growth rate compared to previous phase was observed for conventional pigs, whereas this quantity was sufficient to cover the needs for increased growth rate observed in organic pigs, owing to their lower weight. Growth performance of the pigs can be related to feed composition and feed intake (Tables 2, 3). The organic feed used in the period post-weaning had lower protein level and organic pigs exhibited lower feed intake. In other periods (weeks 11-26), feed intake and the diet were comparable between groups. Because of lower feed intake and growth rate, the organic pigs exhibited lower feed conversion efficiency. Carcass traits Lower growth rate of the organic pigs was reflected in most of the carcass traits (Table 4). In comparison with conventional pigs, organic pigs had lower carcass weight, lower dressing %, smaller LD muscle (DM muscle and LD area), larger fat area over LD, and lighter hams, with no significant difference in subcutaneous fat thickness (DM fat) or carcass leanness. If the influence of organic rearing is estimated for the same carcass weight, an effect is shown for fat thickness (higher than conventional), LD muscle (lower), carcass and belly leanness (lower) and LD meat:fat ratio (lower). Thus it can be speculated that organic pigs would present fatter carcasses than conventional pigs if slaughtered at the same weight. Table 4: Carcass quality traits of pigs raised under conventional and organic rearing systemA At the same age At the same carcass weight Conventional Organic Sig. Conventional Organic Siq. Number of animals 32 35 32 35 Carcass weight, kg 75.8 ± 1.5 65.6 ± 2.0 *** - - - Dressing, % 78.1 ± 0.5 76.2 ± 0.7 * 77.6 ± 0.5 77.2 ± 0.7 ns DM fat, mm 14.9 ± 0.7 15.3 ± 0.9 ns 14.1 ± 0.6 16.9 ± 0.8 * DM muscle, mm 66.7 ± 0.9 61.1 ± 1.2 *** 65.5 ± 0.8 63.5 ± 1.1 ns DM meat, % 58.1 ± 0.5 57.1 ± 0.6 ns 58.5 ± 0.4 56.2 ± 0.6 ** LD area, cm2 44.9 ± 0.9 35.1 ± 1.2 *** 43.5 ± 0.7 37.8 ± 1.0 A** Fat over LD, cm2 13.1 ± 0.4 11.7 ± 0.5 * 12.6 ± 0.3 12.5 ± 0.4 ns LD meat : fat ratio 3.5 ± 0.1 3.1 ± 0.1 * 3.5 ± 0.1 3.1 ± 0.1 * Belly leanness (1-7) 5.0 ± 0.1 4.8 ± 0.2 ns 5.1 ± 0.1 4.6 ± 0.2 * Ham, kg 9.6 ± 0.2 8.3 ± 0.3 *** 9.1 ± 0.1 9.1 ± 0.1 ns Ham in carcass, % 25.2 ± 0.2 25.4 ± 0.2 ns 25.1 ± 0.2 25.5 ± 0.2 ns Ham meat, % 83.4 ± 0.5 82.2 ± 0.6 ns 83.7 ± 0.5 81.7 ± 0.7 * Ham fat, % 16.6 ± 0.5 17.8 ± 0.6 ns 16.3 ± 0.5 18.3 ± 0.7 * Ham meat : fat ratio 5.2 ± 0.2 4.7 ± 0.2 t 5.3 ± 0.2 4.5 ± 0.2 * AValues in the table are LS means ± s.e.; LD - muscle longissimus dorsi, DM fat - minimal fat thickness over the m. gluteus medius; DM muscle - shortest distance between cranial end of m. gluteus medius and dorsal edge of vertebral canal; ns: not significant; ***P<0.001, **P<0.01, *P<0.05; t<0.10 Plasma stress markers and meat quality traits Rearing system was not associated with any significant differences in the plasma levels of cortisol, neopterin or Hsp70 between conventional and organic pigs. However, it is worth noting that the level of Hsp70 was 2-fold higher in organic pigs. With regard to meat quality, the rearing system had a significant effect only on pHD and the intra- muscular fat content of the LD muscle (Table 5). Although significantly higher, the difference in pHD was small from the practical point of view and was not accompanied by any differences in colour or water holding capacity (drip loss) between organic and conventional pigs. Intramuscular fat content of the LD muscle was low in both groups of pigs, and despite lower carcass weight, the organic pigs exhibited higher intramuscular fat content than conventional pigs (1.8 vs. 1.4 %, respectively). Growth, carcass and meat quality traits of pigs raised under organic or conventional rearing systems using commercially. 21 Table 5: Level of plasma stress markers at slaughter and meat quality traits for pigs raised under conventional and organic rearing systemA Item Conventional Organic Sig. Number of animals 32 35 Plasma stress markers Cortisol, |g/dL 10.3 ± 1.1 10.7 ± 1.3 ns Neopterin, nmol/L 3.0 ± 0.5 2.8 ± 0.6 ns Hsp70, ng/mL 3.6 ± 1.4 7.4 ± 1.6 t Meat quality traits SM pH, 6.30 ± 0.06 6.44 ± 0.10 ns SM pHTT 5.64 ± 0.03 5.66 ± 0.04 ns LD pH1 6.03 ± 0.08 6.17 ± 0.12 ns LD pH„ 5.52 ± 0.01 5.58 ± 0.02 ** LD colour (1-6) 3.6 ± 0.1 3.6 ± 0.1 ns Minolta L* 54.1 ± 0.5 54.4 ± 0.7 ns Minolta a* 6.1 ± 0.2 6.2 ± 0.3 ns Minolta b* 2.7 ± 0.1 2.8 ± 0.2 ns Drip loss 24h , % 4.3 ± 0.5 4.9 ± 0.7 ns Drip loss 48h, % 6.4 ± 0.5 7.2 ± 0.7 ns Intramuscular fat, % 1.40 ± 0.10 1.77 ± 0.13 * AValues in the table are LS means ± s.e.; SM - muscle semimembranosus; LD - muscle longissimus dorsi; pH: - pH measured one hour after slaughter; pHT - pH measured 24 hours after slaughter; ns - not significant; **P<0.01, *P<0.05; t P<0. 10; Frequency of PSE meat (fast pH fall shortly post-mortem) was 18% in conventional and 20% in organic pigs Discussion Growth performance The lower growth rate of the organic pigs started during the last stage of lactation and persisted until 22 weeks of age. To explain growth retardation several factors should be considered, which may have interacted, the prolonged lactation, the lower post-weaning feed intake (due to less palatable organic feed or its lower protein level) and larger space provided to organic pigs, which will be discussed below. The observed growth retardation in weeks 4-6 can be related to the prolonged lactation of organic pigs, which results from the diminished milk yield of the sows at the end of lactation, although all the pigs were supplied with additional concentrate. The most noticeable decrease of growth rate in organic pigs was observed post-weaning and it coincides with lower feed intake of organic pigs. The importance of feed intake for growth traits has previously been demonstrated (22). A possible reason for the lower feed intake, given that we observed no particular problem with diarrhoea in the organic pigs, could be the lower palatability of the organic feed mixture (1). It is also possible that the animals felt satiated earlier on the organic diet. The regulation of the ap- petite is a complex mechanism which acts via ghre-lin, a 28-amino acid peptide. Ghrelin is involved in tryptophan-mediated appetite stimulation in swine (23) and has been shown to be stimulated by protein ingestion in men (24,25). Deficiency of tryptophan has been shown to reduce appetite and feed intake in pigs (26,27). Moreover, the organic feed concentrate used for organic pigs in the growing phase had lower concentrations of protein and lysine than the conventional diet (Table 2), which is critical in the first stage of fattening. The availability of essential amino acids adequate for growth is known to be the main difficulty in organic feeding of pigs (28). Organic standards, and especially the ban on synthetic amino acids, make it difficult to meet the amino acid requirements of young pigs. Organic sources rich in amino acids and in lysine could be produced, but are expensive and therefore rarely produced and used for economical reasons. In the present study, commercially available diet was used containing 8.0 g/kg of lysine (Table 2), whereas according to Sundrum (29) requirements for suckling piglets and weaners can be covered with diets containing at least 12 g lysine/kg. Underfeeding or nutrient deprivation during the post-weaning period has a major impact on subse- 22 M. Prevolnik, M. Ocepek, M. Candek-Potokar, M. Bavec, D. Skorjanc quent growth performance (30). Accelerated growth following a period of slower growth, as a result of restricted feeding or nutrient deprivation, is defined as compensatory growth. The use of feeding strategies for obtaining compensatory growth in organic pigs has been of interest (11,31), especially because of the problems with protein supply in organic pig production. In the finishing period (weeks 22-26) the growth rate of the organic and conventional pigs was comparable. Requirements for amino acids, especially lysine, decrease as animals get older and heavier (32), and the lysine content of both diets in the later phases of this experiment was sufficient (29). In the finishing period the organic pigs received the same quantity of feed, and of similar composition, as the conventional pigs, which was sufficient to cover the needs for increased growth rate of organic pigs due to lower body weight. Due to lower feed intake, the organic pigs had lower feed conversion efficiency. However, we cannot exclude the possibility that feed efficiency might have been affected by other parameters such as feed composition, pen space, and outdoor access. The greater physical activity of pigs, which were allocated a larger space and access to an outdoor area, may have resulted in higher energy expenditure. Comparison of the results of the present study with similar literature reports (studies using feeding of organic concentrates) shows inconsistency. In agreement with our results, Enfalt et al. (6) observed a delay in reaching slaughter weight for pigs grown under organic conditions with outdoor access, as compared to conventional pigs, although in their study pigs were fed ad libitum throughout the experiment and had more outdoor space as in the present study. On the contrary, Millet et al. (8) observed a better growth rate for pigs in organic housing that were fed organic feed concentrate ad libitum, as compared to conventional pigs. Several other studies have also reported a higher feed intake and growth rate for organic pigs, as compared to conventional pigs (1,2,10). Strudsholm and Her-mansen (11) reported that when pigs had been fed ad libitum with concentrates, indoor compared with outdoor rearing did not affect growth rate, only feed consumption (which increased). The value of the present study is a demonstration of possible problems with commercially available organic feed mixtures for piglets in early life, which was reflected in growth performance. Carcass quality Due to slower growth of organic as compared to conventional pigs, carcass weight and consequently other carcass traits were affected. Differences between the organic and conventional pigs were observed mainly for the traits of muscularity, for which the most influential explanatory factors seem reduced growth rate due to lower feed intake and limited supply of proteins in organic feed concentrate. The comparison between organic and conventional pigs simulated for the same carcass weight indicated that organic pigs would be fatter if slaughtered at the same weight as conventional pigs. This result can be related to the compensatory growth of organic pigs affecting the deposition of adipose rather than muscle tissue. Published studies dealing with pig carcass traits under organic production regimes (1,2,6,8,10,11) show significant effects of housing and feeding, but the results are difficult to compare and inconsistent since they reflect the variability in the feeding and housing systems applied in different studies. One study (2) however can be highlighted which showed no difference in carcass quality when organic and conventional pigs were reared ad libitum, while 70% restriction of concentrates given to organic pigs reduced their growth rate and increased the lean meat percentage. Plasma stress markers and meat quality In the present experiment the main purpose of assessing plasma stress markers at slaughter was to monitor for the effect of slaughter day (and pre-slaughter susceptibility to stress), owing to its importance for meat quality. The biochemical markers used (cortisol, neopterin and Hsp70) reflect different aspects of stress. Cortisol is the main hormone of hypothalamic-pituitary-adrenocortical axis and is released by the adrenal cortex in response to stress. It influences feeding behaviour, pancreatic hormone secretion, energy expenditure, and pro-tein/lipid balance (33). In the present study, plasma cortisol levels at slaughter were similar in pigs from both rearing systems. The comparable level of cortisol indicates a similar stress response to the pre-slaughter procedures for both rearing systems. Studies that deal with the cortisol level in different rearing systems are rare. In accordance with our results, Barton-Gade (7) and Lebret et al. (28) reported no effect of rearing system on the plasma Growth, carcass and meat quality traits of pigs raised under organic or conventional rearing systems using commercially. 23 Cortisol level prior to slaughter. A comparable level of neopterin in organic and conventional pigs is an indicator of similar health status, because studies suggest it is a good marker of cellular immune activation, with increased concentrations detected in infections, autoimmune diseases and animals with tumours (34). Hsp70 is a stress-limiting factor that is involved in the response to stress at a cellular level (35). The results of the present study suggest that rearing system was not associated with plasma Hsp70 level. Despite this, it is interesting to observe that the level of Hsp70 was 2-fold higher in organic pigs. A possible explanation for the lack of significance was the high variation within groups. Nevertheless, we can consider this result indicative of a stronger response of the organic pigs to stress in terms of cell protein protection. Regarding meat quality an effect of rearing system was observed only for pHD and LD intramuscular fat content, both being higher in organic pigs. Differences in pHD were not big enough to be reflected in LD colour or drip loss, which are properties important for the consumer, and highly correlated to pHD (36). In contrast to our results, higher pHD values in conventional as compared to organic pigs (6,8), or no differences (2,9), have been reported. Studies show, that intramuscular fat is higher in older and heavier pigs and follows general body adiposity (37,38). Despite a considerably lower carcass weight, higher intramuscular fat was observed in organic pigs in the present study, which can be related to feeding. A strategy of restriction and re-alimentation has been suggested to increase intramuscular fat content (38). Contrary to our result, organic feeding has often been shown to reduce intramuscular fat (1,6,10), or no effect has been observed (2). On the whole, the results of the present study corroborate previous reports which show limited effect of organic production on meat quality. Conclusions In comparison to conventional rearing, in the present study, organic pigs exhibited post-weaning growth retardation and as a consequence lower carcass quality. The increased growth rate of organic pigs in the last fattening phase, following the initial growth retardation, explains lower carcass quality and higher intramuscular fat content, the latest being of interest in terms of improved meat quality. The results of the present study indicate possible problems associated with the use of commercially available organic diets for piglets. Acknowledgements The authors acknowledge the financial support by Slovenian Research Agency (project J4-9532, project V4-0338, program P4-0072) and partly by the Ministry of Agriculture Forestry and Food. References 1. Millet S, Hesta M, Seynaeve M, et al. Performance, meat and carcass traits of fattening pigs with organic versus conventional housing and nutrition. Livest Prod Sci 2004; 87: 109-19. 2. 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Candek-Potokar, M. Bavec, D. Skorjanc RASTNOST, KLAVNE LASTNOSTI IN KAKOVOST MESA pRAŠIČEV, VZREJENIH V EKOLOŠKEM ALI KONVENCIONALNEM NAČINu REJE Z upORABO Komercialno dostopnih krmnih mešanic M. Prevolnik, M. Ocepek, M. Čandek-Potokar, M. Bavec, D. Škorjanc povzetek: Cilj raziskave je bil oceniti rastnost pujskov od rojstva do zakola, klavne lastnosti in kakovost mesa prašičev, vzrejenih bodisi konvencionalno (n = 32) ali upoštevajoč standarde ekološke reje (n = 35) ob uporabi ekoloških krmnih mešanic, ki so na voljo na trgu. V tej raziskavi je bila poraba krme od odstavitve do 11. tedna za 22 % nižja pri prašičih iz ekološke reje. Posledično so ekološko vzrejeni prašiči imeli nižje priraste vse do 22. tedna starosti. V zadnji fazi pitanja (od 22. do 26. tedna), ko je bila razpoložljiva krma omejena, se je prirast pri konvencionalnih pitancih zmanjšal, pri ekoloških pa povečal. Vsi prašiči so šli v zakol pri običajni komercialni starosti (26 tednov). Zaradi počasnejše rasti so imeli ekološko vzrejeni prašiči manjšo maso trupa, manjši klavni izplen, manjšo mišico longissimus dorsi (LD) in lažja stegna, medtem, ko pri debelini podkožne maščobe in mesnatosti trupov (z izjemo površine podkožne maščobe nad LD) ni bilo razlik. Ob korekciji na maso trupov je analiza pokazala, da bi ekološki prašiči imeli bolj zamaščene trupe kot konvencionalni, če bi bili zaklani pri enaki masi. V zvezi s kakovostjo mesa je bila pri ekološko vzrejenih prašičih ugotovljena višja vrednost pH po 24 urah in višja vsebnost mišične maščobe. Rezultati kažejo, da pitanje prašičev z uporabo komercialno dostopnih ekoloških krmnih mešanic za pujske lahko vodi v slabše proizvodne rezultate (manjše zaužitje krme, nižji prirasti), po drugi strani pa lahko takšna dieta poveča vsebnost mišične maščobe, ki je zanimiva zaradi izboljšanja kakovosti mesa. Ključne besede: ekološka reja; rast; klavna kakovost; kakovost mesa; stres; prašič Slov Vet Res 2011; 48 (1): 27-34 UDC 636.5.083:636.087.7:637.51 Original Scientific Article EFFECT OF SuppLEMENTATION OF pHYTOGENIC FEED ADDITIVES ON performance parameters and meat quality of broiler chickens Slavomír Marcincák1*, Peter Popelka1, Nevijo Zdolec2, Mária Mártonová1, Jana Simková1, Dana Marcincáková1 1University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81, Kosice, Slovak Republic; 2Department of Hygiene and Technology of Foodstuffs of Animal Origin, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia Corresponding author, E-mail: marcincak@uvm.sk Summary: The aim of the experiment was to study the effects of clove powder (Syzygium aromaticum L), extracts of agrimony (Agrimonia eupatoria L.) and lemon balm (Melissa officinalis L.) on broiler chicken performance, carcass and meat quality. Three treatment combinations were prepared: C - control group without any feed or water additive; AC - feed supplemented with clove powder and drinking water supplemented with agrimony extract; LC - feed supplemented with clove and drinking water supplemented with lemon balm extract. AC group chickens had significantly higher body weight (P < 0.05) compared to the control group within 14 days of fattening. On the 42nd day higher body weight, better feed conversion, higher carcass yield and higher breast and thigh weight were determined in broiler chickens from groups AC and LC. However, significant differences (P > 0.05) in selected parameters were not recorded in experimental groups AC and LC compared to control. Dry matter of thigh muscles derived from AC and LC broiler chickens were significantly higher (P < 0.05) compared to controls. In groups AC and LC lower total fat content was recorded in thigh muscles (P > 0.05) when compared with control and significantly higher (P < 0.05) proportion of total protein was found in AC group. In the sensory evaluation of meat experimental groups AC and LC were rated better, despite no significant differences (P > 0.05) in comparison with control group. Keywords: agrimony; clove; growth of broilers; lemon balm; meat quality Introduction A ban on the use of antibiotics as growth promoters in the European Union in 2006 due to increase bacterial resistance in human population encouraged the search for replacement alternatives, which would be useful to improve the production parameters of fattening pigs and poultry (1). A new group of potential feed additives are herbs, their extracts or essential oils (2). Phytogenic feed additives are usually defined as products derived from plants and added to animal feed for fattening to increase productivity, improving the quality of feed and animal hygiene conditions Received: 17 January 2011 Accepted for publication: 24 February 2011 and not least to improve the quality of produced food. Feed additives produced from plants have often a significant antibacterial effect, thereby suppressing pathogenic microflora in the gastrointestinal tract of animals and thus reducing mortality during the fattening period, especially in stress period (3). Plant additives are often added into feedstuff as they improve the taste and smell of feed and thus improve intake and growth of animals (1). Several herbal additives contain substances which increase the production of digestive juices (saliva, gastric juices, pancreatic and intestinal secretion) and thereby enhance appetite and digestion (4). Several plants or their essential oils, especially those with aromatic character such as cloves, rosemary, cinnamon (1), anise (5), but also oregano (6) and sage (7) have been used in fattening. Plant ar- 28 S. Marcincák, P. Popelka, N. Zdolec, M. Mártonová, J. Simková, D. Marcincáková omatic oils as well as probiotics and organic acids can be used to support growth in order to achieve improvements in breeding broilers in organic farming system (8). Supplementation of feed or drinking water with plant aromatic oils increases body weight of chickens and favourably has an effect on feed conversion (9). For a wider range of action the most suitable combination of extracts is researched. The role of research is to find the most effective concentrations and combinations of plants and essential oils. According to Ertas et al. (10), the combination of some plants extracts has a better effect on the growth performance in poultry than their individual supplementation. Synergism between some herbal constituents was highlighted in the studies of Hernandez et al. (7) and Burt (11). Clove (Syzygium aromaticum L.) is considered as one of the most versatile herbs due to its analgesic and anti-inflammatory, anaesthetic, antimicrobial and antifungal effect (12), antiseptic, appetite and digestion stimulation (13), anti-inflammatory, anticarcinogenic, antiparasitic as well as antioxi-dant properties (6, 14, 15). The major constituent of clove is an essential oil (up to 20 %), which is characterized by presence of up to 85.5 % of eugenol (16). Lemon balm (Mellisa officinalis L.) and agrimony (Agrimonia eupatoria L.) are the most common herbs used in our traditional folk herbal medicine (17). However, little is known about the antioxidant properties of their extracts and essential oils in poultry. The alcohol extracts of both herbs are a potent source of polyphenols (6, 17, 18). In in vitro experiments, it was found that active substances extracted from agrimony possess a significant radical scavenging activity as well as potential antioxi-dant capacity (18, 19). The aim of our work was to study the effect of combination of cloves added to the feed and agrimony or lemon balm extracts administered into water on growth of broiler chickens and quality parameters of produced meat. Materials and methods Plant material Pulverized extracts of agrimony (Agrimonia eupatoria L.) and lemon balm (Mellisa officinalis L.) were prepared by Calendula (Nova Bana, Slovak Republic) and both herbs were collected in East Slovakia. The plant material consisted of leaves, flowered tops and stalks that was dried at 30 - 35 °C, ground, extracted with 50 % ethanol and evaporated to obtain a powder (prescription and protocol of Calendula, Slovak Republic). Clove (Syzygium aromaticum L.) powder was purchased from Maspoma (Zvolen, Slovak Republic). Experimental animals, diets and treatments The experiment was carried out on 120 one-day-old unsexed hybrid broiler chickens Ross 308 which were randomly allotted to 3 groups (40 birds per group). Broilers were kept in large pens with wood shavings. On the day of hatching, the initial room temperature was 32 °C. It was gradually decreased by 3 °C weekly to a final temperature of 23 °C on the 21st day and then kept constant. During the entire fattening period, the lighting regimen was 24 h of continuous light per day. Broilers were reared in the air environment with 70 % humidity. The experiment was approved by the Ethics Committee of the University of Veterinary Medicine and Pharmacy in Kosice, Slovak Republic. All birds were fed with commercial basal diets (BD) for broilers "Starter" for days 1 to 14, "Grower" for days 15 to 29 and "Finisher" for days 30 to 42. The composition of all BDs is presented in Table 1. The control group of chickens received the basal diet (BD) only. The second (LC) group was fed with the same BD enriched with 1 % clove buds powder combined with 0.2 % lemon balm extract diluted in drinking water for 42 days. Third group (AC) was also fed with BD enriched with 1 % clove buds powder combined with 0.2 % agrimony extract diluted in drinking water. The broilers had a free access to the feed and water. Data collection and analysis Body weights were recorded weekly. Feed intake per group was measured throughout the experiment and the feed conversion ratio was calculated at the end of experiment. Broiler chickens were slaughtered on the 42nd day of fattening. After the carcass processing, chickens in each group were weighed, boned and chilled at 4 °C. The yield of carcasses was calculated by dividing the body weight of the animal before slaughter and carcass weight after evisceration. The proportion of breast and thigh muscle was calculated from a weight of individual parts and body weight after evisceration. Effect of supplementation of phytogenic feed additives on performance parameters and meat quality of broiler chickens 29 Chemical composition of meat samples Determination of water content, dry matter content and fat content in % was performed according to Veterinary laboratory methods (20). Sensory evaluation For sensory analysis of meat samples thigh and breast muscle were packed into containers and evaluated 24 hours after slaughtering. Professional evaluation committee was represented by a panel of 7 assessors who worked according to Methods intended for meat sensory evaluation (21). The samples were boiled and 5-point scheme was used while the maximum number of evaluation points was 20 (22). Table 1: Composition of basal diets given to the broilers during the entire experiment Starter Grower Finisher Ingredients (%) (1 to 14 day) (15 to 29 day) (30 to 42 day) Maize 51.3 49.0 52.8 Wheat 8.0 10.0 20.0 Wheat meal 7.0 4.00 - Soybean meal (46.5 % CP, 1.5 % fat) 29.9 31.6 23.5 Wheat bran - 2.25 - Limestone 1.90 1.25 1.75 Monocalcium phosphate 0.89 1.00 0.90 Vitamin-mineral premix a 0.30 0.3 0.30 NaCl 0.36 0.30 0.35 L-lysine 0.25 0.15 0.15 DL-methionine 0.10 0.15 0.25 Nutrient level (%) Linoleic acid 1.0 1.0 1.0 Metabolizable energy (MJ/kg) 11.5 12.0 12.0 Crude protein 17.5 19.0 17.0 Crude fibre 5.0 4.0 4.0 Ash 80.0 70.0 70.0 L-lysine 0.80 0.95 0.95 DL-methionine 0.35 0.40 0.40 Methionine + cysteine 0.70 0.75 0.70 Calcium 0.80 0.70 0.70 Phosphorus 0.50 0.50 0.50 a supplied per kg of basal diet: vitamin A 8,000,000 IU; vitamin D3 1,200,000 IU; vitamin E 15,000 mg; vitamin K3 3,000 mg; vitamin 1,500 mg; vitamin B6 8,000 mg; niacin 15,000 mg; choline chloride 50,000 mg; pantothenic acid 50 mg; pyridoxine 5 mg; folic acid 2 mg; cyanocobalamine 30 ^g; biotin 0.2 mg; I 2 mg; Co 1 mg, K 8.6 g; Cl- 2 g; Cu 6.0 mg; Fe 60 mg; Zn 50 mg; Mn 50 mg Statistical analysis Statistical treatment of results was performed using statistical program GraphPad Prism, version 4.00 (23). Results are expressed as arithmetic mean (x) and standard deviation (sd). Results in each group were compared with each other by one-way ANOVA test. To compare the statistical differences between values Tukey's comparison test was used and P < 0.05 was regarded as statistically significant. Results Average weights of chickens during the whole fattening period are shown in Table 2. The addition of clove (1%) in the feed and agrimony extract (0.2%) into water had the greatest impact on weight of broiler chickens at the beginning of fattening period. At the age of 14 days, the weight of chickens was significantly higher compared with control (P < 0.05). On the 28th and 42nd day, the weights of both 30 S. Marcincák, P. Popelka, N. Zdolec, M. Mártonová, J. Simková, D. Marcincáková experimental groups were higher but no significant differences were recorded when compared with control (P > 0.05). However, chickens in experimental groups were balanced and were seen less fluctuation in weight than control. The total feed consumption in all three groups was approximately the same (Table 2, P > 0.05). Feed conversion, i.e. conversion of feed to gain 1 g, was lower in both experimental groups in comparison with control. The best values were seen in group AC (1.83). Carcass weight in all groups was correlated with weight of living animals before slaughter (Table 3). The average weights of carcasses from experimental groups were higher compared to controls (P > 0.05). The highest average weight of carcasses and also carcass yield percentage was recorded in experimental group AC supplemented with 1% clove in feed and 0.2% extract of agrimony in water. Highest average weights of thigh and breast muscles were recorded in experimental group AC (P > 0.05) again. Table 2: Effect of supplementation of combination of clove (Syzygium aromaticum L) and agrimony (Agrimonia eupatorio. L) or lemon balm (Melissa officinalis L) on broiler performance Parameters Treatments C LC AC LBW 0, g 41 ± 4 42 ± 3 41± 4 LBW14, g 253 ±13a 260 ±16ab 270 ±18b LBW 28, g 1025 ±133 1046 ±104 1118 ± 129 LBW 42, g 2112 ± 262 2156 ± 222 2232 ± 172 ADWG, g 49.30 50.33 52.16 FC 1 - 42, g (g) 4108 4137 4086 FCR (0 - 42) 1.94 1.91 1.83 C - control, LC - Clove (1 %) + Lemon balm (0.2 %), AC - Clove (1 %) + Agrimony (0.2%), LBW- live body weight, ADWG -average daily weight gain, FC - feed consumption, FCR - Feed conversion ratio. ab - values with different labelling in row are statistically different. Table 3: Effect of suplementation of combination of clove (Syzygium aromaticum L) and agrimony (Agrimonia eupatoria L) or lemon balm (Melissa officinalis L) on slaughter characteristics and meat cat-ups C LC AC Final body weight (g) 2112 ± 262 2156 ± 222 2232 ± 172 Carcass yield (g) 1449 ± 231 1473 ± 109 1558 ± 149 Carcass yield (%) 68.60 68.32 69.80 Breast (g) 294 ± 52 303 ± 31 342 ± 33 Breast yield (%) 20.29 20.57 21.95 Thighs (g) 364 ± 44 370 ± 35 410 ± 27 Thighs (%) 25.55 25.52 26.31 C - control, LC - Clove (1 %) + Lemon balm (0.2%), AC - Clove (1 %) + Agrimony (0.2%) Results of chemical composition and sensory analysis of breast and thigh muscles are shown in Table 4. Added extracts had no effect on the chemical composition of breast muscle and experimental groups were comparable with control. Only a smaller proportion of crude protein (P > 0.05) was recorded in the breast muscles of experimental groups. Dry matter content of thigh muscle was significantly lower in the experimental groups (AC, LC) compared with control (P < 0.05). In the experimental group AC a significantly higher proportion of crude protein was recorded compared to other groups (P < 0.05). Lower proportion of fat in thigh muscles was analyzed in experimental groups (AC, LC) compared with control (P > 0.05). Adding plant additives to feed had also a positive impact on the sensory evaluation of breast and thigh muscle (Table 4). Breast and thigh muscles of experimental groups were scored better in comparison with control (P > 0.05). Breast muscle samples were evaluated as the best after feeding cloves and lemon balm extract (LC). As for thigh muscle the best evaluation of a sample was seen after feeding the cloves and agrimony extract (AC). Effect of supplementation of phytogenic feed additives on performance parameters and meat quality of broiler chickens 31 Table 4: Chemical composition and sensory evaluation of meat Treatments Dry matter Crude fat Crude protein Sensory evaluation C Breast 25.76 ± 054 1.60 ± 0.45 23.11 ± 0.14 15.40 ± 1.63 Thigh 28.37 ±0.27a 9.34 ± 0.20 17.59 ± 0.02a 16.80 ± 1.90 LC Breast 25.65 ± 0.28 1.75 ± 0.31 22.91 ± 0.14 16.60 ± 2.01 Thigh 27.51 ± 0.11b 8.80 ± 0.13 17.70 ± 0.07a 16.82 ± 1.70 AC Breast 25.89 ± 0.23 1.65 ± 0.35 22.88 ± 0.17 15.87 ± 1.68 Thigh 27.12 ± 0.25b 8.75 ±0.70 17.90 ± 0.09b 17.60 ± 1.63 C - control, LC - Clove (1 %) + Lemon balm (0.2%), AC - Clove (1 %) + Agrimony (0.2%) ab - values with different labelling in column are statistically different. Discussion In recent years, an interest in plant feed additives as alternative growth a promoter has increased because of the prohibition of the use of antibiotic feed additives. Plant (phytogenic) growth promoters act primarily as regulators of intestinal flora suppressing the growth of potential pathogens in the intestinal tract, especially in critical period of stress (1, 2). In poultry critical period is mainly at the beginning of feeding, which can result in reduced growth and mortality of animals (24). Even in our experiment, the most significant increase in weight was achieved for 14 days of fattening. On the 28th and the 42nd day, the weight of both experimental groups was higher but no significant differences were observed when compared with control (P > 0.05). The effect of plants and their essential oils on the final weight of chickens has been described in several works (1, 2, 4, 5, 7, 10). However, their effect on increasing total weight of chickens is inconsistent (1). The resulting effect depends on plant extracts used in feeding and on their proper concentrations. Adding only one plant extract to the feed or water does not favourably influence the growth parameters of poultry (4). This problem could be overcome by a combined supplementation of different herb constituents with the synergistic effect (2, 8, 25). Hernandez et al. (7) indicate higher weight gains of chickens after feeding a combination of sage, thyme and rosemary extracts in a dose of 5 g per kilogram of feeding mixture. After feeding a combination of these extracts feed conversion was also decreased by 4%. Equal or lower consumption of feed during fattening after addition of plants essential oils is mentioned by several authors (2, 26). Although even in these indicators are not uniform scientific results, generally there is a perception that plant additives have neutral or positive impact on consumption and feed conversion (1, 2, 24). Higher carcass yield was also recorded after the addition of extracts of coneflower, thyme (27) in feed, and in a combination of Nigella sativa extract in feed and coneflower extract administered in water (28). Even in our experiment, higher carcass weight and higher weight of breast and thigh muscles were recorded in experimental groups (AC, LC). Although differences in weight are not statistically significantly higher when compared with control, weight of carcass, breast and thigh muscles of poultry fed with cloves in feedstuff and agrimony extract in water may be interesting for poultry producers. Plumbless importance of plant feeding additives is also in terms of quality of produced meat. Most of the plants and their extracts have strong antioxi-dant properties, significantly resulting in lower fat oxidation during storage of meat (29). Added plant additives often have a positive effect on the sensory evaluation of produced meat (2). Our results showed that feeding a combination of cloves in the feed and agrimony or lemon balm extracts in water to broiler chickens had a slightly positive effect on the sensory evaluation of produced meat. Several authors mention better sensory characteristics of poultry meat after adding plants with antioxidant activity (25, 30). Positively improved taste and smell were evaluated in meat from chickens supplemented with rosemary powder (31). Acknowledgements The work was carried out with the financial support of grant project VEGA 1/0648/11. References 1. Windisch W, Schedle K, Plitzner C, Kroismayr A. Use of phytogenic products as feed additives for swine and poultry. J Anim Sci 2008; 86: 140-8. 32 S. Marcincák, P. Popelka, N. Zdolec, M. Mártonová, J. Simková, D. Marcincáková 2. Brenes A, Roura E. Essential oils in poultry nutrition: main effects and modes of action. Anim Feed Sci Technol 2010; 158: 1-14. 3. Schone, F. Vetter A, Hartung H, et al. Effects of essential oils from fennel (Foeniculi aetheroleum) and caraway (Carvi aetheroleum) in pigs. J Physiol Anim Nutr 2006; 90: 500-10. 4. Barreto MSR, Menten JFM, Racanici AMC, Pereira PWZ, Rizzo PV. Plant extracts used as growth promoters in broilers. Braz J Poultry Sci 2008; 10: 109-15. 5. Al-Kassie GAM. The effect of anise and rosemary on broiler performance. Int J Poultry Sci 2008; 7: 243-5. 6. Marcincak S, Popelka P, Soltysova L. Polyphenols and antioxidative activity of extracts from selected slovakian plants. Acta Sci Pol, Med Vet 2008; 7: 9-14. 7. Hernandez F, Madrid J, Garcia V, Orengo J, Megias MD. Influence of two plant extracts on broiler performance, digestibility and digestive organ size. Poultry Sci 2004; 83: 169-74. 8. Alcicek A, Bozkurt M, Cabuk M. The effect of a mixture of herbal essential oils, an organic acid or a probiotic on broiler performance. South Afr J Anim Sci 2004; 34: 217-22. 9. Basset R. Oregano's positive impact on poultry production. World Poultry 2000; 16: 31-4. 10. Ertas ON, Guler T, Ciftci M, Dalkilic B, Sim-sek UG. The effect of an essential oil mix derived from oregano, clove and anise on broiler performance. Int J Poultry Sci 2005; 4: 879-84. 11. Burt S. Essential oils: their antibacterial properties and potential applications in foods: a review. Int J Food Microbiol 2004; 94: 223-53. 12. Dalkilic B, Guler T. The effects of clove extract supplementation on performance and digestibility of nutrients in boilers. F U Sag Bil Vet Derg 2009; 23: 161-8. 13. Kamel KC. Tracing modes of action and the roles of plant extracts in non-ruminants. In: Garns-worthy PC, Wiseman J, eds. Recent advances in animal nutrition. Nottingham: University Press, 2001: 135-50. 14. Dorman HJD, Surai P, Deans SG. In vitro antioxidant activity of a number of plant essential oils and phytoconstituents. J Essent Oil Res 2000; 12: 241-8. 15. Dragland S, Senoo H, Wake K, Holte K, Blom-hoff R. Several culinary and medicinal herbs are important sources of dietary antioxidants. J Nutr 2003; 133: 1286-90. 16. Viuda-Martos M, Navajas YR, Zapata ES, Fernandez-Lopez J, Perez-Alvarez JA. Antioxidant activity of essential oils of five spice plants widely used in a Mediterranean diet. Flavour Fragrance J 2010; 25: 13-9. 17. Heilerova E, Buckova M, Tarapcik P, Silhar S, Labuda J. Comparison of antioxidative activity data for aqueous extracts of lemon balm, oregano, thyme and agrimony obtained by conventional methods and the DNA- based biosensor. Czech J Food Sci 2003; 21: 78-84. 18. Correia HS, Batista MT, Dinis TCP. The activity of an extract and fraction of Agrimonia eupatoria L. against reactive species. Biofactors 2007; 29: 91104. 19. Giao MS, Gonzalez-Sanjose ML, Muniz P, et al. Protection of deoxyribose and DNA from degradation brought about by aqueous extracts of several wild plants. J Sci Food Agric 2008; 88: 633-40. 20. Veterinary laboratory methods: food chemistry (In Slovak). Bratislava: Statna veterinarna sprava Press, 1990: 130-5. 21. Nagy J. Sensory evaluation of poultry and poultry products In: Pribela A, ed. Sensory evaluation of foodstuff, additives and food products (In Slovak). Kosice: IVVL Press, 2001: 112-6. 22. Pribela A. Sensory evaluation of foodstuff, additives and food products (In Slovak). Kosice: Institut vzdelavania veterinarnych lekarov Press, 2001: 191. 23. GraphPad Prism version 4.00 for Windows, GraphPad Software. San Diego, California: Graph-Pad, 2003. 24. Jang IS, Ko YH, Kang SY, Lee CY. Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Anim Feed Sci Tech 2007; 134: 304-15. 25. Lee KW, Everts H, Beynen AC. Essential oils in broiler nutrition. Int J Poultry Sci 2004; 12: 73852. 26. Symeon GK, Zintilas C, Demiris N, Bizelis IA, Deligeorgis SG. Effects of oregano essential oil dietary supplementation on the feeding and drinking behaviour as well as the activity of broilers. Int J Poutry Sci 2010; 9: 401-6. 27. Koreleski J, Swiatkiewicz S. Dietary supplementation with plant extract, xantophylls and syn-tetic antioxidants: effect on fatty acid profile and oxidative stability of frozen stored chicken breast meat. J Anim Feed Sci 2007; 16: 463-71. 28. Nasir Z, Grashorn MA. Effects of Echinacea purpurea and Nigella sativa supplementation on Effect of supplementation of phytogenic feed additives on performance parameters and meat quality of broiler chickens 33 broiler performance, carcass and meat quality. J Anim Feed Sci 2010; 19: 94-104. 29. Marcincäk S, Cabadaj R Popelka P, Soltyso-vä L. Antioxidative effect of oregano supplemented to broilers on oxidative stability of poultry meat. Slov Vet Res 2008; 45: 61-6. 30. Marcincak S, Popelka P, Nagy J, Hussein K, Hornanovä E. The impact of vitamin E and rosemary on the oxidative stability of poultry meat and ground meat products. Folia Vet 2004; 48: 165-8. 31. Spernakova D, Maté D, Rozanska H, Kovac G. Effects of dietary use of rosemary powder and a-tocopherol on performance of chicken, inhibition of lipid oxidation during storage at chilling conditions and increasing of meat quality. Bull Vet Inst Pulawy 2007; 51: 585-9. 34 S. Marcincák, P. Popelka, N. Zdolec, M. Mártonová, J. Simková, D. Marcincáková učinek fitogenih prehrambenih dodatkov na proizvodne parametre in KAKOVOSTI MESA pRI BROJLERSKIH piŠČANCIH S. Marcincák, P. Popelka, N. Zdolec, M. Mártonová, J. Šimková, D. Marcincáková Povzetek: Cilj poskusa je bil proučiti vplive prahu nageljnovih žbic (Syzygium aromaticum L) ter izvlečkov repika (Agrimonia eupatoria L) in navadne melise (Melissa officinalis L) na proizvodne parameter brojlerskih piščancev, njihove klavne lastnosti in kvaliteto mesa. Pripravljene so bile tri kombinacije dodatkov k prehrani: C - kontrolna skupina brez prehrambenega dodatka in dodatka v prehrani; AC - prehrambeni dodatek s prahom nageljnovih žbic in dodatkom izvlečka repika v pitni vodi; LC - hrana z dodatkom prahu nageljnovih žbic in pitna voda z dodatkom izvlečka melise. Skupina AC brojlerskih piščancev je imela po 14 dneh krmljenja statistično značilno povečano težo (P < 0.05) v primerjavi s kontrolno skupino. 42. dan krmljenja je bil pri skupinah brojlerskih piščancev AC in LC opažen boljši izkoristek krme, večji izplen pri klanju ter večja teža prsi in stegen. Statistično značilnih razlik (P < 0.05) v izbranih parametrih v poskusnih skupinah AC in LC v primerjavi s kontrolno skupino ni bilo. Suha snov v stegenskih mišicah brojlerskih piščancev skupin AC in LC je bila statistično značilno višja (P < 0.05) v primerjavi s kontrolno skupino. V skupinah AC in LC je bila v stegenskih mišicah ugotovljena manjša količina maščobe (P > 0.05) kot v kontrolni skupini in statistično značilno (P < 0.05) višje razmerje celotnih proteinov v skupini AC. Pri senzoričnem ocenjevanju mesa sta bili skupini AC in LC ocenjeni bolje, čeprav ni bilo opaznih statistično značilnih razlik (P > 0.05) v primerjavi s kontrolno skupino. Ključne besede: repik; nageljnove žbice; rast brojlerskih piščancev; navadna melisa; kakovost mesa ©MD Svetovanje Finančne storitve wvuw.vasef¡nance.s¡ Izterjava dolgov in upravljanje s terjatvami ^ Namen ustanovitve in delovanja podjetja MD svetovanje d. o. o. je pomagati podjetjem pri poslovanju z nudenjem produktov in storitev, ki ne spadajo v osnovno dejavnost podjetja. To dosegamo s celovita ponudbo predstavljenih produktov in storitev. Zato smo naš moto Skupaj bomo uspešnejši! nadgradili še z motom in sloganom Vse za Vas na enem mestu! Vizija Postati vodilna neodvisna družba s celotno ponudbo za podjetja in posameznike na enem mestu in na ta način prihraniti podjetjem in posameznikom čas in denar. Vse to nam bo uspelo s trdim delom in kakovostno izvedbo storitev in zaupanih nam nalog, predvsem če bomo sledili naslednjim načelom: zagotavljanje celovite ponudbe, vedno delo v dobro stranke, strokoven razvoj, organizacijsko izpopolnjevanje, zagotavljanje visoke stopnje kakovosti storitev z upoštevanjem predlogov naših strank, ustvarjanje novih delovnih mest, povečanje produktivnosti in dobičkonosnosti, visoko motiviran in usposobljen kader s primernim vodenjem, kar zagotavlja kakovost izvajanja storitev, postati vodilno podjetje, ki ponuja rešitve, ki podjetju omogočajo da si na enem mestu zagotovi vse dejavnosti, ki ne spadajo v njegovo osnovno dejavnost. 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If the source referred to was written by six authors or less, all of them should be cited; in the case of seven or more authors, only the first three should be cited, followed by 'et al.'. Any errata should be submitted to the editor-in-chief in good time after publication so that they may be published in the next issue. Examples of references Book: Hawkins JD. Gene structure and expression. Cambridge: University Press, 1991: 16. Chapterorar ticle in a book: Baldessarini RJ. Dopamine receptors and clinical medicine. In: Neve KA, Neve RL, eds. The dopamine receptors. Totowa: Human Press, 1996: 475-98. Article in a journal or newspaper: Fuji J, Otsu K, Zorzato F, et al. Identification of mutation in porcine ryanodine receptor asociated with malignant hyperthermia. Science 1991; 253: 448-51. Article in proceedings of a meeting or symposium: 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. Slovenski veterinarski zbornik (Slovenian Veterinary Research) objavlja izvirne prispevke, ki še niso bili objavljeni oz. poslani v objavo drugam. Za vse navedbe v prispevkih so odgovorni avtorji. Uredniška politika obsega publiciranje znanstvenih člankov, preglednih znanstvenih člankov, strokovnih člankov, povzetkov disertacij in drugih prispevkov, kot so kritične presoje o vsebini razprav, objavljenih v zborniku, kratke znanstvene prispevke, pisma uredniku in drugo. Avtorji pošljejo prispevke na naslov uredništva. Glavni urednik pregleda vse prispevke. Za vse članke je obvezna strokovna recenzija, za katero poskrbi uredništvo. Prispevki naj bodo napisani v angleškem jeziku, z naslovom, povzetkom in ključnimi besedami tudi v slovenščini. Obsegajo naj največ 12 strani, kar pomeni 27 vrstic na stran s približno 75 znaki v vrstici. Prispevki naj bodo poslani v elektronski obliki v katerem koli urejevalniku besedil za okensko okolje. 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 2011; 48 (1) Review Article phytogenic feed additives on performance parameters and meat quality of broiler chickens............. ... 27 o oo